Effects of Simiao Pill on rheumatoid arthritis complicated with interstitial lung disease via the ATX/LPA/LPA1 and RhoA/ROCK2 signaling pathways

Effects of Simiao Pill on rheumatoid arthritis complicated with interstitial lung disease via the ATX/LPA/LPA1 and RhoA/ROCK2 signaling pathways | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of Simiao Pill on rheumatoid arthritis complicated with interstitial lung disease via the ATX/LPA/LPA1 and RhoA/ROCK2 signaling pathways Jiahui Yan, Liang Han, Xin Ba, Pan Shen, Weiji Lin, Tingting Li, and 16 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6597839/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: In Traditional Chinese Medicine theory, Simiao Pill represents a traditional herbal formulation used for rheumatoid arthritis (RA) management. Initial research indicates that Simiao Pill might have therapeutic benefits for RA linked with interstitial lung disease (RA-ILD), although the mechanisms are not yet understood. Thus, this research seeks to explore the therapeutic potential of Simiao Pill in treating RA-ILD and uncover its molecular mechanisms. Methods: DBA/1 mice were used as animal models, with a collagen-induced arthritis model (CIA) and a bleomycin (BLM)-induced pulmonary fibrosis model (CIA-BLM). Ultra-high-performance liquid chromatography-tandem mass spectrometry was applied to profile the chemical composition of Simiao Pill. The therapeutic effects on arthritis and pulmonary fibrosis were evaluated through microcomputed tomography, histopathological examination, immunohistochemical staining, Western blot, ELISA assays, etc. Lipidomics analysis was performed to screen potential metabolic targets of Simiao Pill, followed by validation experiments on the identified targets. Results: Simiao Pill significantly reduced the arthritis index and decreased bone and cartilage damage in CIA-BLM mice. Additionally, it inhibited the expression of the inflammatory cytokines TNF-α, IL-6, CXCL1, and CXCL2 and reduced inflammatory cell infiltration in lung tissue. Most importantly, it downregulated the levels of the key profibrotic factor TGF-β1, the myofibroblast marker α-SMA, and fibrotic extracellular matrix components such as fibronectin, collagen-1, and collagen-3. These findings suggest that Simiao Pill may inhibits the transition of pulmonary fibroblasts to pulmonary myofibroblasts. Pulmonary lipidomics analysis revealed that lysophosphatidic acid (LPA) may serve as metabolic targets of Simiao Pill. Further validation experiments demonstrated elevated levels of autotaxin (ATX) and LPA in the serum and bronchoalveolar lavage fluid of CIA-BLM mice, accompanied by upregulation of LPA receptor 1 (LPA1) and downstream RhoA/ROCK2 signaling molecules in lung tissues. Notably, Simiao Pill effectively reversed these pathological alterations. Conclusions: Simiao Pill not only alleviated arthritis and bone destruction but also reduced pulmonary inflammation and fibrosis in CIA-BLM mice. The observed lung-protective properties may be attributed to inhibition of the ATX/LPA/LPA1 cascade and subsequent suppression of the RhoA/ROCK2 signaling axis, suggesting that Simiao Pill has potential therapeutic value for RA-ILD. Simiao Pill Rheumatoid arthritis Interstitial lung disease Autotaxin Lysophosphatidic acid Lysophosphatidic acid receptor 1 RhoA ROCK Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Rheumatoid arthritis (RA) is an autoimmune systemic inflammatory arthritis with a prevalence of approximately 0.5–1%[ 1 ]. The key pathological features of RA include synovial membrane inflammation, destruction of cartilage and bone tissues, and pannus proliferation, all of which contribute to progressive and irreversible joint deformities and loss of mobility[ 2 , 3 ]. RA is often accompanied by various extra-articular manifestations, among which RA-associated interstitial lung disease (RA-ILD) is the most prevalent. High-resolution computed tomography (HRCT) can detect ILD in up to 60% of RA patients, though only 10–18% exhibit clinically significant disease[ 4 , 5 ]. Bongartz et al . reported patients with RA alone had a median survival of 9.9 years, compared to only 2.6 years in those with RA-ILD [ 6 ]. Statistics show that ILD increases the mortality risk of RA patients by 2–10 times, ranking as the second leading cause of death in this population [ 7 , 8 ]. Established risk factors for RA-ILD include advanced age, male sex, tobacco exposure, exposure to air pollutants, active synovitis, rheumatoid factor (RF) and/or anti-citrullinated peptide antibodies (ACPAs) seropositivity, and MUC5B polymorphism[ 5 , 9 ]. In RA-ILD, usual interstitial pneumonia (UIP) is the most frequently observed histopathological pattern, with nonspecific interstitial pneumonia (NSIP) being the next most common. UIP exhibits a heterogeneous spatial distribution of pathological features, including patchy inflammation, fibroblast foci, collagen deposition, and honeycombing[ 9 ]. In contrast, NSIP is characterized by inflammation and fibrosis with a relatively homogeneous and diffuse distribution, usually lacking or showing only minimal fibroblast foci[ 9 , 10 ]. UIP is also a histopathological pattern of idiopathic pulmonary fibrosis (IPF). RA-UIP and IPF share many similar clinical and histopathological features, suggesting common pathogenic pathways and potential therapeutic targets[ 11 , 12 ]. Many fibrotic lung diseases, including RA-ILD and IPF, undergo several stages, regardless of etiology, involving lung tissue injury, inflammatory responses, and fibrosis due to impaired repair[ 10 ]. In the late stages, cells from various sources undergo functional and/or phenotypic switching, amplifying fibrotic responses. Among these factors, the activation of fibroblasts into myofibroblasts and their escape from quiescence, defined as the fibroblast–myofibroblast transition (FMT), is a key step in fibrogenesis[ 9 ]. Fibroblasts, myofibroblasts, and the excessive extracellular matrix (ECM) produced by the latter collectively contribute to the formation of fibroblast foci, driving the progression of pulmonary fibrosis[ 13 ]. Lysophosphatidic acid (LPA) is a bioactive phospholipid that regulates various biological processes, such as cell migration, proliferation, differentiation, and cytoskeletal organization[ 14 , 15 ]. LPA is widely distributed throughout the body, and circulating LPA is generated primarily by the autotaxin (ATX) enzyme, which catalyzes lysophospholipids[ 16 ]. In both RA patients and animal models, the expression levels of ATX and LPA in joint synovial fibroblasts are increased, promoting arthritis development[ 17 ]. In 2008, Tager et al . first demonstrated that LPA levels were elevated in the bronchoalveolar lavage fluid (BALF) of IPF patients and bleomycin (BLM)-induced pulmonary fibrosis mouse model[ 18 ]. Numerous subsequent studies confirmed that upregulation of the ATX/LPA axis is closely associated with various autoimmune diseases (including RA) and several pulmonary fibrotic diseases (including RA-ILD)[ 17 , 19 – 23 ]. LPA receptors 1–6 (LPA1–6) have been identified, with LPA shown to regulate the formation of pulmonary fibrosis in BLM-induced mice via LPA1 and LPA2[ 18 , 24 – 26 ]. Currently, the LPA1 antagonists BMS-986278 (NCT04308681) and BMS-986020 (NCT01766817) are undergoing phase II clinical trials for treating human IPF and progressive pulmonary fibrosis, while the involvement of other LPA receptors in pulmonary fibrosis has yet to be confirmed[ 27 ]. These findings suggest that the ATX/LPA/LPA1 pathway plays an important role in the process of pulmonary fibrosis. LPA promotes pulmonary fibrosis by activating multiple downstream signaling pathways through LPA1, particularly the RhoA/ROCK pathway [ 25 , 28 ]. Ras homolog family member A (RhoA), a key member of the Rho GTPase family, induces the formation of actin stress fibers and focal adhesions, thereby regulating cell morphology, adhesion, and motility[ 29 , 30 ]. RhoA activity is modulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and guanine nucleotide dissociation inhibitors (GDIs)[ 31 ]. RhoA cycles between an inactive GDP-bound state and an active GTP-bound state. Upon GTP binding, RhoA activates its downstream effector, Rho-associated coiled-coil-containing protein kinase (ROCK), which subsequently phosphorylates target substrates to regulate cell motility[ 32 – 34 ]. ROCK has two subtypes, ROCK1 and ROCK2, which are structurally and functionally similar. Studies have demonstrated that the RhoA/ROCK pathway is activated in the lungs of both BLM-induced pulmonary fibrosis model mice and IPF patients. Inhibition of this pathway has been shown to attenuate fibrotic phenotypes in the lung. Thus, targeting the RhoA/ROCK signaling pathway represents a promising therapeutic strategy for fibrotic lung diseases[ 35 , 36 ]. Currently, there is no optimal therapeutic strategy for RA-ILD patients. One major challenge in treating RA-ILD lies in the potential pulmonary toxicity of many RA therapies, including disease-modifying antirheumatic drugs (DMARDs) and biologic agents[ 4 ]. Traditional Chinese medicine (TCM) shows considerable potential in treating RA-ILD. According to TCM theory, RA is categorized as “Bi syndrome,” caused by the obstruction of muscles, bones, and joints by pathogenic wind, cold, and dampness. As the disease progresses, these external pathogens may invade the internal organs, including the lungs, disrupting their dispersing and descending functions. This leads to the accumulation of phlegm and blood stasis in the lungs, resulting in symptoms such as chest tightness, shortness of breath, coughing, and wheezing—manifesting as “Pulmonary Bi syndrome.” RA-ILD corresponds to this classification. Simiao Pill (SM) is a classic TCM formula for treating RA. It consists of four herbal components: Phellodendri Chinensis Cortex (Huang Bo), Atractylodis Rhizoma (Cang Zhu), Achyranthis Bidentatae Radix (Niu Xi), and Coicis Semen (Yiyi Ren). Cang Zhu strengthens the spleen, removes dampness, and dissolves phlegm; Huang Bo clears heat and eliminates dampness; Yiyi Ren promotes spleen function, dispels dampness, and alleviates Bi syndrome; and Niu Xi promotes blood circulation, dispels stasis, strengthens the liver and kidneys, and fortifies bones and muscles. The synergistic use of these herbs exerts both pathogen-expelling and body-strengthening effects, which may be beneficial in the treatment of RA-ILD. The purpose of this study was to create an RA-ILD animal model and evaluate the treatment effectiveness of SM using techniques such as ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), lipidomics, micro-computed tomography (micro-CT), western blotting, and immunohistochemical staining. Special attention will be paid to the regulatory effects of SM on the ATX/LPA/LPA1 and RhoA/ROCK signaling pathways. Materials and methods Construction of the CIA-BLM Mouse Model A combined mouse model of collagen-induced arthritis and BLM-induced pulmonary fibrosis (CIA-BLM) was established to simulate clinical RA-ILD. A total of 24 male DBA/1 mice, each 6 weeks old and weighing between 16 and 18 grams, were acquired from Beijing Vital River Laboratory Animal Technology Co., Ltd. (License No. SCXK [Beijing] 2016–0011) and kept in the specific pathogen-free (SPF)-grade animal barrier facility at Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China. All animal experiments and procedures were approved by the Institutional Animal Care and Use Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China (Approval No. TJH-202108002). The facility was maintained at a temperature of 20–26°C, relative humidity of 40–70%, with a 12-hour light/dark cycle, and five mice per cage. The mice were fed ad libitum. Following a week of acclimatization, the mice were divided randomly into four groups as control (CTL), CIA-BLM model, low-dose Simiao Pill (SM-L), and high-dose Simiao Pill (SM-H), with each group containing six mice. Except for the control group, the remaining three groups underwent CIA and BLM induction to establish the RA-ILD model. Construction of the CIA Animal Model: On day 0, 10 mg of type II bovine collagen powder (Chondrex, Cat. 20021) was dissolved in 5 ml of 2 mg/mL acetic acid solution containing type II bovine collagen (Chondrex, Cat. 20022) to obtain a final collagen concentration of 4 mg/mL. The collagen mixture was emulsified with an equal volume of 10 mg/mL complete Freund's adjuvant (Chondrex, Cat. 7027), and 100 µL of the emulsion was injected intradermally at the base of the tail for primary immunization. On day 21, 2 mg/mL type II bovine collagen (Chondrex, Cat. 20022) was emulsified with an equal volume of incomplete Freund's adjuvant (Chondrex, Cat. 7002) to perform secondary immunization. An equivalent volume of physiological saline was administered to control animals using identical delivery protocols. Arthritis severity was evaluated using a visual scoring scale based on paw erythema, swelling, and ankylosis. The arthritis index (AI) was graded as follows: 0 = normal; 1 = swelling and/or redness of the paw or one digit; 2 = involvement of two digits; 3 = three digits; and 4 = severe arthritis involving the entire paw and digits, accompanied by skin desquamation and wrist/ankle swelling[ 37 ]. Each mouse had a maximum score of 16 on the basis of its four paws. Mice were considered to have a relatively successful arthritis model if their AI score was 6 or greater. Construction of the BLM-induced animal model: On day 33, BLM powder (MedChemExpress, Cat. HY-17565A) was dissolved in saline to obtain a 1 mg/mL solution. The solution was administered intratracheally to three groups of CIA mice at a dose of 2 mg/kg using a microsprayer aerosolizer and a mouse laryngoscope (both from Shanghai Yuyan Instruments Co., Ltd.). Animals assigned to the control group received isovolumetric sterile 0.9% NaCl solution delivered via the same transoral intratracheal administration method. Preparation and Component Identification of SM SM is composed of Atractylodis Rhizoma (Cang Zhu), Achyranthis Bidentatae Radix (Niu Xi), Phellodendri Chinensis Cortex (Huang Bo), and Coicis Semen (Yiyi Ren) in a ratio of 1:1:2:2. The formulation used in this study was identical to that employed in our previous research[ 38 ], which included its fingerprint chromatogram (see Additional file 1). All herbs were purchased from Beijing Traditional Chinese Medicine Co., Ltd. The extraction and concentration of the herbal granules were conducted in accordance with Good Manufacturing Practices (GMPs) (FDA, 2010). The therapeutic effect of 1 g of SM granules is equivalent to that of 6 g of traditional decoction. SM granules were dissolved in saline and administered orally at doses equivalent to 2.16 g/kg⋅d⁻¹ and 8.63 g/kg⋅d⁻¹, designated the SM-L and SM-H groups, respectively. Therapeutic intervention commenced on day 30, with daily treatments maintained for 28 days. Vehicle control groups received equivalent volumes of physiological saline via identical gavage procedures. The administration method, concentration, dosage, and frequency of SM were based on our team's previous study[ 38 , 39 ]. In addition, the prepared SM was subjected to three rounds of analysis using UHPLC-MS/MS. In addition, the prepared SM was analyzed in three sequential steps using UHPLC-MS/MS. First, the metabolites of SM were extracted. SM granules were ground into powder, and approximately 50 ± 2 mg of the powder was lyophilized, mixed with beads, and suspended in 500 µL of extraction solvent (MeOH:ACN:H₂O = 2:2:1, v/v/v) containing deuterated internal standards. The mixture was vortexed for 30 s, homogenized at 35 Hz for 240 s, and sonicated in a 4°C water bath for 5 min. This homogenization and sonication cycle was repeated three times. Subsequently, the mixture was incubated at − 40°C for 30 min to precipitate proteins. The samples were then centrifuged at 12,000 rpm (RCF = 13,800 ×g, R = 8.6 cm) for 15 min at 4°C. The resulting supernatants were transferred to fresh vials and incubated for 10 min, followed by a second centrifugation under the same conditions. The final supernatants were collected and transferred to fresh glass vials for subsequent analysis. A quality control (QC) sample was prepared by pooling equal aliquots of the supernatants from all samples. Second, LC-MS/MS analysis was performed on the extracted metabolites. The chromatographic separation was achieved using a Thermo Vanquish UHPLC platform equipped with a Phenomenex Kinetex C18 analytical column (2.1 mm × 100 mm, 2.6 µm particle size), coupled to an Orbitrap Exploris 120 high-resolution mass spectrometer. The mobile phase system comprised: (A) aqueous 0.01% acetic acid and (B) an isopropanol-acetonitrile (1:1, v/v) mixture. The autosampler was maintained at a temperature of 4°C, and the injection volume was set to 2 µL. The Orbitrap Exploris 120 utilized Xcalibur software from Thermo Fisher Scientific to gather MS/MS spectra in information-dependent acquisition mode. In this mode, full-scan MS spectra were continuously evaluated to trigger MS/MS acquisition. The ESI source was operated under optimized conditions with the following settings: sheath gas at 50 Arb, auxiliary gas maintained at 15 Arb, and the capillary heater set to 320°C. Mass spectrometric analyses were performed with a full scan resolution of 60,000 and a MS/MS resolution of 15,000. A stepped normalized collision energy (NCE) of 20, 30, and 40 was applied for fragmentation. The spray voltage was configured at + 3.8 kV for positive ion mode detection and − 3.4 kV for negative ion mode analysis. Finally, the acquired data were preprocessed and annotated. Raw data were converted to mzXML format using ProteoWizard and subsequently processed using an in-house R-based program built on the XCMS platform for peak detection, extraction, alignment, and integration. Metabolite identification was performed using an R-based package and the BiotreeDB (v3.0). Micro-CT After the treatment period, all mice were fasted for 12 hours. One mouse from each group was randomly selected for lung and joint micro-CT scanning to visually assess differences in pulmonary fibrosis and bone destruction among groups. Mice were anesthetized with 1% pentobarbital, and lung scans were performed. The ankle joints were dissected, fixed in 4% paraformaldehyde for 48 hours, and subsequently scanned. Micro-CT scanning was performed using the Bruker SkyScan 1276 system (Kontich, Belgium) under the following conditions: voxel size, 6.534 µm; medium resolution; voltage, 70 kV; current, 200 µA; aluminum filter, 1 mm; and integration time, 525 ms. Density calibration was conducted using a calcium hydroxyapatite (CaHA) phantom provided by the manufacturer. Image reconstruction was performed using NRecon software (version 1.7.4.2), and 3D images were generated from 2D grayscale contour images via distance transformation (CTvox, version 3.3.0). Both 2D and 3D analyses were carried out using CTAn software (version 1.18.8.0). Hematoxylin and Eosin (H&E) Staining Fresh lung, kidney, spleen, and liver tissues were fixed in 4% paraformaldehyde at room temperature for over 24 hours. Ankle joints were decalcified in EDTA solution on a constant-temperature shaker. After embedding the tissues in paraffin, they were sectioned and stained with H&E solution (BaiQianDu Biotechnology, Cat. B1003). Sections were first deparaffinized in xylene (3 × 15 min), followed by rehydration in absolute ethanol (2 × 5 min), then in 95% and 85% ethanol (5 min each). Subsequently, hematoxylin staining, differentiation, bluing, eosin staining, and dehydration were performed. Finally, the slides were mounted with neutral resin. An Olympus BX51 microscope (Olympus, Japan) was used to capture images, with blue-stained nuclei and pink-stained cytoplasm. Image analysis was conducted using ImageJ software. Masson Staining Lung tissue sections were stained using a Masson’s trichrome staining kit (Servicebio, Cat. G1006). Following standard deparaffinization and hydration steps as performed in H&E staining, the sections were incubated in Masson A solution overnight, then sequentially immersed in a 1:1 mixture of Masson B and C solutions for 1 minute, Masson D solution for 6 minutes, Masson E solution for 1 minute, and Masson F solution for 2–30 seconds. Finally, the sections were rinsed with 1% acetic acid, dehydrated, and mounted. An Olympus BX51 microscope (Olympus, Japan) was used to capture images. Collagen fibers appeared blue, while muscle fibers, fibrin, and red blood cells appeared red. Image analysis was performed using ImageJ software. Sirius Red Staining Lung tissue sections were stained using a Sirius Red staining kit (Servicebio, Cat. G1078). Following deparaffinization and hydration, the sections were briefly air-dried and immersed in modified Sirius Red A solution for 2 minutes. After rinsing twice with tap water, the sections were immersed in modified Sirius Red B solution for 30 minutes, followed by dehydration and mounting. An Olympus BX51 microscope (Olympus, Japan) was used to capture images. Collagen fibers appeared red, and the background appeared yellow under bright-field microscopy. Image analysis was conducted using ImageJ software. Safranin O-Fast Green (SO/FG) Staining Sections of the ankle joint were stained with a Safranin O-Fast Green staining kit (Biossci, Cat. BBL-0209). Following deparaffinization and hydration, the sections were stained with Safranin O solution for approximately 1–5 minutes, rinsed with water, and subsequently stained with Fast Green solution for another 1–5 minutes. The sections were then differentiated in absolute ethanol for 5–10 minutes, followed by clearing and mounting. An Olympus BX51 microscope (Olympus, Japan) was used to capture images. Under light microscopy, cartilage appeared bright red to orange-red, bone tissue appeared green, and some connective tissues also appeared red. ELISA Serum and BALF samples were collected from mice for ELISA analysis. The following ELISA kits were used: Mouse CXCL1/KC (ABclonal, Cat. RK00038), Mouse MIP-2/CXCL2 (ABclonal, Cat. RK04208), Mouse TNF-α (ABclonal, Cat. RK00027), Mouse LPA (Bioswamp, Cat. MU30816), Mouse TGF-β1 (Boster, Cat. EK0515), and Mouse ATX (JINGMEI BIOTECHNOLOGY, Cat. JM-13131M1). Cytokine concentrations in serum and BALF were quantified according to the manufacturers’ standard protocols. Immunohistochemical staining Immunohistochemical staining was performed on lung and ankle joint tissue sections collected from mice. The procedure included deparaffinization and hydration, antigen retrieval, quenching of endogenous peroxidase activity, serum blocking, primary antibody incubation, and dehydration followed by mounting. A DAB chromogenic kit (Servicebio, Cat. G1212-200T) was used for signal detection, and normal goat serum (Servicebio, Cat. G1208-5ML) was applied as the blocking agent. The secondary antibodies were goat anti-rabbit (Servicebio, Cat. GB23303) and goat anti-mouse (Servicebio, Cat. GB23301). The primary antibodies were TNF-α rabbit pAb (ABclonal, Cat. A0277), IL-6 rabbit pAb (Servicebio, Cat. GB11117), and EDG2 polyclonal antibody ( tech, Cat. 20442-1-AP). An Olympus BX51 microscope (Olympus, Japan) was used to capture images, which were then analyzed with ImageJ software. Immunofluorescence Immunofluorescence staining was performed on lung tissue sections collected from mice. The procedure included deparaffinization and hydration, antigen retrieval, serum blocking, antibody incubation, and dehydration followed by mounting. Normal goat serum (Servicebio, Cat. G1208-5ML) was used as the blocking agent. The primary antibodies were as follows: α-smooth muscle actin (ACTA2) rabbit monoclonal antibody (CST, Cat. #19245), smooth muscle actin-specific mouse monoclonal antibody (Proteintech, Cat. 67735-1-Ig), fibronectin mouse monoclonal antibody (Proteintech, Cat. 66042-1-Ig), and ROCK2 (middle) rabbit polyclonal antibody (Proteintech, Cat. 21645-1-AP). The secondary antibodies were Cy3-labeled goat anti-mouse IgG (Servicebio, Cat. GB21301) and FITC-labeled goat anti-rabbit IgG (Servicebio, Cat. GB22303). Nuclei were counterstained with DAPI staining solution (ready-to-use; Servicebio, Cat. G1012-10ML), and sections were mounted using anti-fade mounting medium (Servicebio, Cat. G1401-5ML). An Olympus BX51 microscope (Olympus, Japan) was used to capture images, which were then analyzed with ImageJ software. Western blot analysis Western blot analysis was performed on mouse ankle joint and lung tissues. Proteins were extracted using strong RIPA lysis buffer (Servicebio, Cat. G2002-100ML) supplemented with phosphatase inhibitor (Servicebio, Cat. G2007-1ML) and 50× protease inhibitor cocktail (Servicebio, Cat. G2006-250UL). The levels of protein were measured with a BCA protein quantification kit (Servicebio, Cat. G2026-200T). Protein samples with equivalent loading quantities were resolved via SDS-PAGE and subsequently electroblotted onto PVDF membranes employing a wet transfer system. Following transfer, the membranes were first incubated with NcmBlot blocking solution (NCM Biotech, Cat. P30500) to prevent nonspecific binding, then incubated (16h, 4°C) with the following primary antibodies: β-actin mouse mAb (Proteintech, Cat. 66009-1-Ig, 1:10,000), fibronectin mouse mAb (Proteintech, Cat. 66042-1-Ig, 1:5,000), collagen I/COL1A1 rabbit pAb (ABclonal, Cat. A1352, 1:1,000), collagen III (N-terminal) rabbit pAb (Proteintech, Cat. 22734-1-AP, 1:1,000), EDG2 rabbit pAb (Proteintech, Cat. 20442-1-AP, 1:1,000), ENPP2 rabbit pAb (Proteintech, Cat. 14243-1-AP, 1:1,000), RhoA [KD Validated] rabbit pAb (ABclonal, Cat. A18695, 1:1,000), ROCK1 rabbit mAb (ABclonal, Cat. A11158, 1:5,000), and ROCK2 rabbit mAb (ABclonal, Cat. A2395, 1:1,000). The immunoblots were subjected to 1-hour room temperature incubation with species-matched HRP-conjugated secondary antibodies: goat anti-rabbit IgG (H + L) (ABclonal, Cat. AS014) or goat anti-mouse IgG (H + L) (ABclonal, Cat. AS003). Immunoreactive bands were detected using the SuperFemto ECL Chemiluminescence Kit (Vazyme Biotech Co., Ltd, Cat. E423-01) and visualized with the Tanon chemiluminescent imaging system. Protein band intensities were quantified using ImageJ software. Widely targeted lipidomics The whole right lung lobe of the mouse was removed, put into a cryopreservation tube, and quickly frozen in liquid nitrogen before being stored in a freezer at -80°C. Subsequently, the frozen mouse lung tissue was thawed and homogenized to prepare the supernatant. The sample extracts were subjected to analysis via an LC-ESI-MS/MS setup, comprising a UPLC (ExionLC