Analysis of the regulating PI3K/AKT/mTOR signaling pathway and anti-apoptosis activity of Shenqi granule through Network Pharmacology and in vitro experiments

Analysis of the regulating PI3K/AKT/mTOR signaling pathway and anti-apoptosis activity of Shenqi granule through Network Pharmacology and in vitro experiments | 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 Analysis of the regulating PI3K/AKT/mTOR signaling pathway and anti-apoptosis activity of Shenqi granule through Network Pharmacology and in vitro experiments Lifeng Wei, Xiaoping Guo, Yiyun Zhu, Yong Jun, Shixiu Chen, Rui Xu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3800699/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 Idiopathic membranous nephropathy (IMN), a common pathological type of nephrotic syndrome. Shenqi granule(SQ) is a traditional Chinese medical formula that has been used for decades to treat IMN, and there is a large amount of clinical data confirming its effectiveness,but the mechanism is unclear. This study explores the potential mechanisms and targets of action of SQ through network pharmacology and validates them through in vitro experiments and molecular docking techniques. Network pharmacology is a method that can determine how TCM works through pharmacokinetic evaluation, allowing us to study its molecular mechanisms. Through in vitro experiments, MPC5 cells are used to establish puromycin aminonucleoside (PAN)-induced podocytes damage models to extract cell protein, western blot detection signal pathway protein and related target proteins, molecular docking was performed for the validation. The network pharmacology study results indicate that SQ has 106 compounds, and 195 shared targets with MN. The treatment of IMN with SQ is mainly related to the apoptosis, PI3K/AKT/mTOR signaling pathway and other significant signaling pathways. In vitro experiments showed that SQ could effectively inhibit the activity of the PI3K/AKT/mTOR signaling pathway, increase the expression of Bcl2, and suppress the expression levels of apoptosis-related proteins such as Calaspase-3 and Bax in MPC5 cells. This study initially investigated the pharmacological effects of SQ, which effectively ameliorates IMN by potentially regulating the phosphorylation level of the AKT/mTOR pathway, inhibiting apoptotic activity, and restoring skeletal proteins. Shenqi granule(SQ) Membranous nephropathy(MN) Network pharmacology Molecular docking MPC5 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Chronic kidney disease (CKD) is emerging as a significant public health concern, with glomerulopathies being identified as one of the primary causes of end-stage renal disease[ 1 ]. Membranous nephropathy (MN), a glomerular disorder that can manifest at any age, is traditionally diagnosed as either primary MN, in the absence of any underlying disease association, or secondary MN, in the presence of an associated condition such as autoimmune disease, infection, malignancy, or drug toxicity[ 2 ]. Secondary MN, also referred to as idiopathic membranous nephropathy (IMN), represents a prevalent pathological subtype of nephrotic syndrome, with IMN constituting approximately 80% of the overall MN incidence. This condition is characterized by immune-mediated mechanisms involving the deposition of immune complexes beneath the epithelial layer and alterations in the glomerular basement membrane[ 3 ]. IMN is characterized by the occurrence of spontaneous remission and recurrence. Approximately 40% of individuals with IMN experience spontaneous remissions, while relapses are observed in 15–30% of cases, furthermore, it has been found that 50% of patients progress to develop the nephrotic syndrome, with 30% ultimately advancing to end-stage renal disease[ 4 ]. With an increasing prevalence, IMN has received considerable attention in China. Notably, the frequency of IMN cases identified through renal biopsy has recently shown a substantial rise in China. A study revealed a significant increase in the frequency of MN, nearly doubling from 10.4% in the period of 2003–2006 to 24.1% in the period of 2011–2014[ 5 ]. Xu et al conducted a comprehensive review of glomerular disease types in a Chinese population spanning an 11-year period. Their findings revealed that IgA nephropathy remained the predominant pathological type, accounting for 28.1% of cases, followed by membranous nephropathy at 23.4%[ 6 ]. After data calibration, it was observed that IMN increased 13% annually, and its incidence had a tendency to exceed that of IgA nephropathy[ 6 ]. To far, the variable natural course of IMN has made its treatment controversial and discussed. If a partial or complete remission is not achieved by the final stage, it is often recommended for many patients to undergo conservative treatment and continue with immunosuppressive therapy after a period of 6 to 12 months[ 7 ]. The KDIGO 2021 guidelines recommends rituximab, cyclophosphamide combined with glucocorticoids therapy, or tacrolimus[ 7 ]. Despite the long-standing use of immunosuppressive therapy, clinicians still face challenges in balancing the potential benefits and safety concerns associated with serious adverse events[ 8 ]. Due to all of the above factors, alternative treatments are being actively sought. The utilization of Traditional Chinese Medicine (TCM) in the MN presents distinctive advantages in symptom alleviation, mitigation of immunosuppressive drug side effects, prevention and treatment of complications, as well as deceleration of disease progression. Consequently, TCM-based interventions have emerged as a novel alternative therapeutic approach to address MN in China in recent years. SQ was selected based on TCM theories and extensive clinical experience of experienced practitioners who have used TCM to treat patients with kidney disease in recent decades. Comprised of 13 distinct TCM herbs, SQ has been verified as an effective prescription for treating MN with minimal adverse effects in a multicenter, randomized, controlled study in China[ 9 ]. SQ granule have demonstrated significant clinical efficacy in the treatment of MN, unfortunately, the specific mechanisms underlying their therapeutic properties are unidentified. Network pharmacology is a new method that can determine how TCM works through pharmacokinetic evaluation, it is an interdisciplinary field that integrates systems biology and network informatics, and has gained significant traction in the realm of novel drug development in recent times[ 10 , 11 ]. Li[ 12 ]introduced the innovative notion of a "network target", which expands the traditional understanding of a drug target from a single molecule to encompass its broader influence on a biological network. This approach also enhances drug efficacy and diminishes adverse effects[ 13 ]. This concept proves particularly valuable in addressing complex systems like herbal formulae. Consequently, network pharmacology presents a promising avenue for comprehending the intricacies of multi-component drugs. Molecular docking is an advanced computational technique employed to simulate the atomic-level interaction between molecules and proteins, enabling the prediction of ligand and receptor conformations, as well as the calculation of various parameters, including affinity, crucial for evaluating combination scenarios. This precise and cost-effective technology finds primary application in drug design and the elucidation of biochemical pathways[ 14 ]. This study aims to investigate the potential molecular mechanism of SQ in the treatment of MN through network pharmacology and in vitro experiments and molecular docking techniques, thereby offering a novel approach for clinical intervention(Fig. 1 ) . Material and methods Herbal materials and SQ preparation The thirteen herbs contained in SQ granule were supplied by Wanshicheng Pharmaceutical Co., Ltd. (Shanghai, Wanshicheng, China). The thirteen herbs were consistent with some published literature[ 9 , 15 ]. Specific methods of drug dissolution and freezing have been described in detail in previous articles, using high performance liquid chromatography (HPLC) and control standards, it was confirmed that SQ granule complied with the authentic drug regulations of the Chinese Pharmacopoeia (version 2020). Collection of main bioactive components and potential targets of SQ All chemical ingredients and their related targets of each herb of SQ were collected from the TCMSP( http://lsp.nwsuaf.edu.cn/tcmsp.Php ). Ingredients were screened conditional on oral availability (OB) ≥ 30% and drug-like (DL) ≥ 0.18. The chemical structures and general information of the main components of the drug were got from the Chinese Academy of Sciences Shanghai Institute of Organic Medicine and Chemical Composition database( https://organchem.csdb.cn/scdb/main/tcm ). Identifification of the direct protein targets The UniProt Knowledgebase (UniProtKB) is a protein database partially curated by experts and contains 54, 247, 468 sequence entries. Gene information, including the gene name and the gene ID, was confifirmed by the UniProt database ( https://www.uniprot.org ). Screening disease-associated targets The GeneCards database ( https://www.genecards.org/ ) and selection according to the criterion of Risk Score > 1, the Therapeutic Target Database (TTD, https://db.idrblab.org/ttd/ ) and OMIM ( https://omim.org/)wer e used to collect information on MN related to target genes. The association of MN with SQ was then gathered as the core targets of SQ for MN. Obtain SQ-MN genes and draw a Venn diagram Validated human species–associated target genes were selected in the Uniprot database,and gene names were obtained with R language Perl scripts. The drug-related genes were intersected with disease-related genes with a Perl script, and an intersection Venn diagram was drawn. Finally, potential targets of SQ for IMN treatment were obtained. Construction of SQ–active ingredients–MN target network Drug components were numbered and paired with their corresponding target genes. A SQ-active ingredients-MN targets target network was constructed using Cytoscape 3.8.2, and the active ingredients and intersectional targets were acquired simultaneously. Sorting and graphing based on the size of the degree values. Degree indicated the number of edges between a single node and other nodes in a network. Construction of the Protein–Protein Interaction (PPI) network In order to clarify the interaction between the targets of SQ and MN, the candidate targets of SQ for MN treatment were imported into the STRING database ( https://string-db.org/ ) to construct a PPI network.The network analysis plug-in in Cytoscape software was used to analyze network topological features to screen the hub nodes in the PPI network. Degree centrality denotes several direct connections of a node to all other nodes in the network. GO functional enrichment and KEGG pathway analysis Importing gene files into the OmicShare tools, a free online platform for data analysis ( https://www.omicshare.com/tools ), set q value = 0.05, and ran the program to draw a bubble chart. GO and KEGG enrichment analyses of the target genes of SQ in the treatment of MN were performed to elucidate the possible molecular mechanism. Cell culture and viability assay The MPC5 cells were donated by Dr. Baoli Liu (Beijing Hospital of Traditional Chinese Medicine Affiliated to Capital Medical University). Cells were resuspended and inoculated in L-DMEM containing and 10% FBS and cultured at 37°C with 5% CO 2 for proliferation and passaging, then allowed to differentiate for 7–10 days at 37°C, after which performed group experiments. Cells (1.0×10 4 ) were seeded in wells of a 96-well plate, and 100 mL of L-DMEM was added