Lysophosphatidic acid promotes colitis-associated intestinal fibrosis by suppressing autophagy via LPAR3/ AMPK/mTOR pathway

Lysophosphatidic acid promotes colitis-associated intestinal fibrosis by suppressing autophagy via LPAR3/ AMPK/mTOR pathway | 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 Lysophosphatidic acid promotes colitis-associated intestinal fibrosis by suppressing autophagy via LPAR3/ AMPK/mTOR pathway Junjie Lin, Shu Wang, Le Cao, Lu Wang, Jiajia Li, Nana Tang, Chunhua Jiao, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7509054/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 7 You are reading this latest preprint version Abstract Background Intestinal fibrosis, which causes bowel stricture and obstruction, is a serious complication of Crohn’s disease (CD). Lysophosphatidic acid (LPA), a bioactive lipid mediator, exerts pro-fibrotic effects in various chronic diseases through its six cognate receptors (LPAR1-6). While both elevated LPA levels and impaired autophagy have been observed in CD patients, their specific contributions to intestinal fibrogenesis remain poorly understood. Methods In this study, the plasma levels of LPA were measured in CD patients with or without intestinal fibrosis by ELISA. Fibrotic and non-fibrotic intestinal tissues were obtained from patients with CD undergoing surgical resection. Masson's staining was performed to evaluate the fibrosis index score. The mRNA expressions of LPAR3 , collagen I and α-SMA were detected in intestinal tissues from CD patients with or without intestinal fibrosis by reverse-transcriptase polymerase chain reaction (qRT-PCR). DSS-induced chronic colitis model with intestinal fibrosis were established in C57BL/6J mice. These mice were given or not given intraperitoneal injection of an LPAR3 inhibitor (Ki16425) and evaluated the fibrosis indices in the colon. Human intestinal fibroblasts (HIFs) were isolated from surgically resected fibrotic intestines of CD patients and the effects of LPA on autophagy, activation, proliferation, and migration of HIFs were determined. Results Clinical analyses revealed significantly elevated plasma LPA levels and increased LPAR3 expression in fibrotic intestinal tissues of CD patients compared to non-fibrotic controls. The elevated plasma LPA levels were observed in mice with DSS-induced chronic colitis, LPAR3 inhibition attenuated both intestinal inflammation and fibrosis. In vitro experiments, Mechanistically, we demonstrated that LPA promotes HIF migration, activation and proliferation through LPAR3-dependent suppression of autophagy via the AMPK-mTOR pathway. These pro-fibrotic effects were reversed by pharmacological intervention at multiple levels—including LPAR3 antagonism (Ki16425), AMPK activation (A-769662) or mTOR inhibition (rapamycin). Conclusion Our findings establish a novel LPA-LPAR3-AMPK-mTOR signaling axis that drives intestinal fibrosis by suppressing autophagy in HIFs. This pathway represents a promising therapeutic target for preventing fibrosis progression in CD. Lysophosphatidic acid Crohn’s disease Intestinal fibrosis autophagy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Crohn's disease (CD) is characterized by inflammation that extends through the entire thickness of the bowel wall and can affect any segment of the gastrointestinal tract, leading to progressive and destructive disease behavior. Notably, the global prevalence of CD has been steadily increasing in recent decades. [ 1 , 2 ] Intestinal fibrosis frequently occurs as a complication in CD, affecting over one-third of patients throughout their disease progression. [ 3 ] This fibrotic process leads to bowel stricture formation and subsequent obstruction, frequently necessitating surgical intervention due to the lack of effective medical therapies. Therefore, elucidating the molecular pathogenesis of intestinal fibrosis and developing novel therapeutic interventions have become urgent clinical priorities. Emerging evidence has demonstrated that intestinal fibrosis results from a complex interplay of multiple pathogenic factors, including activated mesenchymal cells, pro-fibrotic cytokines, microbial-derived metabolites, and dysfunctional mesenteric adipocytes. [ 4 ] Among them, the combined increase of mesenchymal cell numbers including fibroblasts and myofibroblasts and excessive extracellular matrix (ECM) secretion are the hallmark features of intestinal fibrosis. [ 5 ] Recently, studies have shown that lipids significantly promote the proliferation of human intestinal fibroblasts (HIFs). [ 6 ] This finding is particularly relevant given the well-documented dysregulation of lipid metabolism in inflammatory bowel disease (IBD) patients, which manifests as significant alterations in specific lipid species including lysophosphatidic acid (LPA), phosphatidylserine (PS), and singosin-1-phosphate (S1P). [ 7 ] LPA, a bioactive glycerophospholipid predominantly secreted by platelets, fat cells and fibroblasts, exerts its biological effects through six identified G protein-coupled receptors (Lysophosphatidic acid receptor 1–6, LPAR 1–6 ). [ 8 ] Substantial evidence has established that LPA signaling regulates diverse cellular processes including enhanced proliferation, inhibited apoptosis, and promoted migration. [ 9 – 11 ] Research indicates that LPA is crucial in the development of various chronic fibrotic diseases, including scleroderma fibrosis, [ 12 ] lung fibrosis, [ 13 ] and kidney fibrosis. [ 14 ] Studies have demonstrated elevated LPA levels in patients with CD. [ 7 ] It has been shown that LPA can suppress autophagy by binding to LPAR3. [ 15 ] Moreover, autophagy inhibition has also been observed in patients with CD. [ 16 , 17 ] Despite references, the role of these factors in CD-associated intestinal fibrosis remains largely unexplored. In this study, we investigated the pathogenic role of LPA signaling in intestinal fibrosis and uncovered its underlying molecular mechanisms. Our findings indicate that LPA significantly contributes to intestinal fibrosis in CD. LPA promoted the migration, activation, and proliferation of HIFs by suppressing autophagy through activation of LPAR3 and mTOR pathways. 2. Materials and Methods 2.1 Patients samples Intestinal tissue specimens were prospectively collected from patients with CD undergoing surgical resection at The First Affiliated Hospital with Nanjing Medical University. Intestinal tissues, both fibrotic and non-fibrotic, were collected from each patient. Following collection, the samples were immediately processed with one portion frozen at -80°C for subsequent analysis and another portion fixed in 4% paraformaldehyde for histological examination. The Ethics Committee of Nanjing Medical University approved the study protocol (Approval No 2023-SR-852), with all participants providing written informed consent before sample collection. 2.2 Histological evaluation Masson's trichrome staining was conducted systematically to evaluate intestinal fibrosis levels. Fibrosis severity was graded according to established histological criteria [ 18 ] as follows: grade 0 indicated either no fibrosis or fibrosis confined to the submucosal layer; grade 1 represented significant submucosal fibrosis with maintained architectural integrity of the intestinal wall layers; and grade 2 denoted extensive transmural fibrosis with complete disruption of the normal tissue architecture.. 2.3 Mice C57BL/6J mice were obtained from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and maintained under stringent specific pathogen-free (SPF) conditions at Nanjing Medical University's animal facility. All experimental procedures complied with institutional guidelines and were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University [IACUC-231200]. 2.4 Induction of Chronic Colitis Chronic colitis was induced in mice using a well-established dextran sulfate sodium (DSS) model (MP Biomedicals, USA). A 1.5% DSS solution was provided in drinking water for 7 days, followed by 7 days of regular water, with this cycle repeated three times (over a total of 42 days). Mice were observed twice daily for body weight changes, and disease activity was assessed using a standardized Disease Activity Index (DAI) that includes weight loss percentage, stool consistency, and bleeding (occult/gross). To explore LPA/LPAR3 pathway's impact on intestinal fibrosis, mice were divided into two groups (n = 5/group): 1) DSS control group receiving DSS alone, and 2) DSS + LPAR3 antagonist group receiving DSS with daily intraperitoneal ki16425 (10 mg/kg) during DSS exposure. On day 42, all animals were humanely euthanized for endpoint analyses. The entire colon was excised from ileocecal junction to anal verge for length measurement. Tissue samples were processed by fixing in 4% paraformaldehyde for histological analyses, including H&E and Masson's trichrome staining, and prepared for immunofluorescence detection of α-smooth muscle actin (α-SMA). Histopathological evaluation was performed using a standardized scoring system assessing two key parameters: (1) epithelial damage (scored 0–4) and (2) inflammatory cell infiltration (scored 0–4) [ 19 ] . Intestinal fibrosis was quantitatively assessed through Masson's trichrome staining for collagen deposition and immunofluorescence analysis of α-smooth muscle actin (α-SMA) expression as a marker of myofibroblast activation. 