AD, details available at https://sciex.com.cn/ ) and a QTRAP® System mass spectrometer (further information accessible at https://sciex.com/ ). The conditions set for the analysis were as follows: for the UPLC, a Thermo Accucore™ C30 column (dimensions: 2.1 mm internal diameter by 100 mm length, particle size: 2.6 µm) was utilized. The solvent system was composed of two phases: Phase A consisted of acetonitrile and water (60:40, v/v) supplemented with 0.1% formic acid and 10 mmol/L ammonium formate, while Phase B comprised a mixture of acetonitrile and isopropanol (10:90, v/v), also containing 0.1% formic acid and 10 mmol/L ammonium formate. Gradient elution began with an A/B ratio of 80:20 (v/v) and was adjusted sequentially as follows: 70:30 at 2.0 minutes, 40:60 at 4.0 minutes, 15:85 at 9.0 minutes, 10:90 at 14.0 minutes, and 5:95 at 15.5 minutes. This 5:95 ratio was maintained until 17.3 minutes, after which the system returned to 80:20 and was held constant until 20.0 minutes. The flow rate was maintained at 0.35 ml/min, with the column temperature set at 45°C, and an injection volume of 2 µL. The eluent was then directed to an ESI-triple quadrupole-linear ion trap (QTRAP)-MS for further analysis. The linear ion trap (LIT) and triple quadrupole (QQQ) scans were conducted using a QTRAP® LC-MS/MS System, which integrates a triple quadrupole-linear ion trap mass spectrometer (QTRAP) with an ESI Turbo Ion-Spray interface. The system was operated in both positive and negative ion modes and managed by Analyst 1.6.3 software (Sciex). The operational settings for the ESI source included: a turbo spray ion source, a source temperature maintained at 500°C, and ion spray voltages of 5500 V for positive mode and − 4500 V for negative mode. The mass spectrometry system was operated with optimized gas parameters: ion source gas 1 (GS1, 45 psi), ion source gas 2 (GS2, 55 psi), curtain gas (CUR, 35 psi), and collision gas (CAD, medium setting). For instrument calibration, polypropylene glycol standard solutions were employed at 10 µmol/L (QQQ mode) and 100 µmol/L (LIT mode). The QQQ scans utilized multiple reaction monitoring (MRM) experiments, employing nitrogen as the collision gas at a pressure of 5 psi. Each MRM transition had optimized parameters like declustering potential (DP) and collision energy (CE). During each analysis period, a specific group of MRM transitions was monitored, corresponding to the metabolites eluting within that timeframe. Finally, qualitative analysis was performed based on the in-house targeted reference database MWDB (MetWare Database), utilizing the retention time (RT), parent-daughter ion pairs, and secondary mass spectrometry data of the detected compounds. Metabolite quantification was conducted using MRM mode on a triple quadrupole mass spectrometer. Statistical analysis All experimental data were analysed via GraphPad Prism 8.0.2 (263). The Shapiro‒Wilk test was first used to assess the normality of the data. For normally distributed data, homogeneity of variance was tested via one-way ANOVA. If both normality and homogeneity of variance were satisfied, Dunnett's multiple comparisons test was used for group comparisons. In cases of heteroscedasticity, the Brown‒Forsythe ANOVA test was used for intergroup comparisons. For nonnormally distributed data, the Kruskal‒Wallis test was applied. All experimental data are expressed as the means ± SEMs, and results yielding p < 0.05 were deemed statistically significant. Results Component Analysis of SM The metabolites in the SM were identified by matching the UHPLC-MS/MS results with the RT, precursor ion mass-to-charge ratio (MS1), and secondary mass spectra (MS2) of either reference standards or entries from public metabolite databases. A total of 1,227 chemical constituents were identified in SM (Table S1 ), Representative total ion chromatograms (TICs) obtained in positive and negative ion modes are shown in Figs. 1 A and 1 B, respectively. Based on the descending order of secondary mass spectral matching scores (MS2_score), the top ten compounds identified in positive ion mode were classified into the following categories: shikimates and phenylpropanoids, terpenoids, amino acids and peptides, polyketides, carbohydrates, and alkaloids (Table 1 ). In negative ion mode, the top ten compounds were mainly categorized as fatty acids, shikimates and phenylpropanoids, and carbohydrates (Table 2 ). According to existing data in the HERB database, among the 1,227 compounds, 29 originated from Atractylodis Rhizoma, 29 from Achyranthis Bidentatae Radix, 54 from Phellodendri Chinensis Cortex, and 20 from Coicis Semen (Table S2 ). These findings indicate that the identified metabolites represent the extractable chemical components from the four constituent herbs of SM. Table 1 Peak table of total ion chromatogram (TIC) in positive ion mode. ID MS2.name Formula mz rt 1 Schisandrin C24H32O7 415.2116 379.9 2 (2S,4aS,6aS,6bR,10S,12aS,14bS)-10-hydroxy-2,4a,6a,6b,9,9,12a-heptamethyl-13-oxo-3,4,5,6,6a,7,8,8a,10,11,12,14b-dodecahydro-1H-picene-2-carboxylic acid C30H46O4 471.3459 485.9 3 DEHP C24H38O4 391.2836 568.5 4 Schisandrin A C24H32O6 417.2265 461.1 5 Methylpyroglutamate C6H9NO3 126.0546 117.5 6 Kojic acid C6H6O4 143.0335 96.3 7 Galactose C6H12O6 203.052 42.7 8 Fructose C6H12O6 203.052 42.7 9 (9S,10S)-3,4,5,14,15,16-hexamethoxy-9,10-dimethyl-tricyclo [10.4.0.02,7] hexadeca-1(16),2,4,6,12,14-hexaen-9-ol C24H32O7 415.2116 379.9 10 N-Methylflindersine C15H15NO2 242.1171 420.9 Table 2 Peak table of total ion chromatogram (TIC) in negative ion mode. ID MS2.name Formula mz rt 1 trans-11-Eicosenoic acid C20H38O2 309.2797 588 2 cis-11.14-Eicosadienoic acid C20H36O2 307.2642 570.9 3 cis-9-Palmitoleic acid C16H30O2 253.2171 545.8 4 Myristic acid C14H28O2 227.2015 542.3 5 Undecanoic acid C11H22O2 185.1546 493.2 6 (E)-8-hydroxy-2,6-dimethyl-oct-2-enoic acid C10H18O3 185.1183 330.6 7 Salicylic acid C7H6O3 137.0245 291.3 8 Isoscopoletin C10H8O4 191.035 281.6 9 3-Hydroxybenzoic acid C7H6O3 137.0244 248.9 10 Vidarabine C10H13N5O4 266.0894 192.8 Effects of SM Intervention on CIA-BLM Mice As illustrated in Fig. 2 A, the schematic diagram presents the experimental workflow for the establishment of the CIA-BLM mouse model and subsequent SM intervention. Beginning on day 0, body weights were recorded every three days throughout the experimental period. As shown in Fig. 2 B, the trend in body weight change (mean ± SEM) over time is depicted for each group. Mice in the control group exhibited a gradual increase in body weight, while those in the CIA-BLM group displayed progressive weight loss, consistent with clinical observations in patients with active RA [ 40 ]. As shown in Fig. 2 C, there were no significant differences in the initial average body weight among the groups. However, following CIA induction and prior to SM treatment (Fig. 2 D), the average body weights of the three CIA groups were significantly lower than that of the control group, corroborating the trends observed in the line graph. After SM treatment (Fig. 2 E), there were no significant differences in body weight between the SM-treated groups and the untreated CIA-BLM group, indicating that SM administration did not markedly affect body weight in CIA-BLM mice. The spleen index, which reflects immune activation and functional status, was significantly elevated in the CIA-BLM group relative to untreated controls (Figs. 2 F– 2 G). SM-H administration exhibited a trend toward spleen index reduction. Furthermore, H&E staining of kidney and liver tissues (Fig. 2 H) revealed no notable histopathological differences among the groups, suggesting that SM treatment in CIA-BLM mice was relatively safe. SM Alleviates Joint Inflammation and Bone Destruction in CIA-BLM Mice As shown in Fig. 3 A, compared with the control mice, the CIA-BLM mice presented noticeable swelling in the paw, plantar, and ankle joints, accompanied by limping when walking, whereas ankle swelling was significantly reduced in the SM-treated mice. Beginning on day 30 of SM administration, all mice were evaluated every three days for the AI (Fig. 3 B). Prior to SM treatment, there were no significant differences in AI among the groups (Fig. 3 C), indicating a comparable baseline severity of arthritis. Over time, AI scores in the CIA-BLM group gradually increased, whereas SM treatment, especially in the SM-H group, significantly attenuated this progression (Fig. 3 D). At the end of the experiment, micro-CT scans and histopathological analyses were conducted on the right ankle joints from each group (one mouse randomly selected per group). The micro-CT results revealed severe bone erosion in the CIA-BLM group compared with the control group (Fig. 3 E), with notable reductions in bone density, bone surface density, and bone volume percentage (Figs. 3 F- 3 H). These parameters were significantly improved following SM treatment, especially in the SM-H group. H&E staining (Fig. 3 I) and SO/FG staining (Fig. 3 J) of the ankle joint sections revealed increased inflammatory cell infiltration around the joints, cartilage proliferation invading bone tissue, bone tissue damage, and incomplete or missing joint cavities in the CIA-BLM group. SM treatment noticeably alleviated these conditions. Collectively, these findings demonstrate that SM effectively alleviates joint inflammation and mitigates bone destruction in CIA-BLM mice. SM improves lung inflammation in CIA-BLM mice In various pulmonary fibrosis diseases, including RA-ILD, lung inflammation is a precursor to fibrosis[ 41 ]. To investigate whether SM can alleviate lung inflammation in CIA-BLM mice, we performed H&E staining on lung tissue and ELISA to measure the levels of the inflammatory factors TNF-α, CXCL1, and CXCL2 in the BALF. Additionally, immunohistochemistry was used to assess the expression levels of the inflammatory markers TNF-α and IL-6 in lung tissue. H&E staining results (Figs. 4 A- 4 B) revealed that, compared with the control group, the CIA-BLM group presented significant lung fibrosis, thickened alveolar walls, alveolar collapse, disordered structure, and extensive inflammatory cell infiltration in the alveolar spaces. In contrast, the SM-L and SM-H treatment groups presented marked reductions in alveolar inflammatory cell infiltration, as well as improvements in alveolar wall thickening and alveolar collapse. Compared with that in the control group (Fig. 4 C), TNF-α secretion in the BALF of CIA-BLM mice was significantly elevated, while SM-L and SM-H treatments notably reduced TNF-α secretion. Immunohistochemical staining for TNF-α confirmed these findings (Figs. 4 D& 4 F). Similarly, immunohistochemical staining for IL-6 (Figs. 4 E& 4 G) revealed that IL-6 expression levels in the lungs of CIA-BLM mice were elevated compared with those in the control group, and both the SM-L and SM-H treatments significantly inhibited IL-6 expression. Finally, compared with those in the control group, CXCL1 and CXCL2 levels in the BALF of CIA-BLM mice were markedly increased, whereas the expression of CXCL1 and CXCL2 was reduced to varying degrees in the SM-L and SM-H groups (Figs. 4 H- 4 I). In summary, SM treatment significantly inhibited the expression of inflammatory cytokines in the lungs of CIA-BLM mice, improving lung inflammation and reducing lung tissue damage. SM reduces lung fibrosis in CIA-BLM mice Micro-CT can visually assess the degree of lung fibrosis in mice, as lung CT values (measured in Hounsfield units, HU) reflect lung tissue density. Therefore, HU values indicate the extent of lung fibrosis. Figure 5 A shows that lung fibrosis in CIA-BLM mice was distributed centrally around the trachea and was mainly concentrated in the upper lobes of both lungs, with increased HU values. SM treatment reduced HU values (Fig. 5 D), indicating an improvement in lung fibrosis. A hallmark of lung fibrosis is tissue remodelling (including airway and lung parenchyma), which involves abnormal ECM deposition[ 42 ]. Collagen is a major component of the ECM. Sirius Red staining stains collagen red, while Masson staining stains collagen fibres blue. Figures 5 B and 5 C show a significant increase in collagen in the lung tissue of the CIA-BLM group compared with that in the control group, whereas SM treatment effectively reduced collagen deposition (Fig. 5 E- 5 F). SM May Regulates the FMT Process in CIA-BLM Mice α-Smooth muscle actin (α-SMA) is expressed primarily in the cytoplasm and is a key marker of myofibroblasts[ 43 ]. Increased α-SMA expression is a recognized indicator of the FMT process[ 44 ]. During fibrosis, myofibroblasts continuously proliferate, survive longer, and abnormally secrete large amounts of ECM[ 45 , 46 ]. Among these, type I collagen (Col 1), type III collagen (Col 3), and fibronectin (FN) are major ECM components[ 42 , 47 ]. Immunofluorescence staining revealed that α-SMA and FN expression in the lungs, especially in fibrotic foci, was significantly greater in the CIA-BLM group than in the control group. Both the SM-L and SM-H treatments markedly reduced α-SMA and FN expression levels (Fig. 6 A), suggesting that SM may alleviate lung fibrosis by inhibiting the FMT process. TGF-β1 signaling is a key mechanism underlying FMT and fibrosis and is closely associated with myofibroblast activation[ 48 ]. The ELISA results revealed a significant increase in TGF-β1 levels in the BALF of CIA-BLM mice, whereas SM treatment reduced these levels (Fig. 6 B), indicating that SM may inhibit myofibroblast activation. Western blot analysis revealed a marked increase in Col 1, Col 3, and FN expression in the lung tissue of CIA-BLM mice, whereas SM-L and SM-H treatments reduced Col 1 and Col 3 expression, with SM-H also reducing FN expression (Fig. 6 C- 6 F), indicating that SM treatment can inhibit ECM secretion by myofibroblasts. In summary, SM treatment reduces the expression of the fibrosis-driving factor TGF-β1 in the BALF of CIA-BLM mice and may alleviate lung fibrosis by inhibiting the FMT process. Lipidomics Reveals SM May Ameliorate RA-ILD Pulmonary Lesions via LPA Modulation Orthogonal partial least squares-discriminant analysis (OPLS-DA) modeling was implemented to both visualize intergroup discrimination and characterize significantly altered metabolites. To visualize group separations and identify significantly differential metabolites, was employed. Based on preliminary findings suggesting superior anti-fibrotic efficacy of the SM-H group, this study focused particularly on this treatment group. As shown in Figs. 7 A and 7 B, OPLS-DA score plots demonstrated distinct separations between: (1) CTRL and CIA-BLM groups, and (2) CIA-BLM and SM-H groups, indicating that SM-H treatment significantly altered pulmonary metabolic profiles in CIA-BLM model mice. Subsequent model validation (Figs. 7 C- 7 D) revealed R 2 Y = 0.993/ 0.998, with Q 2 = 0.887/0.617, confirming the OPLS-DA model's reliability, stability, predictive capacity, and absence of overfitting. Using variable importance in projection (VIP) scores from the OPLS-DA model, we preliminarily screened differential metabolites (VIP > 1, p < 0.05). Figure 7 E shows 461 differential metabolites between CTRL and CIA-BLM groups (201 downregulated, 260 upregulated), and 351 between CIA-BLM and SM-H groups (348 downregulated, 3 upregulated). Volcano plots illustrating these differences are presented in Figs. 7 F (CTRL vs CIA-BLM) and 7G (CIA-BLM vs SM-H). To elucidate the interrelationships of differential metabolites among groups, comparative analysis was performed and it revealed 173 shared differential metabolites across all three groups (Fig. 8 A). Further investigation identified 58 metabolites that were upregulated in CIA-BLM but downregulated after SM-H treatment. Cluster analysis (Fig. 8 B) showed these predominantly belonged to glycerophospholipids (GP), with additional sphingolipids (SP) and glycerolipids (GL). The violin plots were drawn for 58 common differential metabolites (Fig. 8 C). The results showed that 3 differential metabolites were LPA(Figure 8 D) in glycerophosphates and 34 differential metabolites were precursors of lysophosphatidic acid, which were phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), N-acyl-lysophosphatidylethanolamines (LNAPEs)、Lysophosphatidylcholines (LPCs)、and Lysophosphatidylethanolamines (LPEs) respectively. This implicates LPA as a potential metabolic target of SM intervention in CIA-BLM mice. KEGG pathway analysis (Fig. 8 E) of all differential metabolites identified 15 significantly enriched pathways. Ten pathways were common to both comparisons: Vitamin digestion and absorption、Thermogenesis、Regulation of lipolysis in adipocytes、Regulation of actin cytoskeleton、Metabolic pathways、Lipid and atherosclerosis、Insulin resistance、Glycerolipid metabolism、Fat digestion and absorption and Cholesterol metabolism. Notably, LPA was enriched in five pathways (Vitamin digestion and absorption、Regulation of actin cytoskeleton、Metabolic pathways、Glycerolipid metabolism and Fat digestion and absorption), suggesting its potential involvement in both RA-ILD pathogenesis and SM's therapeutic mechanism through these biological processes. SM Inhibits the ATX/LPA/LPA1 Signaling Pathway To further investigate the correlation between LPA and RA-ILD as well as SM treatment efficacy, validation experiments were conducted. It is well-established that LPA is present in all cells and biological fluids, including plasma, serum, and BALF. Accumulating evidence indicates that LPA concentrations are significantly increased in both biological fluids and pulmonary tissues from patients with fibrotic lung disease, as well as in bleomycin-challenged murine models of pulmonary fibrosis[ 28 ]. The ELISA results (Figs. 9 A- 9 B) indicated that, compared with those in the control group, LPA levels in the BALF and serum of CIA-BLM mice were significantly increased, whereas SM treatment, particularly at the high dose (SM-H), significantly reduced LPA levels. LPA is primarily generated by the catalytic action of ATX, which is expressed in bronchial epithelial cells and alveolar inflammatory macrophages and can be secreted into the BALF[ 17 ]. In serum, ATX mainly originates from adipose tissue. As shown in Figs. 9 C- 9 D, ATX levels in the BALF and serum of CIA-BLM mice were significantly elevated compared with those in the control group, and SM treatment markedly downregulated ATX expression. The western blot results for lung tissue were consistent with these findings (Figs. 9 E & 9 G), suggesting that the reduction in LPA levels in the BALF and circulation caused by SM is at least partially due to the inhibition of ATX production. LPA1 plays a crucial role in driving pulmonary fibrosis and is highly expressed on the surface of fibroblasts and myofibroblasts[ 23 ]. Western blot analysis (Figs. 9 E‒9F) revealed that LPA1 receptor expression in the lung tissue of CIA-BLM mice was significantly upregulated compared with that in the control group, whereas both SM-L and SM-H treatments significantly downregulated LPA1 expression. Figures 9 H and 9 I show representative images and quantification of LPA1 immunohistochemical staining in lung tissue, which was aligned with the immunoblotting data. In summary, SM may improve lung lesions by inhibiting the expression of the LPA-generating enzyme ATX, reducing LPA levels, and downregulating the expression of the downstream receptor LPA1 in lung tissue. SM Regulates the LPA1-Mediated RhoA/ROCK2 Pathway RhoA and ROCK are key regulators of cell locomotion mediated by reorganization of the actin cytoskeleton[ 49 ]. Studies have shown that inhibiting the RhoA/ROCK1/ROCK2 pathway not only suppresses the pulmonary fibrosis phenotype but also inhibits fibroblast activation, proliferation, and transformation into myofibroblasts, i.e., the FMT process[ 35 , 36 , 50 , 51 ]. Aligning with previous reports, immunoblot analysis (Figs. 10 A- 10 B) confirmed RhoA overexpression in CIA-BLM pulmonary tissues, which was markedly attenuated by SM intervention. However, unlike ROCK1, ROCK2 expression was markedly upregulated in the lung tissue of CIA-BLM mice (Figs. 10 A& 10 C- 10 D), and SM treatment was able to reduce ROCK2 expression levels. Immunofluorescence staining of lung tissue (Figs. 10 E- 10 G) revealed that α-SMA (a marker of myofibroblasts) and ROCK2 expression were elevated in CIA-BLM lung tissue, whereas fluorescence expression levels were decreased in the SM-treated group. In summary, SM may alleviate lung damage and tissue remodelling in CIA-BLM mice by regulating the LPA1 downstream RhoA/ROCK2 signaling pathway to inhibit the FMT process. Discussion Consequently, there are two hypotheses regarding the pathogenesis of lung involvement in RA: either immune tolerance to citrullinated proteins is first lost in synovial tissue, and these proteins subsequently cross-react with similar antigens in the lungs, or lung tolerance is first lost, leading to subsequent spread to the joints[ 12 ]. In any case, as the most common and severe manifestation of RA-related lung involvement, ILD generally presents with subtle symptoms in its early stages, often leading to severe lung damage at the time of diagnosis. This significantly delays the early diagnosis and treatment of RA-ILD. For example, a study of 167 RA-ILD patients revealed that at the time of diagnosis, 14% had a forced vital capacity (FVC) less than 50% of the predicted value, and 29% had a diffusing capacity of the lungs for carbon monoxide (DLco) less than 40% of the predicted value[ 52 ]. Such diagnostic delays increase the mortality rate of RA-ILD[ 53 ]. Therefore, emphasizing the early diagnosis and treatment of RA-ILD is crucial. Currently, there is no clearly effective treatment regimen for RA-ILD from any international scientific associations[ 5 ]. A prospective cohort study revealed that poor control of arthritis was associated with the development of RA-ILD[ 54 ]. Therefore, especially for high-risk populations, arthritis inflammation should be controlled to prevent potential future RA-ILD. However, a challenge arises in that some medications used to treat RA may have pulmonary toxicity, with methotrexate (MTX) being of particular concern. A meta-analysis involving 22 studies and 8,584 participants reported that MTX was associated with an increased risk of all adverse respiratory events and respiratory infections, although no increased mortality risk was observed[ 55 ]. However, some studies contradict these findings, and there is currently no compelling evidence suggesting the need to discontinue MTX in the presence of ILD[ 56 , 57 ]. Additionally, some studies have reported that the use of immunosuppressants can increase susceptibility to respiratory infections and mortality in RA-ILD patients[ 58 , 59 ]. Given the similarities between RA-ILD and IPF, the use of antifibrotic drugs such as nintedanib and pirfenidone can improve lung function in RA-ILD patients to some extent[ 57 , 60 , 61 ]. TCM may hold great potential for treating RA-ILD. The herbal formula SM is a classic TCM prescription for RA, supported by various studies: SM has been shown to reduce hind paw swelling and bone erosion in CIA and to prevent adjuvant induced arthritis in arthritic rats and significantly decrease the serum levels of the inflammatory factors TNF-α, IL-1β, and IL-6[ 39 , 62 ]. The mechanisms by which SM exerts its therapeutic effects on RA may involve multiple signaling pathways, including the TLR4/MyD88/IRAK4/MAPK, ATX/LPA, MAPK, JAK2/STAT3, VEGF, and PI3K/AKT pathways, along with several metabolic pathways including arachidonic acid metabolism, glycerophospholipid metabolism, and tryptophan metabolism[ 38 , 39 , 63 ]. Additionally, research suggests that SM may improve ILD in parallel with treating RA. Findings from metabolomics and network pharmacology indicate that SM may achieve early prevention of RA-ILD by regulating the ferroptosis pathway[ 64 ]. SM may also reduce pulmonary fibrosis by modulating the TGF-β/Smad2/3 pathway[ 38 ]. Our findings indicate that SM comprises 1,227 distinct chemical constituents. It not only significantly alleviates joint inflammation and mitigates bone and cartilage destruction in CIA-BLM mice, but also reduces pulmonary inflammation and fibrosis. According to the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and published literature, the therapeutic effects of SM may be attributed to key bioactive compounds such as berberine, coptisine, quercetin, vanillin, kaempferol, rutaecarpine, and ferulic acid[ 65 – 71 ]. The lipid-modulating properties of SM may be partially attributable to its oleic acid content, which could influence metabolic pathway regulation[ 72 ]. Lipidomic analysis of lung tissue further suggests that LPA and its precursors are likely key targets of SM in the treatment of RA-ILD. Subsequent experimental validation confirmed that SM significantly downregulates the ATX/LPA/LPA1 signaling axis and its downstream effectors, including the RhoA/ROCK2 pathway. Moreover, SM exhibited no apparent hepatotoxicity or nephrotoxicity, indicating a favorable safety profile. Taken together, these findings indicate SM's potential as a novel therapeutic candidate for managing RA-ILD. Various immune cytokines are known to jointly contribute to the development of lung inflammation in RA-ILD. During lung inflammation, M1 macrophages secrete multiple proinflammatory factors, such as TNF-α, IL-6, and IL-1α, and the chemokines CXCL1-3, CXCL5, and CXCL8-10, promoting lung tissue inflammation and damage[ 73 ]. TNF-α can increase corticosteroid insensitivity in airway smooth muscle, leading to increased expression of inflammatory genes and cytokine secretion[ 74 , 75 ]. As a key player in the cytokine network, IL-6 promotes B-cell differentiation into plasma cells and plays a critical role in cytokine storms[ 76 – 78 ]. Conversely, IL-6 inhibition can reduce LPS-induced acute lung injury[ 79 ]. CXCL1 and CXCL2 function primarily to recruit immune cells, especially neutrophils[ 80 ]. Studies have shown that inhibiting the expression of CXCL1 and CXCL2 can decrease inflammatory cell infiltration and alleviate LPS-induced lung injury[ 81 ]. According to Zhang and colleagues, SM can lower the serum concentrations of TNF-α, IL-6, and IL-1β in mice suffering from hyperuricemia[ 82 ]. Similarly, Jie et al. reported that Simiao Yong'an Decoction significantly decreased the levels of TNF-α, IL-6, IL-1β, as well as the chemokines CXCL1 and CXCL2 in the joints of CIA mice[ 83 ]. Our findings indicate that SM treatment downregulates the expression levels of the inflammatory cytokines TNF-α, IL-6, CXCL1, and CXCL2 in the lung