to each well. A CCK-8 assay kit (BioSharp, Hefei, China) was used to determine cell viability according to the manufacturer’s instructions. Cell groups and treatments MPC5 cells were divided into the following groups: Control group (L-DMEM and 10% FBS); CsA group (0.5 µg/mL CsA + PAN + L-DMEM and 10% FBS); SQ groups (administered 0.5, 1.0, 2.0 mg/mL SQ + PAN + L-DMEM and 10% FBS). Immunofluorescence in MPC5 cells(Identification of the cytoskeletal proteins) Synaptopodin and F-actin immunofluorescence in MPC5 cells was analysed using an immunofluorescence method. Cells were fixed in 4% formalin buffered with PBS for 30 mins and permeabilize cells with 0.1% Triton-X-100 for 15 minutes. After blocking with 5% BSA at RT for 60 min, the slides were incubated with FITC-labelled primary antibodies overnight. Nuclei were counterstained with DAPI. After washing, an anti-fluorescence quencher was added to the slides, which were subsequently observed under an inverted fluorescence microscope. Image J (software version 1.46, National Institutes of Health (NIH), Bethesda, MA, USA) was used to determine the amount of fluorescence and compare the results between different groups. Western bloting Analysis MPC5 cells from each group were dissolved, and total protein was extracted using RIPA lysis buffer. Supernatants were collected after centrifugation, and protein concentrations were measured. Proteins were loaded, electrophoresed, transferred to 0.45-µm PVDF membranes, incubated with the primary antibodies (PI3K, p-PI3K, AKT, p-AKT, mTOR, p-mTOR, Bcl2, Bax, cleaved Caspase-3) overnight at 4℃, then with secondary antibody for 60 min at RT. Bands were detected on a gel imaging system using enhanced chemiluminescence. The grey values were analysed using Image J (software version 1.46 ,National Institutes of Health (NIH),Bethesda, MA, USA). Molecular docking verification According to the PPI interaction network, the important targets were screened and the corresponding chemical components in the drug were verified by molecular docking. Four drug components with high OB and DL values and clear active ingredients were selected, and 3D structures of these compounds were downloaded from the PubChem database, calculated energy optimized and saved. The 3D structures of the MN related target protein was downloaded from the PDB( https://www.rcsb.org/ ). The pdbid are Bax and Bcl2. Molecular docking was performed using AutoDock Vina, and the docking results were plotted with Pymol. Statistical analysis Statistical analyses were performed using SPSS version 25.0 (SPSS Inc., Chicago, IL, USA). Data are presented as means ± SD. Student’s t-test was used to examine differences between two groups. For comparisons of multiple groups, ANOVA was used followed by Dunnett's post hoc test in the absence of equivalent variance. A P value < 0.05 was considered statistically significant. Results The SQ composition and the major chemical components Table 1 Chinese Name Latin Name Molecule Name Chemical formula Huang Qi Astragalus membranaceus (Fisch.) Bunge Astragaloside IV C41H68O14 Dang Gui Angelicae Sinensis Radix β-sitosterol C29H50O Cang Shu Atractylis chinensis (Bunge) DC. Wogonin C16H12O5 Bai shu Atractylis macrocephala (Koidz.) Hand.-Mazz. Salvianolic acid B C36H30O16 Shan Yao Dioscorea oppositifolia L. Diosgenin C27H42O3 Zhu Ling Polyporus umbellatus (Pers.) Fr. Chlorogenic acid C25H24O12 Fu Ling Poria cocos (Schw.) Wolf Neochlorogenic acid C16H18O9 Jiang Can Bombyx micranthus (L.f.) I.Riedl Cryptochlorogenic acid C16H18O9 She Shecao Hedyotis diffusa Willd. Quercetin C15H10O7 Yi Yiren Coix lacryma-jobi L. Stigmasterol C29H48O Dang Shen Codonopsis pilosula (Franch.) Nannf. Polyporusterone B C28H44O6 Dan Shen Salvia miltiorrhiza Bunge Pachymic acid C33H52O5 Shui Zhi Hirudo nipponica Whitman Linoleic Acid C18H32O2 Table 1 The SQ composition and the major chemical components Chinese Name Latin Name Molecule Name Chemical formula Huang Qi Astragalus membranaceus (Fisch.) Bunge Astragaloside IV C41H68O14 Dang Gui Angelicae Sinensis Radix β-sitosterol C29H50O Cang Shu Atractylis chinensis (Bunge) DC. Wogonin C16H12O5 Bai shu Atractylis macrocephala (Koidz.) Hand.-Mazz. Salvianolic acid B C36H30O16 Shan Yao Dioscorea oppositifolia L. Diosgenin C27H42O3 Zhu Ling Polyporus umbellatus (Pers.) Fr. Chlorogenic acid C25H24O12 Fu Ling Poria cocos (Schw.) Wolf Neochlorogenic acid C16H18O9 Jiang Can Bombyx micranthus (L.f.) I.Riedl Cryptochlorogenic acid C16H18O9 She Shecao Hedyotis diffusa Willd. Quercetin C15H10O7 Yi Yiren Coix lacryma-jobi L. Stigmasterol C29H48O Dang Shen Codonopsis pilosula (Franch.) Nannf. Polyporusterone B C28H44O6 Dan Shen Salvia miltiorrhiza Bunge Pachymic acid C33H52O5 Shui Zhi Hirudo nipponica Whitman Linoleic Acid C18H32O2 Network pharmacology Obtaining disease targets of MN, targets of SQ and compound common targets The common targets of 13 Chinese herbs in SQ were obtained from TCMSP and Shanghai Institute of Organic Medicine database, and 419 targets were obtained after de-duplication; the disease targets of MN were obtained from GeneCards database, and a total of 1729 targets were obtained after integration and de-duplication 195 common targets were obtained(Fig. 2 ) . SQ-active ingredient-MN target network diagram The main active ingredients of SQ are astragaloside, peroxyergosterol, dousterol, β-sitosterol, quercetin, stigmasterol, atractylenolide III, 4-Pcoumaric acid, salvianolic acid, adenosine, ferulic acid, wogonin, diosgenin, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, polyporusterone B, pachymic acid and linoleic acid, etc. 13 herbs in SQ, and 106 of the most relevant active ingredients in SQ were selected to make the association plot with MN. The "SQ-active ingredient-MNtarget network diagram" was constructed by Cytoscape 3.8.2 system(Fig. 3 ) . Common target protein interaction map (PPI) All 195 genes in the gene set for SQ treatment of MN were imported into STRING, selected the highest confidence level of 0.9, hide disconnected nodes in the network, and the PPI network of genes for SQ treatment of IMN was constructed, as shown in Fig. 3 A. Based on the Cytoscape 3.8.3, the degree was ranked from high to low. The key targets in PPI network were AKT1, TNF, STAT3, IL6, IL18, BCL2, CASP3 and JUN, etc, as shown in Fig. 4 A-B(Fig. 4 A-B ) . GO and KEGG analysis for obtaining the 15 most relevant pathways for SQ treatment of MN By the OmicShare tools analysis, We found that SQ treatment for MN involves molecular function, biological process, cellular component(Fig. 5 A-B ) . Molecular function (MF): function in 905, including enzyme binding, signaling receptor binding, kinase binding, molecular function regulator,, etc. Biological process (BP): involving 6,385 cases, mainly including response to oxygen-containing compound, response to lipid, regulation of cell death, cell proliferation, apoptotic process, and etc. Cellular Component(CC): extracellular space, extracellular region part,extracellular region,vesicle,etc. Details are as shown in Supplementary Fig. 1. The KEGG results suggest that SQ treatment for IMN may involve 266 signaling pathways, and the top 15 signaling pathways most relevant to the disease, which contain Lipid and atherosclerosis, AGE-RAGE signaling pathway in diabetic complications, HIF-1 signaling pathway, PI3K-Akt signaling pathway, Endocrine resistance, TNF signaling pathway, Apoptosis, mTOR signaling pathway, and etc(Fig. 5 C-E ) . Major Molecular docking verification To verify the network pharmacology results, the important targets were screened and the corresponding chemical components in the drug were verified by molecular docking, as shown in the Fig. 6 . The targets Bax and Bcl2 selected. The required files were obtained from the PDB database, all receptor files were treated with organic matter, water molecules and hydrogenated charge distribution. To demonstrate the docking mode, typical dockings were visualized using PyMol and AutoDockVina and attached with binding energy and K i values. This result can further prove that the active components of SQ, such as wogonin and quercetin, act on Bax and Bcl2 receptors and other related core targets, and have favorable affinity with key targets, reflecting that the active components of SQ can treat or alleviate IMN through these targets. It confirmed the strong binding affinity of the active components in SQ with the disease targets( Supplementary Table 2 ). Validation of in vitro experiments Successful MPC5 were identified by immunofluorescence techniques Synaptopodin is a signature skeletal protein unique to MPC5 cells. By immunofluorescence, in Fig. 7 we can see that the cells are highly positive for synaptopodin protein expression, confirming that this cell line is MPC5( Supplementary Table 1 ). SQ is able to protect the MPC5 cytoskeletal protein F-actin polymerization and expression The results of immunofluorescence showed that, compared with the Con group, after PAN damaged MPC-5, F-actin cleavage and expression decreased. SQ could protect F-actin, reduced its cleavage, increased expression, increased polymerization of F-actin, and this effect was concentration dependent, and the high dose of SQ was optimal, as displayed in Fig. 8 . SQ granule limits the phosphorylation level of PI3K/AKT/mTOR signaling pathway To explore the specific link between signaling pathway and MPC5 cells, and the possible mechanism of SQ granule action, the upstream signaling pathway of PI3K/AKT/mTOR was examined. The results suggested that the expressions of p-PI3K, p-AKT and p-mTOR were all enhanced in PAN-induced MPC5 cell injury ( P < 0.05), and SQ granule could inhibit the phosphorylation expression of p-AKT and p-mTOR ( P < 0.05), SQ showed no significant effect in regulating the expression of PI3K phosphorylation(Fig. 9 ) .These findings indicate that SQ granule may be inhibited the AKT/mTOR phosphorylation. SQ decreased the Bax and cleave-caspase3 expression and increased the Bcl 2 protein expression To verify whether PAN-induced cytoskeletal protein damage is related to apoptosis in MPC5 cells, we observed the expression of apoptosis proteins. In Fig. 10 , we can see that the Bcl2 was reduced in the Model group, while Bax and cleave-caspase3 expression were upregulated ( P < 0.05), yet SQ granule, in contrast, could upregulate the Bcl2 ratio and inhibit Bax and cleave-caspase3 expression( P < 0.05). These findings may indicate that SQ granule has an depressed apoptosis activity in PAN-induced MPC5 cells. Discussion MN as a prominent subtype of CKD, is a pathologically defined renal glomerular disorder characterized by the presence of immune complexes on the outer surface of the basement membrane[ 16 , 17 ]. This autoimmune disease is typified by the deposition of immunoglobulin G (IgG), relevant antigens, and complement components, including the membrane attack complex (MAC)[ 18 , 19 ]. The immunological conflict results in significant protein loss in the urine, known as proteinuria, primarily due to the disruption of podocyte structure caused by immune complex deposition and MAC formation[ 19 ]. Recent research indicates a significant rise in the prevalence of IMN in China[ 20 ]. Therefore, actively seeking a treatment for MN, especially for IMN, is our clinical challenge and the direction of our research. Network pharmacology analyzed the intersection of the active components of SQ and the disease targets of IMN, as depicted in Fig. 2 of the Wayne diagram, yielded a total of 195 common targets( Figure. 2 ). By identifying the overlapping targets between the active components of SQ and the gene targets of MN, it was postulated that SQ may possess therapeutic potential through its intervention on these shared targets and thus made the SQ-active ingredient-MN target network and PPI diagram( Figure. 