2.5 Cell culture Primary human intestinal fibroblasts (HIFs) were isolated from fibrotic tissues collected during surgical resection from CD patients, following an established protocol. [ 20 ] Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Meisen CTCC, China) under standard conditions (37°C, 5% CO₂, humidified environment). For all experiments, HIFs between passages 3 and 10 were utilized to ensure consistent cellular characteristics. 2.6 RNA isolation followed by quantitative real-time PCR (qPCR) analysis Total RNA extraction was performed with TRIzol reagent (Invitrogen, USA) according to the manufacturer's protocol. The homogenized samples underwent phase separation with chloroform, followed by RNA precipitation from the aqueous phase using isopropanol. The RNA pellet was washed, air-dried, and resuspended in RNase-free water. Spectrophotometry was used to assess RNA concentration and purity. Using a Reverse Transcription Kit (Vazyme Biotech, China), 1 µg of total RNA was converted into first-strand cDNA. Quantitative PCR was conducted on the ABI 7300 Real-Time PCR System (Thermo Scientific, USA) utilizing SYBR Green QPCR Master Mix (Vazyme Biotech, China). Refer to Supplementary Table 2 for primer sequences. 2.7 Protein extraction and Western blot (WB) Tissues or cells were rinsed with phosphate-buffered saline (PBS, Servicebio, China) and lysed on ice using RIPA Lysis Buffer (Beyotime, China) containing the protease inhibitor phenylmethanesulfonyl fluoride (PMSF, Beyotime, China). Protein levels were measured with a BCA Protein Assay Kit (Beyotime, China). Equal protein quantities were then subjected to SDS-PAGE and transferred to a PVDF membrane (Millipore, USA). The membranes were first blocked with 5% BSA (Biosharp, China) for 2 hours at room temperature, followed by overnight incubation at 4°C with the following primary antibodies: GAPDH (1:10000, Proteintech, USA), α-SMA (1:1000, Santa, USA), p-PI3K, PI3K, p-AKT, AKT (all 1:1000, CST, Germany), p-AMPK, AMPK (both 1:2000, abmart, China), p-mTOR, mTOR (both 1:1000, abmart, China), and p-MEK (1:2000, MCE, USA), MEK (1:1000, MCE, USA). Following washing, membranes were incubated for 2 hours at room temperature with the corresponding secondary antibodies: goat anti-mouse and goat anti-rabbit (1:10000, Bioworld Technology, USA). Protein bands were detected using an Enhanced Chemiluminescence (ECL) detection kit (Biosharp, USA) and quantified using ImageJ software. For sequential detection of different targets, membranes were stripped with blot stripping buffer (Thermo, USA) before reprobing. 2.8 Enzyme-linked immunosorbent assay (ELISA) The blood samples were collected with EDTA as an anticoagulant. Subsequently, the samples were centrifuged at a speed of 1000rpm and a temperature of 4°C for 15 minutes. Then the supernatant was aliquoted with care and stored at -80°C. Plasma LPA levels were measured using commercial ELISA kits (Hengyuan, Shanghai) following the manufacturer's guidelines. 2.9 Immunocytofluorescense Cells were treated with 4% paraformaldehyde for 15 minutes at room temperature and subsequently permeabilized using 0.1% Triton-X (Beyotime, China) for 20 minutes. Following 1 hour blocking with 10% goat serum (Biosharp, USA), cells were incubated overnight at 4°C with the following primary antibodies: α-SMA (1:100, Santa Cruz) and Ki67 (1:1000, Abcam). The following day, cells were incubated for 1 hour at room temperature in the dark with Alexa Fluor 594 (or 488)-labeled secondary antibody (1:1000, Proteintech, USA). Nuclei were stained with DAPI (Biosharp, Germany) for 10 minutes. Finally, the images were photographed by Thunder Imager microscope (Thunder, DMi8 Germany). 2.10 Assay for Wound-Healing Migration Cell migration ability was examined by Wound-Healing Migration Assay. were seeded in a six-well plate at a density of 1x10 ^6 cells per well and grown until reaching 80–90% confluence. Cells were pretreated for 2 h with either Ki16425 (LPAR3 inhibitor, 10 µM; MCE, USA) or rapamycin (mTOR inhibitor, 50 nM; MCE, USA) prior to LPA (MCE, USA) stimulation. A sterile 200 µl pipette tip was used to create a uniform wound in the confluent monolayer, and the area was gently washed three times with PBS to eliminate cellular debris. Wound closure was observed and documented at specified intervals using an Olympus CKX41 inverted phase-contrast microscope. 2.11 Statistical analysis Quantitative data are presented as means ± standard deviation (SD) and analyzed with GraphPad Prism (version 9.0; Canada). Two-tailed Student's t-tests were used for comparisons between two groups, and one-way ANOVA was applied for analyses involving multiple groups. A P-value of less than 0.05 was deemed statistically significant. 3. Results 3.1 Elevated plasma levels of LPA and LPAR3 were identified in CD patients with intestinal fibrosis. We collected plasma samples from CD patients, both with and without intestinal fibrosis, to examine LPA's role in the pathogenesis of intestinal fibrosis. Moreover, surgical intestinal tissue samples from CD patients, both fibrotic and non-fibrotic, were collected in pairs. Histopathological assessment revealed significantly higher fibrosis index scores in fibrotic intestinal tissues compared to non-fibrotic regions (Fig. 1 A). Fibrotic tissues exhibited increased mRNA expression of collagen genes (COL1A1, COL6A1, and COL6A3; Figure S1 A) and higher protein levels of α-smooth muscle actin (α-SMA; Figure S1 B), consistent with the fibrotic phenotype. ELISA analysis of plasma samples showed markedly higher LPA concentrations in fibrostenotic CD patients compared to non-fibrotic controls (Fig. 1 B). Comprehensive evaluation of LPA receptor expression patterns revealed that among the four LPAR subtypes (LPAR1-4), LPAR2, LPAR3 and LPAR4 mRNA levels were significantly elevated in fibrotic tissues, with LPAR3 showing the most pronounced increase (Fig. 1 C). Western blot analysis confirmed the upregulation of LPAR3 protein expression in fibrotic areas (Fig. 1 D). The findings suggest a significant role of the LPA-LPAR3 axis in the development of intestinal fibrosis in CD. 3.2 The LPA/LPAR3 pathway exacerbates inflammation and intestinal fibrosis in mice with DSS-induced chronic colitis. We established a DSS-induced chronic colitis model with intestinal fibrosis. DSS-treated mice developed characteristic features of severe colitis, including significant body weight loss (Figure S2 A), marked colonic shortening (Figure S2 B), elevated histopathological colitis scores (Fig. 2 A) and increased proinflammatory cytokine levels (Fig. 2 B). Furthermore, these animals exhibited clear evidence of intestinal fibrosis, demonstrated by increased collagen deposition (Masson's trichrome staining; Fig. 2 C), upregulated mRNA expression of collagen genes ( Col1a1, Col6a1 , and Col6a3 ; Fig. 2 D), and both increased thickness of the α-SMA-positive muscular layer and elevated α-SMA protein expression (Figs. 2 E and 2 F). We initially assessed plasma LPA levels in a DSS-induced chronic colitis model to explore the involvement of the LPA/LPAR3 pathway in intestinal fibrosis. Figure 3 A shows increased plasma LPA levels in mice with DSS-induced chronic colitis. To directly assess LPAR3's role, we administered the selective LPAR3 antagonist Ki16425 (10 mg/kg, i.p. daily) during DSS treatment (n = 5 per group). Pharmacological inhibition of LPAR3 significantly improved therapeutic outcomes in mice, evidenced by decreased inflammatory responses, reduced body weight loss (Figure S3A), maintained colon length [Figure S3B], enhanced histopathological scores (Fig. 3 B), and diminished levels of proinflammatory cytokines (IL-1β, IL-6, TNF-α, Fig. 3 C). Meanwhile, fibrosis markers were also significantly attenuated, as evidenced by reduced collagenase deposition (Fig. 3 D), decreased mRNA expressions of collagen such as Col1a1, Col6a1 and Col6a3 (Fig. 3 E), thinner α-SMA + muscular layer (Fig. 3 F), and lower α-SMA protein level (Fig. 3 G). These findings demonstrate that LPA exacerbates intestinal inflammation and fibrosis primarily through LPAR3 signaling, identifying this pathway as a potential therapeutic target for IBD-related fibrosis. 3.3 LPA promotes the migration, activation and proliferation of HIFs We examined the impact of LPA on HIFs derived from CD patients. When treated with varying LPA (0 1, 5, and 10 and 20µM) for 24 or 48 hours, HIFs exhibited dose-dependent responses. The scratch wound-healing assay revealed that LPA significantly accelerated wound closure, with maximal effect observed at 10 µM (Fig. 4 A). Western blot and immunofluorescence analyses demonstrated that 10 µM LPA treatment for 48 hours markedly increased α-SMA expression, indicating fibroblast activation (Fig. 4 B-D). Furthermore, LPA treatment elevated the proportion of proliferating (Ki67 + ) HIFs (Fig. 4 D) and upregulated COL1A1, COL6A1 and COL6A3 mRNA expression of extracellular matrix components (Fig. 4 E). These findings suggest LPA promotes intestinal fibrosis by enhancing HIF migration, activation, proliferation, and collagen production, which are critical factors in the development of intestinal fibrosis. To elucidate the receptor specificity underlying these effects, we employed the selective LPAR3 antagonist Ki16425. Pharmacological inhibition of LPAR3 effectively attenuated LPA-induced profibrotic responses in human intestinal fibroblasts, including impaired cell migration (Fig. 5 A), reduced α-SMA expression (Fig. 5 B-C), decreased cell proliferation (Fig. 5 C), and suppressed collagen production (Fig. 5 D). These results establish LPAR3 as the primary mediator of LPA's profibrotic actions on human intestinal fibroblasts. 