tissue of CIA-BLM mice, reduces inflammatory cell infiltration, and exacerbates structural damage to the lungs. The pathogenic mechanisms of various pulmonary fibrotic diseases, including RA-ILD and IPF, share certain similarities. Under the influence of susceptibility factors, repeated microinjuries to lung tissue lead to the death of alveolar epithelial cells (AECs)[ 84 ]. Type 2 alveolar epithelial cells (AEC2s), which possess stem cell-like functions, are activated to participate in repair, but dysfunctional AEC2s result in abnormal repair processes. At this stage, abnormally activated AECs secrete various growth factors and chemokines, recruiting resident fibroblasts and bone marrow-derived fibroblasts to the site of injury. These activated epithelial cells also secrete TGF-β1, promoting epithelial‒mesenchymal transition (EMT) and FMT[ 85 ]. Together, fibroblasts, myofibroblasts, and newly formed collagen form “fibroblast foci.” Within these foci, myofibroblasts secrete excessive ECM proteins, driving the development of lung fibrosis. Myofibroblast activation is the central step in lung fibrosis, with myofibroblasts originating from various cells, including resident fibroblasts, epithelial cells, endothelial cells, fibrocytes, pericytes, and macrophages[ 86 ]. FMT is considered a primary source of myofibroblasts[ 87 ]. Studies have shown that TGF-β1 stimulation increases the transition of human lung fibroblast-1 cells into myofibroblasts and induces mouse embryonic fibroblasts to produce elevated levels of Col, FN, and α-SMA[ 46 , 88 ]. Chen et al. suggested that SM can alleviate pulmonary interstitial thickening and inflammatory cell infiltration in the lungs of CIA rats[ 64 ]. Similarly, Ba et al. reported that SM suppresses pulmonary collagen deposition and the EMT process in CIA-BLM mice[ 38 ]. In our study, CIA-BLM mice exhibited significantly elevated levels of TGF-β1 in BALF, along with pronounced pulmonary fibrosis. This was accompanied by increased expression of the myofibroblast marker α-SMA and ECM components, including FN, Col 1, and Col 3. SM treatment downregulated the expression of α-SMA and ECM proteins and attenuated the extent of pulmonary fibrosis. These findings suggest that the anti-fibrotic effect of SM in RA-ILD may be mediated, at least in part, by its ability to inhibit the FMT process. LPA is a simple phospholipid consisting of a phosphate head group, a glycerol moiety, and a single fatty acid chain. It is not a single molecule but exists in multiple forms, such as 1-acyl-LPA, 2-acyl-LPA, and alkyl-LPA. Biologically active LPA is produced through only two pathways: the first involves secreted ATX, which catalyzes other lysophospholipids (mainly LPC) outside the cell membrane to generate LPA, which is the primary source of extracellular LPA. The second pathway involves membrane-bound PA-selective PLA1α (mPA-PLA1α), which catalyzes PA within the cell membrane to produce LPA[ 89 ]. ATX was initially identified in 1992 in human melanoma cells as an effective motility factor and is encoded by the Enpp2 gene[ 90 ]. It was later shown to be a secreted lysophospholipase D (lysoPLD)[ 89 ]. ATX exhibits catalytic activity in most body fluids, including serum and BALF[ 23 ]. LPA functions by binding to six different LPA receptor subtypes (LPA1-LPA6), which play various roles in numerous biological processes. LPA receptors are specific GPCRs, with LPA1 primarily coupling to Gαi, Gαq, and Gα13. LPA1 belongs to the EDG family and is also known as EDG2 and Vzg-1[ 89 ]. Growing evidence implicates the ATX-LPA-LPA1 signaling cascade in the pathogenesis of diverse disorders spanning oncology (malignancies), hepatology (hepatitis), cardiology (acute coronary syndrome), rheumatology (RA), and IPF[ 91 – 95 ]. The role of the ATX‒LPA‒LPA‒1 axis in pulmonary fibrosis has been extensively studied. ATX is significantly elevated in the BALF of BLM-induced mice, and conditional gene deletion of ATX in bronchial epithelial cells or alveolar macrophages reduces ATX activity in BALF, decreases vascular leakage and inflammatory cell infiltration, and reduces the expression of TGF-β and collagen in BLM-induced mice[ 96 , 97 ]. This effect is attributed to reduced LPA production. Elevated LPA levels in the BALF of IPF patients stimulate fibroblast migration, mediate fibroblast recruitment and vascular leakage, and induce TGF-β, FN, α-SMA, and Col expression in lung fibroblasts through multiple signaling pathways[ 18 , 26 , 98 ]. Further experiments have shown that LPA primarily activates, proliferates, migrates, and induces resistance to apoptosis in lung fibroblasts via LPA1 rather than LPA2/3[ 18 , 28 , 99 – 102 ]. This process strongly promotes FMT and fibroblast focus formation. Moreover, the absence of LPA1 not only provides significant protection against fibrosis and mortality in BLM-induced pulmonary fibrosis mouse models but also reduces fibroblast proliferation, migration, and vascular leakage in the lungs. Additionally, LPA1 is involved in several stages of fibrosis progression, including ECM accumulation, epithelial cell apoptosis, myofibroblast differentiation, and endothelial barrier disruption[ 18 , 103 ]. Therefore, LPA1 is an effective target for regulating fibroblast activation and the FMT process. Earlier research has demonstrated that SM can influence lipid metabolism issues in mice with high lipid levels and enhance insulin resistance and liver lipid buildup in diabetic mice, suggesting its potential in regulating metabolic dysregulation[ 104 , 105 ]. Our findings further demonstrate that, in addition to alleviating pulmonary fibrosis in CIA-BLM mice, SM effectively reverses the upregulated ATX/LPA/LPA1 signaling pathway observed in this model. LPA connects to the downstream Gα13-dependent Rho GTPase/ROCK pathway via LPA1[ 103 ]. RhoA, one of the most prominent members of the Rho GTPase family, acts as a molecular switch regulating cytoskeletal protein activation[ 106 ]. As a key downstream effector of RhoA, ROCK belongs to the serine/threonine kinase family and is closely related to a range of cellular functions, including activation, migration, proliferation, apoptosis, differentiation, and contraction[ 107 ]. The ROCK signaling pathway is a critical regulator of profibrotic signal transduction, as it influences actin-myosin organization and ECM remodelling[ 108 ]. ROCK has two paralogues, ROCK1 and ROCK2[ 109 ]. Previously, Watts et al . demonstrated that the overexpression of connective tissue growth factor and myofibroblast formation in IPF cell lines are RhoA-dependent[ 110 ]. The Rho kinase inhibitor fasudil reduces the extent of BLM-induced pulmonary fibrosis[ 111 ]. Recent preclinical studies have shown that the selective ROCK2 inhibitor GNS-3595 effectively inhibits ROCK2-mediated myosin light chain phosphorylation in vitro; reduces fibrosis-related protein expression (e.g., Col, FN, and α-SMA) in various in vitro cell models; and prevents TGF-β1-induced FMT[ 108 ]. Our study revealed that total RhoA protein and ROCK2 were significantly upregulated in the lung tissue of CIA-BLM mice, whereas ROCK1 expression was seemingly unaffected. SM treatment downregulated the RhoA/ROCK2 signaling pathway, suggesting that SM may alleviate pulmonary interstitial lesions in CIA-BLM mice by regulating the RhoA/ROCK2 signaling pathway. Conclusion This study demonstrates that SM, a classic anti-rheumatic formula, achieves dual therapeutic effects on both arthritis and pulmonary fibrosis in the CIA-BLM mouse model by modulating the ATX/LPA/LPA1-RhoA/ROCK2 axis, thereby providing mechanistic and pharmacodynamic evidence for TCM in treating RA-ILD—a major clinical challenge. In summary, our findings confirm that SM not only alleviates arthritis symptoms but also mitigates pulmonary inflammation and fibrosis in CIA-BLM mice, likely through downregulating the ATX/LPA/LPA1-RhoA/ROCK2 signaling pathway. These results offer a translational candidate with integrated TCM-Western mechanistic insights for RA-ILD management, while also validating the multi-target therapeutic principle of TCM from the perspective of "treating different diseases with the same method." Although further in vitro studies are needed to delineate SM’s bioactive compounds and target interactions, this work establishes a novel framework for developing anti-fibrotic drugs from traditional herbal repositories. Declarations Ethics approval and consent to participate All animal experiments and procedures were approved by the Institutional Animal Care and Use Committee of Tongji Hospital, affiliated with Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China (TJH-202108002). Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Funding This work was funded by grants from the National Natural Science Foundation of China (82074267, 82474285, 82174185, 82204872, 82104639, 82204871), the Hubei Provincial Natural Science Foundation Joint Fund (2025AFD590), the Hubei Provincial Department of Science and Technology (2022CFB742), and the Jingmen Science and Technology Bureau Key Projects (2022YFZD014). Authors' contributions ZC: resources, conceptualization; ST: resources, conceptualization, writing review and editing; YJ: methodology, formal analysis, writing-original draft, writing-review & editing; LH: resources, conceptualization, writing review and editing; XB: conceptualization, writing-review and editing; RZ: conceptualization, writing-review and editing; YY: conceptualization, writing-review and editing; YL: conceptualization, writing-review and editing; CL: conceptualization, writing-review and editing; GL: methodology; YH: resources, conceptualization; resources, conceptualization; YH: resources, conceptualization; resources, conceptualization; KQ: methodology; SY: resources, conceptualization; resources, conceptualization PS: methodology; WL: methodology; TL: methodology. YW: methodology; YG: conceptualization; YL: resources; HH: conceptualization; LZ: methodology. All authors contributed to the article and approved the submitted version. Acknowledgements We would like to express our gratitude to the Experimental Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology for providing the Tanon chemiluminescence imaging system for Western blot analysis. We also extend our sincere appreciation to Biotree Biotech Co., Ltd. (Shanghai, China) for their contribution to UHPLC-MS/MS analysis. Figure 11 was created using BioRender (authorization ID: VI2865W8ZI). Availability of data and materials Data used to support the fndings of this study are available from the corresponding author upon request. References Smolen JS, Aletaha D, McInnes IB. Rheumatoid arthritis. Lancet (London England). 2016;388(10055):2023–38. Smith MH, Berman JR. What Is Rheumatoid Arthritis? Jama. 2022;327(12):1194. Aletaha D, Smolen JS. Diagnosis and Management of Rheumatoid Arthritis: A Review. JAMA. 2018;320(13):1360–72. Kadura S, Raghu G. Rheumatoid arthritis-interstitial lung disease: manifestations and current concepts in pathogenesis and management. Eur respiratory review: official J Eur Respiratory Soc. 2021;30(160). Koduri G, Solomon JJ, Arthritis. & rheumatology (Hoboken, NJ). 2023;75(12):2067–77. Bongartz T, Nannini C, Medina-Velasquez YF, Achenbach SJ, Crowson CS, Ryu JH, et al. Incidence and mortality of interstitial lung disease in rheumatoid arthritis: a population-based study. Arthritis Rheum. 2010;62(6):1583–91. Hyldgaard C, Hilberg O, Pedersen AB, Ulrichsen SP, Løkke A, Bendstrup E, et al. A population-based cohort study of rheumatoid arthritis-associated interstitial lung disease: comorbidity and mortality. Ann Rheum Dis. 2017;76(10):1700–6. Pinheiro FA, Souza DC, Sato EI. A Study of Multiple Causes of Death in Rheumatoid Arthritis. J Rhuematol. 2015;42(12):2221–8. Kim Y, Yang HI, Kim KS. Etiology and Pathogenesis of Rheumatoid Arthritis-Interstitial Lung Disease. Int J Mol Sci. 2023;24(19). Thannickal VJ, Toews GB, White ES, Lynch JP 3rd, Martinez FJ. Mechanisms of pulmonary fibrosis. Annu Rev Med. 2004;55:395–417. Leavy OC, Kawano-Dourado L, Stewart ID, Quint JK, Solomon JJ, Borie R, et al. Rheumatoid arthritis and idiopathic pulmonary fibrosis: a bidirectional Mendelian randomisation study. Thorax. 2024;79(6):538–44. Spagnolo P, Lee JS, Sverzellati N, Rossi G, Cottin V, Arthritis. & rheumatology (Hoboken, NJ). 2018;70(10):1544–54. Nishioka Y, Araya J, Tanaka Y, Kumanogoh A. Pathological mechanisms and novel drug targets in fibrotic interstitial lung disease. Inflamm regeneration. 2024;44(1):34. Salgado-Polo F, Borza R, Matsoukas MT, Marsais F, Jagerschmidt C, Waeckel L, et al. Autotaxin facilitates selective LPA receptor signaling. Cell Chem biology. 2023;30(1):69–e8414. Volkmann ER, Denton CP, Kolb M, Wijsenbeek-Lourens MS, Emson C, Hudson K et al. Lysophosphatidic acid receptor 1 inhibition: a potential treatment target for pulmonary fibrosis. Eur respiratory review: official J Eur Respiratory Soc. 2024;33(172). Cao P, Walker NM, Braeuer RR, Mazzoni-Putman S, Aoki Y, Misumi K, et al. Loss of FOXF1 expression promotes human lung-resident mesenchymal stromal cell migration via ATX/LPA/LPA1 signaling axis. Sci Rep. 2020;10(1):21231. Magkrioti C, Galaris A, Kanellopoulou P, Stylianaki EA, Kaffe E, Aidinis V. Autotaxin and chronic inflammatory diseases. J Autoimmun. 2019;104:102327. Tager AM, LaCamera P, Shea BS, Campanella GS, Selman M, Zhao Z, et al. The lysophosphatidic acid receptor LPA1 links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak. Nat Med. 2008;14(1):45–54. Bohdziewicz A, Pawlik KK, Maciejewska M, Sikora M, Alda-Malicka R, Czuwara J et al. Future Treatment Options in Systemic Sclerosis-Potential Targets and Ongoing Clinical Trials. J Clin Med. 2022;11(5). Orosa B, García S, Conde C. The autotaxin-lysophosphatidic acid pathway in pathogenesis of rheumatoid arthritis. Eur J Pharmacol. 2015;765:228–33. Tsuchida Y, Shoda H, Sawada T, Fujio K. Role of autotaxin in systemic lupus erythematosus. Front Med. 2023;10:1166343. Ninou I, Magkrioti C, Aidinis V. Autotaxin in Pathophysiology and Pulmonary Fibrosis. Front Med. 2018;5:180. Kano K, Aoki J, Hla T. Lysophospholipid Mediators in Health and Disease. Annu Rev Pathol. 2022;17:459–83. Zulfikar S, Mulholland S, Adamali H, Barratt SL. Inhibitors of the Autotaxin-Lysophosphatidic Acid Axis and Their Potential in the Treatment of Interstitial Lung Disease: Current Perspectives. Clin pharmacology: Adv Appl. 2020;12:97–108. Hemmings DG, Brindley DN. Signalling by lysophosphatidate and its health implications. Essays Biochem. 2020;64(3):547–63. Huang LS, Fu P, Patel P, Harijith A, Sun T, Zhao Y, et al. Lysophosphatidic acid receptor-2 deficiency confers protection against bleomycin-induced lung injury and fibrosis in mice. Am J Respir Cell Mol Biol. 2013;49(6):912–22. Corte TJ, Lancaster L, Swigris JJ, Maher TM, Goldin JG, Palmer SM et al. Phase 2 trial design of BMS-986278, a lysophosphatidic acid receptor 1 (LPA(1)) antagonist, in patients with idiopathic pulmonary fibrosis (IPF) or progressive fibrotic interstitial lung disease (PF-ILD). BMJ open respiratory Res. 2021;8(1). Suryadevara V, Ramchandran R, Kamp DW, Natarajan V. Lipid Mediators Regulate Pulmonary Fibrosis: Potential Mechanisms and Signaling Pathways. Int J Mol Sci. 2020;21(12). Xiang SY, Dusaban SS, Brown JH. Lysophospholipid receptor activation of RhoA and lipid signaling pathways. Biochim Biophys Acta. 2013;1831(1):213–22. Etienne-Manneville S, Hall A. Rho GTPases in cell biology. Nature. 2002;420(6916):629–35. Jaffe AB, Hall A. Rho GTPases: biochemistry and biology. Annu Rev Cell Dev Biol. 2005;21:247–69. Riento K, Ridley AJ. Rocks: multifunctional kinases in cell behaviour. Nat Rev Mol Cell Biol. 2003;4(6):446–56. Amin E, Dubey BN, Zhang SC, Gremer L, Dvorsky R, Moll JM, et al. Rho-kinase: regulation, (dys)function, and inhibition. Biol Chem. 2013;394(11):1399–410. Amano M, Nakayama M, Kaibuchi K. Rho-kinase/ROCK: A key regulator of the cytoskeleton and cell polarity. Cytoskeleton (Hoboken NJ). 2010;67(9):545–54. Monaghan-Benson E, Wittchen ES, Doerschuk CM, Burridge K. A Rnd3/p190RhoGAP pathway regulates RhoA activity in idiopathic pulmonary fibrosis fibroblasts. Mol Biol Cell. 2018;29(18):2165–75. Knipe RS, Probst CK, Lagares D, Franklin A, Spinney JJ, Brazee PL, et al. The Rho Kinase Isoforms ROCK1 and ROCK2 Each Contribute to the Development of Experimental Pulmonary Fibrosis. Am J Respir Cell Mol Biol. 2018;58(4):471–81. Paoletti A, Ly B, Cailleau C, Gao F, de Ponfilly-Sotier MP, Pascaud J, et al. Liposomal AntagomiR-155-5p Restores Anti-Inflammatory Macrophages and Improves Arthritis in Preclinical Models of Rheumatoid Arthritis. Volume 76. Hoboken, NJ: Arthritis & rheumatology; 2024. pp. 18–31. 1. Ba X, Wang H, Huang Y, Yan J, Han L, Lin W, et al. Simiao pill attenuates collagen-induced arthritis and bleomycin-induced pulmonary fibrosis in mice by suppressing the JAK2/STAT3 and TGF-β/Smad2/3 signalling pathway. J Ethnopharmacol. 2023;309:116274. Shen P, Tu S, Wang H, Qin K, Chen Z. Simiao Pill Attenuates Collagen-Induced Arthritis in Rats through Suppressing the ATX-LPA and MAPK Signalling Pathways. Evidence-based complementary and alternative medicine: eCAM. 2019;2019:7498527. England BR, Baker JF, Sayles H, Michaud K, Caplan L, Davis LA, et al. Body Mass Index, Weight Loss, and Cause-Specific Mortality in Rheumatoid Arthritis. Arthritis Care Res. 2018;70(1):11–8. Savin IA, Zenkova MA, Sen'kova AV. Pulmonary Fibrosis as a Result of Acute Lung Inflammation: Molecular Mechanisms, Relevant In Vivo Models, Prognostic and Therapeutic Approaches. Int J Mol Sci. 2022;23(23). Liu G, Philp AM, Corte T, Travis MA, Schilter H, Hansbro NG, et al. Therapeutic targets in lung tissue remodelling and fibrosis. Pharmacol Ther. 2021;225:107839. Xu H, Yang J, Tu M, Weng J, Xie M, Zhou Z, et al. Vincristine Promotes Transdifferentiation of Fibroblasts Into Myofibroblasts via P38 and ERK Signal Pathways. Front Pharmacol. 2022;13:901000. Lin X, Lei Y, Pan M, Hu C, Xie B, Wu W, et al. Augmentation of scleral glycolysis promotes myopia through histone lactylation. Cell Metabol. 2024;36(3):511–e257. Montero P, Milara J, Roger I, Cortijo J. Role of JAK/STAT in Interstitial Lung Diseases; Molecular and Cellular Mechanisms. Int J Mol Sci. 2021;22(12). Tanner L, Single AB, Bhongir RKV, Heusel M, Mohanty T, Karlsson CAQ, et al. Small-molecule-mediated OGG1 inhibition attenuates pulmonary inflammation and lung fibrosis in a murine lung fibrosis model. Nat Commun. 2023;14(1):643. Wang J, Jiang M, Xiong A, Zhang L, Luo L, Liu Y, et al. Integrated analysis of single-cell and bulk RNA sequencing reveals pro-fibrotic PLA2G7(high) macrophages in pulmonary fibrosis. Pharmacol Res. 2022;182:106286. Györfi AH, Matei AE, Distler JHW. Targeting TGF-β signaling for the treatment of fibrosis. Matrix biology: J Int Soc Matrix Biology. 2018;68–69:8–27. Shimizu Y, Dobashi K, Iizuka K, Horie T, Suzuki K, Tukagoshi H, et al. Contribution of small GTPase Rho and its target protein rock in a murine model of lung fibrosis. Am J Respir Crit Care Med. 2001;163(1):210–7. Tsoyi K, Chu SG, Patino-Jaramillo NG, Wilder J, Villalba J, Doyle-Eisele M, et al. Syndecan-2 Attenuates Radiation-induced Pulmonary Fibrosis and Inhibits Fibroblast Activation by Regulating PI3K/Akt/ROCK Pathway via CD148. Am J Respir Cell Mol Biol. 2018;58(2):208–15. Rackow AR, Nagel DJ, Zapas G, Clough RS, Sime PJ, Kottmann RM. The Novel Small Molecule BTB Inhibits Pro-Fibrotic Fibroblast Behavior though Inhibition of RhoA Activity. Int J Mol Sci. 2022;23(19). Zamora-Legoff JA, Krause ML, Crowson CS, Ryu JH, Matteson EL, Arthritis. & rheumatology (Hoboken, NJ). 2017;69(3):542–9. Cano-Jiménez E, Vázquez Rodríguez T, Martín-Robles I, Castillo Villegas D, Juan García J, Bollo, de Miguel E et al. Diagnostic delay of associated interstitial lung disease increases mortality in rheumatoid arthritis. Scientific reports. 2021;11(1):9184. Sparks JA, He X, Huang J, Fletcher EA, Zaccardelli A, Friedlander HM et al. Rheumatoid Arthritis Disease Activity Predicting Incident Clinically Apparent Rheumatoid Arthritis-Associated Interstitial Lung Disease: A Prospective Cohort Study. Arthritis & rheumatology (Hoboken, NJ). 2019;71(9):1472–82. Conway R, Low C, Coughlan RJ, O'Donnell MJ, Carey JJ. Methotrexate and lung disease in rheumatoid arthritis: a meta-analysis of randomized controlled trials. Arthritis Rheumatol (Hoboken NJ). 2014;66(4):803–12. Dawson JK, Graham DR, Desmond J, Fewins HE, Lynch MP. Investigation of the chronic pulmonary effects of low-dose oral methotrexate in patients with rheumatoid arthritis: a prospective study incorporating HRCT scanning and pulmonary function tests. Rheumatology (Oxford). 2002;41(3):262–7. Pugashetti JV, Lee JS. Overview of Rheumatoid Arthritis-Associated Interstitial Lung Disease and Its Treatment. Semin Respir Crit Care Med. 2024;45(3):329–41. Mena-Vázquez N, Redondo-Rodriguez R, Rojas-Gimenez M, Romero-Barco CM, Fuego-Varela C, Perez-Gómez N, et al. Rate of severe and fatal infections in a cohort of patients with interstitial lung disease associated with rheumatoid arthritis: a multicenter prospective study. Front Immunol. 2024;15:1341321. Kim JW, Shin K, Jung J, Suh CH, Kim J, Lee SK, et al. Uncovering risk factors for adverse events and infections in rheumatoid arthritis and rheumatoid arthritis with interstitial lung disease under treatment with biologics or targeted synthetic DMARDs: insights from the KOBIO Registry. Clin Exp Rheumatol. 2024;42(9):1781–91. Matteson EL, Aringer M, Burmester GR, Mueller H, Moros L, Kolb M. Effect of nintedanib in patients with progressive pulmonary fibrosis associated with rheumatoid arthritis: data from the INBUILD trial. Clin Rheumatol. 2023;42(9):2311–9. Solomon JJ, Danoff SK, Woodhead FA, Hurwitz S, Maurer R, Glaspole I, et al. Safety, tolerability, and efficacy of pirfenidone in patients with rheumatoid arthritis-associated interstitial lung disease: a randomised, double-blind, placebo-controlled, phase 2 study. Lancet Respiratory Med. 2023;11(1):87–96. Wang X, Zhang X, Zhang L, Li Y. [Effects and mechanisms of Simiao pill on adjuvant arthritis rats model]. Zhongguo Zhong yao za zhi = Zhongguo zhongyao zazhi = China. J Chin materia Med. 2010;35(21):2889–92. Wang Y, Zhang F, Li X, Li X, Wang J, He J, et al. Integrated Multi-Omics Techniques and Network Pharmacology Analysis to Explore the Material Basis and Mechanism of Simiao Pill in the Treatment of Rheumatoid Arthritis. ACS omega. 2023;8(12):11138–50. Chen Y, Liu H, Han R, Lin J, Yang J, Guo M, et al. Analyzing how SiMiao Wan regulates ferroptosis to prevent RA-ILD using metabolomics and cyberpharmacology. Phytomedicine: Int J phytotherapy phytopharmacology. 2024;133:155912. Huang DN, Wu FF, Zhang AH, Sun H, Wang XJ. Efficacy of berberine in treatment of rheumatoid arthritis: From multiple targets to therapeutic potential. Pharmacol Res. 2021;169:105667. Xu F, Shen C, Zhang S, Liu Y, Liu D, Kuang Y, et al. Coptisine inhibits aggressive and proliferative actions of fibroblast like synoviocytes and exerts a therapeutic potential for rheumatoid arthritis. Int Immunopharmacol. 2024;128:111433. Shen P, Lin W, Deng X, Ba X, Han L, Chen Z, et al. Potential Implications of Quercetin in Autoimmune Diseases. Front Immunol. 2021;12:689044. Tao Y, Pu J, Wang P. Ethnobotany, phytochemistry, pharmacology and quality control of Peucedanum decursivum (Miq.) Maxim: A critical review. J Ethnopharmacol. 2024;334:118542. He X, Wu T, He H, Chen L, Han K, Zheng J, et al. Study of kaempferol in the treatment of rheumatoid arthritis through modulation of the NLRP3/CASP1/GSDMD axis and T-cell activation: Based on network pharmacology, single-cell analysis, and experimental validation. Int Immunopharmacol. 2024;143(Pt 1):113357. Guo B, Zhao C, Zhang C, Xiao Y, Yan G, Liu L, et al. Elucidation of the anti-inflammatory mechanism of Er Miao San by integrative approach of network pharmacology and experimental verification. Pharmacol Res. 2022;175:106000. Doss HM, Samarpita S, Ganesan R, Rasool M. Ferulic acid, a dietary polyphenol suppresses osteoclast differentiation and bone erosion via the inhibition of RANKL dependent NF-κB signalling pathway. Life Sci. 2018;207:284–95. Lei T, Jiang C, Zhao L, Zhang J, Xiao Q, Chen Y et al. Exploring the Mechanism of Topical Application of Clematis Florida in the Treatment of Rheumatoid Arthritis through Network Pharmacology and Experimental Validation. Pharmaceuticals (Basel Switzerland). 2024;17(7). Arabpour M, Saghazadeh A, Rezaei N. Anti-inflammatory and M2 macrophage polarization-promoting effect of mesenchymal stem cell-derived exosomes. Int Immunopharmacol. 2021;97:107823. Britt RD Jr., Ruwanpathirana A, Ford ML, Lewis BW. Macrophages Orchestrate Airway Inflammation, Remodeling, and Resolution in Asthma. Int J Mol Sci. 2023;24(13). Savin IA, Zenkova MA, Sen'kova AV. Bronchial Asthma, Airway Remodeling and Lung Fibrosis as Successive Steps of One Process. Int J Mol Sci. 2023;24(22). Jones SA, Jenkins BJ. Recent insights into targeting the IL-6 cytokine family in inflammatory diseases and cancer. Nat Rev Immunol. 2018;18(12):773–89. Tanaka T, Narazaki M, Kishimoto T. Immunotherapeutic implications of IL-6 blockade for cytokine storm. Immunotherapy. 2016;8(8):959–70. Uciechowski P, Dempke WCM. Interleukin-6: A Masterplayer in the Cytokine Network. Oncology. 2020;98(3):131–7. Chen IC, Wang SC, Chen YT, Tseng HH, Liu PL, Lin TC et al. Corylin Ameliorates LPS-Induced Acute Lung Injury via Suppressing the MAPKs and IL-6/STAT3 Signaling Pathways. Pharmaceuticals (Basel, Switzerland). 2021;14(10). Zhou C, Gao Y, Ding P, Wu T, Ji G. The role of CXCL family members in different diseases. Cell death discovery. 2023;9(1):212. Song Y, Xu C, Wu J, Shu J, Sheng H, Shen Y. Palmatine alleviates LPS-induced acute lung injury via interfering the interaction of TAK1 and TAB1. Biochem Pharmacol. 2022;202:115120. Zhang Y, Wang S, Dai X, Liu T, Liu Y, Shi H, et al. Simiao San alleviates hyperuricemia and kidney inflammation by inhibiting NLRP3 inflammasome and JAK2/STAT3 signaling in hyperuricemia mice. J Ethnopharmacol. 2023;312:116530. Jie SS, Sun HJ, Liu JX, Gao Y, Bai D, Zhu LL, et al. Simiao Yong'an decoction ameliorates murine collagen-induced arthritis by modulating neutrophil activities: An in vitro and in vivo study. J Ethnopharmacol. 2023;305:116119. King TE Jr., Pardo A, Selman M. Idiopathic pulmonary fibrosis. Lancet (London England). 2011;378(9807):1949–61. Richeldi L, Collard HR, Jones MG. Idiopathic pulmonary fibrosis. Lancet (London England). 