3–4 ). Furthermore, GO and KEGG analyses suggested that SQ primarily engages in enzyme binding, signaling receptor binding, kinase binding, response to lipid, regulation of cell death, cell proliferation, apoptotic process, extracellular space, etc, and disease of the most closely related signaling pathways containing Lipid and atherosclerosis, HIF-1 signaling pathway, PI3K-Akt signaling pathway, TNF signaling pathway, Apoptosis, mTOR signaling and so on( Figure. 5 ). Podocytes, the smallest and most fundamental cell of the glomerulus[ 21 , 22 ], were utilized to establish the cell model of MN through the use of PAN. Podocyte function is dependent on actin cytoskeleton regulation within the foot processes, structures that link podocytes to the glomerular basement membrane. Actin cytoskeleton dynamics in podocyte foot processes are complex and regulated by multiple proteins and other factors[ 23 ]. Changes in the actin cytoskeleton within a cell are necessary for maintenance of cell shape, cell motility and intracellular transport[ 24 ]. Our previous study demonstrated that SQ offers protection against podocyte cytoskeletal proteins, including CD2AP and α-actinin4[ 15 , 25 , 26 ]. In this study, upon further examination of the podocytes' cytoskeletal protein, it was observed that PAN induces impairment in the F-actin protein, resulting in polymerization breakdown. However, it was found that SQ has the ability to restore the integrity of the skeletal cytoskeleton proteins. The effectiveness of this protective effect and its dependence on drug concentration are illustrated in Figure. 8 . This investigation revealed that both SQ and CsA possess the ability to restore F-actin expression and enhance polymerization. Notably, the effectiveness of SQ in this regard is contingent upon the concentration of the drug. Phosphoinositide 3-kinase (PI3K), AKT serine/threonine kinase (Akt), and mammalian target of rapamycin (mTOR) are kinases that mediate cellular signaling pathways involved in the regulation of various cellular processes, including the cell cycle, cell survival, cytoskeleton rearrangements, metabolism, and protein synthesis[ 27 ], ectopic or too intense activation of this signaling is a causal event in diseases characterized by uncontrolled cellular expansion[ 28 , 29 ]. PI3K plays a crucial role in controlling diverse cellular processes such as growth, survival, metabolism, apoptosis, and autophagy[ 30 ]. AKT serves as a primary effector of PI3K, upon activation, AKT phosphorylates tuberous sclerosis complex 2, which disrupts the formation of inhibitory TSC1/TSC2 heterodimers, subsequently activating TOR complex 1, this complex, consisting of mTOR, performs distinct functions within the cell[ 31 ]. mTOR is a serine/threonine protein kinase that forms the catalytic subunit of two distinct protein complexes, known as mTOR Complex 1 (mTORC1) and 2 (mTORC2), the two complexes exert their activity towards distinct substrates and, as a consequence, regulate different cellular functions[ 32 ]. Our network pharmacology demonstrates that SQ for IMN may be involved in the pathway. In vitro experiments, we have confirmed this inference by observing that PAN intervention in MPC5 activates the PI3K/AKT/mTOR signaling pathway, leading to a significant increase in AKT/mTOR phosphorylation levels( Figure. 9 ). However, we have observed that various concentrations of SQ exhibit remarkable effects in reducing the expression of p-AKT and p-mTOR, as well as inhibiting the activation of this signaling pathway's phosphorylation. These findings may potentially induce subsequent alterations in downstream apoptosis and autophagic activity. Apoptosis is characterised by distinct morphological alterations in cellular structure, accompanied by enzyme-dependent biochemical mechanisms[ 33 ]. Bcl-2/Bax and cleaved caspase-3 are crucial factors in the advancement of cellular apoptosis. The Bcl-2 family significantly influences the facilitation or inhibition of the intrinsic apoptotic pathway, which is initiated by mitochondrial dysfunction[ 34 , 35 , 36 ]. Certain investigations propose that kidney-related apoptosis is mediated by Bcl2 proteins and could potentially be controlled through systemic intervention aimed at impeding conformational bax activation and/or reduction in Bcl2 levels induced by renal stress[ 37 ]. Caspase-dependent apoptosis is responsible for approximately 90% of cellular turnover in homeostatic conditions[ 38 ]. According to the structure and the roles of caspase-3 in various cellular mechanisms, mainly in apoptosis, this enzyme was specifically considered as a therapeutic target to combat apoptosis-related diseases[ 39 ]. Cleaved caspase3, a downstream protein of the Caspase family, serves as the final product of apoptosis[ 40 , 41 , 42 , 43 ]. In our study, depicted in Figure. 10 , it is evident that PAN-induced cells exhibited suppressed Bcl2 expression and heightened expression of Bax and cleaved Caspase-3, indicating an increase in apoptotic activity. Conversely, SQ was found to activate Bcl2 expression, decrease Bax and cleaved Caspase-3 expression, and decelerate cellular apoptosis. These findings imply that certain active components within the drug composition of SQ possess the capability to inhibit apoptosis and regulate certain signaling pathway and facilitate cytoskeleton structure repair. Consequently, these results support the alignment between our SQ and network pharmacology, as the in vitro experiments substantiated the conjecture made by network pharmacology. We through network pharmacology found that SQ and IMN have the same therapeutic targets, and dig out the potential mechanism of SQ treatment of IMN, earlier studies have shown that SQ can activate autophagy activity, improve cytoskeletal protein expression, has the effect of protecting damaged podocytes. Our study has yielded novel findings, indicating that SQ exerts a regulatory effect on cell apoptosis and has significant feedback implications for the associated signaling pathways( Figure. 11 ). These findings were validated through in vitro studies and molecular docking technology, which confirmed the strong binding affinity of the active components in SQ with the disease targets( Figure. 6, Supplementary Table 2 ). Conclusion TCM treatment of diseases involves multiple targets, signaling pathways, and intricate mechanisms of action. The application of network pharmacology methods and in vitro experiments provided evidence that SQ effectively ameliorates IMN by potentially regulating the phosphorylation level of the AKT/mTOR pathway, inhibiting apoptotic activity, and restoring skeletal proteins. These findings were further substantiated through molecular docking analysis. Abbreviations AJKD American Journal of Kidney Diseases Akt AKT serine/threonine kinase BSA Bovine serum albumin CKD Chronic kidney disease CsA Cyclosporine ESRD End-stage renal disease FBS Foetal bovine serum IFN-γ Interferon-gamma IMN Idiopathic membranous nephropathy KDIGO The Kidney Disease: Improving Global Outcomes L-DMEM Low-Dulbecco’s Modified Eagle Medium MPC5 Mouse podocyte cell line mTOR mammalian target of rapamycin PAN Puromycin aminonucleoside PI3K Phosphoinositide 3-kinase RT Room temperature SQ Shenqi granule TCM Traditional Chinese medicine. Declarations Declaration of Interest The authors declare that the research was performed in the absence of any commercial or financial relationships that may be construed as a potential conflict of interest. CRediT author statement Li-feng Wei : Writing-Original draft preparation and Editing. Xiao-ping Guo : Writing-Original draft preparation and Editing. Yi-yun Zhu : Methodology and Validation. Jun Yong : : Resources and Data curation. Rui Xu : Resources and Data curation. Shi-xiu Chen : Visualization. Yi-ping Chen : Conceptualization and Resources. Lin Wang : Supervision and Writing- Reviewing . Acknowledgement None Ethical Approval Not applicable. Funding This study was supported by grants from Shanghai Municipality Further accelerates the Three-year Action Plan for TCM Inheritance, Innovation and Development (2021-2023) (ZY (2021-2023) -0403) TCM High-level Talents Leading Plan, the Clinical Research Plan of SHDC (SHDC2020CR2048B), National Natural Science Foundation of China (No. 81273730), Shanghai Municipal Health and Family Planning Commission project (202040163), Shanghai Municipal Health and Family Planning Commission further accelerating the three-year action plan project of the development of Chinese medicine [ZY (2018–2020)-ZYBZ-02]. Availability of data and materials The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. References Santos JE, Fiel D, Santos R, Vicente R, Aguiar R, Santos I, Amoedo M, Pires C (2020) Rituximab use in adult glomerulopathies and its rationale. J Bras Nefrol 42(1):77–93 S.Sethi, New 'Antigens' in Membranous Nephropathy. J Am Soc Nephrol 32(2) (2021) 268–278 Couser WG, Nephropathy PM (2017) Clin J Am Soc Nephrol 12(6):983–997 Keri KC, Blumenthal S, Kulkarni V, Beck L, Chongkrairatanakul T (2019) Primary membranous nephropathy: comprehensive review and historical perspective. Postgrad Med J 95(1119):23–31 Hou JH, Zhu HX, Zhou ML, Le WB, Zeng CH, Liang SS, Xu F, Liang DD, Shao SJ, Liu Y, Liu ZH (2018) Changes in the Spectrum of Kidney Diseases: An Analysis of 40,759 Biopsy-Proven Cases from 2003 to 2014 in China. Kidney Dis (Basel) 4(1):10–19 Xu X, Wang G, Chen N, Lu T, Nie S, Xu G, Zhang P, Luo Y, Wang Y, Wang X, Schwartz J, Geng J, Hou FF (2016) Long-Term Exposure to Air Pollution and Increased Risk of Membranous Nephropathy in China. J Am Soc Nephrol 27(12):3739–3746 Disease GK (2021) Improving Global Outcomes Glomerular Diseases Work, KDIGO 2021 Clinical Practice Guideline for the Management of Glomerular Diseases. Kidney Int 100(4S):S1–S276 Hofstra JM, Wetzels JF (2010) Alkylating agents in membranous nephropathy: efficacy proven beyond doubt. Nephrol Dial Transplant 25(6):1760–1766 Chen Y, Deng Y, Ni Z, Chen N, Chen X, Shi W, Zhan Y, Yuan F, Deng W, Zhong Y (2013) Efficacy and safety of traditional chinese medicine (Shenqi particle) for patients with idiopathic membranous nephropathy: a multicenter randomized controlled clinical trial. Am J Kidney Dis 62(6):1068–1076 Hopkins AL (2008) Network pharmacology: the next paradigm in drug discovery. Nat Chem Biol 4(11):682–690 Li S, Zhang B (2013) Traditional Chinese medicine network pharmacology: theory, methodology and application. Chin J Nat Med 11(2):110–120 Li S (2011) Network target: a starting point for traditional Chinese medicine network pharmacology. China J Chin Med 36(15):2017–2020 Zhang J, Huang K, Zhong G, Huang Y, Li S, Qu S, Zhang J (2016) Acupuncture Decreases NF-κB p65, miR-155, and miR-21 and Increases miR-146a Expression in Chronic Atrophic Gastritis Rats, Evid Based Complement Alternat Med (2016) 9404629 Meng X, Zhang H, Mezei M, Cui M (2011) Molecular docking: a powerful approach for structure-based drug discovery. Curr Comput Aided Drug Des 7(2):146–157 Wei L, Yong J, Zhang X, Ling C, Wu Y, Xu Z, Zhang H, Cao X, Sheng L, Zhang Q, Chen Y, Wang L (2023) Shenqi granule upregulates CD2AP and alpha-actinin4 and activates autophagy through regulation of mTOR/ULK1 pathway in MPC5 cells. J Ethnopharmacol 303:115942 Petrosyan A, Cravedi P, Villani V, Angeletti A, Manrique J, Renieri A, De Filippo RE, Perin L, Da Sacco S (2019) A glomerulus-on-a-chip to recapitulate the human glomerular filtration barrier. Nat Commun 10(1):3656 Xiaofan H, Jing X, Chenni G, Yifan W, Xialian Y, Li L, Hong R, Wen Z, Weiming W, Xiaoxia P, Jingyuan X, Nan C (2019) New risk score for predicting progression of membranous nephropathy. J