3.5 LPA promoting migration, activation and proliferation of HIFs by suppressing autophagy via mTOR pathways Emerging evidence indicates that autophagy impairment contributes to intestinal fibrosis in CD, and pharmacological induction of autophagy attenuates fibrotic progression in mice. [ 17 , 21 ] Given that LPA has been shown to inhibit autophagy, [ 15 ] we evaluated its effects on autophagy in HIFs. Western blot analysis revealed that treatment with 10 µM LPA significantly reduced the LC3-II/LC3-I ratio while increasing p62/SQSTM1 accumulation, indicative of autophagy suppression (Fig. 6 A). Furthermore, LPA robustly enhanced mTOR phosphorylation (Fig. 6 A), an effect that was abolished by pretreatment with the LPAR3 antagonist Ki16425 (Fig. 6 B), suggesting LPAR3-dependent mTOR activation. To further investigate the role of autophagy in LPA-mediated fibrogenic responses, we pharmacologically inhibited mTOR using rapamycin, a well-characterized autophagy inducer. Notably, rapamycin treatment effectively attenuated LPA's profibrotic effects, as evidenced by several key observations: significant inhibition of LPA-induced cell migration (Fig. 6 C), marked reduction in fibroblast activation, as evidenced by decreased α-SMA expression (Fig. 6 D&E), substantial suppression of cellular proliferation (Fig. 6 E), and attenuated collagen production (Fig. 6 F). These comprehensive results establish that LPA promotes HIF migration, activation and proliferation through an mTOR-dependent autophagy suppression mechanism. 3.6. LPAR3-AMPK-mTOR axis mediates LPA-induced autophagy inhibition in HIF activation The mTOR pathway can be regulated through multiple upstream signals, including RTK-PI3K-AKT, RAS-RAF-MEK-ERK, and AMPK pathways, [ 22 ] RAS-RAF-MEK-ERK, [ 23 ] and AMP-activated protein kinase (AMPK). [ 24 ] To determine the specific mechanism underlying LPA/LPAR3-mediated mTOR activation in HIFs, we performed comprehensive phosphoprotein analysis. Western blotting revealed that treatment with 10 µM LPA did not significantly enhance PI3K or AKT phosphorylation (Fig. 7 A). However, LPA induced an increase in MEK1/2 and ERK1/2 phosphorylation, while decreasing AMPK phosphorylation (Fig. 7 A). Notably, pretreatment with the LPAR3 antagonist Ki16425 completely abolished LPA-induced AMPK suppression while having no effect on MEK-ERK pathway activation (Fig. 7 B). To functionally validate AMPK's role, we employed the specific AMPK activator A-769662. Pharmacological AMPK activation significantly attenuated all LPA-induced profibrotic responses, including reduced cell migration (Fig. 7 C), activation (Fig. 7 D & E), proliferation (Fig. 7 E) and collagen production (Fig. 7 F). These results establish that LPA promotes HIF activation and fibrogenesis primarily through an LPAR3-AMPK-mTOR signaling axis. 4. Discussion Intestinal fibrosis, marked by excessive extracellular matrix (ECM) accumulation, is a significant complication of CD, frequently resulting in strictures and necessitating surgical intervention. [ 25 ] Fibroblasts and myofibroblasts are the primary effector cells driving this pathological ECM accumulation [ 26 ] . While chronic inflammation is known to promote fibrogenesis through sustained activation of these cells, growing research reveals that aberrant lipid homeostasis significantly contributes to fibrogenesis across multiple organs, including the kidney, [ 27 ] liver, [ 28 ] lung [ 29 ] and heart. [ 30 ] Notably, lipid metabolic disturbances have also been implicated in IBD pathogenesis [ 31 , 32 ] and are increasingly recognized as key modulators of intestinal fibroblast function. [ 6 , 33 ] Among lipid mediators, LPA has garnered attention as a potent signaling molecule produced by platelets, adipocytes, and fibroblasts. LPA influences multiple cellular activities including cell proliferation, migration, and inflammation by interacting with six G protein-coupled receptors (LPAR1-6). [ 34 – 36 ] Elevated LPA levels have been reported in patients with CD, [ 7 ] with most studies focusing on its pro-inflammatory effects [ 37 ] , such as promoting monocyte-to-macrophage differentiation, M1 polarization, and lymphocyte chemotaxis. [ 38 ] [ 39 ] . Consistent with these findings, our study observed increased plasma LPA levels in murine models of DSS-induced chronic colitis, where LPAR3 inhibition attenuated intestinal inflammation, further supporting LPA’s role in IBD pathogenesis. Beyond inflammation, LPA has been implicated in fibrotic disorders of the lungs, liver, and kidneys. [ 40 , 41 ] [ 42 ] [ 43 ] . However, its contribution to CD-associated intestinal fibrosis remains poorly understood. Our study revealed significantly higher LPA levels in CD patients with fibrosis compared to those without, alongside a notable upregulation of LPAR3 expression in fibrotic intestinal tissues. Importantly, pharmacological LPAR3 blockade not only reduced inflammation but also ameliorated fibrosis in DSS-treated mice, as evidenced by decreased collagen deposition. Mechanistically, we identified that LPA promotes intestinal fibrosis by suppressing autophagy in HIFs via LPAR3-mediated mTOR activation. Autophagy, a lysosomal degradation pathway critical for cellular homeostasis, is genetically linked to CD, with risk variants in autophagy-related genes predisposing to aberrant immune responses and barrier dysfunction. [ 17 , 44 ] [ 45 ] [ 46 , 47 ] . While autophagy induction has been shown to mitigate intestinal inflammation, its role in fibrosis remains underexplored. Numerous studies have shown a link between dysregulated autophagy and fibrotic diseases.I mpaired autophagy significantly accelerates the progression of heart failure and interstitial fibrosis [ 48 ] . In idiopathic pulmonary fibrosis inhibition of autophagy promoted fibrogenesis in the lungs [ 49 ] . Regulating signaling pathway that is essential in mediating autophagy included AMPK/mTOR, PI3K/AKT, and MER/ERK [ 22 , 23 , 50 ] , and regulating autophagy through AMPK/mTOR signaling pathway has been proved to be useful in renal tubulointerstitial fibrosis [ 51 ] , the liver fibrosis nonalcoholic fatty liver disease [ 51 ] and silica-induced pulmonary fibrosis [ 52 ] . Our findings reveal that LPA disrupts autophagic flux by inhibiting AMPK phosphorylation, thereby activating mTOR-a key negative regulator of autophagy. This LPAR3-AMPK-mTOR axis drives HIF migration, activation, proliferation, and collagen production, all of which were reversed by AMPK activation. These results align with studies in other fibrotic diseases, where AMPK/mTOR modulation has proven therapeutic potential. While this study provides important insights into LPA-mediated intestinal fibrosis, several limitations should be acknowledged. First, our investigation of the autophagy regulatory mechanism in LPA-induced colitis-associated fibrosis was primarily conducted in vitro, and warrants further validation using in vivo models, such as fibroblast-specific autophagy-deficient mice. Second, while our focus was on HIFs as the main effector cells, recent evidence indicates that processes like epithelial- epithelial-to-mesenchymal transformation (EMT) and vascular endothelial-mesenchymal conversion (EndoMT) also play a significant role in intestinal fibrogenesis. [ 53 , 54 ] The potential involvement of LPA in regulating these alternative fibrogenic pathways remains to be elucidated. Future studies should systematically examine LPA's effects on epithelial and endothelial cells, as well as its possible role in facilitating cell-type transitions during fibrosis development. Conclusion In conclusion, our study establishes LPA as a critical mediator of intestinal fibrosis in CD, acting through LPAR3 to inhibit AMPK/mTOR-regulated autophagy and thereby promoting profibrotic HIF responses. Targeting the LPA-LPAR3-AMPK-mTOR axis may offer a novel therapeutic strategy to mitigate fibrosis progression in patients with CD. Declarations Author Contributions Conceptualization: J.L., S.W., X.Z. and H.Z.; Methodology: J.L., L.W., L.C and S.W.; Investigation: J.L., S.W., L.W., L.C. and H.Z.; Resources: N.T., C.J., J.M., X.Z. and H.Z.; Writing – original draft preparation: J.L., H.Z. and X.Z.; Writing – review and editing: J.L., X.Z. and H.Z. with input from all other authors; Supervision: X.Z. and H.Z.; Funding acquisition: X.Z. and H.Z. Funding This research received funding from the National Natural Science Foundation of China under Grant Numbers 82370535 and 82200582. Data Availability The authors can provide data upon reasonable requests. Conflict of interest: The authors declare no conflict of interests . Clinical trial number: not applicable. References Dolinger, M., J. Torres, and S. Vermeire. 2024. Crohn's disease. Lancet Mar 23(10432):1177–1191. Ng, S. C., H. Y. Shi, and N. Hamidi et al. 2017. 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Identification of Endothelial-to-Mesenchymal Transition as a Potential Participant in Radiation Proctitis. Am J Pathol Sep 185(9):2550–2562. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.docx Supplementary Figure 1. Evaluation of intestinal fibrosis of patients with CD. Surgical intestinal tissues were taken from nonfibrotic and fibrotic sites from CD patients with intestinal fibrosis [n=8]. [A] Levels of COL1A1、COL6A1 and COL6A3 in colonic tissues were measured by qRT-PCR. [B] Expressions of α-SMA were detected by Western blot. P < 0.05, P < 0.01, P < 0.001. Supplementary Figure 2. DSS-induced chronic colitis model in C57BL/6J mice. Mice (n = 5/group) received three cycles of 1.5% DSS treatment (7 days DSS + 7 days water recovery per cycle) and were sacrificed on day 42. [A] Longitudinal monitoring of body weight changes (%) throughout the experimental timeline. [B] Macroscopic evaluation of colon morphology and quantification of colon shortening. P < 0.05, P < 0.01, P < 0.001 Supplementary Figure 3. LPAR3 inhibition attenuates LPA-exacerbated intestinal inflammation in DSS-induced colitis. C57BL/6J mice (n=5 per group) were subjected to three cycles of colitis induction, each consisting of 7 days of 1.5% (w/v) DSS in drinking water followed by 7 days of regular water recovery, with concurrent intraperitoneal administration of the LPAR3 inhibitor Ki16425 (10 mg/kg) throughout the DSS treatment periods, and all mice were humanely euthanized for tissue collection on day 42. [A] Body weight dynamics (% change from baseline) during cyclic DSS administration with or without LPAR3 antagonist treatment. [B] Macroscopic evaluation of colon morphology and quantitative analysis of colon shortening. 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07:10:48","extension":"html","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":134853,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7509054/v1/ff5e7cfa3d50128fcbd8307c.html"},{"id":94945637,"identity":"46d0d0b8-8623-4ce8-86bf-cf94ab3a4540","added_by":"auto","created_at":"2025-11-02 09:23:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3310196,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElevated plasma levels of LPA and LPAR3 were identified in CD patients with intestinal fibrosis. \u003c/strong\u003e[A] Masson staining was performed on the colon tissues and the score of fibrosis index were evaluated (Scale bar=500 μm). [B] LPA levels in the plasma were detected by ELISA [C] Levels of \u003cem\u003eLPAR1, LPAR2, LPAR3 and LLPAR4\u003c/em\u003e in colonic tissues were measured by qRT-PCR. [D]Expression level of LPAR3 in intestinal tissues were measured by WB. All values are expressed as Mean ± SD. ns, no significance, \u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01,\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7509054/v1/4f3aff49983b3616412eedab.png"},{"id":94945630,"identity":"533eb869-32c9-41bd-be4e-1071178ac418","added_by":"auto","created_at":"2025-11-02 09:23:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":10347261,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMice with experimentally-induced chronic colitis exhibited characteristic features of intestinal fibrosis. \u003c/strong\u003e[A] Histopathological assessment by H\u0026amp;E staining (200× magnification, scale bar = 50 μm) with corresponding inflammation scoring. [B] Proinflammatory cytokines levels of \u003cem\u003eIl-1β, Il-6\u003c/em\u003e and \u003cem\u003eTnf-α\u003c/em\u003e in colons were detected by qRT-PCR. [C] Colon tissues were stained with Masson, and percentage of Masson staining positive area was calculated Scale bar=10 μm. [D] Levels of \u003cem\u003eCol1a1 Col6a1\u003c/em\u003e and \u003cem\u003eCol6a1\u003c/em\u003e in colonic tissues were measured by qRT-PCR. [E] Colon tissues were stained with immunofluorescence and a-SMA layer thickness were measured. Scale bar=200 μm. [F] Protein levels of α-SMA in each group were determined by WB. All values are expressed as Mean ± SD .\u003csup\u003e*\u003c/sup\u003eP \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003eP \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003eP \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7509054/v1/a77794f6962fed0e6833b360.png"},{"id":94945632,"identity":"ad01b43a-e3b3-4664-9022-e7f2c9dfe224","added_by":"auto","created_at":"2025-11-02 09:23:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":11438696,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe LPA/LPAR3 pathway exacerbates inflammation and intestinal fibrosis in mice with DSS-induced chronic colitis.\u003c/strong\u003e C57BL/6J mice (n=5/group) received three cycles of 1.5% DSS treatment (7 days DSS + 7 days recovery per cycle) with or without concurrent LPAR3 inhibitor administration (10 mg/kg, i.p.). All animals were sacrificed on day 42. [A] Plasma LPA levels measured by ELISA. [B] Histopathological evaluation by H\u0026amp;E staining (scale bar = 100 μm) with corresponding inflammation scoring. [C] Proinflammatory cytokines levels of \u003cem\u003eIl-1β, Il-6\u003c/em\u003e and \u003cem\u003eTnf-α\u003c/em\u003e in colons were detected by qRT-PCR. [D] Fibrosis assessment by Masson's trichrome staining (scale bar = 100 μm) with quantitative analysis of collagen deposition area. [E] Levels of \u003cem\u003eCol1a1 Col6a1\u003c/em\u003e and \u003cem\u003eCol6a1\u003c/em\u003e in colonic tissues were measured by qRT-PCR. [F] Immunofluorescence staining of α-SMA (scale bar = 200 μm) with quantitative measurement of α-SMA layer thickness. [G] Western blot analysis of α-SMA protein expression. All values are expressed as Mean ± SD .\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7509054/v1/f794ed53b0ca1ec2d0f7643b.png"},{"id":94988854,"identity":"9a493811-dd6d-4edb-98da-7888528b2e90","added_by":"auto","created_at":"2025-11-03 07:11:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":310089,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLPA promotes the migration, activation and proliferation of HIFs.\u003c/strong\u003e [A] Scratch wound assay demonstrating HIF migration following treatment with LPA (5, 10, 20 μM; scale bar = 500 μm). [B] Expression of α-SMA in fibroblasts were detected by Western blot. [C] Dual immunofluorescence staining for Ki67 (proliferation marker) and α-SMA (scale bar = 100 μm) with quantification of Ki67-positive cells. [D] Expression of \u003cem\u003eCOL1A1 COL6A1 and COL6A3\u003c/em\u003e were measured by qRT-PCR. All values are expressed as Mean ± SD. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7509054/v1/7ce509bd4771095344593b2b.png"},{"id":94945634,"identity":"e71a8142-7cb5-4eb8-9274-aaf82499179a","added_by":"auto","created_at":"2025-11-02 09:23:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":281229,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLPA promotes human intestinal fibroblasts activation, proliferation and migration via activating LPAR3\u003c/strong\u003e. Human intestinal fibroblasts treated with 10 μM LPA or 10 μM LPA+10 μM LPAR3 inhibitor (Ki16425). [A] Representative images and quantification of wound closure in scratch assays following treatments with 10 μM LPA alone or in combination with 10 μM LPAR3 antagonist (Ki16425). [B] Western blot analysis of α-smooth muscle actin (α-SMA) protein expression. [C] Immunofluorescence co-staining of Ki67 (proliferation marker) and α-SMA (scale bar = 100 μm) with quantification of Ki67\u003csup\u003e+\u003c/sup\u003e cells. [D] Expression of \u003cem\u003eCOL1A1 COL6A1 \u003c/em\u003eand \u003cem\u003eCOL6A3 \u003c/em\u003ewere measured by qRT-PCR. All values are expressed as Mean ± SD.\u003csup\u003e*\u003c/sup\u003eP \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7509054/v1/2d90860cd1b551d696b72826.png"},{"id":94945647,"identity":"969323af-e6ba-47c3-9f25-59894adb7770","added_by":"auto","created_at":"2025-11-02 09:23:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4820477,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLPA inhibits autophagy via mTOR-dependent mechanism to promote HIF activation. \u003c/strong\u003e[A] Human intestinal fibroblasts treated with the 10 μM LPA, expression of LC3B, P62, phosphorylation of mTOR and total mTOR were detected by Western blot. [B] Human intestinal fibroblasts treated 10 μM LPA with or without 10 μM Ki16425 (LPAR3 inhibitor), expression of LC3B, P62, phosphorylation of mTOR and total mTOR were detected by Western blot. [C-F] Human intestinal fibroblasts treated with 10 μ0 LPA or 10 μM LPA+50nM mTOR inhibitor (Rapamycin): [C] Cell migration by scratch assay. [D] Expression of α-SMA in fibroblasts was detected by Western blot. [E] Cell proliferation (Ki67/α-SMA co-staining; scale bar = 100 μm). [F] Expression of \u003cem\u003eCOL1A1 COL6A1 \u003c/em\u003eand\u003cem\u003e COL6A3\u003c/em\u003e were measured by qRT-PCR. All values are expressed as Mean ± SD.\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7509054/v1/b3970f2081ce0f28ea3b524e.png"},{"id":94945641,"identity":"ab5a1548-73e6-4503-b6c7-4afa2747efbb","added_by":"auto","created_at":"2025-11-02 09:23:42","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5142916,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLPA/LPAR3 regulates the activity of mTOR via suppressing the phosphorylation of AMPK. \u003c/strong\u003e[A] Human intestinal fibroblasts treated with 10 μ0 LPA , expression of phosphorylation of PI3K, AKT, AMPK and MEK were detected by Western blot. [B] Human intestinal fibroblasts treated 10 μs LPA with or without 10 μL Ki16425(LPAR3 inhibitor), expression of phosphorylation of AMPK, MEK1/2, ERK1/2 were detected by Western blot. [C] Cell migration by scratch assay. [D] Expression of α-SMA in fibroblasts was detected by Western blot. [E] Immunofluorescence co-staining of Ki67 (proliferation marker) and α-SMA (scale bar = 100 μm) with quantification of Ki67\u003csup\u003e+\u003c/sup\u003e cells. [F] Expression of \u003cem\u003eCOL1A1, COL6A1\u003c/em\u003e and\u003cem\u003e COL6A3\u003c/em\u003e were measured by qRT-PCR. All values are expressed as Mean ± SD.\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7509054/v1/6e49a132639159ff25bdb1f6.png"},{"id":94991079,"identity":"8a467817-ec4a-4ccf-8bb2-cb8b4d108275","added_by":"auto","created_at":"2025-11-03 07:19:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":34467744,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7509054/v1/1a625830-7484-4225-98bb-fd719721ee0e.pdf"},{"id":94987759,"identity":"044121f9-bed2-4110-b0d5-4aef46c7bbc0","added_by":"auto","created_at":"2025-11-03 07:02:29","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":497058,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Figure 1\u003c/strong\u003e. \u003cstrong\u003eEvaluation of intestinal fibrosis of patients with CD.