2017;389(10082):1941–52. Rockey DC, Bell PD, Hill JA. Fibrosis–a common pathway to organ injury and failure. N Engl J Med. 2015;372(12):1138–49. Zhang H, Zhou Y, Wen D, Wang J. Noncoding RNAs: Master Regulator of Fibroblast to Myofibroblast Transition in Fibrosis. Int J Mol Sci. 2023;24(2). Tanner L, Bergwik J, Single AB, Bhongir RKV, Erjefält JS, Egesten A. Zoledronic Acid Targeting of the Mevalonate Pathway Causes Reduced Cell Recruitment and Attenuates Pulmonary Fibrosis. Front Pharmacol. 2022;13:899469. Yanagida K, Shimizu T. Lysophosphatidic acid, a simple phospholipid with myriad functions. Pharmacol Ther. 2023;246:108421. Stracke ML, Krutzsch HC, Unsworth EJ, Arestad A, Cioce V, Schiffmann E, et al. Identification, purification, and partial sequence analysis of autotaxin, a novel motility-stimulating protein. J Biol Chem. 1992;267(4):2524–9. Bese T, Barbaros M, Baykara E, Guralp O, Cengiz S, Demirkiran F, et al. Comparison of total plasma lysophosphatidic acid and serum CA-125 as a tumor marker in the diagnosis and follow-up of patients with epithelial ovarian cancer. J gynecologic Oncol. 2010;21(4):248–54. Abdul Rahman M, Mohamad Haron DE, Hollows RJ, Abdul Ghani ZDF, Ali Mohd M, Chai WL, et al. Profiling lysophosphatidic acid levels in plasma from head and neck cancer patients. PeerJ. 2020;8:e9304. Dohi T, Miyauchi K, Ohkawa R, Nakamura K, Kishimoto T, Miyazaki T, et al. Increased circulating plasma lysophosphatidic acid in patients with acute coronary syndrome. Clin Chim Acta. 2012;413(1–2):207–12. Pattanaik D, Postlethwaite AE. A role for lysophosphatidic acid and sphingosine 1-phosphate in the pathogenesis of systemic sclerosis. Discov Med. 2010;10(51):161–7. Watanabe N, Ikeda H, Nakamura K, Ohkawa R, Kume Y, Tomiya T, et al. Plasma lysophosphatidic acid level and serum autotaxin activity are increased in liver injury in rats in relation to its severity. Life Sci. 2007;81(12):1009–15. Black KE, Berdyshev E, Bain G, Castelino FV, Shea BS, Probst CK, et al. Autotaxin activity increases locally following lung injury, but is not required for pulmonary lysophosphatidic acid production or fibrosis. FASEB journal: official publication Federation Am Soc Experimental Biology. 2016;30(6):2435–50. Oikonomou N, Mouratis MA, Tzouvelekis A, Kaffe E, Valavanis C, Vilaras G, et al. Pulmonary autotaxin expression contributes to the pathogenesis of pulmonary fibrosis. Am J Respir Cell Mol Biol. 2012;47(5):566–74. Sakai T, Peyruchaud O, Fässler R, Mosher DF. Restoration of beta1A integrins is required for lysophosphatidic acid-induced migration of beta1-null mouse fibroblastic cells. J Biol Chem. 1998;273(31):19378–82. Qian Y, Hamilton M, Sidduri A, Gabriel S, Ren Y, Peng R, et al. Discovery of highly selective and orally active lysophosphatidic acid receptor-1 antagonists with potent activity on human lung fibroblasts. J Med Chem. 2012;55(17):7920–39. Wen J, Lin X, Gao W, Qu B, Zuo Y, Liu R, et al. Inhibition of LPA(1) Signaling Impedes Conversion of Human Tenon's Fibroblasts into Myofibroblasts Via Suppressing TGF-β/Smad2/3 Signaling. J ocular Pharmacol therapeutics: official J Association Ocular Pharmacol Ther. 2019;35(6):331–40. Hama K, Aoki J, Fukaya M, Kishi Y, Sakai T, Suzuki R, et al. Lysophosphatidic acid and autotaxin stimulate cell motility of neoplastic and non-neoplastic cells through LPA1. J Biol Chem. 2004;279(17):17634–9. Swaney JS, Chapman C, Correa LD, Stebbins KJ, Bundey RA, Prodanovich PC, et al. A novel, orally active LPA(1) receptor antagonist inhibits lung fibrosis in the mouse bleomycin model. Br J Pharmacol. 2010;160(7):1699–713. Fukushima N, Kimura Y, Chun J. A single receptor encoded by vzg-1/lpA1/edg-2 couples to G proteins and mediates multiple cellular responses to lysophosphatidic acid. Proc Natl Acad Sci USA. 1998;95(11):6151–6. Wang K, Xu W, He W, Ding M, Xia T, Tan X. Simiao Wan attenuates high-fat diet-induced hyperlipidemia in mice by modulating the gut microbiota-bile acid axis. J Ethnopharmacol. 2025;337(Pt 2):118868. Chen Y, Zhu L, Hu W, Wang Y, Wen X, Yang J. Simiao Wan modulates the gut microbiota and bile acid metabolism during improving type 2 diabetes mellitus in mice. Phytomedicine: Int J phytotherapy phytopharmacology. 2022;104:154264. Bros M, Haas K, Moll L, Grabbe S. RhoA as a Key Regulator of Innate and Adaptive Immunity. Cells. 2019;8(7). Bei Y, Hua-Huy T, Duong-Quy S, Nguyen VH, Chen W, Nicco C, et al. Long-term treatment with fasudil improves bleomycin-induced pulmonary fibrosis and pulmonary hypertension via inhibition of Smad2/3 phosphorylation. Pulm Pharmacol Ther. 2013;26(6):635–43. Hwang S, Lee W, Ravi D, Devine W, Yong M, Diebold RB, et al. Novel Small-Molecule ROCK2 Inhibitor GNS-3595 Attenuates Pulmonary Fibrosis in Preclinical Studies. Am J Respir Cell Mol Biol. 2024;71(4):430–41. Pernis AB, Ricker E, Weng CH, Rozo C, Yi W. Rho Kinases in Autoimmune Diseases. Annu Rev Med. 2016;67:355–74. Watts KL, Spiteri MA. Connective tissue growth factor expression and induction by transforming growth factor-beta is abrogated by simvastatin via a Rho signaling mechanism. Am J Physiol Lung Cell Mol Physiol. 2004;287(6):L1323–32. Jiang C, Huang H, Liu J, Wang Y, Lu Z, Xu Z. Fasudil, a Rho-kinase inhibitor, attenuates bleomycin-induced pulmonary fibrosis in mice. Int J Mol Sci. 2012;13(7):8293–307. Supplementary Files Abbreviation.xlsx Additionalfile1.pdf ChineseMedicineAuthorChecklist202503.pdf TableS1.xls.xlsx TableS2.xls.xlsx westernblot.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6597839","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":455986320,"identity":"4af021ba-a65b-4df7-be5f-c400b61e7f1b","order_by":0,"name":"Jiahui Yan","email":"","orcid":"","institution":"Tongji Medical College of HUST: Huazhong University of Science and Technology Tongji Medical College Tongji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jiahui","middleName":"","lastName":"Yan","suffix":""},{"id":455986321,"identity":"013dde19-9b77-465a-90b9-7d7847e78c6a","order_by":1,"name":"Liang Han","email":"","orcid":"","institution":"Tongji Medical College of HUST: Huazhong University of Science and Technology Tongji Medical College Tongji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Han","suffix":""},{"id":455986322,"identity":"97c7cd8a-79e3-496d-9a6b-881cb81f597b","order_by":2,"name":"Xin Ba","email":"","orcid":"","institution":"Tongji Medical College of HUST: Huazhong University of Science and Technology Tongji Medical College Tongji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Ba","suffix":""},{"id":455986323,"identity":"f8270ed8-76ff-4993-a3cd-79756be47bc7","order_by":3,"name":"Pan Shen","email":"","orcid":"","institution":"Tongji Medical College of HUST: Huazhong University of Science and Technology Tongji Medical College Tongji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Pan","middleName":"","lastName":"Shen","suffix":""},{"id":455986324,"identity":"3e3dfbde-56ab-4b60-841a-d17ac36360d7","order_by":4,"name":"Weiji Lin","email":"","orcid":"","institution":"Tongji Medical College of HUST: Huazhong University of Science and Technology Tongji Medical College Tongji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Weiji","middleName":"","lastName":"Lin","suffix":""},{"id":455986325,"identity":"7b7dce13-8000-4e34-b004-65bce62a6c3a","order_by":5,"name":"Tingting Li","email":"","orcid":"","institution":"Tongji Medical College of HUST: Huazhong University of Science and Technology Tongji Medical College Tongji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tingting","middleName":"","lastName":"Li","suffix":""},{"id":455986326,"identity":"e7e0bfe7-9d36-44b1-a65a-50d037bc994b","order_by":6,"name":"Ruiyuan Zhang","email":"","orcid":"","institution":"Tongji Medical College of HUST: Huazhong University of Science and Technology Tongji Medical College Tongji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ruiyuan","middleName":"","lastName":"Zhang","suffix":""},{"id":455986327,"identity":"50dab345-cbdd-4b0e-9d7d-24b6bf58d8e8","order_by":7,"name":"Yuyao Yang","email":"","orcid":"","institution":"Tongji Medical College of HUST: Huazhong University of Science and Technology Tongji Medical College Tongji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yuyao","middleName":"","lastName":"Yang","suffix":""},{"id":455986328,"identity":"387e7030-49b9-4cec-9541-adc4d5644ce8","order_by":8,"name":"Yifei Li","email":"","orcid":"","institution":"Tongji Medical College of HUST: Huazhong University of Science and Technology Tongji Medical College Tongji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yifei","middleName":"","lastName":"Li","suffix":""},{"id":455986329,"identity":"ffa9eb5f-50ed-4bae-9c40-b24b645a14ac","order_by":9,"name":"Chennan Li","email":"","orcid":"","institution":"Tongji Medical College of HUST: Huazhong University of Science and Technology Tongji Medical College Tongji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chennan","middleName":"","lastName":"Li","suffix":""},{"id":455986330,"identity":"a43ffffc-76a2-47b5-81c5-b4f26e064f4b","order_by":10,"name":"Guanghuan Li","email":"","orcid":"","institution":"Tongji Medical College of HUST: Huazhong University of Science and Technology Tongji Medical College Tongji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Guanghuan","middleName":"","lastName":"Li","suffix":""},{"id":455986331,"identity":"7b971e48-c8ca-431d-afb4-5de1e2efff4c","order_by":11,"name":"Yao Huang","email":"","orcid":"","institution":"Tongji Medical College of HUST: Huazhong University of Science and Technology Tongji Medical College Tongji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yao","middleName":"","lastName":"Huang","suffix":""},{"id":455986332,"identity":"291fb90e-aec2-4a43-a254-5d923acb2f1e","order_by":12,"name":"Ying Huang","email":"","orcid":"","institution":"Tongji Medical College of HUST: Huazhong University of Science and Technology Tongji Medical College Tongji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Huang","suffix":""},{"id":455986333,"identity":"7df9614a-6a38-4274-a475-75e96a55dc63","order_by":13,"name":"Kai Qin","email":"","orcid":"","institution":"Tongji Medical College of HUST: Huazhong University of Science and Technology Tongji Medical College Tongji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Qin","suffix":""},{"id":455986334,"identity":"414f1872-536b-4cb1-b7a2-d56b90ad7766","order_by":14,"name":"Yu Wang","email":"","orcid":"","institution":"Tongji Medical College of HUST: Huazhong University of Science and Technology Tongji Medical College Tongji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Wang","suffix":""},{"id":455986335,"identity":"8b57f1be-67e2-414d-8895-b0a82b3f247c","order_by":15,"name":"Yinhong Geng","email":"","orcid":"","institution":"Xinxiang Medical University First Affiliated Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yinhong","middleName":"","lastName":"Geng","suffix":""},{"id":455986336,"identity":"8ddddd99-ae25-4a11-ae9a-13af292edd07","order_by":16,"name":"Yafei Liu","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Yafei","middleName":"","lastName":"Liu","suffix":""},{"id":455986337,"identity":"c9fbfb59-32dd-4a0f-bd03-24d5b06d6734","order_by":17,"name":"Hua Huang","email":"","orcid":"","institution":"Jingmen People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hua","middleName":"","lastName":"Huang","suffix":""},{"id":455986338,"identity":"ce19a8f7-30e9-42d9-a711-09a8a84ecfd2","order_by":18,"name":"Shenghao Tu","email":"","orcid":"","institution":"Tongji Medical College of HUST: Huazhong University of Science and Technology Tongji Medical College Tongji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Shenghao","middleName":"","lastName":"Tu","suffix":""},{"id":455986339,"identity":"5ba669f4-1d2f-4ae9-8347-baaf4df51e33","order_by":19,"name":"Liang Zou","email":"","orcid":"","institution":"Jingmen People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Zou","suffix":""},{"id":455986340,"identity":"870a82db-4cef-461d-a558-d723da462ebf","order_by":20,"name":"Sisi Yang","email":"","orcid":"","institution":"Tongji Medical College of HUST: Huazhong University of Science and Technology Tongji Medical College Tongji Hospital","correspondingAuthor":false,"prefix":"","firstName":"Sisi","middleName":"","lastName":"Yang","suffix":""},{"id":455986341,"identity":"867f79df-2ed2-4d55-b7a1-64e052a10cad","order_by":21,"name":"Zhe Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0ElEQVRIiWNgGAWjYDACCSBOqJDg4WdvbHz4gXgtZ2zkJHsONxtLEK2FsS3N2GBGepsADzE65Gc3H3vwgO1w4gbJh21A/XZyug0EtDDOOZZukMBzOHG7dGLbgwKGZGOzAwS0MEvkmEkkSBxO3Dk7sd1AguFA4jZCWtgk8r9JJBgAHXbzYJsEDzFaeCRy2CQSEoDev8FIpBYJiTRzg4QDoEBOBAayARF+kZ+R/Ozhz3+gqDz+8OGHCjs5glpA3kFiGxBWjq5lFIyCUTAKRgEWAACGFkKHu4Y4igAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-8654-9218","institution":"Tongji Medical College of HUST: Huazhong University of Science and Technology Tongji Medical College Tongji Hospital","correspondingAuthor":true,"prefix":"","firstName":"Zhe","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2025-05-06 01:08:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6597839/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6597839/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82889345,"identity":"70241eca-dba0-40e5-9f90-1f0bca00b5b8","added_by":"auto","created_at":"2025-05-16 12:05:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":282950,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe base peak ion chromatogram obtained by UHPLC-MS/MS analysis of SM samples. \u003c/strong\u003e(A) and (B) represent the total ion current chromatogram of a SM sample in positive and negative ion modes, respectively.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6597839/v1/e2a9ae250501f73a3128369b.png"},{"id":82891599,"identity":"d7b47c62-db5a-43b6-8905-a3b353f44dae","added_by":"auto","created_at":"2025-05-16 12:13:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2287473,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of SM Intervention on CIA-BLM Mice.\u003c/strong\u003e (A) Schematic diagram of CIA-BLM model construction and SM treatment; (B) Body weight changes of each group, measured every three days during the experiment; (C-E) Body weight differences among groups at the start of the experiment (day 0), before SM treatment (day 30), and after SM treatment (day 57); (F) Differences in spleen index among groups; (G) H\u0026amp;E-stained pathological sections of spleen, kidney, and liver tissues for each group. Results are expressed as mean ± SEM (N=6). ns: no significance; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6597839/v1/2c47fd08b3ec71a05625a7f8.png"},{"id":82892944,"identity":"0ab7af36-c352-4aec-b4fc-d80abe7a4835","added_by":"auto","created_at":"2025-05-16 12:21:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1978068,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSM Improves Arthritis Symptoms in CIA-BLM Mice. \u003c/strong\u003e(A) Representative images of hind paws from each group; (B) Time course of arthritis index (AI) for each group, measured every three days from the start of SM intervention (N=6); (C-D) Differences in AI among groups before SM treatment (day 30) and after treatment (day 57) (N=6); (E) Representative images and statistical results of Micro-CT scans of ankle joints for each group, with (F) bone density, (G) bone surface density, and (H) bone volume percentage statistics (N=1); (I) Representative images of H\u0026amp;E staining of ankle joints for each group; (J) Representative images of Safranin O-Fast Green staining of ankle joints for each group. Results are expressed as mean ± SEM. ns: no significance; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6597839/v1/90d6ad3a991a3097d7f0b339.png"},{"id":82889350,"identity":"adf68fcc-634c-4fa0-bf58-364bd51e4e9e","added_by":"auto","created_at":"2025-05-16 12:05:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2769481,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSM Reduces Lung Inflammation in CIA-BLM Mice.\u003c/strong\u003e (A) Histopathological examination of lung tissues using H\u0026amp;E staining across each group; (B) Inflammatory cell accumulation visualized by H\u0026amp;E staining in pulmonary specimens of each group (N=3); (C) TNF-α levels in bronchoalveolar lavage fluid (BALF) for each group (N=5); (D-E) Representative immunohistochemical staining images of TNF-α and IL-6 in lung tissue for each group; (F-G) Statistical graphs of the percentage of positive staining area for TNF-α and IL-6 (N=3); (H-I) Statistical graphs of inflammatory chemokines CXCL1 and CXCL2 levels in BALF for each group (N=5). Results are expressed as mean ± SEM. ns: no significance; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6597839/v1/8ecc28a2179ce9185c8c2200.png"},{"id":82892945,"identity":"a1b3e2d9-0dc7-467e-bb5d-199b599f8034","added_by":"auto","created_at":"2025-05-16 12:21:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3523805,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSM Reduces Lung Fibrosis in CIA-BLM Mice.\u003c/strong\u003e (A) Representative Micro-CT images of lungs from each group; (B) Representative Sirius Red-stained images of lung tissue for each group; (C) Representative Masson-stained images of lung tissue for each group; (D) Differences in lung CT values among groups (N=1); (E) Statistical graph of the percentage of positive area in Sirius Red-stained lung tissue for each group (N=3); (F) Statistical graph of the percentage of positive area in Masson-stained lung tissue for each group (N=3). Results are expressed as mean ± SEM. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6597839/v1/5fde0434782dd06eaa38557a.png"},{"id":82891598,"identity":"63c68308-a2d3-4c5b-9710-ab4b722d529f","added_by":"auto","created_at":"2025-05-16 12:13:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1367402,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSM May Regulates the FMT Process in CIA-BLM Mice. \u003c/strong\u003e(A) Representative immunofluorescence images of myofibroblast marker α-SMA and extracellular matrix component Fibronectin in lung tissue for each group; (B) ELISA detection of profibrotic cytokine TGF-β1 levels in bronchoalveolar lavage fluid (BALF) for each group (N=5); (C) Representative western blot bands of extracellular matrix components Fibronectin, Collagen-1, and Collagen-3 in lung tissue for each group; (D-F) Quantitative analysis of Fibronectin, Collagen-1, and Collagen-3 from western blot results (N=6). Results are expressed as mean ± SEM. ns: no significance; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6597839/v1/744523aa2d810f019998e8d7.png"},{"id":82892946,"identity":"b649e530-169c-4493-a086-45c15dd61fb4","added_by":"auto","created_at":"2025-05-16 12:21:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":507794,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLipidomics-based identification and screening of differential metabolites in mouse lung tissues across groups. \u003c/strong\u003e(A) OPLS-DA score plot showing group separation between the Ctrl group (i.e., CTRL) and CIA-BLM group;\u003cstrong\u003e \u003c/strong\u003e(B) OPLS-DA score plot showing group separation between the CIA-BLM and H-SMW (i.e., SM-H) group;\u003cstrong\u003e \u003c/strong\u003e(C) Validation plot of the OPLS-DA model for Ctrl (CTRL) vs. CIA-BLM;\u003cstrong\u003e \u003c/strong\u003e(D) Validation plot of the OPLS-DA model for CIA-BLM vs. H-SMW (SM-H);\u003cstrong\u003e \u003c/strong\u003e(E) Statistics of differential metabolites among Ctrl (CTRL), CIA-BLM, and H-SMW (SM-H) groups;\u003cstrong\u003e \u003c/strong\u003e(F) Volcano plot of differential metabolites between Ctrl (CTRL) and CIA-BLM;\u003cstrong\u003e \u003c/strong\u003e(G) Volcano plot of differential metabolites between CIA-BLM and H-SMW (SM-H).\u003cstrong\u003e (\u003c/strong\u003eN = 5).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6597839/v1/8c3d03b83a98238381bc8654.png"},{"id":82889347,"identity":"e3550785-5b14-494b-b600-83cbadbfe4f8","added_by":"auto","created_at":"2025-05-16 12:05:38","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":845362,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLipidomics Reveals That SM May Ameliorate RA-ILD Pulmonary Lesions via Regulation of LPA. \u003c/strong\u003e(A) Venn diagram illustrating the relationships among differential metabolites in Ctrl (CTRL), CIA-BLM, and H-SMW (SM-H) groups; (B) Cluster analysis and heatmap of 58 shared differential metabolites upregulated in CIA-BLM but downregulated after SM-H treatment; (C) Violin plots of the 58 shared metabolites; LPA (red) and its precursor (green) are highlighted; (D) Three LPAs that were upregulated in the CIA-BLM group and downregulated in the H-SMW (SM-H) group; (E) KEGG pathway enrichment analysis of differential metabolites. Five key pathways potentially involving LPA are marked in red. Rich factor: ratio of differentially expressed metabolites in a pathway to the total annotated metabolites in that pathway (higher values indicate greater enrichment). P-value: hypergeometric test P-value (closer to 0 indicates stronger significance). Dot size reflects the number of enriched metabolites per pathway. (N = 5).\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-6597839/v1/e650c3bcdc3136d0a571e0bc.png"},{"id":82889346,"identity":"3a26ecae-7b02-42de-b7b7-1deb1389baeb","added_by":"auto","created_at":"2025-05-16 12:05:38","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1060424,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSM Inhibits the ATX/LPA/LPA1 Signaling Pathway.\u003c/strong\u003e (A) Quantitative analysis of LPA levels in bronchoalveolar lavage fluid (BALF) from each group detected by ELISA (N=5); (B) Quantitative analysis of LPA levels in serum from each group detected by ELISA (N=5); (C) Quantitative analysis of Autotaxin (ATX) levels in BALF from each group detected by ELISA (N=5); (D) Quantitative analysis of ATX levels in serum from each group detected by ELISA (N=5); (E) Representative western blot bands of LPA1 and ATX in lung tissue for each group; (F-G) Quantitative analysis of western blot results for LPA1 and ATX (N=6); (H-I) Representative images of LPA1 immunohistochemical staining in lung tissue and quantitative analysis of LPA1-positive area for each group(N=3). Results are expressed as mean ± SEM. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-6597839/v1/473c98fbfa3cdb3f9aebfa20.png"},{"id":82889351,"identity":"0d94e0ce-e188-433f-b6e3-719e7113b49e","added_by":"auto","created_at":"2025-05-16 12:05:38","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":2284604,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSM Regulates the LPA1-Mediated RhoA/ROCK2 Pathway. \u003c/strong\u003e(A) Representative western blot bands of RhoA, ROCK1, and ROCK2 proteins in lung tissue for each group; (B-D) Quantitative analysis of western blot bands for RhoA, ROCK1, and ROCK2 proteins (N=6); (E) Representative immunofluorescence images of α-SMA and ROCK2 in lung tissue for each group; (F-G) Quantitative analysis of the percentage of positive area for α-SMA and ROCK2 in immunofluorescence staining (N=3). Results are expressed as mean ± SEM. ns: no significance; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"Figure10.png","url":"https://assets-eu.researchsquare.com/files/rs-6597839/v1/d06bf5ccb45f6711ff2b9512.png"},{"id":82889354,"identity":"97caa5ef-650e-4277-91ad-ae92146e6c20","added_by":"auto","created_at":"2025-05-16 12:05:38","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":976839,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram of the effects and mechanisms of SM in CIA-BLM mice. \u003c/strong\u003eSM not only effectively ameliorates joint inflammation and bone destruction in CIA-BLM mice, but also attenuates pulmonary inflammation and fibrosis. The latter effect may be attributed to SM's downregulation of the ATX/LPA/LPA1 axis and its downstream RhoA/ROCK2 signaling pathway.\u003c/p\u003e","description":"","filename":"Figure11.png","url":"https://assets-eu.researchsquare.com/files/rs-6597839/v1/2e1e3b4b12b9fb076a227630.png"},{"id":84223225,"identity":"96b01af6-f8fd-4b4e-b9c4-f6d9c57e1a8d","added_by":"auto","created_at":"2025-06-09 12:19:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19330852,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6597839/v1/ca125c3c-5ae5-4321-a95b-e0d7e9b13f6a.pdf"},{"id":82889359,"identity":"f99ff421-34ab-4788-8d2d-0fa5ab2ca309","added_by":"auto","created_at":"2025-05-16 12:05:38","extension":"xlsx","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":12249,"visible":true,"origin":"","legend":"","description":"","filename":"Abbreviation.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6597839/v1/c2e7093c70697f4f0d88b654.xlsx"},{"id":82889361,"identity":"1d34d0f4-0d04-4635-b6e2-6657cf828242","added_by":"auto","created_at":"2025-05-16 12:05:38","extension":"pdf","order_by":16,"title":"","display":"","copyAsset":false,"role":"supplement","size":547437,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6597839/v1/96718c40d6a5288337e36ef8.pdf"},{"id":82891606,"identity":"2864a166-7430-4432-8b8a-f4ee6cbe70eb","added_by":"auto","created_at":"2025-05-16 12:13:38","extension":"pdf","order_by":17,"title":"","display":"","copyAsset":false,"role":"supplement","size":232753,"visible":true,"origin":"","legend":"","description":"","filename":"ChineseMedicineAuthorChecklist202503.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6597839/v1/1fa79acdf51ddf527669d61f.pdf"},{"id":82889368,"identity":"94def4ad-94f3-4b3a-b3df-fb93641cd59d","added_by":"auto","created_at":"2025-05-16 12:05:39","extension":"xlsx","order_by":18,"title":"","display":"","copyAsset":false,"role":"supplement","size":420606,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xls.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6597839/v1/5d6b1124a6fed35588df8c3b.xlsx"},{"id":82889358,"identity":"51b15e4b-8e82-4d77-903f-e8626f650acf","added_by":"auto","created_at":"2025-05-16 12:05:38","extension":"xlsx","order_by":19,"title":"","display":"","copyAsset":false,"role":"supplement","size":15043,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xls.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6597839/v1/9ca458eea4391dbb1a6a44ac.xlsx"},{"id":82891605,"identity":"3d8fee95-2aa5-4b54-9fc2-41802897bc9d","added_by":"auto","created_at":"2025-05-16 12:13:38","extension":"pdf","order_by":20,"title":"","display":"","copyAsset":false,"role":"supplement","size":572668,"visible":true,"origin":"","legend":"","description":"","filename":"westernblot.