Transl Med 17(1):41 Wu L, Lai J, Ling Y, Weng Y, Zhou S, Wu S, Jiang S, Ding X, Jin X, Yu K, Chen Y (2021) A Review of the Current Practice of Diagnosis and Treatment of Idiopathic Membranous Nephropathy in China. Med Sci Monit 27:e930097 Ronco P, Beck L, Debiec H, Fervenza FC, Hou FF, Jha V, Sethi S, Tong A, Vivarelli M, Wetzels J (2021) Membranous nephropathy. Nat Rev Dis Primers 7(1):69 Glassock RJ (2010) The pathogenesis of idiopathic membranous nephropathy: a 50-year odyssey. Am J Kidney Dis 56(1):157–167 Garg P (2018) A Review of Podocyte Biology. Am J Nephrol 47:1 Nagata M (2016) Podocyte injury and its consequences. Kidney Int 89(6):1221–1230 Blaine J, Dylewski J (2020) Regulation of the Actin Cytoskeleton in Podocytes. Cells 9(7) Blanchoin L, Boujemaa-Paterski R, Sykes C, Plastino J (2014) Actin dynamics, architecture, and mechanics in cell motility. Physiol Rev 94(1):235–263 Ling C, Zhang X, Wang L (2018) Chen’s Yiqi Huoxue Huashi Recipe protected PAN cultured podocyte by regulating autophagy. Chin J Integr Nephrol 19(09):760–763 Yong J, Ling C, Zhang X, Wang L (2019) Regulation of Yiqi Huoxue Huashi formula on podocyte slit membrane protein α-actinin-4 and CD2AP in passive Heymann nephritis model rats. Shanghai J Traditional Chin Med 53(05):69–75 Margaria JP, Campa CC, De Santis MC, Hirsch E, Franco I (2020) The PI3K/Akt/mTOR pathway in polycystic kidney disease: A complex interaction with polycystins and primary cilium. Cell Signal 66:109468 Fruman DA, Chiu H, Hopkins BD, Bagrodia S, Cantley LC, Abraham RT (2017) The PI3K Pathway in Human Disease. Cell 170(4):605–635 Goncalves MD, Hopkins BD, Cantley LC (2018) Phosphatidylinositol 3-Kinase, Growth Disorders, and Cancer. N Engl J Med 379(21):2052–2062 Xu Z, Han X, Ou D, Liu T, Li Z, Jiang G, Liu J, Zhang J (2020) Targeting PI3K/AKT/mTOR-mediated autophagy for tumor therapy. Appl Microbiol Biotechnol 104(2):575–587 Wang L, Li X, Yang Z, Pan X, Liu X, Zhu M, Xie J (2017) Crotonaldehyde induces autophagy-mediated cytotoxicity in human bronchial epithelial cells via PI3K, AMPK and MAPK pathways. Environ Pollut 228:287–296 Saxton RA, Sabatini DM (2017) mTOR Signaling in Growth, Metabolism, and Disease. Cell 168(6):960–976 D'Arcy MS (2019) Cell death: a review of the major forms of apoptosis, necrosis and autophagy. Cell Biol Int 43(6):582–592 Mohan S, Abdelwahab SI, Kamalidehghan B, Syam S, May KS, Harmal NS, Shafifiyaz N, Hadi AH, Hashim NM, Rahmani M, Taha MM, Cheah SC, Zajmi A (2012) Involvement of NF-kappaB and Bcl2/Bax signaling pathways in the apoptosis of MCF7 cells induced by a xanthone compound Pyranocycloartobiloxanthone A. Phytomedicine 19(11):1007–1015 K.Vucicevic V, Jakovljevic N, Colovic N, Tosic T, Kostic I, Glumac S, Pavlovic T, Karan-Djurasevic M, Colovic (2016) Association of Bax Expression and Bcl2/Bax Ratio with Clinical and Molecular Prognostic Markers in Chronic Lymphocytic Leukemia. J Med Biochem 35(2):150–157 Yip KW, Reed JC (2008) Bcl-2 family proteins and cancer. Oncogene 27(50):6398–6406 Borkan SC (2016) The Role of BCL-2 Family Members in Acute Kidney Injury. Semin Nephrol 36(3):237–250 Fuchs Y, Steller H (2011) Programmed cell death in animal development and disease. Cell 147(4):742–758 Asadi M, Taghizadeh S, Kaviani E, Vakili O, Taheri-Anganeh M, Tahamtan M, Savardashtaki A (2022) Caspase-3: Structure, function, and biotechnological aspects. Biotechnol Appl Biochem 69(4):1633–1645 Waterhouse N, Kumar S, Song Q, Strike P, Sparrow L, Dreyfuss G, Alnemri ES, Litwack G, Lavin M, Watters D (1996) Heteronuclear ribonucleoproteins C1 and C2, components of the spliceosome, are specific targets of interleukin 1beta-converting enzyme-like proteases in apoptosis. J Biol Chem 271(46):29335–29341 Chang H, Yang X (2000) Proteases for cell suicide: functions and regulation of caspases. Microbiol Mol Biol Rev 64(4):821–846 Elmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35(4):495–516 Poreba M, Strózyk A, Salvesen GS, Drag M (2013) Caspase substrates and inhibitors. Cold Spring Harb Perspect Biol 5(8):a008680 Additional Declarations No competing interests reported. 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-3800699","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":264661849,"identity":"caf6f053-3ce7-4a6e-96c5-18116abf5d6f","order_by":0,"name":"Lifeng Wei","email":"","orcid":"","institution":"Longhua Hospital Shanghai University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Lifeng","middleName":"","lastName":"Wei","suffix":""},{"id":264661850,"identity":"d20bf872-a011-496f-94a4-5195433e668f","order_by":1,"name":"Xiaoping Guo","email":"","orcid":"","institution":"Longhua Hospital Shanghai University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xiaoping","middleName":"","lastName":"Guo","suffix":""},{"id":264661851,"identity":"fe872c10-98e6-463a-90eb-af1f754cde1f","order_by":2,"name":"Yiyun Zhu","email":"","orcid":"","institution":"Longhua Hospital Shanghai University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yiyun","middleName":"","lastName":"Zhu","suffix":""},{"id":264661852,"identity":"e4f157db-bc21-44c8-8dce-e5235a933064","order_by":3,"name":"Yong Jun","email":"","orcid":"","institution":"Longhua Hospital Shanghai University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"Jun","suffix":""},{"id":264661853,"identity":"831fb26d-9c80-4105-be71-c800db2bbdf4","order_by":4,"name":"Shixiu Chen","email":"","orcid":"","institution":"Longhua Hospital Shanghai University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shixiu","middleName":"","lastName":"Chen","suffix":""},{"id":264661854,"identity":"309247e9-75a9-4b2e-9a79-e7a1ea7d332f","order_by":5,"name":"Rui Xu","email":"","orcid":"","institution":"Longhua Hospital Shanghai University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Rui","middleName":"","lastName":"Xu","suffix":""},{"id":264661855,"identity":"b32929c6-01a1-4566-81d9-7087088bcbfc","order_by":6,"name":"Yiping Chen","email":"","orcid":"","institution":"Longhua Hospital Shanghai University of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yiping","middleName":"","lastName":"Chen","suffix":""},{"id":264661856,"identity":"f2e41585-c58b-42fd-915c-205479f27072","order_by":7,"name":"Lin Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYBACAwhlAcTMBw58+EG0lgQJIMGWeHBmD2laeIwPc7ARocWc/eyxx7w/JBK3S/d8OMzAwyDPL3YAvxbLnrx0Y54EicSdc85uOFxgwWA4c3YCAYcdyDGTBmnZcCN3w+EZPAwJBrcJaTn/BqYl58FhHjZitNyA25LDQKyWd+mGc9IkjDfcSDMABrIEEX45n3vswRsbG9kNN5Iff/jww0aeX5qAFmB0sDHxMDA4NkB4EoSUQ7QwApOJPTFKR8EoGAWjYIQCAM0OSOEEpBmgAAAAAElFTkSuQmCC","orcid":"","institution":"Longhua Hospital Shanghai University of Traditional Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Lin","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2023-12-24 14:14:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3800699/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3800699/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49106640,"identity":"46cecaf2-08b2-454b-88ef-1df423046fb5","added_by":"auto","created_at":"2024-01-03 08:13:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":567249,"visible":true,"origin":"","legend":"\u003cp\u003eDiagram of TCM for the treatment of MN techniques based on network pharmacology and in vitro experiments and molecular docking validation.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3800699/v1/5c52e61c1379a77898a951ac.png"},{"id":49106641,"identity":"69715f9e-5f42-44f3-ae53-d339d418e1a5","added_by":"auto","created_at":"2024-01-03 08:13:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":40865,"visible":true,"origin":"","legend":"\u003cp\u003eA-Venn diagram of SQ-MN targets: The overlap in SQ targets and MN potential genes.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3800699/v1/573b6349af7d766552889852.png"},{"id":49107336,"identity":"eef2f800-ccba-47ea-b395-3ab6a1b6c42f","added_by":"auto","created_at":"2024-01-03 08:21:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":325374,"visible":true,"origin":"","legend":"\u003cp\u003eAssociation plots of TCM, active ingredients and diseases and common overlapping targets, where Dangshen labeled Ds and Danshen labeled DS.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3800699/v1/bfc6d6a4123a71b69e5419a9.png"},{"id":49106645,"identity":"e9f969b6-607b-46c2-8d16-73b1128bbcb1","added_by":"auto","created_at":"2024-01-03 08:13:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":979748,"visible":true,"origin":"","legend":"\u003cp\u003eA-B. The PPI networks of relative targets. In Fig 3B, have 172 nodes and 1498 edges.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3800699/v1/4e551f3172622b3f061cb53b.png"},{"id":49106648,"identity":"6c476a69-5e1d-448b-9e3e-d4bc0d82a6a5","added_by":"auto","created_at":"2024-01-03 08:13:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1091040,"visible":true,"origin":"","legend":"\u003cp\u003eA-B. GO and KEGG functional annotation pathway enrichments. C-D. The KEGG signaling pathways, the barplot (C) and the gradient(D). B,E. The first circle is the enriched classification, the circle is the sitting ruler of gene number, different colors represent different classifications, the number of background genes in the second circle and the Q-value or P-value. The more genes, the smaller the bar, the red, the bar represents the number of up-regulated genes, green represents the number of downregulated genes, the specific value is shown in the box.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3800699/v1/5ab901bc4ce1bf6dd988853d.png"},{"id":49107339,"identity":"2f36b6ac-b5ed-424f-9950-cabcd115627b","added_by":"auto","created_at":"2024-01-03 08:21:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":483301,"visible":true,"origin":"","legend":"\u003cp\u003eReceptor-ligand interactions on 3D diagram. A: Molecular docking of Quercetin and Bax; B: Molecular docking of Quercetin and Bcl2; C: Molecular docking of Wogonin and Bax; D: Molecular docking of Wogonin and Bcl2.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3800699/v1/986d50408fb19446d0b12f73.png"},{"id":49106642,"identity":"ec95a3b1-32e6-40cd-9c04-84ea3083f880","added_by":"auto","created_at":"2024-01-03 08:13:16","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":98841,"visible":true,"origin":"","legend":"\u003cp\u003eThe donated cells were strongly positive for synaptopodin expression(200X). Percentage contibution of High Positive: 98.41%.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3800699/v1/a9640c3e38e1982973c55500.png"},{"id":49106649,"identity":"f9eb7456-7283-432d-8cd8-01cb3392772d","added_by":"auto","created_at":"2024-01-03 08:13:16","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":301468,"visible":true,"origin":"","legend":"\u003cp\u003eAverage optical density/pixel of F-actin in each group(200X). The SQ low(0.25mg/ml), medium(0.5mg/ml) and high(1.0mg/ml) dose groups increased, in which high-dose F-actin expression was obvious and the aggregation was enhanced. *\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05 vs. the Control group.\u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt;0.05 vs. the Model group.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-3800699/v1/6ace33ed98bb85ab6520fadf.png"},{"id":49107337,"identity":"97a45ca3-d5c1-4fa1-b5d9-d5248772c37a","added_by":"auto","created_at":"2024-01-03 08:21:16","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":288335,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blotting analysis of the expression of PI3K/AKT/mTOR and their phosphoproteins in MPC5 cells. *\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05 vs. the Control group.\u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05 vs. the Model group.