\u003c/strong\u003e Surgical intestinal tissues were taken from nonfibrotic and fibrotic sites from CD patients with intestinal fibrosis [n=8]. [A] Levels of \u003cem\u003eCOL1A1、COL6A1 and COL6A3\u003c/em\u003e in colonic tissues were measured by qRT-PCR. [B] Expressions of α-SMA were detected by Western blot. \u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e\u003cem\u003e***\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 2\u003c/strong\u003e. \u003cstrong\u003eDSS-induced chronic colitis model in C57BL/6J mice.\u003c/strong\u003e Mice (n = 5/group) received three cycles of 1.5% DSS treatment (7 days DSS + 7 days water recovery per cycle) and were sacrificed on day 42. [A] Longitudinal monitoring of body weight changes (%) throughout the experimental timeline. [B] Macroscopic evaluation of colon morphology and quantification of colon shortening. \u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01,\u003csup\u003e \u003c/sup\u003e\u003csup\u003e\u003cem\u003e***\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Figure 3. LPAR3 inhibition attenuates LPA-exacerbated intestinal inflammation in DSS-induced colitis\u003c/strong\u003e. C57BL/6J mice (n=5 per group) were subjected to three cycles of colitis induction, each consisting of 7 days of 1.5% (w/v) DSS in drinking water followed by 7 days of regular water recovery, with concurrent intraperitoneal administration of the LPAR3 inhibitor Ki16425 (10 mg/kg) throughout the DSS treatment periods, and all mice were humanely euthanized for tissue collection on day 42. [A] Body weight dynamics (% change from baseline) during cyclic DSS administration with or without LPAR3 antagonist treatment. [B] Macroscopic evaluation of colon morphology and quantitative analysis of colon shortening. \u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e\u003cem\u003e***\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7509054/v1/70eaf664e0dce0618e29a556.docx"},{"id":94945639,"identity":"c3ab070f-a34d-4587-bf58-6815a34eba2b","added_by":"auto","created_at":"2025-11-02 09:23:42","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1236034,"visible":true,"origin":"","legend":"","description":"","filename":"uncroppedwesternblotimages.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7509054/v1/aac6d7bcf864ebbb48bd66d3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Lysophosphatidic acid promotes colitis-associated intestinal fibrosis by suppressing autophagy via LPAR3/ AMPK/mTOR pathway","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCrohn's disease (CD) is characterized by inflammation that extends through the entire thickness of the bowel wall and can affect any segment of the gastrointestinal tract, leading to progressive and destructive disease behavior. Notably, the global prevalence of CD has been steadily increasing in recent decades. \u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e Intestinal fibrosis frequently occurs as a complication in CD, affecting over one-third of patients throughout their disease progression. \u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e This fibrotic process leads to bowel stricture formation and subsequent obstruction, frequently necessitating surgical intervention due to the lack of effective medical therapies. Therefore, elucidating the molecular pathogenesis of intestinal fibrosis and developing novel therapeutic interventions have become urgent clinical priorities.\u003c/p\u003e\u003cp\u003eEmerging evidence has demonstrated that intestinal fibrosis results from a complex interplay of multiple pathogenic factors, including activated mesenchymal cells, pro-fibrotic cytokines, microbial-derived metabolites, and dysfunctional mesenteric adipocytes. \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e Among them, the combined increase of mesenchymal cell numbers including fibroblasts and myofibroblasts and excessive extracellular matrix (ECM) secretion are the hallmark features of intestinal fibrosis. \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e Recently, studies have shown that lipids significantly promote the proliferation of human intestinal fibroblasts (HIFs). \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e This finding is particularly relevant given the well-documented dysregulation of lipid metabolism in inflammatory bowel disease (IBD) patients, which manifests as significant alterations in specific lipid species including lysophosphatidic acid (LPA), phosphatidylserine (PS), and singosin-1-phosphate (S1P). \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eLPA, a bioactive glycerophospholipid predominantly secreted by platelets, fat cells and fibroblasts, exerts its biological effects through six identified G protein-coupled receptors (Lysophosphatidic acid receptor 1\u0026ndash;6, LPAR\u003csub\u003e1\u0026ndash;6\u003c/sub\u003e). \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e Substantial evidence has established that LPA signaling regulates diverse cellular processes including enhanced proliferation, inhibited apoptosis, and promoted migration. \u003csup\u003e[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e Research indicates that LPA is crucial in the development of various chronic fibrotic diseases, including scleroderma fibrosis, \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e lung fibrosis, \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e and kidney fibrosis. \u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e Studies have demonstrated elevated LPA levels in patients with CD. \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e It has been shown that LPA can suppress autophagy by binding to LPAR3. \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e Moreover, autophagy inhibition has also been observed in patients with CD. \u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e Despite references, the role of these factors in CD-associated intestinal fibrosis remains largely unexplored.\u003c/p\u003e\u003cp\u003eIn this study, we investigated the pathogenic role of LPA signaling in intestinal fibrosis and uncovered its underlying molecular mechanisms. Our findings indicate that LPA significantly contributes to intestinal fibrosis in CD. LPA promoted the migration, activation, and proliferation of HIFs by suppressing autophagy through activation of LPAR3 and mTOR pathways.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Patients samples\u003c/h2\u003e\u003cp\u003eIntestinal tissue specimens were prospectively collected from patients with CD undergoing surgical resection at The First Affiliated Hospital with Nanjing Medical University. Intestinal tissues, both fibrotic and non-fibrotic, were collected from each patient. Following collection, the samples were immediately processed with one portion frozen at -80\u0026deg;C for subsequent analysis and another portion fixed in 4% paraformaldehyde for histological examination. The Ethics Committee of Nanjing Medical University approved the study protocol (Approval No 2023-SR-852), with all participants providing written informed consent before sample collection.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Histological evaluation\u003c/h2\u003e\u003cp\u003eMasson's trichrome staining was conducted systematically to evaluate intestinal fibrosis levels. Fibrosis severity was graded according to established histological criteria \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e as follows: grade 0 indicated either no fibrosis or fibrosis confined to the submucosal layer; grade 1 represented significant submucosal fibrosis with maintained architectural integrity of the intestinal wall layers; and grade 2 denoted extensive transmural fibrosis with complete disruption of the normal tissue architecture..\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Mice\u003c/h2\u003e\u003cp\u003eC57BL/6J mice were obtained from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and maintained under stringent specific pathogen-free (SPF) conditions at Nanjing Medical University's animal facility. All experimental procedures complied with institutional guidelines and were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University [IACUC-231200].