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6597839/v1/cf92e52df53737644d772d0b.pdf"}],"financialInterests":"","formattedTitle":"Effects of Simiao Pill on rheumatoid arthritis complicated with interstitial lung disease via the ATX/LPA/LPA1 and RhoA/ROCK2 signaling pathways","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRheumatoid arthritis (RA) is an autoimmune systemic inflammatory arthritis with a prevalence of approximately 0.5\u0026ndash;1%[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The key pathological features of RA include synovial membrane inflammation, destruction of cartilage and bone tissues, and pannus proliferation, all of which contribute to progressive and irreversible joint deformities and loss of mobility[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. RA is often accompanied by various extra-articular manifestations, among which RA-associated interstitial lung disease (RA-ILD) is the most prevalent. High-resolution computed tomography (HRCT) can detect ILD in up to 60% of RA patients, though only 10\u0026ndash;18% exhibit clinically significant disease[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Bongartz \u003cem\u003eet al\u003c/em\u003e. reported patients with RA alone had a median survival of 9.9 years, compared to only 2.6 years in those with RA-ILD [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Statistics show that ILD increases the mortality risk of RA patients by 2\u0026ndash;10 times, ranking as the second leading cause of death in this population [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Established risk factors for RA-ILD include advanced age, male sex, tobacco exposure, exposure to air pollutants, active synovitis, rheumatoid factor (RF) and/or anti-citrullinated peptide antibodies (ACPAs) seropositivity, and MUC5B polymorphism[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn RA-ILD, usual interstitial pneumonia (UIP) is the most frequently observed histopathological pattern, with nonspecific interstitial pneumonia (NSIP) being the next most common. UIP exhibits a heterogeneous spatial distribution of pathological features, including patchy inflammation, fibroblast foci, collagen deposition, and honeycombing[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In contrast, NSIP is characterized by inflammation and fibrosis with a relatively homogeneous and diffuse distribution, usually lacking or showing only minimal fibroblast foci[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. UIP is also a histopathological pattern of idiopathic pulmonary fibrosis (IPF). RA-UIP and IPF share many similar clinical and histopathological features, suggesting common pathogenic pathways and potential therapeutic targets[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Many fibrotic lung diseases, including RA-ILD and IPF, undergo several stages, regardless of etiology, involving lung tissue injury, inflammatory responses, and fibrosis due to impaired repair[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In the late stages, cells from various sources undergo functional and/or phenotypic switching, amplifying fibrotic responses. Among these factors, the activation of fibroblasts into myofibroblasts and their escape from quiescence, defined as the fibroblast\u0026ndash;myofibroblast transition (FMT), is a key step in fibrogenesis[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Fibroblasts, myofibroblasts, and the excessive extracellular matrix (ECM) produced by the latter collectively contribute to the formation of fibroblast foci, driving the progression of pulmonary fibrosis[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLysophosphatidic acid (LPA) is a bioactive phospholipid that regulates various biological processes, such as cell migration, proliferation, differentiation, and cytoskeletal organization[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. LPA is widely distributed throughout the body, and circulating LPA is generated primarily by the autotaxin (ATX) enzyme, which catalyzes lysophospholipids[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In both RA patients and animal models, the expression levels of ATX and LPA in joint synovial fibroblasts are increased, promoting arthritis development[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In 2008, Tager \u003cem\u003eet al\u003c/em\u003e. first demonstrated that LPA levels were elevated in the bronchoalveolar lavage fluid (BALF) of IPF patients and bleomycin (BLM)-induced pulmonary fibrosis mouse model[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Numerous subsequent studies confirmed that upregulation of the ATX/LPA axis is closely associated with various autoimmune diseases (including RA) and several pulmonary fibrotic diseases (including RA-ILD)[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR20 CR21 CR22\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. LPA receptors 1\u0026ndash;6 (LPA1\u0026ndash;6) have been identified, with LPA shown to regulate the formation of pulmonary fibrosis in BLM-induced mice via LPA1 and LPA2[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Currently, the LPA1 antagonists BMS-986278 (NCT04308681) and BMS-986020 (NCT01766817) are undergoing phase II clinical trials for treating human IPF and progressive pulmonary fibrosis, while the involvement of other LPA receptors in pulmonary fibrosis has yet to be confirmed[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. These findings suggest that the ATX/LPA/LPA1 pathway plays an important role in the process of pulmonary fibrosis.\u003c/p\u003e \u003cp\u003eLPA promotes pulmonary fibrosis by activating multiple downstream signaling pathways through LPA1, particularly the RhoA/ROCK pathway [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Ras homolog family member A (RhoA), a key member of the Rho GTPase family, induces the formation of actin stress fibers and focal adhesions, thereby regulating cell morphology, adhesion, and motility[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. RhoA activity is modulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and guanine nucleotide dissociation inhibitors (GDIs)[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. RhoA cycles between an inactive GDP-bound state and an active GTP-bound state. Upon GTP binding, RhoA activates its downstream effector, Rho-associated coiled-coil-containing protein kinase (ROCK), which subsequently phosphorylates target substrates to regulate cell motility[\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. ROCK has two subtypes, ROCK1 and ROCK2, which are structurally and functionally similar. Studies have demonstrated that the RhoA/ROCK pathway is activated in the lungs of both BLM-induced pulmonary fibrosis model mice and IPF patients. Inhibition of this pathway has been shown to attenuate fibrotic phenotypes in the lung. Thus, targeting the RhoA/ROCK signaling pathway represents a promising therapeutic strategy for fibrotic lung diseases[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCurrently, there is no optimal therapeutic strategy for RA-ILD patients. One major challenge in treating RA-ILD lies in the potential pulmonary toxicity of many RA therapies, including disease-modifying antirheumatic drugs (DMARDs) and biologic agents[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Traditional Chinese medicine (TCM) shows considerable potential in treating RA-ILD. According to TCM theory, RA is categorized as \u0026ldquo;Bi syndrome,\u0026rdquo; caused by the obstruction of muscles, bones, and joints by pathogenic wind, cold, and dampness. As the disease progresses, these external pathogens may invade the internal organs, including the lungs, disrupting their dispersing and descending functions. This leads to the accumulation of phlegm and blood stasis in the lungs, resulting in symptoms such as chest tightness, shortness of breath, coughing, and wheezing\u0026mdash;manifesting as \u0026ldquo;Pulmonary Bi syndrome.\u0026rdquo; RA-ILD corresponds to this classification. Simiao Pill (SM) is a classic TCM formula for treating RA. It consists of four herbal components: Phellodendri Chinensis Cortex (Huang Bo), Atractylodis Rhizoma (Cang Zhu), Achyranthis Bidentatae Radix (Niu Xi), and Coicis Semen (Yiyi Ren). Cang Zhu strengthens the spleen, removes dampness, and dissolves phlegm; Huang Bo clears heat and eliminates dampness; Yiyi Ren promotes spleen function, dispels dampness, and alleviates Bi syndrome; and Niu Xi promotes blood circulation, dispels stasis, strengthens the liver and kidneys, and fortifies bones and muscles. The synergistic use of these herbs exerts both pathogen-expelling and body-strengthening effects, which may be beneficial in the treatment of RA-ILD. The purpose of this study was to create an RA-ILD animal model and evaluate the treatment effectiveness of SM using techniques such as ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS), lipidomics, micro-computed tomography (micro-CT), western blotting, and immunohistochemical staining. Special attention will be paid to the regulatory effects of SM on the ATX/LPA/LPA1 and RhoA/ROCK signaling pathways.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of the CIA-BLM Mouse Model\u003c/h2\u003e \u003cp\u003eA combined mouse model of collagen-induced arthritis and BLM-induced pulmonary fibrosis (CIA-BLM) was established to simulate clinical RA-ILD. A total of 24 male DBA/1 mice, each 6 weeks old and weighing between 16 and 18 grams, were acquired from Beijing Vital River Laboratory Animal Technology Co., Ltd. (License No. SCXK [Beijing] 2016\u0026ndash;0011) and kept in the specific pathogen-free (SPF)-grade animal barrier facility at Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China. All animal experiments and procedures were approved by the Institutional Animal Care and Use Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China (Approval No. TJH-202108002). The facility was maintained at a temperature of 20\u0026ndash;26\u0026deg;C, relative humidity of 40\u0026ndash;70%, with a 12-hour light/dark cycle, and five mice per cage. The mice were fed ad libitum. Following a week of acclimatization, the mice were divided randomly into four groups as control (CTL), CIA-BLM model, low-dose Simiao Pill (SM-L), and high-dose Simiao Pill (SM-H), with each group containing six mice. Except for the control group, the remaining three groups underwent CIA and BLM induction to establish the RA-ILD model.\u003c/p\u003e \u003cp\u003eConstruction of the CIA Animal Model: On day 0, 10 mg of type II bovine collagen powder (Chondrex, Cat. 20021) was dissolved in 5 ml of 2 mg/mL acetic acid solution containing type II bovine collagen (Chondrex, Cat. 20022) to obtain a final collagen concentration of 4 mg/mL. The collagen mixture was emulsified with an equal volume of 10 mg/mL complete Freund's adjuvant (Chondrex, Cat. 7027), and 100 \u0026micro;L of the emulsion was injected intradermally at the base of the tail for primary immunization. On day 21, 2 mg/mL type II bovine collagen (Chondrex, Cat. 20022) was emulsified with an equal volume of incomplete Freund's adjuvant (Chondrex, Cat. 7002) to perform secondary immunization. An equivalent volume of physiological saline was administered to control animals using identical delivery protocols. Arthritis severity was evaluated using a visual scoring scale based on paw erythema, swelling, and ankylosis. The arthritis index (AI) was graded as follows: 0\u0026thinsp;=\u0026thinsp;normal; 1\u0026thinsp;=\u0026thinsp;swelling and/or redness of the paw or one digit; 2\u0026thinsp;=\u0026thinsp;involvement of two digits; 3\u0026thinsp;=\u0026thinsp;three digits; and 4\u0026thinsp;=\u0026thinsp;severe arthritis involving the entire paw and digits, accompanied by skin desquamation and wrist/ankle swelling[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Each mouse had a maximum score of 16 on the basis of its four paws. Mice were considered to have a relatively successful arthritis model if their AI score was 6 or greater.\u003c/p\u003e \u003cp\u003eConstruction of the BLM-induced animal model: On day 33, BLM powder (MedChemExpress, Cat. HY-17565A) was dissolved in saline to obtain a 1 mg/mL solution. The solution was administered intratracheally to three groups of CIA mice at a dose of 2 mg/kg using a microsprayer aerosolizer and a mouse laryngoscope (both from Shanghai Yuyan Instruments Co., Ltd.). Animals assigned to the control group received isovolumetric sterile 0.9% NaCl solution delivered via the same transoral intratracheal administration method.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation and Component Identification of SM\u003c/h3\u003e\n\u003cp\u003eSM is composed of Atractylodis Rhizoma (Cang Zhu), Achyranthis Bidentatae Radix (Niu Xi), Phellodendri Chinensis Cortex (Huang Bo), and Coicis Semen (Yiyi Ren) in a ratio of 1:1:2:2. The formulation used in this study was identical to that employed in our previous research[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], which included its fingerprint chromatogram (see Additional file 1). All herbs were purchased from Beijing Traditional Chinese Medicine Co., Ltd. The extraction and concentration of the herbal granules were conducted in accordance with Good Manufacturing Practices (GMPs) (FDA, 2010). The therapeutic effect of 1 g of SM granules is equivalent to that of 6 g of traditional decoction. SM granules were dissolved in saline and administered orally at doses equivalent to 2.16 g/kg\u0026sdot;d⁻\u0026sup1; and 8.63 g/kg\u0026sdot;d⁻\u0026sup1;, designated the SM-L and SM-H groups, respectively. Therapeutic intervention commenced on day 30, with daily treatments maintained for 28 days. Vehicle control groups received equivalent volumes of physiological saline via identical gavage procedures. The administration method, concentration, dosage, and frequency of SM were based on our team's previous study[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition, the prepared SM was subjected to three rounds of analysis using UHPLC-MS/MS. In addition, the prepared SM was analyzed in three sequential steps using UHPLC-MS/MS. First, the metabolites of SM were extracted. SM granules were ground into powder, and approximately 50\u0026thinsp;\u0026plusmn;\u0026thinsp;2 mg of the powder was lyophilized, mixed with beads, and suspended in 500 \u0026micro;L of extraction solvent (MeOH:ACN:H₂O\u0026thinsp;=\u0026thinsp;2:2:1, v/v/v) containing deuterated internal standards. The mixture was vortexed for 30 s, homogenized at 35 Hz for 240 s, and sonicated in a 4\u0026deg;C water bath for 5 min. This homogenization and sonication cycle was repeated three times. Subsequently, the mixture was incubated at \u0026minus;\u0026thinsp;40\u0026deg;C for 30 min to precipitate proteins. The samples were then centrifuged at 12,000 rpm (RCF\u0026thinsp;=\u0026thinsp;13,800 \u0026times;g, R\u0026thinsp;=\u0026thinsp;8.6 cm) for 15 min at 4\u0026deg;C. The resulting supernatants were transferred to fresh vials and incubated for 10 min, followed by a second centrifugation under the same conditions. The final supernatants were collected and transferred to fresh glass vials for subsequent analysis. A quality control (QC) sample was prepared by pooling equal aliquots of the supernatants from all samples. Second, LC-MS/MS analysis was performed on the extracted metabolites. The chromatographic separation was achieved using a Thermo Vanquish UHPLC platform equipped with a Phenomenex Kinetex C18 analytical column (2.1 mm \u0026times; 100 mm, 2.6 \u0026micro;m particle size), coupled to an Orbitrap Exploris 120 high-resolution mass spectrometer. The mobile phase system comprised: (A) aqueous 0.01% acetic acid and (B) an isopropanol-acetonitrile (1:1, v/v) mixture. The autosampler was maintained at a temperature of 4\u0026deg;C, and the injection volume was set to 2 \u0026micro;L. The Orbitrap Exploris 120 utilized Xcalibur software from Thermo Fisher Scientific to gather MS/MS spectra in information-dependent acquisition mode. In this mode, full-scan MS spectra were continuously evaluated to trigger MS/MS acquisition. The ESI source was operated under optimized conditions with the following settings: sheath gas at 50 Arb, auxiliary gas maintained at 15 Arb, and the capillary heater set to 320\u0026deg;C. Mass spectrometric analyses were performed with a full scan resolution of 60,000 and a MS/MS resolution of 15,000. A stepped normalized collision energy (NCE) of 20, 30, and 40 was applied for fragmentation. The spray voltage was configured at +\u0026thinsp;3.8 kV for positive ion mode detection and \u0026minus;\u0026thinsp;3.4 kV for negative ion mode analysis. Finally, the acquired data were preprocessed and annotated. Raw data were converted to mzXML format using ProteoWizard and subsequently processed using an in-house R-based program built on the XCMS platform for peak detection, extraction, alignment, and integration. Metabolite identification was performed using an R-based package and the BiotreeDB (v3.0).\u003c/p\u003e\n\u003ch3\u003eMicro-CT\u003c/h3\u003e\n\u003cp\u003eAfter the treatment period, all mice were fasted for 12 hours. One mouse from each group was randomly selected for lung and joint micro-CT scanning to visually assess differences in pulmonary fibrosis and bone destruction among groups. Mice were anesthetized with 1% pentobarbital, and lung scans were performed. The ankle joints were dissected, fixed in 4% paraformaldehyde for 48 hours, and subsequently scanned. Micro-CT scanning was performed using the Bruker SkyScan 1276 system (Kontich, Belgium) under the following conditions: voxel size, 6.534 \u0026micro;m; medium resolution; voltage, 70 kV; current, 200 \u0026micro;A; aluminum filter, 1 mm; and integration time, 525 ms. Density calibration was conducted using a calcium hydroxyapatite (CaHA) phantom provided by the manufacturer. Image reconstruction was performed using NRecon software (version 1.7.4.2), and 3D images were generated from 2D grayscale contour images via distance transformation (CTvox, version 3.3.0). Both 2D and 3D analyses were carried out using CTAn software (version 1.18.8.0).\u003c/p\u003e\n\u003ch3\u003eHematoxylin and Eosin (H\u0026E) Staining\u003c/h3\u003e\n\u003cp\u003eFresh lung, kidney, spleen, and liver tissues were fixed in 4% paraformaldehyde at room temperature for over 24 hours. Ankle joints were decalcified in EDTA solution on a constant-temperature shaker. After embedding the tissues in paraffin, they were sectioned and stained with H\u0026amp;E solution (BaiQianDu Biotechnology, Cat. B1003). Sections were first deparaffinized in xylene (3 \u0026times; 15 min), followed by rehydration in absolute ethanol (2 \u0026times; 5 min), then in 95% and 85% ethanol (5 min each). Subsequently, hematoxylin staining, differentiation, bluing, eosin staining, and dehydration were performed. Finally, the slides were mounted with neutral resin. An Olympus BX51 microscope (Olympus, Japan) was used to capture images, with blue-stained nuclei and pink-stained cytoplasm. Image analysis was conducted using ImageJ software.\u003c/p\u003e\n\u003ch3\u003eMasson Staining\u003c/h3\u003e\n\u003cp\u003eLung tissue sections were stained using a Masson\u0026rsquo;s trichrome staining kit (Servicebio, Cat. G1006). Following standard deparaffinization and hydration steps as performed in H\u0026amp;E staining, the sections were incubated in Masson A solution overnight, then sequentially immersed in a 1:1 mixture of Masson B and C solutions for 1 minute, Masson D solution for 6 minutes, Masson E solution for 1 minute, and Masson F solution for 2\u0026ndash;30 seconds. Finally, the sections were rinsed with 1% acetic acid, dehydrated, and mounted. An Olympus BX51 microscope (Olympus, Japan) was used to capture images. Collagen fibers appeared blue, while muscle fibers, fibrin, and red blood cells appeared red. Image analysis was performed using ImageJ software.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSirius Red Staining\u003c/h2\u003e \u003cp\u003eLung tissue sections were stained using a Sirius Red staining kit (Servicebio, Cat. G1078). Following deparaffinization and hydration, the sections were briefly air-dried and immersed in modified Sirius Red A solution for 2 minutes. After rinsing twice with tap water, the sections were immersed in modified Sirius Red B solution for 30 minutes, followed by dehydration and mounting. An Olympus BX51 microscope (Olympus, Japan) was used to capture images. Collagen fibers appeared red, and the background appeared yellow under bright-field microscopy. Image analysis was conducted using ImageJ software.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSafranin O-Fast Green (SO/FG) Staining\u003c/h3\u003e\n\u003cp\u003eSections of the ankle joint were stained with a Safranin O-Fast Green staining kit (Biossci, Cat. BBL-0209). Following deparaffinization and hydration, the sections were stained with Safranin O solution for approximately 1\u0026ndash;5 minutes, rinsed with water, and subsequently stained with Fast Green solution for another 1\u0026ndash;5 minutes. The sections were then differentiated in absolute ethanol for 5\u0026ndash;10 minutes, followed by clearing and mounting. An Olympus BX51 microscope (Olympus, Japan) was used to capture images. Under light microscopy, cartilage appeared bright red to orange-red, bone tissue appeared green, and some connective tissues also appeared red.\u003c/p\u003e\n\u003ch3\u003eELISA\u003c/h3\u003e\n\u003cp\u003eSerum and BALF samples were collected from mice for ELISA analysis. The following ELISA kits were used: Mouse CXCL1/KC (ABclonal, Cat. RK00038), Mouse MIP-2/CXCL2 (ABclonal, Cat. RK04208), Mouse TNF-α (ABclonal, Cat. RK00027), Mouse LPA (Bioswamp, Cat. MU30816), Mouse TGF-β1 (Boster, Cat. EK0515), and Mouse ATX (JINGMEI BIOTECHNOLOGY, Cat. JM-13131M1). Cytokine concentrations in serum and BALF were quantified according to the manufacturers\u0026rsquo; standard protocols.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical staining\u003c/h2\u003e \u003cp\u003eImmunohistochemical staining was performed on lung and ankle joint tissue sections collected from mice. The procedure included deparaffinization and hydration, antigen retrieval, quenching of endogenous peroxidase activity, serum blocking, primary antibody incubation, and dehydration followed by mounting. A DAB chromogenic kit (Servicebio, Cat. G1212-200T) was used for signal detection, and normal goat serum (Servicebio, Cat. G1208-5ML) was applied as the blocking agent. The secondary antibodies were goat anti-rabbit (Servicebio, Cat. GB23303) and goat anti-mouse (Servicebio, Cat. GB23301). The primary antibodies were TNF-α rabbit pAb (ABclonal, Cat. A0277), IL-6 rabbit pAb (Servicebio, Cat. GB11117), and EDG2 polyclonal antibody ( tech, Cat. 20442-1-AP). An Olympus BX51 microscope (Olympus, Japan) was used to capture images, which were then analyzed with ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eImmunofluorescence staining was performed on lung tissue sections collected from mice. The procedure included deparaffinization and hydration, antigen retrieval, serum blocking, antibody incubation, and dehydration followed by mounting. Normal goat serum (Servicebio, Cat. G1208-5ML) was used as the blocking agent. The primary antibodies were as follows: α-smooth muscle actin (ACTA2) rabbit monoclonal antibody (CST, Cat. #19245), smooth muscle actin-specific mouse monoclonal antibody (Proteintech, Cat. 67735-1-Ig), fibronectin mouse monoclonal antibody (Proteintech, Cat. 66042-1-Ig), and ROCK2 (middle) rabbit polyclonal antibody (Proteintech, Cat. 21645-1-AP). The secondary antibodies were Cy3-labeled goat anti-mouse IgG (Servicebio, Cat. GB21301) and FITC-labeled goat anti-rabbit IgG (Servicebio, Cat. GB22303). Nuclei were counterstained with DAPI staining solution (ready-to-use; Servicebio, Cat. G1012-10ML), and sections were mounted using anti-fade mounting medium (Servicebio, Cat. G1401-5ML). An Olympus BX51 microscope (Olympus, Japan) was used to capture images, which were then analyzed with ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eWestern blot analysis was performed on mouse ankle joint and lung tissues. Proteins were extracted using strong RIPA lysis buffer (Servicebio, Cat. G2002-100ML) supplemented with phosphatase inhibitor (Servicebio, Cat. G2007-1ML) and 50\u0026times; protease inhibitor cocktail (Servicebio, Cat. G2006-250UL). The levels of protein were measured with a BCA protein quantification kit (Servicebio, Cat. G2026-200T). Protein samples with equivalent loading quantities were resolved via SDS-PAGE and subsequently electroblotted onto PVDF membranes employing a wet transfer system. Following transfer, the membranes were first incubated with NcmBlot blocking solution (NCM Biotech, Cat. P30500) to prevent nonspecific binding, then incubated (16h, 4\u0026deg;C) with the following primary antibodies: β-actin mouse mAb (Proteintech, Cat. 66009-1-Ig, 1:10,000), fibronectin mouse mAb (Proteintech, Cat. 66042-1-Ig, 1:5,000), collagen I/COL1A1 rabbit pAb (ABclonal, Cat. A1352, 1:1,000), collagen III (N-terminal) rabbit pAb (Proteintech, Cat. 22734-1-AP, 1:1,000), EDG2 rabbit pAb (Proteintech, Cat. 20442-1-AP, 1:1,000), ENPP2 rabbit pAb (Proteintech, Cat. 14243-1-AP, 1:1,000), RhoA [KD Validated] rabbit pAb (ABclonal, Cat. A18695, 1:1,000), ROCK1 rabbit mAb (ABclonal, Cat. A11158, 1:5,000), and ROCK2 rabbit mAb (ABclonal, Cat. A2395, 1:1,000). The immunoblots were subjected to 1-hour room temperature incubation with species-matched HRP-conjugated secondary antibodies: goat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) (ABclonal, Cat. AS014) or goat anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L) (ABclonal, Cat. AS003). Immunoreactive bands were detected using the SuperFemto ECL Chemiluminescence Kit (Vazyme Biotech Co., Ltd, Cat. E423-01) and visualized with the Tanon chemiluminescent imaging system. Protein band intensities were quantified using ImageJ software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eWidely targeted lipidomics\u003c/h2\u003e \u003cp\u003eThe whole right lung lobe of the mouse was removed, put into a cryopreservation tube, and quickly frozen in liquid nitrogen before being stored in a freezer at -80\u0026deg;C. Subsequently, the frozen mouse lung tissue was thawed and homogenized to prepare the supernatant. The sample extracts were subjected to analysis via an LC-ESI-MS/MS setup, comprising a UPLC (ExionLC AD, details available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://sciex.com.cn/\u003c/span\u003e\u003cspan address=\"https://sciex.com.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and a QTRAP\u0026reg; System mass spectrometer (further information accessible at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://sciex.com/\u003c/span\u003e\u003cspan address=\"https://sciex.