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-3800699/v1/19965d805a007ef01a152b55.png"},{"id":49107481,"identity":"55dad921-379f-4f56-8b3a-47ec2d70dfb2","added_by":"auto","created_at":"2024-01-03 08:29:16","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":144837,"visible":true,"origin":"","legend":"\u003cp\u003eWestern blotting analysis of the expression of apoptosisl proteins in MPC5 cells. A-B. Determined the protein expression levels of Bcl2, Bax and Cleaved Caspase-3. *\u003cem\u003eP\u003c/em\u003e \u0026lt;0.05 vs. the Control group.\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 vs. the Model group.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-3800699/v1/2808611ad5ede1c345e12612.png"},{"id":49106644,"identity":"846f62b5-cd73-42ec-a7c7-0171e94f1a83","added_by":"auto","created_at":"2024-01-03 08:13:16","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":284267,"visible":true,"origin":"","legend":"\u003cp\u003eSQ reduced PI3K / AKT / mTOR phosphorylation, and subsequently inhibited apoptosis activity.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-3800699/v1/9d87dd110cd1910598fe1fd9.png"},{"id":49272154,"identity":"3bca6b68-62d4-4a1d-88fd-2417506e5b02","added_by":"auto","created_at":"2024-01-07 08:07:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4575284,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3800699/v1/05d27be6-0160-4c59-bd6a-cb017196efef.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Analysis of the regulating PI3K/AKT/mTOR signaling pathway and anti-apoptosis activity of Shenqi granule through Network Pharmacology and in vitro experiments","fulltext":[{"header":"Introduction","content":"\u003cp\u003eChronic kidney disease (CKD) is emerging as a significant public health concern, with glomerulopathies being identified as one of the primary causes of end-stage renal disease[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Membranous nephropathy (MN), a glomerular disorder that can manifest at any age, is traditionally diagnosed as either primary MN, in the absence of any underlying disease association, or secondary MN, in the presence of an associated condition such as autoimmune disease, infection, malignancy, or drug toxicity[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Secondary MN, also referred to as idiopathic membranous nephropathy (IMN), represents a prevalent pathological subtype of nephrotic syndrome, with IMN constituting approximately 80% of the overall MN incidence. This condition is characterized by immune-mediated mechanisms involving the deposition of immune complexes beneath the epithelial layer and alterations in the glomerular basement membrane[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. IMN is characterized by the occurrence of spontaneous remission and recurrence. Approximately 40% of individuals with IMN experience spontaneous remissions, while relapses are observed in 15\u0026ndash;30% of cases, furthermore, it has been found that 50% of patients progress to develop the nephrotic syndrome, with 30% ultimately advancing to end-stage renal disease[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. With an increasing prevalence, IMN has received considerable attention in China. Notably, the frequency of IMN cases identified through renal biopsy has recently shown a substantial rise in China. A study revealed a significant increase in the frequency of MN, nearly doubling from 10.4% in the period of 2003\u0026ndash;2006 to 24.1% in the period of 2011\u0026ndash;2014[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Xu et al conducted a comprehensive review of glomerular disease types in a Chinese population spanning an 11-year period. Their findings revealed that IgA nephropathy remained the predominant pathological type, accounting for 28.1% of cases, followed by membranous nephropathy at 23.4%[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. After data calibration, it was observed that IMN increased 13% annually, and its incidence had a tendency to exceed that of IgA nephropathy[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo far, the variable natural course of IMN has made its treatment controversial and discussed. If a partial or complete remission is not achieved by the final stage, it is often recommended for many patients to undergo conservative treatment and continue with immunosuppressive therapy after a period of 6 to 12 months[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The KDIGO 2021 guidelines recommends rituximab, cyclophosphamide combined with glucocorticoids therapy, or tacrolimus[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Despite the long-standing use of immunosuppressive therapy, clinicians still face challenges in balancing the potential benefits and safety concerns associated with serious adverse events[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDue to all of the above factors, alternative treatments are being actively sought. The utilization of Traditional Chinese Medicine (TCM) in the MN presents distinctive advantages in symptom alleviation, mitigation of immunosuppressive drug side effects, prevention and treatment of complications, as well as deceleration of disease progression. Consequently, TCM-based interventions have emerged as a novel alternative therapeutic approach to address MN in China in recent years. SQ was selected based on TCM theories and extensive clinical experience of experienced practitioners who have used TCM to treat patients with kidney disease in recent decades. Comprised of 13 distinct TCM herbs, SQ has been verified as an effective prescription for treating MN with minimal adverse effects in a multicenter, randomized, controlled study in China[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. SQ granule have demonstrated significant clinical efficacy in the treatment of MN, unfortunately, the specific mechanisms underlying their therapeutic properties are unidentified.\u003c/p\u003e \u003cp\u003eNetwork pharmacology is a new method that can determine how TCM works through pharmacokinetic evaluation, it is an interdisciplinary field that integrates systems biology and network informatics, and has gained significant traction in the realm of novel drug development in recent times[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Li[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]introduced the innovative notion of a \"network target\", which expands the traditional understanding of a drug target from a single molecule to encompass its broader influence on a biological network. This approach also enhances drug efficacy and diminishes adverse effects[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This concept proves particularly valuable in addressing complex systems like herbal formulae. Consequently, network pharmacology presents a promising avenue for comprehending the intricacies of multi-component drugs.\u003c/p\u003e \u003cp\u003eMolecular docking is an advanced computational technique employed to simulate the atomic-level interaction between molecules and proteins, enabling the prediction of ligand and receptor conformations, as well as the calculation of various parameters, including affinity, crucial for evaluating combination scenarios. This precise and cost-effective technology finds primary application in drug design and the elucidation of biochemical pathways[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study aims to investigate the potential molecular mechanism of SQ in the treatment of MN through network pharmacology and in vitro experiments and molecular docking techniques, thereby offering a novel approach for clinical intervention(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHerbal materials and SQ preparation\u003c/h2\u003e \u003cp\u003eThe thirteen herbs contained in SQ granule were supplied by Wanshicheng Pharmaceutical Co., Ltd. (Shanghai, Wanshicheng, China). The thirteen herbs were consistent with some published literature[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Specific methods of drug dissolution and freezing have been described in detail in previous articles, using high performance liquid chromatography (HPLC) and control standards, it was confirmed that SQ granule complied with the authentic drug regulations of the Chinese Pharmacopoeia (version 2020).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCollection of main bioactive components and potential targets of SQ\u003c/h2\u003e \u003cp\u003eAll chemical ingredients and their related targets of each herb of SQ were collected from the TCMSP(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://lsp.nwsuaf.edu.cn/tcmsp.Php\u003c/span\u003e\u003cspan address=\"http://lsp.nwsuaf.edu.cn/tcmsp.Php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Ingredients were screened conditional on oral availability (OB)\u0026thinsp;\u0026ge;\u0026thinsp;30% and drug-like (DL)\u0026thinsp;\u0026ge;\u0026thinsp;0.18. The chemical structures and general information of the main components of the drug were got from the Chinese Academy of Sciences Shanghai Institute of Organic Medicine and Chemical Composition database( \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://organchem.csdb.cn/scdb/main/tcm\u003c/span\u003e\u003cspan address=\"https://organchem.csdb.cn/scdb/main/tcm\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eIdentifification of the direct protein targets\u003c/h2\u003e \u003cp\u003eThe UniProt Knowledgebase (UniProtKB) is a protein database partially curated by experts and contains 54, 247, 468 sequence entries. Gene information, including the gene name and the gene ID, was confifirmed by the UniProt database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eScreening disease-associated targets\u003c/h2\u003e \u003cp\u003eThe GeneCards database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genecards.org/\u003c/span\u003e\u003cspan address=\"https://www.genecards.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and selection according to the criterion of Risk Score\u0026thinsp;\u0026gt;\u0026thinsp;1, the Therapeutic Target Database (TTD, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://db.idrblab.org/ttd/\u003c/span\u003e\u003cspan address=\"https://db.idrblab.org/ttd/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and OMIM (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://omim.org/)wer\u003c/span\u003e\u003cspan address=\"https://omim.org/)wer\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003ee used to collect information on MN related to target genes. The association of MN with SQ was then gathered as the core targets of SQ for MN.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eObtain SQ-MN genes and draw a Venn diagram\u003c/h2\u003e \u003cp\u003eValidated human species\u0026ndash;associated target genes were selected in the Uniprot database,and gene names were obtained with R language Perl scripts. The drug-related genes were intersected with disease-related genes with a Perl script, and an intersection Venn diagram was drawn. Finally, potential targets of SQ for IMN treatment were obtained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of SQ\u0026ndash;active ingredients\u0026ndash;MN target network\u003c/h2\u003e \u003cp\u003eDrug components were numbered and paired with their corresponding target genes. A SQ-active ingredients-MN targets target network was constructed using Cytoscape 3.8.2, and the active ingredients and intersectional targets were acquired simultaneously. Sorting and graphing based on the size of the degree values. Degree indicated the number of edges between a single node and other nodes in a network.