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Induction of Chronic Colitis\u003c/h2\u003e\u003cp\u003eChronic colitis was induced in mice using a well-established dextran sulfate sodium (DSS) model (MP Biomedicals, USA). A 1.5% DSS solution was provided in drinking water for 7 days, followed by 7 days of regular water, with this cycle repeated three times (over a total of 42 days). Mice were observed twice daily for body weight changes, and disease activity was assessed using a standardized Disease Activity Index (DAI) that includes weight loss percentage, stool consistency, and bleeding (occult/gross). To explore LPA/LPAR3 pathway's impact on intestinal fibrosis, mice were divided into two groups (n\u0026thinsp;=\u0026thinsp;5/group): 1) DSS control group receiving DSS alone, and 2) DSS\u0026thinsp;+\u0026thinsp;LPAR3 antagonist group receiving DSS with daily intraperitoneal ki16425 (10 mg/kg) during DSS exposure. On day 42, all animals were humanely euthanized for endpoint analyses. The entire colon was excised from ileocecal junction to anal verge for length measurement. Tissue samples were processed by fixing in 4% paraformaldehyde for histological analyses, including H\u0026amp;E and Masson's trichrome staining, and prepared for immunofluorescence detection of α-smooth muscle actin (α-SMA). Histopathological evaluation was performed using a standardized scoring system assessing two key parameters: (1) epithelial damage (scored 0\u0026ndash;4) and (2) inflammatory cell infiltration (scored 0\u0026ndash;4) \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Intestinal fibrosis was quantitatively assessed through Masson's trichrome staining for collagen deposition and immunofluorescence analysis of α-smooth muscle actin (α-SMA) expression as a marker of myofibroblast activation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Cell culture\u003c/h2\u003e\u003cp\u003ePrimary human intestinal fibroblasts (HIFs) were isolated from fibrotic tissues collected during surgical resection from CD patients, following an established protocol. \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e Cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; Meisen CTCC, China) under standard conditions (37\u0026deg;C, 5% CO₂, humidified environment). For all experiments, HIFs between passages 3 and 10 were utilized to ensure consistent cellular characteristics.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 RNA isolation followed by quantitative real-time PCR (qPCR) analysis\u003c/h2\u003e\u003cp\u003eTotal RNA extraction was performed with TRIzol reagent (Invitrogen, USA) according to the manufacturer's protocol. The homogenized samples underwent phase separation with chloroform, followed by RNA precipitation from the aqueous phase using isopropanol. The RNA pellet was washed, air-dried, and resuspended in RNase-free water. Spectrophotometry was used to assess RNA concentration and purity. Using a Reverse Transcription Kit (Vazyme Biotech, China), 1 \u0026micro;g of total RNA was converted into first-strand cDNA. Quantitative PCR was conducted on the ABI 7300 Real-Time PCR System (Thermo Scientific, USA) utilizing SYBR Green QPCR Master Mix (Vazyme Biotech, China). Refer to Supplementary Table\u0026nbsp;2 for primer sequences.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Protein extraction and Western blot (WB)\u003c/h2\u003e\u003cp\u003eTissues or cells were rinsed with phosphate-buffered saline (PBS, Servicebio, China) and lysed on ice using RIPA Lysis Buffer (Beyotime, China) containing the protease inhibitor phenylmethanesulfonyl fluoride (PMSF, Beyotime, China). Protein levels were measured with a BCA Protein Assay Kit (Beyotime, China). Equal protein quantities were then subjected to SDS-PAGE and transferred to a PVDF membrane (Millipore, USA). The membranes were first blocked with 5% BSA (Biosharp, China) for 2 hours at room temperature, followed by overnight incubation at 4\u0026deg;C with the following primary antibodies: GAPDH (1:10000, Proteintech, USA), α-SMA (1:1000, Santa, USA), p-PI3K, PI3K, p-AKT, AKT (all 1:1000, CST, Germany), p-AMPK, AMPK (both 1:2000, abmart, China), p-mTOR, mTOR (both 1:1000, abmart, China), and p-MEK (1:2000, MCE, USA), MEK (1:1000, MCE, USA). Following washing, membranes were incubated for 2 hours at room temperature with the corresponding secondary antibodies: goat anti-mouse and goat anti-rabbit (1:10000, Bioworld Technology, USA). Protein bands were detected using an Enhanced Chemiluminescence (ECL) detection kit (Biosharp, USA) and quantified using ImageJ software. For sequential detection of different targets, membranes were stripped with blot stripping buffer (Thermo, USA) before reprobing.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Enzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e\u003cp\u003eThe blood samples were collected with EDTA as an anticoagulant. Subsequently, the samples were centrifuged at a speed of 1000rpm and a temperature of 4\u0026deg;C for 15 minutes. Then the supernatant was aliquoted with care and stored at -80\u0026deg;C. Plasma LPA levels were measured using commercial ELISA kits (Hengyuan, Shanghai) following the manufacturer's guidelines.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Immunocytofluorescense\u003c/h2\u003e\u003cp\u003eCells were treated with 4% paraformaldehyde for 15 minutes at room temperature and subsequently permeabilized using 0.1% Triton-X (Beyotime, China) for 20 minutes. Following 1 hour blocking with 10% goat serum (Biosharp, USA), cells were incubated overnight at 4\u0026deg;C with the following primary antibodies: α-SMA (1:100, Santa Cruz) and Ki67 (1:1000, Abcam). The following day, cells were incubated for 1 hour at room temperature in the dark with Alexa Fluor 594 (or 488)-labeled secondary antibody (1:1000, Proteintech, USA). Nuclei were stained with DAPI (Biosharp, Germany) for 10 minutes. Finally, the images were photographed by Thunder Imager microscope (Thunder, DMi8 Germany).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Assay for Wound-Healing Migration\u003c/h2\u003e\u003cp\u003eCell migration ability was examined by Wound-Healing Migration Assay. were seeded in a six-well plate at a density of 1x10\u003csup\u003e^6\u003c/sup\u003e cells per well and grown until reaching 80\u0026ndash;90% confluence. Cells were pretreated for 2 h with either Ki16425 (LPAR3 inhibitor, 10 \u0026micro;M; MCE, USA) or rapamycin (mTOR inhibitor, 50 nM; MCE, USA) prior to LPA (MCE, USA) stimulation. A sterile 200 \u0026micro;l pipette tip was used to create a uniform wound in the confluent monolayer, and the area was gently washed three times with PBS to eliminate cellular debris. Wound closure was observed and documented at specified intervals using an Olympus CKX41 inverted phase-contrast microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Statistical analysis\u003c/h2\u003e\u003cp\u003eQuantitative data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) and analyzed with GraphPad Prism (version 9.0; Canada). Two-tailed Student's t-tests were used for comparisons between two groups, and one-way ANOVA was applied for analyses involving multiple groups. A P-value of less than 0.05 was deemed statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Elevated plasma levels of LPA and LPAR3 were identified in CD patients with intestinal fibrosis.\u003c/h2\u003e\u003cp\u003eWe collected plasma samples from CD patients, both with and without intestinal fibrosis, to examine LPA's role in the pathogenesis of intestinal fibrosis. Moreover, surgical intestinal tissue samples from CD patients, both fibrotic and non-fibrotic, were collected in pairs. Histopathological assessment revealed significantly higher fibrosis index scores in fibrotic intestinal tissues compared to non-fibrotic regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Fibrotic tissues exhibited increased mRNA expression of collagen genes (COL1A1, COL6A1, and COL6A3; Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA) and higher protein levels of α-smooth muscle actin (α-SMA; Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB), consistent with the fibrotic phenotype. ELISA analysis of plasma samples showed markedly higher LPA concentrations in fibrostenotic CD patients compared to non-fibrotic controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Comprehensive evaluation of LPA receptor expression patterns revealed that among the four LPAR subtypes (LPAR1-4), \u003cem\u003eLPAR2, LPAR3\u003c/em\u003e and \u003cem\u003eLPAR4\u003c/em\u003e mRNA levels were significantly elevated in fibrotic tissues, with LPAR3 showing the most pronounced increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Western blot analysis confirmed the upregulation of LPAR3 protein expression in fibrotic areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). The findings suggest a significant role of the LPA-LPAR3 axis in the development of intestinal fibrosis in CD.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.2 The LPA/LPAR3 pathway exacerbates inflammation and intestinal fibrosis in mice with DSS-induced chronic colitis.\u003c/h2\u003e\u003cp\u003eWe established a DSS-induced chronic colitis model with intestinal fibrosis. DSS-treated mice developed characteristic features of severe colitis, including significant body weight loss (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA), marked colonic shortening (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB), elevated histopathological colitis scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and increased proinflammatory cytokine levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Furthermore, these animals exhibited clear evidence of intestinal fibrosis, demonstrated by increased collagen deposition (Masson's trichrome staining; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), upregulated mRNA expression of collagen genes (\u003cem\u003eCol1a1, Col6a1\u003c/em\u003e, and \u003cem\u003eCol6a3\u003c/em\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), and both increased thickness of the α-SMA-positive muscular layer and elevated α-SMA protein expression (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe initially assessed plasma LPA levels in a DSS-induced chronic colitis model to explore the involvement of the LPA/LPAR3 pathway in intestinal fibrosis. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA shows increased plasma LPA levels in mice with DSS-induced chronic colitis. To directly assess LPAR3's role, we administered the selective LPAR3 antagonist Ki16425 (10 mg/kg, i.p. daily) during DSS treatment (n\u0026thinsp;=\u0026thinsp;5 per group). Pharmacological inhibition of LPAR3 significantly improved therapeutic outcomes in mice, evidenced by decreased inflammatory responses, reduced body weight loss (Figure S3A), maintained colon length [Figure S3B], enhanced histopathological scores (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), and diminished levels of proinflammatory cytokines (IL-1β, IL-6, TNF-α, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Meanwhile, fibrosis markers were also significantly attenuated, as evidenced by reduced collagenase deposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), decreased mRNA expressions of collagen such as \u003cem\u003eCol1a1, Col6a1\u003c/em\u003eand \u003cem\u003eCol6a3\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), thinner α-SMA\u003csup\u003e+\u003c/sup\u003e muscular layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF), and lower α-SMA protein level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). These findings demonstrate that LPA exacerbates intestinal inflammation and fibrosis primarily through LPAR3 signaling, identifying this pathway as a potential therapeutic target for IBD-related fibrosis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.3 LPA promotes the migration, activation and proliferation of HIFs\u003c/h2\u003e\u003cp\u003eWe examined the impact of LPA on HIFs derived from CD patients. When treated with varying LPA (0 1, 5, and 10 and 20\u0026micro;M) for 24 or 48 hours, HIFs exhibited dose-dependent responses. The scratch wound-healing assay revealed that LPA significantly accelerated wound closure, with maximal effect observed at 10 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Western blot and immunofluorescence analyses demonstrated that 10 \u0026micro;M LPA treatment for 48 hours markedly increased α-SMA expression, indicating fibroblast activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-D). Furthermore, LPA treatment elevated the proportion of proliferating (Ki67\u003csup\u003e+\u003c/sup\u003e) HIFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) and upregulated \u003cem\u003eCOL1A1, COL6A1\u003c/em\u003e and \u003cem\u003eCOL6A3\u003c/em\u003e mRNA expression of extracellular matrix components (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). These findings suggest LPA promotes intestinal fibrosis by enhancing HIF migration, activation, proliferation, and collagen production, which are critical factors in the development of intestinal fibrosis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo elucidate the receptor specificity underlying these effects, we employed the selective LPAR3 antagonist Ki16425. Pharmacological inhibition of LPAR3 effectively attenuated LPA-induced profibrotic responses in human intestinal fibroblasts, including impaired cell migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), reduced α-SMA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-C), decreased cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), and suppressed collagen production (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). These results establish LPAR3 as the primary mediator of LPA's profibrotic actions on human intestinal fibroblasts.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.5 LPA promoting migration, activation and proliferation of HIFs by suppressing autophagy via mTOR pathways\u003c/h2\u003e\u003cp\u003eEmerging evidence indicates that autophagy impairment contributes to intestinal fibrosis in CD, and pharmacological induction of autophagy attenuates fibrotic progression in mice. \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e Given that LPA has been shown to inhibit autophagy, \u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e we evaluated its effects on autophagy in HIFs. Western blot analysis revealed that treatment with 10 \u0026micro;M LPA significantly reduced the LC3-II/LC3-I ratio while increasing p62/SQSTM1 accumulation, indicative of autophagy suppression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Furthermore, LPA robustly enhanced mTOR phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), an effect that was abolished by pretreatment with the LPAR3 antagonist Ki16425 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), suggesting LPAR3-dependent mTOR activation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further investigate the role of autophagy in LPA-mediated fibrogenic responses, we pharmacologically inhibited mTOR using rapamycin, a well-characterized autophagy inducer. Notably, rapamycin treatment effectively attenuated LPA's profibrotic effects, as evidenced by several key observations: significant inhibition of LPA-induced cell migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), marked reduction in fibroblast activation, as evidenced by decreased α-SMA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD\u0026amp;E), substantial suppression of cellular proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), and attenuated collagen production (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). These comprehensive results establish that LPA promotes HIF migration, activation and proliferation through an mTOR-dependent autophagy suppression mechanism.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.6. LPAR3-AMPK-mTOR axis mediates LPA-induced autophagy inhibition in HIF activation\u003c/h2\u003e\u003cp\u003eThe mTOR pathway can be regulated through multiple upstream signals, including RTK-PI3K-AKT, RAS-RAF-MEK-ERK, and AMPK pathways, \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e RAS-RAF-MEK-ERK, \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e and AMP-activated protein kinase (AMPK). \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e To determine the specific mechanism underlying LPA/LPAR3-mediated mTOR activation in HIFs, we performed comprehensive phosphoprotein analysis. Western blotting revealed that treatment with 10 \u0026micro;M LPA did not significantly enhance PI3K or AKT phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). However, LPA induced an increase in MEK1/2 and ERK1/2 phosphorylation, while decreasing AMPK phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Notably, pretreatment with the LPAR3 antagonist Ki16425 completely abolished LPA-induced AMPK suppression while having no effect on MEK-ERK pathway activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). To functionally validate AMPK's role, we employed the specific AMPK activator A-769662. Pharmacological AMPK activation significantly attenuated all LPA-induced profibrotic responses, including reduced cell migration (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD \u0026amp; E), proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE) and collagen production (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). These results establish that LPA promotes HIF activation and fibrogenesis primarily through an LPAR3-AMPK-mTOR signaling axis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIntestinal fibrosis, marked by excessive extracellular matrix (ECM) accumulation, is a significant complication of CD, frequently resulting in strictures and necessitating surgical intervention. \u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e Fibroblasts and myofibroblasts are the primary effector cells driving this pathological ECM accumulation \u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. While chronic inflammation is known to promote fibrogenesis through sustained activation of these cells, growing research reveals that aberrant lipid homeostasis significantly contributes to fibrogenesis across multiple organs, including the kidney, \u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e liver, \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e lung \u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e and heart. \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e Notably, lipid metabolic disturbances have also been implicated in IBD pathogenesis \u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e and are increasingly recognized as key modulators of intestinal fibroblast function. \u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eAmong lipid mediators, LPA has garnered attention as a potent signaling molecule produced by platelets, adipocytes, and fibroblasts. LPA influences multiple cellular activities including cell proliferation, migration, and inflammation by interacting with six G protein-coupled receptors (LPAR1-6). \u003csup\u003e[\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e Elevated LPA levels have been reported in patients with CD, \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e with most studies focusing on its pro-inflammatory effects \u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e, such as promoting monocyte-to-macrophage differentiation, M1 polarization, and lymphocyte chemotaxis.\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. Consistent with these findings, our study observed increased plasma LPA levels in murine models of DSS-induced chronic colitis, where LPAR3 inhibition attenuated intestinal inflammation, further supporting LPA\u0026rsquo;s role in IBD pathogenesis.\u003c/p\u003e\u003cp\u003eBeyond inflammation, LPA has been implicated in fibrotic disorders of the lungs, liver, and kidneys. \u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. However, its contribution to CD-associated intestinal fibrosis remains poorly understood. Our study revealed significantly higher LPA levels in CD patients with fibrosis compared to those without, alongside a notable upregulation of LPAR3 expression in fibrotic intestinal tissues. Importantly, pharmacological LPAR3 blockade not only reduced inflammation but also ameliorated fibrosis in DSS-treated mice, as evidenced by decreased collagen deposition.