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The conditions set for the analysis were as follows: for the UPLC, a Thermo Accucore\u0026trade; C30 column (dimensions: 2.1 mm internal diameter by 100 mm length, particle size: 2.6 \u0026micro;m) was utilized. The solvent system was composed of two phases: Phase A consisted of acetonitrile and water (60:40, v/v) supplemented with 0.1% formic acid and 10 mmol/L ammonium formate, while Phase B comprised a mixture of acetonitrile and isopropanol (10:90, v/v), also containing 0.1% formic acid and 10 mmol/L ammonium formate. Gradient elution began with an A/B ratio of 80:20 (v/v) and was adjusted sequentially as follows: 70:30 at 2.0 minutes, 40:60 at 4.0 minutes, 15:85 at 9.0 minutes, 10:90 at 14.0 minutes, and 5:95 at 15.5 minutes. This 5:95 ratio was maintained until 17.3 minutes, after which the system returned to 80:20 and was held constant until 20.0 minutes. The flow rate was maintained at 0.35 ml/min, with the column temperature set at 45\u0026deg;C, and an injection volume of 2 \u0026micro;L. The eluent was then directed to an ESI-triple quadrupole-linear ion trap (QTRAP)-MS for further analysis. The linear ion trap (LIT) and triple quadrupole (QQQ) scans were conducted using a QTRAP\u0026reg; LC-MS/MS System, which integrates a triple quadrupole-linear ion trap mass spectrometer (QTRAP) with an ESI Turbo Ion-Spray interface. The system was operated in both positive and negative ion modes and managed by Analyst 1.6.3 software (Sciex). The operational settings for the ESI source included: a turbo spray ion source, a source temperature maintained at 500\u0026deg;C, and ion spray voltages of 5500 V for positive mode and \u0026minus;\u0026thinsp;4500 V for negative mode. The mass spectrometry system was operated with optimized gas parameters: ion source gas 1 (GS1, 45 psi), ion source gas 2 (GS2, 55 psi), curtain gas (CUR, 35 psi), and collision gas (CAD, medium setting). For instrument calibration, polypropylene glycol standard solutions were employed at 10 \u0026micro;mol/L (QQQ mode) and 100 \u0026micro;mol/L (LIT mode). The QQQ scans utilized multiple reaction monitoring (MRM) experiments, employing nitrogen as the collision gas at a pressure of 5 psi. Each MRM transition had optimized parameters like declustering potential (DP) and collision energy (CE). During each analysis period, a specific group of MRM transitions was monitored, corresponding to the metabolites eluting within that timeframe. Finally, qualitative analysis was performed based on the in-house targeted reference database MWDB (MetWare Database), utilizing the retention time (RT), parent-daughter ion pairs, and secondary mass spectrometry data of the detected compounds. Metabolite quantification was conducted using MRM mode on a triple quadrupole mass spectrometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experimental data were analysed via GraphPad Prism 8.0.2 (263). The Shapiro‒Wilk test was first used to assess the normality of the data. For normally distributed data, homogeneity of variance was tested via one-way ANOVA. If both normality and homogeneity of variance were satisfied, Dunnett's multiple comparisons test was used for group comparisons. In cases of heteroscedasticity, the Brown‒Forsythe ANOVA test was used for intergroup comparisons. For nonnormally distributed data, the Kruskal‒Wallis test was applied. All experimental data are expressed as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEMs, and results yielding p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were deemed statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eComponent Analysis of SM\u003c/h2\u003e \u003cp\u003eThe metabolites in the SM were identified by matching the UHPLC-MS/MS results with the RT, precursor ion mass-to-charge ratio (MS1), and secondary mass spectra (MS2) of either reference standards or entries from public metabolite databases. A total of 1,227 chemical constituents were identified in SM (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), Representative total ion chromatograms (TICs) obtained in positive and negative ion modes are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, respectively. Based on the descending order of secondary mass spectral matching scores (MS2_score), the top ten compounds identified in positive ion mode were classified into the following categories: shikimates and phenylpropanoids, terpenoids, amino acids and peptides, polyketides, carbohydrates, and alkaloids (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In negative ion mode, the top ten compounds were mainly categorized as fatty acids, shikimates and phenylpropanoids, and carbohydrates (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). According to existing data in the HERB database, among the 1,227 compounds, 29 originated from Atractylodis Rhizoma, 29 from Achyranthis Bidentatae Radix, 54 from Phellodendri Chinensis Cortex, and 20 from Coicis Semen (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). These findings indicate that the identified metabolites represent the extractable chemical components from the four constituent herbs of SM.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePeak table of total ion chromatogram (TIC) in positive ion mode.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMS2.name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFormula\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003emz\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ert\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchisandrin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC24H32O7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e415.2116\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e379.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(2S,4aS,6aS,6bR,10S,12aS,14bS)-10-hydroxy-2,4a,6a,6b,9,9,12a-heptamethyl-13-oxo-3,4,5,6,6a,7,8,8a,10,11,12,14b-dodecahydro-1H-picene-2-carboxylic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC30H46O4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e471.3459\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e485.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDEHP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC24H38O4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e391.2836\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e568.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSchisandrin A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC24H32O6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e417.2265\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e461.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMethylpyroglutamate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC6H9NO3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e126.0546\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e117.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKojic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC6H6O4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e143.0335\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e96.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGalactose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC6H12O6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e203.052\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e42.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFructose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC6H12O6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e203.052\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e42.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(9S,10S)-3,4,5,14,15,16-hexamethoxy-9,10-dimethyl-tricyclo [10.4.0.02,7] hexadeca-1(16),2,4,6,12,14-hexaen-9-ol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC24H32O7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e415.2116\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e379.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN-Methylflindersine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC15H15NO2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e242.1171\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e420.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePeak table of total ion chromatogram (TIC) in negative ion mode.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMS2.name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFormula\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003emz\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ert\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003etrans-11-Eicosenoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC20H38O2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e309.2797\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e588\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ecis-11.14-Eicosadienoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC20H36O2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e307.2642\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e570.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ecis-9-Palmitoleic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC16H30O2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e253.2171\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e545.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMyristic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC14H28O2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e227.2015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e542.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUndecanoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC11H22O2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e185.1546\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e493.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(E)-8-hydroxy-2,6-dimethyl-oct-2-enoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC10H18O3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e185.1183\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e330.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSalicylic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC7H6O3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e137.0245\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e291.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIsoscopoletin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC10H8O4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e191.035\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e281.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3-Hydroxybenzoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC7H6O3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e137.0244\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e248.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVidarabine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC10H13N5O4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e266.0894\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e192.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eEffects of SM Intervention on CIA-BLM Mice\u003c/h2\u003e \u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, the schematic diagram presents the experimental workflow for the establishment of the CIA-BLM mouse model and subsequent SM intervention. Beginning on day 0, body weights were recorded every three days throughout the experimental period. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, the trend in body weight change (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM) over time is depicted for each group. Mice in the control group exhibited a gradual increase in body weight, while those in the CIA-BLM group displayed progressive weight loss, consistent with clinical observations in patients with active RA [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, there were no significant differences in the initial average body weight among the groups. However, following CIA induction and prior to SM treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), the average body weights of the three CIA groups were significantly lower than that of the control group, corroborating the trends observed in the line graph. After SM treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE), there were no significant differences in body weight between the SM-treated groups and the untreated CIA-BLM group, indicating that SM administration did not markedly affect body weight in CIA-BLM mice. The spleen index, which reflects immune activation and functional status, was significantly elevated in the CIA-BLM group relative to untreated controls (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF\u0026ndash;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). SM-H administration exhibited a trend toward spleen index reduction. Furthermore, H\u0026amp;E staining of kidney and liver tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH) revealed no notable histopathological differences among the groups, suggesting that SM treatment in CIA-BLM mice was relatively safe.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eSM Alleviates Joint Inflammation and Bone Destruction in CIA-BLM Mice\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, compared with the control mice, the CIA-BLM mice presented noticeable swelling in the paw, plantar, and ankle joints, accompanied by limping when walking, whereas ankle swelling was significantly reduced in the SM-treated mice. Beginning on day 30 of SM administration, all mice were evaluated every three days for the AI (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Prior to SM treatment, there were no significant differences in AI among the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), indicating a comparable baseline severity of arthritis. Over time, AI scores in the CIA-BLM group gradually increased, whereas SM treatment, especially in the SM-H group, significantly attenuated this progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). At the end of the experiment, micro-CT scans and histopathological analyses were conducted on the right ankle joints from each group (one mouse randomly selected per group). The micro-CT results revealed severe bone erosion in the CIA-BLM group compared with the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), with notable reductions in bone density, bone surface density, and bone volume percentage (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). These parameters were significantly improved following SM treatment, especially in the SM-H group. H\u0026amp;E staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI) and SO/FG staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ) of the ankle joint sections revealed increased inflammatory cell infiltration around the joints, cartilage proliferation invading bone tissue, bone tissue damage, and incomplete or missing joint cavities in the CIA-BLM group. SM treatment noticeably alleviated these conditions. Collectively, these findings demonstrate that SM effectively alleviates joint inflammation and mitigates bone destruction in CIA-BLM mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eSM improves lung inflammation in CIA-BLM mice\u003c/h2\u003e \u003cp\u003eIn various pulmonary fibrosis diseases, including RA-ILD, lung inflammation is a precursor to fibrosis[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. To investigate whether SM can alleviate lung inflammation in CIA-BLM mice, we performed H\u0026amp;E staining on lung tissue and ELISA to measure the levels of the inflammatory factors TNF-α, CXCL1, and CXCL2 in the BALF. Additionally, immunohistochemistry was used to assess the expression levels of the inflammatory markers TNF-α and IL-6 in lung tissue. H\u0026amp;E staining results (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) revealed that, compared with the control group, the CIA-BLM group presented significant lung fibrosis, thickened alveolar walls, alveolar collapse, disordered structure, and extensive inflammatory cell infiltration in the alveolar spaces. In contrast, the SM-L and SM-H treatment groups presented marked reductions in alveolar inflammatory cell infiltration, as well as improvements in alveolar wall thickening and alveolar collapse. Compared with that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), TNF-α secretion in the BALF of CIA-BLM mice was significantly elevated, while SM-L and SM-H treatments notably reduced TNF-α secretion. Immunohistochemical staining for TNF-α confirmed these findings (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u0026amp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Similarly, immunohistochemical staining for IL-6 (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE\u0026amp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG) revealed that IL-6 expression levels in the lungs of CIA-BLM mice were elevated compared with those in the control group, and both the SM-L and SM-H treatments significantly inhibited IL-6 expression. Finally, compared with those in the control group, CXCL1 and CXCL2 levels in the BALF of CIA-BLM mice were markedly increased, whereas the expression of CXCL1 and CXCL2 was reduced to varying degrees in the SM-L and SM-H groups (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). In summary, SM treatment significantly inhibited the expression of inflammatory cytokines in the lungs of CIA-BLM mice, improving lung inflammation and reducing lung tissue damage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eSM reduces lung fibrosis in CIA-BLM mice\u003c/h2\u003e \u003cp\u003eMicro-CT can visually assess the degree of lung fibrosis in mice, as lung CT values (measured in Hounsfield units, HU) reflect lung tissue density. Therefore, HU values indicate the extent of lung fibrosis. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA shows that lung fibrosis in CIA-BLM mice was distributed centrally around the trachea and was mainly concentrated in the upper lobes of both lungs, with increased HU values. SM treatment reduced HU values (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), indicating an improvement in lung fibrosis. A hallmark of lung fibrosis is tissue remodelling (including airway and lung parenchyma), which involves abnormal ECM deposition[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Collagen is a major component of the ECM. Sirius Red staining stains collagen red, while Masson staining stains collagen fibres blue. Figures\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC show a significant increase in collagen in the lung tissue of the CIA-BLM group compared with that in the control group, whereas SM treatment effectively reduced collagen deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eSM May Regulates the FMT Process in CIA-BLM Mice\u003c/h2\u003e \u003cp\u003eα-Smooth muscle actin (α-SMA) is expressed primarily in the cytoplasm and is a key marker of myofibroblasts[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Increased α-SMA expression is a recognized indicator of the FMT process[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. During fibrosis, myofibroblasts continuously proliferate, survive longer, and abnormally secrete large amounts of ECM[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Among these, type I collagen (Col 1), type III collagen (Col 3), and fibronectin (FN) are major ECM components[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Immunofluorescence staining revealed that α-SMA and FN expression in the lungs, especially in fibrotic foci, was significantly greater in the CIA-BLM group than in the control group. Both the SM-L and SM-H treatments markedly reduced α-SMA and FN expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), suggesting that SM may alleviate lung fibrosis by inhibiting the FMT process. TGF-β1 signaling is a key mechanism underlying FMT and fibrosis and is closely associated with myofibroblast activation[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The ELISA results revealed a significant increase in TGF-β1 levels in the BALF of CIA-BLM mice, whereas SM treatment reduced these levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), indicating that SM may inhibit myofibroblast activation. Western blot analysis revealed a marked increase in Col 1, Col 3, and FN expression in the lung tissue of CIA-BLM mice, whereas SM-L and SM-H treatments reduced Col 1 and Col 3 expression, with SM-H also reducing FN expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC-\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF), indicating that SM treatment can inhibit ECM secretion by myofibroblasts. In summary, SM treatment reduces the expression of the fibrosis-driving factor TGF-β1 in the BALF of CIA-BLM mice and may alleviate lung fibrosis by inhibiting the FMT process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eLipidomics Reveals SM May Ameliorate RA-ILD Pulmonary Lesions via LPA Modulation\u003c/h2\u003e \u003cp\u003eOrthogonal partial least squares-discriminant analysis (OPLS-DA) modeling was implemented to both visualize intergroup discrimination and characterize significantly altered metabolites. To visualize group separations and identify significantly differential metabolites, was employed. Based on preliminary findings suggesting superior anti-fibrotic efficacy of the SM-H group, this study focused particularly on this treatment group. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, OPLS-DA score plots demonstrated distinct separations between: (1) CTRL and CIA-BLM groups, and (2) CIA-BLM and SM-H groups, indicating that SM-H treatment significantly altered pulmonary metabolic profiles in CIA-BLM model mice. Subsequent model validation (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC-\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD) revealed R\u003csup\u003e2\u003c/sup\u003eY\u0026thinsp;=\u0026thinsp;0.993/ 0.998, with Q\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.887/0.617, confirming the OPLS-DA model's reliability, stability, predictive capacity, and absence of overfitting.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUsing variable importance in projection (VIP) scores from the OPLS-DA model, we preliminarily screened differential metabolites (VIP\u0026thinsp;\u0026gt;\u0026thinsp;1, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE shows 461 differential metabolites between CTRL and CIA-BLM groups (201 downregulated, 260 upregulated), and 351 between CIA-BLM and SM-H groups (348 downregulated, 3 upregulated). Volcano plots illustrating these differences are presented in Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF (CTRL vs CIA-BLM) and 7G (CIA-BLM vs SM-H).\u003c/p\u003e \u003cp\u003eTo elucidate the interrelationships of differential metabolites among groups, comparative analysis was performed and it revealed 173 shared differential metabolites across all three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Further investigation identified 58 metabolites that were upregulated in CIA-BLM but downregulated after SM-H treatment. Cluster analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB) showed these predominantly belonged to glycerophospholipids (GP), with additional sphingolipids (SP) and glycerolipids (GL). The violin plots were drawn for 58 common differential metabolites (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). The results showed that 3 differential metabolites were LPA(Figure \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD) in glycerophosphates and 34 differential metabolites were precursors of lysophosphatidic acid, which were phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), N-acyl-lysophosphatidylethanolamines (LNAPEs)、Lysophosphatidylcholines (LPCs)、and Lysophosphatidylethanolamines (LPEs) respectively. This implicates LPA as a potential metabolic target of SM intervention in CIA-BLM mice. KEGG pathway analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE) of all differential metabolites identified 15 significantly enriched pathways. Ten pathways were common to both comparisons: Vitamin digestion and absorption、Thermogenesis、Regulation of lipolysis in adipocytes、Regulation of actin cytoskeleton、Metabolic pathways、Lipid and atherosclerosis、Insulin resistance、Glycerolipid metabolism、Fat digestion and absorption and Cholesterol metabolism. Notably, LPA was enriched in five pathways (Vitamin digestion and absorption、Regulation of actin cytoskeleton、Metabolic pathways、Glycerolipid metabolism and Fat digestion and absorption), suggesting its potential involvement in both RA-ILD pathogenesis and SM's therapeutic mechanism through these biological processes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eSM Inhibits the ATX/LPA/LPA1 Signaling Pathway\u003c/h2\u003e \u003cp\u003eTo further investigate the correlation between LPA and RA-ILD as well as SM treatment efficacy, validation experiments were conducted. It is well-established that LPA is present in all cells and biological fluids, including plasma, serum, and BALF. Accumulating evidence indicates that LPA concentrations are significantly increased in both biological fluids and pulmonary tissues from patients with fibrotic lung disease, as well as in bleomycin-challenged murine models of pulmonary fibrosis[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The ELISA results (Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA-\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB) indicated that, compared with those in the control group, LPA levels in the BALF and serum of CIA-BLM mice were significantly increased, whereas SM treatment, particularly at the high dose (SM-H), significantly reduced LPA levels. LPA is primarily generated by the catalytic action of ATX, which is expressed in bronchial epithelial cells and alveolar inflammatory macrophages and can be secreted into the BALF[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In serum, ATX mainly originates from adipose tissue. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC-\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD, ATX levels in the BALF and serum of CIA-BLM mice were significantly elevated compared with those in the control group, and SM treatment markedly downregulated ATX expression. The western blot results for lung tissue were consistent with these findings (Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE \u0026amp; \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eG), suggesting that the reduction in LPA levels in the BALF and circulation caused by SM is at least partially due to the inhibition of ATX production. LPA1 plays a crucial role in driving pulmonary fibrosis and is highly expressed on the surface of fibroblasts and myofibroblasts[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Western blot analysis (Figs.