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of the Protein\u0026ndash;Protein Interaction (PPI) network\u003c/h2\u003e \u003cp\u003eIn order to clarify the interaction between the targets of SQ and MN, the candidate targets of SQ for MN treatment were imported into the STRING database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/\u003c/span\u003e\u003cspan address=\"https://string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to construct a PPI network.The network analysis plug-in in Cytoscape software was used to analyze network topological features to screen the hub nodes in the PPI network. Degree centrality denotes several direct connections of a node to all other nodes in the network.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eGO functional enrichment and KEGG pathway analysis\u003c/h2\u003e \u003cp\u003eImporting gene files into the OmicShare tools, a free online platform for data analysis (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.omicshare.com/tools\u003c/span\u003e\u003cspan address=\"https://www.omicshare.com/tools\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), set q value\u0026thinsp;=\u0026thinsp;0.05, and ran the program to draw a bubble chart. GO and KEGG enrichment analyses of the target genes of SQ in the treatment of MN were performed to elucidate the possible molecular mechanism.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003eCell culture and viability assay\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe MPC5 cells were donated by Dr. Baoli Liu (Beijing Hospital of Traditional Chinese Medicine Affiliated to Capital Medical University). Cells were resuspended and inoculated in L-DMEM containing and 10% FBS and cultured at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e for proliferation and passaging, then allowed to differentiate for 7\u0026ndash;10 days at 37\u0026deg;C, after which performed group experiments. Cells (1.0\u0026times;10\u003csup\u003e4\u003c/sup\u003e) were seeded in wells of a 96-well plate, and 100 mL of L-DMEM was added to each well. A CCK-8 assay kit (BioSharp, Hefei, China) was used to determine cell viability according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell groups and treatments\u003c/h2\u003e \u003cp\u003eMPC5 cells were divided into the following groups: Control group (L-DMEM and 10% FBS); CsA group (0.5 \u0026micro;g/mL CsA\u0026thinsp;+\u0026thinsp;PAN\u0026thinsp;+\u0026thinsp;L-DMEM and 10% FBS); SQ groups (administered 0.5, 1.0, 2.0 mg/mL SQ\u0026thinsp;+\u0026thinsp;PAN\u0026thinsp;+\u0026thinsp;L-DMEM and 10% FBS).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence in MPC5 cells(Identification of the cytoskeletal proteins)\u003c/h2\u003e \u003cp\u003eSynaptopodin and F-actin immunofluorescence in MPC5 cells was analysed using an immunofluorescence method. Cells were fixed in 4% formalin buffered with PBS for 30 mins and permeabilize cells with 0.1% Triton-X-100 for 15 minutes. After blocking with 5% BSA at RT for 60 min, the slides were incubated with FITC-labelled primary antibodies overnight. Nuclei were counterstained with DAPI. After washing, an anti-fluorescence quencher was added to the slides, which were subsequently observed under an inverted fluorescence microscope. Image J (software version 1.46, National Institutes of Health (NIH), Bethesda, MA, USA) was used to determine the amount of fluorescence and compare the results between different groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eWestern bloting Analysis\u003c/h2\u003e \u003cp\u003eMPC5 cells from each group were dissolved, and total protein was extracted using RIPA lysis buffer. Supernatants were collected after centrifugation, and protein concentrations were measured. Proteins were loaded, electrophoresed, transferred to 0.45-\u0026micro;m PVDF membranes, incubated with the primary antibodies (PI3K, p-PI3K, AKT, p-AKT, mTOR, p-mTOR, Bcl2, Bax, cleaved\u0026ensp;Caspase-3) overnight at 4℃, then with secondary antibody for 60 min at RT. Bands were detected on a gel imaging system using enhanced chemiluminescence. The grey values were analysed using Image J (software version 1.46 ,National Institutes of Health (NIH),Bethesda, MA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMolecular docking verification\u003c/h2\u003e \u003cp\u003eAccording to the PPI interaction network, the important targets were screened and the corresponding chemical components in the drug were verified by molecular docking. Four drug components with high OB and DL values and clear active ingredients were selected, and 3D structures of these compounds were downloaded from the PubChem database, calculated energy optimized and saved. The 3D structures of the MN related target protein was downloaded from the PDB(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.rcsb.org/\u003c/span\u003e\u003cspan address=\"https://www.rcsb.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The pdbid are Bax and Bcl2. Molecular docking was performed using AutoDock Vina, and the docking results were plotted with Pymol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using SPSS version 25.0 (SPSS Inc., Chicago, IL, USA). Data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Student\u0026rsquo;s t-test was used to examine differences between two groups. For comparisons of multiple groups, ANOVA was used followed by Dunnett's post hoc test in the absence of equivalent variance. A P value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eThe SQ composition and the major chemical components\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"4\"\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=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChinese Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLatin Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMolecule Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChemical formula\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHuang Qi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAstragalus membranaceus (Fisch.) Bunge\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAstragaloside IV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC41H68O14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDang Gui\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAngelicae Sinensis Radix\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eβ-sitosterol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC29H50O\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCang Shu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAtractylis chinensis (Bunge) DC.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWogonin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC16H12O5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBai shu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAtractylis macrocephala (Koidz.) Hand.-Mazz.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSalvianolic acid B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC36H30O16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShan Yao\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eDioscorea oppositifolia L.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDiosgenin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC27H42O3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZhu Ling\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePolyporus umbellatus (Pers.) Fr.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChlorogenic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC25H24O12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFu Ling\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePoria cocos\u0026nbsp;(Schw.) Wolf\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNeochlorogenic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC16H18O9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJiang Can\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eBombyx\u0026nbsp;micranthus\u0026nbsp;(L.f.) I.Riedl\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCryptochlorogenic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC16H18O9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShe Shecao\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eHedyotis\u0026nbsp;diffusa\u0026nbsp;Willd.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eQuercetin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC15H10O7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYi Yiren\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCoix lacryma-jobi L.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStigmasterol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC29H48O\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDang Shen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCodonopsis pilosula (Franch.) Nannf.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePolyporusterone B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC28H44O6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDan Shen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSalvia miltiorrhiza Bunge\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePachymic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC33H52O5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShui Zhi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eHirudo nipponica Whitman\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLinoleic Acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC18H32O2\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=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe SQ composition and the major chemical components\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChinese Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLatin Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMolecule Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChemical formula\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHuang Qi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAstragalus membranaceus (Fisch.) Bunge\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAstragaloside IV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC41H68O14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDang Gui\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAngelicae Sinensis Radix\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eβ-sitosterol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC29H50O\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCang Shu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAtractylis chinensis (Bunge) DC.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWogonin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC16H12O5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBai shu\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eAtractylis macrocephala (Koidz.) Hand.-Mazz.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSalvianolic acid B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC36H30O16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShan Yao\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eDioscorea oppositifolia L.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDiosgenin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC27H42O3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZhu Ling\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePolyporus umbellatus (Pers.) Fr.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChlorogenic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC25H24O12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFu Ling\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003ePoria cocos\u0026nbsp;(Schw.) Wolf\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNeochlorogenic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC16H18O9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eJiang Can\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eBombyx\u0026nbsp;micranthus\u0026nbsp;(L.f.) I.Riedl\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCryptochlorogenic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC16H18O9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShe Shecao\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eHedyotis\u0026nbsp;diffusa\u0026nbsp;Willd.