\u003c/p\u003e\u003cp\u003eMechanistically, we identified that LPA promotes intestinal fibrosis by suppressing autophagy in HIFs via LPAR3-mediated mTOR activation. Autophagy, a lysosomal degradation pathway critical for cellular homeostasis, is genetically linked to CD, with risk variants in autophagy-related genes predisposing to aberrant immune responses and barrier dysfunction. \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. While autophagy induction has been shown to mitigate intestinal inflammation, its role in fibrosis remains underexplored. Numerous studies have shown a link between dysregulated autophagy and fibrotic diseases.I mpaired autophagy significantly accelerates the progression of heart failure and interstitial fibrosis\u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e. In idiopathic pulmonary fibrosis inhibition of autophagy promoted fibrogenesis in the lungs\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. Regulating signaling pathway that is essential in mediating autophagy included AMPK/mTOR, PI3K/AKT, and MER/ERK\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e, and regulating autophagy through AMPK/mTOR signaling pathway has been proved to be useful in renal tubulointerstitial fibrosis\u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e, the liver fibrosis nonalcoholic fatty liver disease\u003csup\u003e[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]\u003c/sup\u003e and silica-induced pulmonary fibrosis\u003csup\u003e[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]\u003c/sup\u003e. Our findings reveal that LPA disrupts autophagic flux by inhibiting AMPK phosphorylation, thereby activating mTOR-a key negative regulator of autophagy. This LPAR3-AMPK-mTOR axis drives HIF migration, activation, proliferation, and collagen production, all of which were reversed by AMPK activation. These results align with studies in other fibrotic diseases, where AMPK/mTOR modulation has proven therapeutic potential.\u003c/p\u003e\u003cp\u003eWhile this study provides important insights into LPA-mediated intestinal fibrosis, several limitations should be acknowledged. First, our investigation of the autophagy regulatory mechanism in LPA-induced colitis-associated fibrosis was primarily conducted in vitro, and warrants further validation using in vivo models, such as fibroblast-specific autophagy-deficient mice. Second, while our focus was on HIFs as the main effector cells, recent evidence indicates that processes like epithelial- epithelial-to-mesenchymal transformation (EMT) and vascular endothelial-mesenchymal conversion (EndoMT) also play a significant role in intestinal fibrogenesis. \u003csup\u003e[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]\u003c/sup\u003e The potential involvement of LPA in regulating these alternative fibrogenic pathways remains to be elucidated. Future studies should systematically examine LPA's effects on epithelial and endothelial cells, as well as its possible role in facilitating cell-type transitions during fibrosis development.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our study establishes LPA as a critical mediator of intestinal fibrosis in CD, acting through LPAR3 to inhibit AMPK/mTOR-regulated autophagy and thereby promoting profibrotic HIF responses. Targeting the LPA-LPAR3-AMPK-mTOR axis may offer a novel therapeutic strategy to mitigate fibrosis progression in patients with CD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: J.L., S.W., X.Z. and H.Z.; Methodology: J.L., L.W., L.C and S.W.; Investigation: J.L., S.W., L.W., L.C. and H.Z.; Resources: N.T., C.J., J.M., X.Z. and H.Z.; Writing\u0026nbsp;\u0026ndash;\u0026nbsp;original draft preparation: J.L., H.Z. and X.Z.; Writing\u0026nbsp;\u0026ndash;\u0026nbsp;review and editing: J.L., X.Z. and H.Z. with input from all other authors; Supervision: X.Z. and H.Z.; Funding acquisition: X.Z. and H.Z.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received funding from the National Natural Science Foundation of China under Grant Numbers 82370535 and 82200582.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors can provide data upon reasonable requests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflict of interests\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number:\u0026nbsp;\u003c/strong\u003enot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDolinger, M., J. 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Identification of Endothelial-to-Mesenchymal Transition as a Potential Participant in Radiation Proctitis. \u003cem\u003eAm J Pathol Sep\u003c/em\u003e 185(9):2550\u0026ndash;2562.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"inflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ifla","sideBox":"Learn more about [Inflammation](https://www.springer.com/journal/10753)","snPcode":"10753","submissionUrl":"https://submission.nature.com/new-submission/10753/3","title":"Inflammation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Lysophosphatidic acid, Crohn’s disease, Intestinal fibrosis, autophagy","lastPublishedDoi":"10.21203/rs.3.rs-7509054/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7509054/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eIntestinal fibrosis, which causes bowel stricture and obstruction, is a serious complication of Crohn\u0026rsquo;s disease (CD). Lysophosphatidic acid (LPA), a bioactive lipid mediator, exerts pro-fibrotic effects in various chronic diseases through its six cognate receptors (LPAR1-6). While both elevated LPA levels and impaired autophagy have been observed in CD patients, their specific contributions to intestinal fibrogenesis remain poorly understood.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eIn this study, the plasma levels of LPA were measured in CD patients with or without intestinal fibrosis by ELISA. Fibrotic and non-fibrotic intestinal tissues were obtained from patients with CD undergoing surgical resection. Masson's staining was performed to evaluate the fibrosis index score. The mRNA expressions of \u003cem\u003eLPAR3\u003c/em\u003e, \u003cem\u003ecollagen I\u003c/em\u003e and \u003cem\u003eα-SMA\u003c/em\u003e were detected in intestinal tissues from CD patients with or without intestinal fibrosis by reverse-transcriptase polymerase chain reaction (qRT-PCR). DSS-induced chronic colitis model with intestinal fibrosis were established in C57BL/6J mice. These mice were given or not given intraperitoneal injection of an LPAR3 inhibitor (Ki16425) and evaluated the fibrosis indices in the colon. Human intestinal fibroblasts (HIFs) were isolated from surgically resected fibrotic intestines of CD patients and the effects of LPA on autophagy, activation, proliferation, and migration of HIFs were determined.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eClinical analyses revealed significantly elevated plasma LPA levels and increased LPAR3 expression in fibrotic intestinal tissues of CD patients compared to non-fibrotic controls. The elevated plasma LPA levels were observed in mice with DSS-induced chronic colitis, LPAR3 inhibition attenuated both intestinal inflammation and fibrosis. In vitro experiments, Mechanistically, we demonstrated that LPA promotes HIF migration, activation and proliferation through LPAR3-dependent suppression of autophagy via the AMPK-mTOR pathway. These pro-fibrotic effects were reversed by pharmacological intervention at multiple levels\u0026mdash;including LPAR3 antagonism (Ki16425), AMPK activation (A-769662) or mTOR inhibition (rapamycin).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eOur findings establish a novel LPA-LPAR3-AMPK-mTOR signaling axis that drives intestinal fibrosis by suppressing autophagy in HIFs. This pathway represents a promising therapeutic target for preventing fibrosis progression in CD.\u003c/p\u003e","manuscriptTitle":"Lysophosphatidic acid promotes colitis-associated intestinal fibrosis by suppressing autophagy via LPAR3/ AMPK/mTOR pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-02 09:23:36","doi":"10.21203/rs.3.rs-7509054/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-27T23:13:58+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-24T04:35:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"62928357668203696924998728209535783685","date":"2025-10-25T05:52:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-21T23:34:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-08T11:44:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-08T11:42:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Inflammation","date":"2025-09-01T13:35:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"inflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ifla","sideBox":"Learn more about [Inflammation](https://www.springer.com/journal/10753)","snPcode":"10753","submissionUrl":"https://submission.nature.com/new-submission/10753/3","title":"Inflammation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"48bd6c6f-3c01-4fa4-8f5e-63f57640b34e","owner":[],"postedDate":"November 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-01-27T23:23:34+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-02 09:23:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7509054","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7509054","identity":"rs-7509054","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}