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE‒9F) revealed that LPA1 receptor expression in the lung tissue of CIA-BLM mice was significantly upregulated compared with that in the control group, whereas both SM-L and SM-H treatments significantly downregulated LPA1 expression. Figures\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eH and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eI show representative images and quantification of LPA1 immunohistochemical staining in lung tissue, which was aligned with the immunoblotting data. In summary, SM may improve lung lesions by inhibiting the expression of the LPA-generating enzyme ATX, reducing LPA levels, and downregulating the expression of the downstream receptor LPA1 in lung tissue.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eSM Regulates the LPA1-Mediated RhoA/ROCK2 Pathway\u003c/h2\u003e \u003cp\u003eRhoA and ROCK are key regulators of cell locomotion mediated by reorganization of the actin cytoskeleton[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Studies have shown that inhibiting the RhoA/ROCK1/ROCK2 pathway not only suppresses the pulmonary fibrosis phenotype but also inhibits fibroblast activation, proliferation, and transformation into myofibroblasts, i.e., the FMT process[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Aligning with previous reports, immunoblot analysis (Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA-\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB) confirmed RhoA overexpression in CIA-BLM pulmonary tissues, which was markedly attenuated by SM intervention. However, unlike ROCK1, ROCK2 expression was markedly upregulated in the lung tissue of CIA-BLM mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA\u0026amp; \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eC-\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eD), and SM treatment was able to reduce ROCK2 expression levels. Immunofluorescence staining of lung tissue (Figs.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eE-\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eG) revealed that α-SMA (a marker of myofibroblasts) and ROCK2 expression were elevated in CIA-BLM lung tissue, whereas fluorescence expression levels were decreased in the SM-treated group. In summary, SM may alleviate lung damage and tissue remodelling in CIA-BLM mice by regulating the LPA1 downstream RhoA/ROCK2 signaling pathway to inhibit the FMT process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eConsequently, there are two hypotheses regarding the pathogenesis of lung involvement in RA: either immune tolerance to citrullinated proteins is first lost in synovial tissue, and these proteins subsequently cross-react with similar antigens in the lungs, or lung tolerance is first lost, leading to subsequent spread to the joints[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In any case, as the most common and severe manifestation of RA-related lung involvement, ILD generally presents with subtle symptoms in its early stages, often leading to severe lung damage at the time of diagnosis. This significantly delays the early diagnosis and treatment of RA-ILD. For example, a study of 167 RA-ILD patients revealed that at the time of diagnosis, 14% had a forced vital capacity (FVC) less than 50% of the predicted value, and 29% had a diffusing capacity of the lungs for carbon monoxide (DLco) less than 40% of the predicted value[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Such diagnostic delays increase the mortality rate of RA-ILD[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Therefore, emphasizing the early diagnosis and treatment of RA-ILD is crucial. Currently, there is no clearly effective treatment regimen for RA-ILD from any international scientific associations[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. A prospective cohort study revealed that poor control of arthritis was associated with the development of RA-ILD[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Therefore, especially for high-risk populations, arthritis inflammation should be controlled to prevent potential future RA-ILD. However, a challenge arises in that some medications used to treat RA may have pulmonary toxicity, with methotrexate (MTX) being of particular concern. A meta-analysis involving 22 studies and 8,584 participants reported that MTX was associated with an increased risk of all adverse respiratory events and respiratory infections, although no increased mortality risk was observed[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. However, some studies contradict these findings, and there is currently no compelling evidence suggesting the need to discontinue MTX in the presence of ILD[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Additionally, some studies have reported that the use of immunosuppressants can increase susceptibility to respiratory infections and mortality in RA-ILD patients[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Given the similarities between RA-ILD and IPF, the use of antifibrotic drugs such as nintedanib and pirfenidone can improve lung function in RA-ILD patients to some extent[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTCM may hold great potential for treating RA-ILD. The herbal formula SM is a classic TCM prescription for RA, supported by various studies: SM has been shown to reduce hind paw swelling and bone erosion in CIA and to prevent adjuvant induced arthritis in arthritic rats and significantly decrease the serum levels of the inflammatory factors TNF-α, IL-1β, and IL-6[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. The mechanisms by which SM exerts its therapeutic effects on RA may involve multiple signaling pathways, including the TLR4/MyD88/IRAK4/MAPK, ATX/LPA, MAPK, JAK2/STAT3, VEGF, and PI3K/AKT pathways, along with several metabolic pathways including arachidonic acid metabolism, glycerophospholipid metabolism, and tryptophan metabolism[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Additionally, research suggests that SM may improve ILD in parallel with treating RA. Findings from metabolomics and network pharmacology indicate that SM may achieve early prevention of RA-ILD by regulating the ferroptosis pathway[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. SM may also reduce pulmonary fibrosis by modulating the TGF-β/Smad2/3 pathway[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Our findings indicate that SM comprises 1,227 distinct chemical constituents. It not only significantly alleviates joint inflammation and mitigates bone and cartilage destruction in CIA-BLM mice, but also reduces pulmonary inflammation and fibrosis. According to the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and published literature, the therapeutic effects of SM may be attributed to key bioactive compounds such as berberine, coptisine, quercetin, vanillin, kaempferol, rutaecarpine, and ferulic acid[\u003cspan additionalcitationids=\"CR66 CR67 CR68 CR69 CR70\" citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. The lipid-modulating properties of SM may be partially attributable to its oleic acid content, which could influence metabolic pathway regulation[\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Lipidomic analysis of lung tissue further suggests that LPA and its precursors are likely key targets of SM in the treatment of RA-ILD. Subsequent experimental validation confirmed that SM significantly downregulates the ATX/LPA/LPA1 signaling axis and its downstream effectors, including the RhoA/ROCK2 pathway. Moreover, SM exhibited no apparent hepatotoxicity or nephrotoxicity, indicating a favorable safety profile. Taken together, these findings indicate SM's potential as a novel therapeutic candidate for managing RA-ILD.\u003c/p\u003e \u003cp\u003eVarious immune cytokines are known to jointly contribute to the development of lung inflammation in RA-ILD. During lung inflammation, M1 macrophages secrete multiple proinflammatory factors, such as TNF-α, IL-6, and IL-1α, and the chemokines CXCL1-3, CXCL5, and CXCL8-10, promoting lung tissue inflammation and damage[\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. TNF-α can increase corticosteroid insensitivity in airway smooth muscle, leading to increased expression of inflammatory genes and cytokine secretion[\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. As a key player in the cytokine network, IL-6 promotes B-cell differentiation into plasma cells and plays a critical role in cytokine storms[\u003cspan additionalcitationids=\"CR77\" citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. Conversely, IL-6 inhibition can reduce LPS-induced acute lung injury[\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. CXCL1 and CXCL2 function primarily to recruit immune cells, especially neutrophils[\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. Studies have shown that inhibiting the expression of CXCL1 and CXCL2 can decrease inflammatory cell infiltration and alleviate LPS-induced lung injury[\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e]. According to Zhang and colleagues, SM can lower the serum concentrations of TNF-α, IL-6, and IL-1β in mice suffering from hyperuricemia[\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. Similarly, Jie et al. reported that Simiao Yong'an Decoction significantly decreased the levels of TNF-α, IL-6, IL-1β, as well as the chemokines CXCL1 and CXCL2 in the joints of CIA mice[\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. Our findings indicate that SM treatment downregulates the expression levels of the inflammatory cytokines TNF-α, IL-6, CXCL1, and CXCL2 in the lung tissue of CIA-BLM mice, reduces inflammatory cell infiltration, and exacerbates structural damage to the lungs.\u003c/p\u003e \u003cp\u003eThe pathogenic mechanisms of various pulmonary fibrotic diseases, including RA-ILD and IPF, share certain similarities. Under the influence of susceptibility factors, repeated microinjuries to lung tissue lead to the death of alveolar epithelial cells (AECs)[\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]. Type 2 alveolar epithelial cells (AEC2s), which possess stem cell-like functions, are activated to participate in repair, but dysfunctional AEC2s result in abnormal repair processes. At this stage, abnormally activated AECs secrete various growth factors and chemokines, recruiting resident fibroblasts and bone marrow-derived fibroblasts to the site of injury. These activated epithelial cells also secrete TGF-β1, promoting epithelial‒mesenchymal transition (EMT) and FMT[\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]. Together, fibroblasts, myofibroblasts, and newly formed collagen form \u0026ldquo;fibroblast foci.\u0026rdquo; Within these foci, myofibroblasts secrete excessive ECM proteins, driving the development of lung fibrosis. Myofibroblast activation is the central step in lung fibrosis, with myofibroblasts originating from various cells, including resident fibroblasts, epithelial cells, endothelial cells, fibrocytes, pericytes, and macrophages[\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e]. FMT is considered a primary source of myofibroblasts[\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e]. Studies have shown that TGF-β1 stimulation increases the transition of human lung fibroblast-1 cells into myofibroblasts and induces mouse embryonic fibroblasts to produce elevated levels of Col, FN, and α-SMA[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e]. Chen et al. suggested that SM can alleviate pulmonary interstitial thickening and inflammatory cell infiltration in the lungs of CIA rats[\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. Similarly, Ba et al. reported that SM suppresses pulmonary collagen deposition and the EMT process in CIA-BLM mice[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In our study, CIA-BLM mice exhibited significantly elevated levels of TGF-β1 in BALF, along with pronounced pulmonary fibrosis. This was accompanied by increased expression of the myofibroblast marker α-SMA and ECM components, including FN, Col 1, and Col 3. SM treatment downregulated the expression of α-SMA and ECM proteins and attenuated the extent of pulmonary fibrosis. These findings suggest that the anti-fibrotic effect of SM in RA-ILD may be mediated, at least in part, by its ability to inhibit the FMT process.\u003c/p\u003e \u003cp\u003eLPA is a simple phospholipid consisting of a phosphate head group, a glycerol moiety, and a single fatty acid chain. It is not a single molecule but exists in multiple forms, such as 1-acyl-LPA, 2-acyl-LPA, and alkyl-LPA. Biologically active LPA is produced through only two pathways: the first involves secreted ATX, which catalyzes other lysophospholipids (mainly LPC) outside the cell membrane to generate LPA, which is the primary source of extracellular LPA. The second pathway involves membrane-bound PA-selective PLA1α (mPA-PLA1α), which catalyzes PA within the cell membrane to produce LPA[\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e]. ATX was initially identified in 1992 in human melanoma cells as an effective motility factor and is encoded by the Enpp2 gene[\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e]. It was later shown to be a secreted lysophospholipase D (lysoPLD)[\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e]. ATX exhibits catalytic activity in most body fluids, including serum and BALF[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. LPA functions by binding to six different LPA receptor subtypes (LPA1-LPA6), which play various roles in numerous biological processes. LPA receptors are specific GPCRs, with LPA1 primarily coupling to Gαi, Gαq, and Gα13. LPA1 belongs to the EDG family and is also known as EDG2 and Vzg-1[\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e]. Growing evidence implicates the ATX-LPA-LPA1 signaling cascade in the pathogenesis of diverse disorders spanning oncology (malignancies), hepatology (hepatitis), cardiology (acute coronary syndrome), rheumatology (RA), and IPF[\u003cspan additionalcitationids=\"CR92 CR93 CR94\" citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe role of the ATX‒LPA‒LPA‒1 axis in pulmonary fibrosis has been extensively studied. ATX is significantly elevated in the BALF of BLM-induced mice, and conditional gene deletion of ATX in bronchial epithelial cells or alveolar macrophages reduces ATX activity in BALF, decreases vascular leakage and inflammatory cell infiltration, and reduces the expression of TGF-β and collagen in BLM-induced mice[\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e, \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e]. This effect is attributed to reduced LPA production. Elevated LPA levels in the BALF of IPF patients stimulate fibroblast migration, mediate fibroblast recruitment and vascular leakage, and induce TGF-β, FN, α-SMA, and Col expression in lung fibroblasts through multiple signaling pathways[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e]. Further experiments have shown that LPA primarily activates, proliferates, migrates, and induces resistance to apoptosis in lung fibroblasts via LPA1 rather than LPA2/3[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan additionalcitationids=\"CR100 CR101\" citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e]. This process strongly promotes FMT and fibroblast focus formation. Moreover, the absence of LPA1 not only provides significant protection against fibrosis and mortality in BLM-induced pulmonary fibrosis mouse models but also reduces fibroblast proliferation, migration, and vascular leakage in the lungs. Additionally, LPA1 is involved in several stages of fibrosis progression, including ECM accumulation, epithelial cell apoptosis, myofibroblast differentiation, and endothelial barrier disruption[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e]. Therefore, LPA1 is an effective target for regulating fibroblast activation and the FMT process. Earlier research has demonstrated that SM can influence lipid metabolism issues in mice with high lipid levels and enhance insulin resistance and liver lipid buildup in diabetic mice, suggesting its potential in regulating metabolic dysregulation[\u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e, \u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e]. Our findings further demonstrate that, in addition to alleviating pulmonary fibrosis in CIA-BLM mice, SM effectively reverses the upregulated ATX/LPA/LPA1 signaling pathway observed in this model.\u003c/p\u003e \u003cp\u003eLPA connects to the downstream Gα13-dependent Rho GTPase/ROCK pathway via LPA1[\u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e]. RhoA, one of the most prominent members of the Rho GTPase family, acts as a molecular switch regulating cytoskeletal protein activation[\u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e]. As a key downstream effector of RhoA, ROCK belongs to the serine/threonine kinase family and is closely related to a range of cellular functions, including activation, migration, proliferation, apoptosis, differentiation, and contraction[\u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e107\u003c/span\u003e]. The ROCK signaling pathway is a critical regulator of profibrotic signal transduction, as it influences actin-myosin organization and ECM remodelling[\u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e108\u003c/span\u003e]. ROCK has two paralogues, ROCK1 and ROCK2[\u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e]. Previously, Watts \u003cem\u003eet al\u003c/em\u003e. demonstrated that the overexpression of connective tissue growth factor and myofibroblast formation in IPF cell lines are RhoA-dependent[\u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e]. The Rho kinase inhibitor fasudil reduces the extent of BLM-induced pulmonary fibrosis[\u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e111\u003c/span\u003e]. Recent preclinical studies have shown that the selective ROCK2 inhibitor GNS-3595 effectively inhibits ROCK2-mediated myosin light chain phosphorylation in vitro; reduces fibrosis-related protein expression (e.g., Col, FN, and α-SMA) in various in vitro cell models; and prevents TGF-β1-induced FMT[\u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e108\u003c/span\u003e]. Our study revealed that total RhoA protein and ROCK2 were significantly upregulated in the lung tissue of CIA-BLM mice, whereas ROCK1 expression was seemingly unaffected. SM treatment downregulated the RhoA/ROCK2 signaling pathway, suggesting that SM may alleviate pulmonary interstitial lesions in CIA-BLM mice by regulating the RhoA/ROCK2 signaling pathway.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates that SM, a classic anti-rheumatic formula, achieves dual therapeutic effects on both arthritis and pulmonary fibrosis in the CIA-BLM mouse model by modulating the ATX/LPA/LPA1-RhoA/ROCK2 axis, thereby providing mechanistic and pharmacodynamic evidence for TCM in treating RA-ILD\u0026mdash;a major clinical challenge. In summary, our findings confirm that SM not only alleviates arthritis symptoms but also mitigates pulmonary inflammation and fibrosis in CIA-BLM mice, likely through downregulating the ATX/LPA/LPA1-RhoA/ROCK2 signaling pathway. These results offer a translational candidate with integrated TCM-Western mechanistic insights for RA-ILD management, while also validating the multi-target therapeutic principle of TCM from the perspective of \"treating different diseases with the same method.\" Although further in vitro studies are needed to delineate SM\u0026rsquo;s bioactive compounds and target interactions, this work establishes a novel framework for developing anti-fibrotic drugs from traditional herbal repositories.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003e All animal experiments and procedures were approved by the Institutional Animal Care and Use Committee of Tongji Hospital, affiliated with Tongji Medical College of Huazhong University of Science and Technology, Wuhan, China (TJH-202108002).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was funded by grants from the National Natural Science Foundation of China (82074267, 82474285, 82174185, 82204872, 82104639, 82204871), the Hubei Provincial Natural Science Foundation Joint Fund (2025AFD590), the Hubei Provincial Department of Science and Technology (2022CFB742), and the Jingmen Science and Technology Bureau Key Projects (2022YFZD014).\u003c/p\u003e\u003ch2\u003eAuthors' contributions\u003c/h2\u003e \u003cp\u003eZC: resources, conceptualization; ST: resources, conceptualization, writing review and editing; YJ: methodology, formal analysis, writing-original draft, writing-review \u0026amp; editing; LH: resources, conceptualization, writing review and editing; XB: conceptualization, writing-review and editing; RZ: conceptualization, writing-review and editing; YY: conceptualization, writing-review and editing; YL: conceptualization, writing-review and editing; CL: conceptualization, writing-review and editing; GL: methodology; YH: resources, conceptualization; resources, conceptualization; YH: resources, conceptualization; resources, conceptualization; KQ: methodology; SY: resources, conceptualization; resources, conceptualization PS: methodology; WL: methodology; TL: methodology. YW: methodology; YG: conceptualization; YL: resources; HH: conceptualization; LZ: methodology. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe would like to express our gratitude to the Experimental Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology for providing the Tanon chemiluminescence imaging system for Western blot analysis. We also extend our sincere appreciation to Biotree Biotech Co., Ltd. (Shanghai, China) for their contribution to UHPLC-MS/MS analysis. Figure\u0026nbsp;11 was created using BioRender (authorization ID: VI2865W8ZI).\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e \u003cp\u003eData used to support the fndings of this study are available from the corresponding author upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSmolen JS, Aletaha D, McInnes IB. Rheumatoid arthritis. Lancet (London England). 2016;388(10055):2023\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmith MH, Berman JR. What Is Rheumatoid Arthritis? Jama. 2022;327(12):1194.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAletaha D, Smolen JS. Diagnosis and Management of Rheumatoid Arthritis: A Review. JAMA. 2018;320(13):1360\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKadura S, Raghu G. Rheumatoid arthritis-interstitial lung disease: manifestations and current concepts in pathogenesis and management. Eur respiratory review: official J Eur Respiratory Soc. 2021;30(160).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoduri G, Solomon JJ, Arthritis. \u0026amp; rheumatology (Hoboken, NJ). 2023;75(12):2067\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBongartz T, Nannini C, Medina-Velasquez YF, Achenbach SJ, Crowson CS, Ryu JH, et al. Incidence and mortality of interstitial lung disease in rheumatoid arthritis: a population-based study. Arthritis Rheum. 2010;62(6):1583\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHyldgaard C, Hilberg O, Pedersen AB, Ulrichsen SP, L\u0026oslash;kke A, Bendstrup E, et al. A population-based cohort study of rheumatoid arthritis-associated interstitial lung disease: comorbidity and mortality. Ann Rheum Dis. 2017;76(10):1700\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePinheiro FA, Souza DC, Sato EI. A Study of Multiple Causes of Death in Rheumatoid Arthritis. J Rhuematol. 2015;42(12):2221\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim Y, Yang HI, Kim KS. Etiology and Pathogenesis of Rheumatoid Arthritis-Interstitial Lung Disease. Int J Mol Sci. 2023;24(19).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThannickal VJ, Toews GB, White ES, Lynch JP 3rd, Martinez FJ. Mechanisms of pulmonary fibrosis. Annu Rev Med. 2004;55:395\u0026ndash;417.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeavy OC, Kawano-Dourado L, Stewart ID, Quint JK, Solomon JJ, Borie R, et al. Rheumatoid arthritis and idiopathic pulmonary fibrosis: a bidirectional Mendelian randomisation study. Thorax. 2024;79(6):538\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpagnolo P, Lee JS, Sverzellati N, Rossi G, Cottin V, Arthritis. \u0026amp; rheumatology (Hoboken, NJ). 2018;70(10):1544\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNishioka Y, Araya J, Tanaka Y, Kumanogoh A. Pathological mechanisms and novel drug targets in fibrotic interstitial lung disease. Inflamm regeneration. 2024;44(1):34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSalgado-Polo F, Borza R, Matsoukas MT, Marsais F, Jagerschmidt C, Waeckel L, et al. Autotaxin facilitates selective LPA receptor signaling. Cell Chem biology. 2023;30(1):69\u0026ndash;e8414.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVolkmann ER, Denton CP, Kolb M, Wijsenbeek-Lourens MS, Emson C, Hudson K et al. Lysophosphatidic acid receptor 1 inhibition: a potential treatment target for pulmonary fibrosis. Eur respiratory review: official J Eur Respiratory Soc. 2024;33(172).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao P, Walker NM, Braeuer RR, Mazzoni-Putman S, Aoki Y, Misumi K, et al. Loss of FOXF1 expression promotes human lung-resident mesenchymal stromal cell migration via ATX/LPA/LPA1 signaling axis. Sci Rep. 2020;10(1):21231.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMagkrioti C, Galaris A, Kanellopoulou P, Stylianaki EA, Kaffe E, Aidinis V. Autotaxin and chronic inflammatory diseases. J Autoimmun. 2019;104:102327.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTager AM, LaCamera P, Shea BS, Campanella GS, Selman M, Zhao Z, et al. The lysophosphatidic acid receptor LPA1 links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak. Nat Med. 2008;14(1):45\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBohdziewicz A, Pawlik KK, Maciejewska M, Sikora M, Alda-Malicka R, Czuwara J et al. Future Treatment Options in Systemic Sclerosis-Potential Targets and Ongoing Clinical Trials. J Clin Med. 2022;11(5).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOrosa B, Garc\u0026iacute;a S, Conde C. The autotaxin-lysophosphatidic acid pathway in pathogenesis of rheumatoid arthritis. Eur J Pharmacol. 