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eQuercetin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC15H10O7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYi Yiren\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCoix lacryma-jobi L.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eStigmasterol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC29H48O\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDang Shen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eCodonopsis pilosula (Franch.) Nannf.\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePolyporusterone B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC28H44O6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDan Shen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eSalvia miltiorrhiza Bunge\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePachymic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC33H52O5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShui Zhi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eHirudo nipponica Whitman\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLinoleic Acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC18H32O2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv class=\"DuplicateTablecaptionEnd\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eNetwork pharmacology\u003c/h2\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003eObtaining disease targets of MN, targets of SQ and compound common targets\u003c/h2\u003e \u003cp\u003eThe common targets of 13 Chinese herbs in SQ were obtained from TCMSP and Shanghai Institute of Organic Medicine database, and 419 targets were obtained after de-duplication; the disease targets of MN were obtained from GeneCards database, and a total of 1729 targets were obtained after integration and de-duplication 195 common targets were obtained(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eSQ-active ingredient-MN target network diagram\u003c/h2\u003e \u003cp\u003eThe main active ingredients of SQ are astragaloside, peroxyergosterol, dousterol, β-sitosterol, quercetin, stigmasterol, atractylenolide III, 4-Pcoumaric acid, salvianolic acid, adenosine, ferulic acid, wogonin, diosgenin, chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, polyporusterone B, pachymic acid and linoleic acid, etc. 13 herbs in SQ, and 106 of the most relevant active ingredients in SQ were selected to make the association plot with MN. The \"SQ-active ingredient-MNtarget network diagram\" was constructed by Cytoscape 3.8.2 system(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eCommon target protein interaction map (PPI)\u003c/h2\u003e \u003cp\u003eAll 195 genes in the gene set for SQ treatment of MN were imported into STRING, selected the highest confidence level of 0.9, hide disconnected nodes in the network, and the PPI network of genes for SQ treatment of IMN was constructed, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. Based on the Cytoscape 3.8.3, the degree was ranked from high to low. The key targets in PPI network were AKT1, TNF, STAT3, IL6, IL18, BCL2, CASP3 and JUN, etc, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eGO and KEGG analysis for obtaining the 15 most relevant pathways for SQ treatment of MN\u003c/em\u003e \u003c/p\u003e \u003cp\u003eBy the OmicShare tools analysis, We found that SQ treatment for MN involves molecular function, biological process, cellular component(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B\u003cb\u003e)\u003c/b\u003e. Molecular function (MF): function in 905, including enzyme binding, signaling receptor binding, kinase binding, molecular function regulator,, etc. Biological process (BP): involving 6,385 cases, mainly including response to oxygen-containing compound, response to lipid, regulation of cell death, cell proliferation, apoptotic process, and etc. Cellular Component(CC): extracellular space, extracellular region part,extracellular region,vesicle,etc. Details are as shown in Supplementary Fig.\u0026nbsp;1. The KEGG results suggest that SQ treatment for IMN may involve 266 signaling pathways, and the top 15 signaling pathways most relevant to the disease, which contain Lipid and atherosclerosis, AGE-RAGE signaling pathway in diabetic complications, HIF-1 signaling pathway, PI3K-Akt signaling pathway, Endocrine resistance, TNF signaling pathway, Apoptosis, mTOR signaling pathway, and etc(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-E\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eMajor Molecular docking verification\u003c/h2\u003e \u003cp\u003eTo verify the network pharmacology results, the important targets were screened and the corresponding chemical components in the drug were verified by molecular docking, as shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. The targets Bax and Bcl2 selected. The required files were obtained from the PDB database, all receptor files were treated with organic matter, water molecules and hydrogenated charge distribution. To demonstrate the docking mode, typical dockings were visualized using PyMol and AutoDockVina and attached with binding energy and K\u003csub\u003ei\u003c/sub\u003e values. This result can further prove that the active components of SQ, such as wogonin and quercetin, act on Bax and Bcl2 receptors and other related core targets, and have favorable affinity with key targets, reflecting that the active components of SQ can treat or alleviate IMN through these targets. It confirmed the strong binding affinity of the active components in SQ with the disease targets(\u003cb\u003eSupplementary Table\u0026nbsp;2\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eValidation of in vitro experiments\u003c/h2\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eSuccessful MPC5 were identified by immunofluorescence techniques\u003c/h2\u003e \u003cp\u003eSynaptopodin is a signature skeletal protein unique to MPC5 cells. By immunofluorescence, in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e we can see that the cells are highly positive for synaptopodin protein expression, confirming that this cell line is MPC5(\u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eSQ is able to protect the MPC5 cytoskeletal protein F-actin polymerization and expression\u003c/h2\u003e \u003cp\u003eThe results of immunofluorescence showed that, compared with the Con group, after PAN damaged MPC-5, F-actin cleavage and expression decreased. SQ could protect F-actin, reduced its cleavage, increased expression, increased polymerization of F-actin, and this effect was concentration dependent, and the high dose of SQ was optimal, as displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eSQ granule limits the phosphorylation level of PI3K/AKT/mTOR signaling pathway\u003c/h2\u003e \u003cp\u003eTo explore the specific link between signaling pathway and MPC5 cells, and the possible mechanism of SQ granule action, the upstream signaling pathway of PI3K/AKT/mTOR was examined. The results suggested that the expressions of p-PI3K, p-AKT and p-mTOR were all enhanced in PAN-induced MPC5 cell injury (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and SQ granule could inhibit the phosphorylation expression of p-AKT and p-mTOR (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), SQ showed no significant effect in regulating the expression of PI3K phosphorylation(Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.These findings indicate that SQ granule may be inhibited the AKT/mTOR phosphorylation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eSQ decreased the Bax and cleave-caspase3 expression and increased the Bcl 2 protein expression\u003c/h2\u003e \u003cp\u003eTo verify whether PAN-induced cytoskeletal protein damage is related to apoptosis in MPC5 cells, we observed the expression of apoptosis proteins. In Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e, we can see that the Bcl2 was reduced in the Model group, while Bax and cleave-caspase3 expression were upregulated (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), yet SQ granule, in contrast, could upregulate the Bcl2 ratio and inhibit Bax and cleave-caspase3 expression(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These findings may indicate that SQ granule has an depressed apoptosis activity in PAN-induced MPC5 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eMN as a prominent subtype of CKD, is a pathologically defined renal glomerular disorder characterized by the presence of immune complexes on the outer surface of the basement membrane[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. This autoimmune disease is typified by the deposition of immunoglobulin G (IgG), relevant antigens, and complement components, including the membrane attack complex (MAC)[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The immunological conflict results in significant protein loss in the urine, known as proteinuria, primarily due to the disruption of podocyte structure caused by immune complex deposition and MAC formation[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Recent research indicates a significant rise in the prevalence of IMN in China[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Therefore, actively seeking a treatment for MN, especially for IMN, is our clinical challenge and the direction of our research.\u003c/p\u003e \u003cp\u003eNetwork pharmacology analyzed the intersection of the active components of SQ and the disease targets of IMN, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e of the Wayne diagram, yielded a total of 195 common targets(\u003cb\u003eFigure. 2\u003c/b\u003e). By identifying the overlapping targets between the active components of SQ and the gene targets of MN, it was postulated that SQ may possess therapeutic potential through its intervention on these shared targets and thus made the SQ-active ingredient-MN target network and PPI diagram(\u003cb\u003eFigure. 3\u0026ndash;4\u003c/b\u003e). Furthermore, GO and KEGG analyses suggested that SQ primarily engages in enzyme binding, signaling receptor binding, kinase binding, response to lipid, regulation of cell death, cell proliferation, apoptotic process, extracellular space, etc, and disease of the most closely related signaling pathways containing Lipid and atherosclerosis, HIF-1 signaling pathway, PI3K-Akt signaling pathway, TNF signaling pathway, Apoptosis, mTOR signaling and so on(\u003cb\u003eFigure. 5\u003c/b\u003e).\u003c/p\u003e \u003cp\u003ePodocytes, the smallest and most fundamental cell of the glomerulus[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], were utilized to establish the cell model of MN through the use of PAN. Podocyte function is dependent on actin cytoskeleton regulation within the foot processes, structures that link podocytes to the glomerular basement membrane. Actin cytoskeleton dynamics in podocyte foot processes are complex and regulated by multiple proteins and other factors[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Changes in the actin cytoskeleton within a cell are necessary for maintenance of cell shape, cell motility and intracellular transport[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Our previous study demonstrated that SQ offers protection against podocyte cytoskeletal proteins, including CD2AP and α-actinin4[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In this study, upon further examination of the podocytes' cytoskeletal protein, it was observed that PAN induces impairment in the F-actin protein, resulting in polymerization breakdown. However, it was found that SQ has the ability to restore the integrity of the skeletal cytoskeleton proteins. The effectiveness of this protective effect and its dependence on drug concentration are illustrated in \u003cb\u003eFigure. 