2015;765:228\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsuchida Y, Shoda H, Sawada T, Fujio K. Role of autotaxin in systemic lupus erythematosus. Front Med. 2023;10:1166343.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNinou I, Magkrioti C, Aidinis V. Autotaxin in Pathophysiology and Pulmonary Fibrosis. Front Med. 2018;5:180.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKano K, Aoki J, Hla T. Lysophospholipid Mediators in Health and Disease. Annu Rev Pathol. 2022;17:459\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZulfikar S, Mulholland S, Adamali H, Barratt SL. Inhibitors of the Autotaxin-Lysophosphatidic Acid Axis and Their Potential in the Treatment of Interstitial Lung Disease: Current Perspectives. Clin pharmacology: Adv Appl. 2020;12:97\u0026ndash;108.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHemmings DG, Brindley DN. Signalling by lysophosphatidate and its health implications. Essays Biochem. 2020;64(3):547\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang LS, Fu P, Patel P, Harijith A, Sun T, Zhao Y, et al. Lysophosphatidic acid receptor-2 deficiency confers protection against bleomycin-induced lung injury and fibrosis in mice. Am J Respir Cell Mol Biol. 2013;49(6):912\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCorte TJ, Lancaster L, Swigris JJ, Maher TM, Goldin JG, Palmer SM et al. Phase 2 trial design of BMS-986278, a lysophosphatidic acid receptor 1 (LPA(1)) antagonist, in patients with idiopathic pulmonary fibrosis (IPF) or progressive fibrotic interstitial lung disease (PF-ILD). BMJ open respiratory Res. 2021;8(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuryadevara V, Ramchandran R, Kamp DW, Natarajan V. Lipid Mediators Regulate Pulmonary Fibrosis: Potential Mechanisms and Signaling Pathways. Int J Mol Sci. 2020;21(12).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiang SY, Dusaban SS, Brown JH. Lysophospholipid receptor activation of RhoA and lipid signaling pathways. Biochim Biophys Acta. 2013;1831(1):213\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEtienne-Manneville S, Hall A. Rho GTPases in cell biology. Nature. 2002;420(6916):629\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJaffe AB, Hall A. Rho GTPases: biochemistry and biology. Annu Rev Cell Dev Biol. 2005;21:247\u0026ndash;69.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRiento K, Ridley AJ. Rocks: multifunctional kinases in cell behaviour. Nat Rev Mol Cell Biol. 2003;4(6):446\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmin E, Dubey BN, Zhang SC, Gremer L, Dvorsky R, Moll JM, et al. Rho-kinase: regulation, (dys)function, and inhibition. Biol Chem. 2013;394(11):1399\u0026ndash;410.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmano M, Nakayama M, Kaibuchi K. Rho-kinase/ROCK: A key regulator of the cytoskeleton and cell polarity. Cytoskeleton (Hoboken NJ). 2010;67(9):545\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMonaghan-Benson E, Wittchen ES, Doerschuk CM, Burridge K. A Rnd3/p190RhoGAP pathway regulates RhoA activity in idiopathic pulmonary fibrosis fibroblasts. Mol Biol Cell. 2018;29(18):2165\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKnipe RS, Probst CK, Lagares D, Franklin A, Spinney JJ, Brazee PL, et al. The Rho Kinase Isoforms ROCK1 and ROCK2 Each Contribute to the Development of Experimental Pulmonary Fibrosis. Am J Respir Cell Mol Biol. 2018;58(4):471\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePaoletti A, Ly B, Cailleau C, Gao F, de Ponfilly-Sotier MP, Pascaud J, et al. Liposomal AntagomiR-155-5p Restores Anti-Inflammatory Macrophages and Improves Arthritis in Preclinical Models of Rheumatoid Arthritis. Volume 76. Hoboken, NJ: Arthritis \u0026amp; rheumatology; 2024. pp. 18\u0026ndash;31. 1.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBa X, Wang H, Huang Y, Yan J, Han L, Lin W, et al. Simiao pill attenuates collagen-induced arthritis and bleomycin-induced pulmonary fibrosis in mice by suppressing the JAK2/STAT3 and TGF-β/Smad2/3 signalling pathway. J Ethnopharmacol. 2023;309:116274.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen P, Tu S, Wang H, Qin K, Chen Z. Simiao Pill Attenuates Collagen-Induced Arthritis in Rats through Suppressing the ATX-LPA and MAPK Signalling Pathways. Evidence-based complementary and alternative medicine: eCAM. 2019;2019:7498527.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEngland BR, Baker JF, Sayles H, Michaud K, Caplan L, Davis LA, et al. Body Mass Index, Weight Loss, and Cause-Specific Mortality in Rheumatoid Arthritis. Arthritis Care Res. 2018;70(1):11\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSavin IA, Zenkova MA, Sen'kova AV. Pulmonary Fibrosis as a Result of Acute Lung Inflammation: Molecular Mechanisms, Relevant In Vivo Models, Prognostic and Therapeutic Approaches. Int J Mol Sci. 2022;23(23).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu G, Philp AM, Corte T, Travis MA, Schilter H, Hansbro NG, et al. Therapeutic targets in lung tissue remodelling and fibrosis. Pharmacol Ther. 2021;225:107839.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu H, Yang J, Tu M, Weng J, Xie M, Zhou Z, et al. Vincristine Promotes Transdifferentiation of Fibroblasts Into Myofibroblasts via P38 and ERK Signal Pathways. Front Pharmacol. 2022;13:901000.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin X, Lei Y, Pan M, Hu C, Xie B, Wu W, et al. Augmentation of scleral glycolysis promotes myopia through histone lactylation. Cell Metabol. 2024;36(3):511\u0026ndash;e257.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMontero P, Milara J, Roger I, Cortijo J. Role of JAK/STAT in Interstitial Lung Diseases; Molecular and Cellular Mechanisms. Int J Mol Sci. 2021;22(12).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTanner L, Single AB, Bhongir RKV, Heusel M, Mohanty T, Karlsson CAQ, et al. Small-molecule-mediated OGG1 inhibition attenuates pulmonary inflammation and lung fibrosis in a murine lung fibrosis model. Nat Commun. 2023;14(1):643.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J, Jiang M, Xiong A, Zhang L, Luo L, Liu Y, et al. Integrated analysis of single-cell and bulk RNA sequencing reveals pro-fibrotic PLA2G7(high) macrophages in pulmonary fibrosis. Pharmacol Res. 2022;182:106286.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGy\u0026ouml;rfi AH, Matei AE, Distler JHW. Targeting TGF-β signaling for the treatment of fibrosis. Matrix biology: J Int Soc Matrix Biology. 2018;68\u0026ndash;69:8\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShimizu Y, Dobashi K, Iizuka K, Horie T, Suzuki K, Tukagoshi H, et al. Contribution of small GTPase Rho and its target protein rock in a murine model of lung fibrosis. Am J Respir Crit Care Med. 2001;163(1):210\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsoyi K, Chu SG, Patino-Jaramillo NG, Wilder J, Villalba J, Doyle-Eisele M, et al. Syndecan-2 Attenuates Radiation-induced Pulmonary Fibrosis and Inhibits Fibroblast Activation by Regulating PI3K/Akt/ROCK Pathway via CD148. Am J Respir Cell Mol Biol. 2018;58(2):208\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRackow AR, Nagel DJ, Zapas G, Clough RS, Sime PJ, Kottmann RM. The Novel Small Molecule BTB Inhibits Pro-Fibrotic Fibroblast Behavior though Inhibition of RhoA Activity. Int J Mol Sci. 2022;23(19).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZamora-Legoff JA, Krause ML, Crowson CS, Ryu JH, Matteson EL, Arthritis. \u0026amp; rheumatology (Hoboken, NJ). 2017;69(3):542\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCano-Jim\u0026eacute;nez E, V\u0026aacute;zquez Rodr\u0026iacute;guez T, Mart\u0026iacute;n-Robles I, Castillo Villegas D, Juan Garc\u0026iacute;a J, Bollo, de Miguel E et al. Diagnostic delay of associated interstitial lung disease increases mortality in rheumatoid arthritis. Scientific reports. 2021;11(1):9184.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSparks JA, He X, Huang J, Fletcher EA, Zaccardelli A, Friedlander HM et al. Rheumatoid Arthritis Disease Activity Predicting Incident Clinically Apparent Rheumatoid Arthritis-Associated Interstitial Lung Disease: A Prospective Cohort Study. Arthritis \u0026amp; rheumatology (Hoboken, NJ). 2019;71(9):1472\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eConway R, Low C, Coughlan RJ, O'Donnell MJ, Carey JJ. Methotrexate and lung disease in rheumatoid arthritis: a meta-analysis of randomized controlled trials. Arthritis Rheumatol (Hoboken NJ). 2014;66(4):803\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDawson JK, Graham DR, Desmond J, Fewins HE, Lynch MP. Investigation of the chronic pulmonary effects of low-dose oral methotrexate in patients with rheumatoid arthritis: a prospective study incorporating HRCT scanning and pulmonary function tests. Rheumatology (Oxford). 2002;41(3):262\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePugashetti JV, Lee JS. Overview of Rheumatoid Arthritis-Associated Interstitial Lung Disease and Its Treatment. Semin Respir Crit Care Med. 2024;45(3):329\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMena-V\u0026aacute;zquez N, Redondo-Rodriguez R, Rojas-Gimenez M, Romero-Barco CM, Fuego-Varela C, Perez-G\u0026oacute;mez N, et al. Rate of severe and fatal infections in a cohort of patients with interstitial lung disease associated with rheumatoid arthritis: a multicenter prospective study. Front Immunol. 2024;15:1341321.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim JW, Shin K, Jung J, Suh CH, Kim J, Lee SK, et al. Uncovering risk factors for adverse events and infections in rheumatoid arthritis and rheumatoid arthritis with interstitial lung disease under treatment with biologics or targeted synthetic DMARDs: insights from the KOBIO Registry. Clin Exp Rheumatol. 2024;42(9):1781\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatteson EL, Aringer M, Burmester GR, Mueller H, Moros L, Kolb M. Effect of nintedanib in patients with progressive pulmonary fibrosis associated with rheumatoid arthritis: data from the INBUILD trial. Clin Rheumatol. 2023;42(9):2311\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSolomon JJ, Danoff SK, Woodhead FA, Hurwitz S, Maurer R, Glaspole I, et al. Safety, tolerability, and efficacy of pirfenidone in patients with rheumatoid arthritis-associated interstitial lung disease: a randomised, double-blind, placebo-controlled, phase 2 study. Lancet Respiratory Med. 2023;11(1):87\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X, Zhang X, Zhang L, Li Y. [Effects and mechanisms of Simiao pill on adjuvant arthritis rats model]. Zhongguo Zhong yao za zhi\u0026thinsp;=\u0026thinsp;Zhongguo zhongyao zazhi\u0026thinsp;=\u0026thinsp;China. J Chin materia Med. 2010;35(21):2889\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Zhang F, Li X, Li X, Wang J, He J, et al. Integrated Multi-Omics Techniques and Network Pharmacology Analysis to Explore the Material Basis and Mechanism of Simiao Pill in the Treatment of Rheumatoid Arthritis. ACS omega. 2023;8(12):11138\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Y, Liu H, Han R, Lin J, Yang J, Guo M, et al. Analyzing how SiMiao Wan regulates ferroptosis to prevent RA-ILD using metabolomics and cyberpharmacology. Phytomedicine: Int J phytotherapy phytopharmacology. 2024;133:155912.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang DN, Wu FF, Zhang AH, Sun H, Wang XJ. Efficacy of berberine in treatment of rheumatoid arthritis: From multiple targets to therapeutic potential. Pharmacol Res. 2021;169:105667.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu F, Shen C, Zhang S, Liu Y, Liu D, Kuang Y, et al. Coptisine inhibits aggressive and proliferative actions of fibroblast like synoviocytes and exerts a therapeutic potential for rheumatoid arthritis. Int Immunopharmacol. 2024;128:111433.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen P, Lin W, Deng X, Ba X, Han L, Chen Z, et al. Potential Implications of Quercetin in Autoimmune Diseases. Front Immunol. 2021;12:689044.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTao Y, Pu J, Wang P. Ethnobotany, phytochemistry, pharmacology and quality control of Peucedanum decursivum (Miq.) Maxim: A critical review. J Ethnopharmacol. 2024;334:118542.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe X, Wu T, He H, Chen L, Han K, Zheng J, et al. Study of kaempferol in the treatment of rheumatoid arthritis through modulation of the NLRP3/CASP1/GSDMD axis and T-cell activation: Based on network pharmacology, single-cell analysis, and experimental validation. Int Immunopharmacol. 2024;143(Pt 1):113357.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo B, Zhao C, Zhang C, Xiao Y, Yan G, Liu L, et al. Elucidation of the anti-inflammatory mechanism of Er Miao San by integrative approach of network pharmacology and experimental verification. Pharmacol Res. 2022;175:106000.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDoss HM, Samarpita S, Ganesan R, Rasool M. Ferulic acid, a dietary polyphenol suppresses osteoclast differentiation and bone erosion via the inhibition of RANKL dependent NF-κB signalling pathway. Life Sci. 2018;207:284\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLei T, Jiang C, Zhao L, Zhang J, Xiao Q, Chen Y et al. Exploring the Mechanism of Topical Application of Clematis Florida in the Treatment of Rheumatoid Arthritis through Network Pharmacology and Experimental Validation. Pharmaceuticals (Basel Switzerland). 2024;17(7).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArabpour M, Saghazadeh A, Rezaei N. Anti-inflammatory and M2 macrophage polarization-promoting effect of mesenchymal stem cell-derived exosomes. Int Immunopharmacol. 2021;97:107823.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBritt RD Jr., Ruwanpathirana A, Ford ML, Lewis BW. Macrophages Orchestrate Airway Inflammation, Remodeling, and Resolution in Asthma. Int J Mol Sci. 2023;24(13).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSavin IA, Zenkova MA, Sen'kova AV. Bronchial Asthma, Airway Remodeling and Lung Fibrosis as Successive Steps of One Process. Int J Mol Sci. 2023;24(22).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJones SA, Jenkins BJ. Recent insights into targeting the IL-6 cytokine family in inflammatory diseases and cancer. Nat Rev Immunol. 2018;18(12):773\u0026ndash;89.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTanaka T, Narazaki M, Kishimoto T. Immunotherapeutic implications of IL-6 blockade for cytokine storm. Immunotherapy. 2016;8(8):959\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUciechowski P, Dempke WCM. Interleukin-6: A Masterplayer in the Cytokine Network. Oncology. 2020;98(3):131\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen IC, Wang SC, Chen YT, Tseng HH, Liu PL, Lin TC et al. Corylin Ameliorates LPS-Induced Acute Lung Injury via Suppressing the MAPKs and IL-6/STAT3 Signaling Pathways. Pharmaceuticals (Basel, Switzerland). 2021;14(10).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou C, Gao Y, Ding P, Wu T, Ji G. The role of CXCL family members in different diseases. Cell death discovery. 2023;9(1):212.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSong Y, Xu C, Wu J, Shu J, Sheng H, Shen Y. Palmatine alleviates LPS-induced acute lung injury via interfering the interaction of TAK1 and TAB1. Biochem Pharmacol. 2022;202:115120.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Y, Wang S, Dai X, Liu T, Liu Y, Shi H, et al. Simiao San alleviates hyperuricemia and kidney inflammation by inhibiting NLRP3 inflammasome and JAK2/STAT3 signaling in hyperuricemia mice. J Ethnopharmacol. 2023;312:116530.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJie SS, Sun HJ, Liu JX, Gao Y, Bai D, Zhu LL, et al. Simiao Yong'an decoction ameliorates murine collagen-induced arthritis by modulating neutrophil activities: An in vitro and in vivo study. J Ethnopharmacol. 2023;305:116119.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKing TE Jr., Pardo A, Selman M. Idiopathic pulmonary fibrosis. Lancet (London England). 2011;378(9807):1949\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRicheldi L, Collard HR, Jones MG. Idiopathic pulmonary fibrosis. Lancet (London England). 2017;389(10082):1941\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRockey DC, Bell PD, Hill JA. Fibrosis\u0026ndash;a common pathway to organ injury and failure. N Engl J Med. 2015;372(12):1138\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang H, Zhou Y, Wen D, Wang J. Noncoding RNAs: Master Regulator of Fibroblast to Myofibroblast Transition in Fibrosis. Int J Mol Sci. 2023;24(2).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTanner L, Bergwik J, Single AB, Bhongir RKV, Erjef\u0026auml;lt JS, Egesten A. Zoledronic Acid Targeting of the Mevalonate Pathway Causes Reduced Cell Recruitment and Attenuates Pulmonary Fibrosis. Front Pharmacol. 2022;13:899469.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYanagida K, Shimizu T. Lysophosphatidic acid, a simple phospholipid with myriad functions. Pharmacol Ther. 2023;246:108421.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStracke ML, Krutzsch HC, Unsworth EJ, Arestad A, Cioce V, Schiffmann E, et al. Identification, purification, and partial sequence analysis of autotaxin, a novel motility-stimulating protein. J Biol Chem. 1992;267(4):2524\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBese T, Barbaros M, Baykara E, Guralp O, Cengiz S, Demirkiran F, et al. Comparison of total plasma lysophosphatidic acid and serum CA-125 as a tumor marker in the diagnosis and follow-up of patients with epithelial ovarian cancer. J gynecologic Oncol. 2010;21(4):248\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdul Rahman M, Mohamad Haron DE, Hollows RJ, Abdul Ghani ZDF, Ali Mohd M, Chai WL, et al. Profiling lysophosphatidic acid levels in plasma from head and neck cancer patients. PeerJ. 2020;8:e9304.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDohi T, Miyauchi K, Ohkawa R, Nakamura K, Kishimoto T, Miyazaki T, et al. Increased circulating plasma lysophosphatidic acid in patients with acute coronary syndrome. Clin Chim Acta. 2012;413(1\u0026ndash;2):207\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePattanaik D, Postlethwaite AE. A role for lysophosphatidic acid and sphingosine 1-phosphate in the pathogenesis of systemic sclerosis. Discov Med. 2010;10(51):161\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWatanabe N, Ikeda H, Nakamura K, Ohkawa R, Kume Y, Tomiya T, et al. Plasma lysophosphatidic acid level and serum autotaxin activity are increased in liver injury in rats in relation to its severity. Life Sci. 2007;81(12):1009\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlack KE, Berdyshev E, Bain G, Castelino FV, Shea BS, Probst CK, et al. Autotaxin activity increases locally following lung injury, but is not required for pulmonary lysophosphatidic acid production or fibrosis. FASEB journal: official publication Federation Am Soc Experimental Biology. 2016;30(6):2435\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOikonomou N, Mouratis MA, Tzouvelekis A, Kaffe E, Valavanis C, Vilaras G, et al. Pulmonary autotaxin expression contributes to the pathogenesis of pulmonary fibrosis. Am J Respir Cell Mol Biol. 2012;47(5):566\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSakai T, Peyruchaud O, F\u0026auml;ssler R, Mosher DF. Restoration of beta1A integrins is required for lysophosphatidic acid-induced migration of beta1-null mouse fibroblastic cells. J Biol Chem. 1998;273(31):19378\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQian Y, Hamilton M, Sidduri A, Gabriel S, Ren Y, Peng R, et al. Discovery of highly selective and orally active lysophosphatidic acid receptor-1 antagonists with potent activity on human lung fibroblasts. J Med Chem. 2012;55(17):7920\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWen J, Lin X, Gao W, Qu B, Zuo Y, Liu R, et al. Inhibition of LPA(1) Signaling Impedes Conversion of Human Tenon's Fibroblasts into Myofibroblasts Via Suppressing TGF-β/Smad2/3 Signaling. J ocular Pharmacol therapeutics: official J Association Ocular Pharmacol Ther. 2019;35(6):331\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHama K, Aoki J, Fukaya M, Kishi Y, Sakai T, Suzuki R, et al. Lysophosphatidic acid and autotaxin stimulate cell motility of neoplastic and non-neoplastic cells through LPA1. J Biol Chem. 2004;279(17):17634\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSwaney JS, Chapman C, Correa LD, Stebbins KJ, Bundey RA, Prodanovich PC, et al. A novel, orally active LPA(1) receptor antagonist inhibits lung fibrosis in the mouse bleomycin model. Br J Pharmacol. 2010;160(7):1699\u0026ndash;713.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFukushima N, Kimura Y, Chun J. A single receptor encoded by vzg-1/lpA1/edg-2 couples to G proteins and mediates multiple cellular responses to lysophosphatidic acid. Proc Natl Acad Sci USA. 1998;95(11):6151\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang K, Xu W, He W, Ding M, Xia T, Tan X. Simiao Wan attenuates high-fat diet-induced hyperlipidemia in mice by modulating the gut microbiota-bile acid axis. J Ethnopharmacol. 2025;337(Pt 2):118868.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Y, Zhu L, Hu W, Wang Y, Wen X, Yang J. Simiao Wan modulates the gut microbiota and bile acid metabolism during improving type 2 diabetes mellitus in mice. Phytomedicine: Int J phytotherapy phytopharmacology. 2022;104:154264.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBros M, Haas K, Moll L, Grabbe S. RhoA as a Key Regulator of Innate and Adaptive Immunity. Cells. 2019;8(7).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBei Y, Hua-Huy T, Duong-Quy S, Nguyen VH, Chen W, Nicco C, et al. Long-term treatment with fasudil improves bleomycin-induced pulmonary fibrosis and pulmonary hypertension via inhibition of Smad2/3 phosphorylation. Pulm Pharmacol Ther. 2013;26(6):635\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHwang S, Lee W, Ravi D, Devine W, Yong M, Diebold RB, et al. Novel Small-Molecule ROCK2 Inhibitor GNS-3595 Attenuates Pulmonary Fibrosis in Preclinical Studies. Am J Respir Cell Mol Biol. 2024;71(4):430\u0026ndash;41.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePernis AB, Ricker E, Weng CH, Rozo C, Yi W. Rho Kinases in Autoimmune Diseases. Annu Rev Med. 2016;67:355\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWatts KL, Spiteri MA. Connective tissue growth factor expression and induction by transforming growth factor-beta is abrogated by simvastatin via a Rho signaling mechanism. Am J Physiol Lung Cell Mol Physiol. 2004;287(6):L1323\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang C, Huang H, Liu J, Wang Y, Lu Z, Xu Z. Fasudil, a Rho-kinase inhibitor, attenuates bleomycin-induced pulmonary fibrosis in mice. Int J Mol Sci. 2012;13(7):8293\u0026ndash;307.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Simiao Pill, Rheumatoid arthritis, Interstitial lung disease, Autotaxin, Lysophosphatidic acid, Lysophosphatidic acid receptor 1, RhoA, ROCK","lastPublishedDoi":"10.21203/rs.3.rs-6597839/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6597839/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground:\u003c/h2\u003e \u003cp\u003eIn Traditional Chinese Medicine theory, Simiao Pill represents a traditional herbal formulation used for rheumatoid arthritis (RA) management. Initial research indicates that Simiao Pill might have therapeutic benefits for RA linked with interstitial lung disease (RA-ILD), although the mechanisms are not yet understood. Thus, this research seeks to explore the therapeutic potential of Simiao Pill in treating RA-ILD and uncover its molecular mechanisms.\u003c/p\u003e\u003ch2\u003eMethods:\u003c/h2\u003e \u003cp\u003eDBA/1 mice were used as animal models, with a collagen-induced arthritis model (CIA) and a bleomycin (BLM)-induced pulmonary fibrosis model (CIA-BLM). Ultra-high-performance liquid chromatography-tandem mass spectrometry was applied to profile the chemical composition of Simiao Pill. The therapeutic effects on arthritis and pulmonary fibrosis were evaluated through microcomputed tomography, histopathological examination, immunohistochemical staining, Western blot, ELISA assays, etc. Lipidomics analysis was performed to screen potential metabolic targets of Simiao Pill, followed by validation experiments on the identified targets.\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e \u003cp\u003eSimiao Pill significantly reduced the arthritis index and decreased bone and cartilage damage in CIA-BLM mice. Additionally, it inhibited the expression of the inflammatory cytokines TNF-α, IL-6, CXCL1, and CXCL2 and reduced inflammatory cell infiltration in lung tissue. Most importantly, it downregulated the levels of the key profibrotic factor TGF-β1, the myofibroblast marker α-SMA, and fibrotic extracellular matrix components such as fibronectin, collagen-1, and collagen-3. These findings suggest that Simiao Pill may inhibits the transition of pulmonary fibroblasts to pulmonary myofibroblasts. Pulmonary lipidomics analysis revealed that lysophosphatidic acid (LPA) may serve as metabolic targets of Simiao Pill. Further validation experiments demonstrated elevated levels of autotaxin (ATX) and LPA in the serum and bronchoalveolar lavage fluid of CIA-BLM mice, accompanied by upregulation of LPA receptor 1 (LPA1) and downstream RhoA/ROCK2 signaling molecules in lung tissues. Notably, Simiao Pill effectively reversed these pathological alterations.\u003c/p\u003e\u003ch2\u003eConclusions:\u003c/h2\u003e \u003cp\u003eSimiao Pill not only alleviated arthritis and bone destruction but also reduced pulmonary inflammation and fibrosis in CIA-BLM mice. The observed lung-protective properties may be attributed to inhibition of the ATX/LPA/LPA1 cascade and subsequent suppression of the RhoA/ROCK2 signaling axis, suggesting that Simiao Pill has potential therapeutic value for RA-ILD.\u003c/p\u003e","manuscriptTitle":"Effects of Simiao Pill on rheumatoid arthritis complicated with interstitial lung disease via the ATX/LPA/LPA1 and RhoA/ROCK2 signaling pathways","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-16 12:05:33","doi":"10.21203/rs.3.rs-6597839/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"90ca2b92-c0ca-41c6-b20e-6ae2e825106f","owner":[],"postedDate":"May 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-09T12:11:19+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-16 12:05:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6597839","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6597839","identity":"rs-6597839","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}