8\u003c/b\u003e. This investigation revealed that both SQ and CsA possess the ability to restore F-actin expression and enhance polymerization. Notably, the effectiveness of SQ in this regard is contingent upon the concentration of the drug.\u003c/p\u003e \u003cp\u003ePhosphoinositide 3-kinase (PI3K), AKT serine/threonine kinase (Akt), and mammalian target of rapamycin (mTOR) are kinases that mediate cellular signaling pathways involved in the regulation of various cellular processes, including the cell cycle, cell survival, cytoskeleton rearrangements, metabolism, and protein synthesis[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], ectopic or too intense activation of this signaling is a causal event in diseases characterized by uncontrolled cellular expansion[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. PI3K plays a crucial role in controlling diverse cellular processes such as growth, survival, metabolism, apoptosis, and autophagy[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. AKT serves as a primary effector of PI3K, upon activation, AKT phosphorylates tuberous sclerosis complex 2, which disrupts the formation of inhibitory TSC1/TSC2 heterodimers, subsequently activating TOR complex 1, this complex, consisting of mTOR, performs distinct functions within the cell[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. mTOR is a serine/threonine protein kinase that forms the catalytic subunit of two distinct protein complexes, known as mTOR Complex 1 (mTORC1) and 2 (mTORC2), the two complexes exert their activity towards distinct substrates and, as a consequence, regulate different cellular functions[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Our network pharmacology demonstrates that SQ for IMN may be involved in the pathway. In vitro experiments, we have confirmed this inference by observing that PAN intervention in MPC5 activates the PI3K/AKT/mTOR signaling pathway, leading to a significant increase in AKT/mTOR phosphorylation levels(\u003cb\u003eFigure. 9\u003c/b\u003e). However, we have observed that various concentrations of SQ exhibit remarkable effects in reducing the expression of p-AKT and p-mTOR, as well as inhibiting the activation of this signaling pathway's phosphorylation. These findings may potentially induce subsequent alterations in downstream apoptosis and autophagic activity.\u003c/p\u003e \u003cp\u003eApoptosis is characterised by distinct morphological alterations in cellular structure, accompanied by enzyme-dependent biochemical mechanisms[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Bcl-2/Bax and cleaved caspase-3 are crucial factors in the advancement of cellular apoptosis. The Bcl-2 family significantly influences the facilitation or inhibition of the intrinsic apoptotic pathway, which is initiated by mitochondrial dysfunction[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Certain investigations propose that kidney-related apoptosis is mediated by Bcl2 proteins and could potentially be controlled through systemic intervention aimed at impeding conformational bax activation and/or reduction in Bcl2 levels induced by renal stress[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Caspase-dependent apoptosis is responsible for approximately 90% of cellular turnover in homeostatic conditions[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. According to the structure and the roles of caspase-3 in various cellular mechanisms, mainly in apoptosis, this enzyme was specifically considered as a therapeutic target to combat apoptosis-related diseases[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Cleaved caspase3, a downstream protein of the Caspase family, serves as the final product of apoptosis[\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]. In our study, depicted in \u003cb\u003eFigure. 10\u003c/b\u003e, it is evident that PAN-induced cells exhibited suppressed Bcl2 expression and heightened expression of Bax and cleaved Caspase-3, indicating an increase in apoptotic activity. Conversely, SQ was found to activate Bcl2 expression, decrease Bax and cleaved Caspase-3 expression, and decelerate cellular apoptosis.\u003c/p\u003e \u003cp\u003eThese findings imply that certain active components within the drug composition of SQ possess the capability to inhibit apoptosis and regulate certain signaling pathway and facilitate cytoskeleton structure repair. Consequently, these results support the alignment between our SQ and network pharmacology, as the in vitro experiments substantiated the conjecture made by network pharmacology.\u003c/p\u003e \u003cp\u003eWe through network pharmacology found that SQ and IMN have the same therapeutic targets, and dig out the potential mechanism of SQ treatment of IMN, earlier studies have shown that SQ can activate autophagy activity, improve cytoskeletal protein expression, has the effect of protecting damaged podocytes. Our study has yielded novel findings, indicating that SQ exerts a regulatory effect on cell apoptosis and has significant feedback implications for the associated signaling pathways(\u003cb\u003eFigure. 11\u003c/b\u003e). These findings were validated through in vitro studies and molecular docking technology, which confirmed the strong binding affinity of the active components in SQ with the disease targets(\u003cb\u003eFigure. 6, Supplementary Table\u0026nbsp;2\u003c/b\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTCM treatment of diseases involves multiple targets, signaling pathways, and intricate mechanisms of action. The application of network pharmacology methods and in vitro experiments provided evidence that SQ effectively ameliorates IMN by potentially regulating the phosphorylation level of the AKT/mTOR pathway, inhibiting apoptotic activity, and restoring skeletal proteins. These findings were further substantiated through molecular docking analysis.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAJKD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAmerican Journal of Kidney Diseases\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAkt\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAKT serine/threonine kinase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBSA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBovine serum albumin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCKD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eChronic kidney disease\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCsA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCyclosporine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eESRD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEnd-stage renal disease\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eFBS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFoetal bovine serum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIFN-γ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInterferon-gamma\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIMN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIdiopathic membranous nephropathy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKDIGO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThe Kidney Disease: Improving Global Outcomes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eL-DMEM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLow-Dulbecco\u0026rsquo;s Modified Eagle Medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMPC5\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMouse podocyte cell line\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emTOR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emammalian target of rapamycin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePAN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePuromycin aminonucleoside\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePI3K\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphoinositide 3-kinase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRoom temperature\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSQ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eShenqi granule\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTCM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTraditional Chinese medicine.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDeclaration of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was performed in the absence of any commercial or financial relationships that may be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT author statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLi-feng Wei\u003c/strong\u003e: Writing-Original draft preparation and Editing. \u003cstrong\u003eXiao-ping Guo\u003c/strong\u003e: Writing-Original draft preparation and Editing. \u003cstrong\u003eYi-yun Zhu\u003c/strong\u003e: Methodology and Validation. \u003cstrong\u003eJun Yong\u003c/strong\u003e: : Resources and Data curation. \u003cstrong\u003eRui Xu\u003c/strong\u003e: Resources and Data curation. \u003cstrong\u003eShi-xiu Chen\u003c/strong\u003e: Visualization. \u003cstrong\u003eYi-ping Chen\u003c/strong\u003e: Conceptualization and Resources. \u003cstrong\u003eLin Wang\u003c/strong\u003e:\u0026nbsp;Supervision and Writing- Reviewing .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgement \u003c/strong\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval \u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding \u003c/strong\u003eThis study was supported by grants from Shanghai Municipality Further accelerates the Three-year Action Plan for TCM Inheritance, Innovation and Development (2021-2023) (ZY (2021-2023) -0403) TCM High-level Talents Leading Plan, the Clinical Research Plan of SHDC (SHDC2020CR2048B), National Natural Science Foundation of China (No. 81273730), Shanghai Municipal Health and Family Planning Commission project (202040163), Shanghai Municipal Health and Family Planning Commission further accelerating the three-year action plan project of the development of Chinese medicine [ZY (2018\u0026ndash;2020)-ZYBZ-02].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSantos JE, Fiel D, Santos R, Vicente R, Aguiar R, Santos I, Amoedo M, Pires C (2020) Rituximab use in adult glomerulopathies and its rationale. J Bras Nefrol 42(1):77\u0026ndash;93\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS.Sethi, New 'Antigens' in Membranous Nephropathy. J Am Soc Nephrol 32(2) (2021) 268\u0026ndash;278\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCouser WG, Nephropathy PM (2017) Clin J Am Soc Nephrol 12(6):983\u0026ndash;997\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKeri KC, Blumenthal S, Kulkarni V, Beck L, Chongkrairatanakul T (2019) Primary membranous nephropathy: comprehensive review and historical perspective. Postgrad Med J 95(1119):23\u0026ndash;31\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHou JH, Zhu HX, Zhou ML, Le WB, Zeng CH, Liang SS, Xu F, Liang DD, Shao SJ, Liu Y, Liu ZH (2018) Changes in the Spectrum of Kidney Diseases: An Analysis of 40,759 Biopsy-Proven Cases from 2003 to 2014 in China. Kidney Dis (Basel) 4(1):10\u0026ndash;19\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu X, Wang G, Chen N, Lu T, Nie S, Xu G, Zhang P, Luo Y, Wang Y, Wang X, Schwartz J, Geng J, Hou FF (2016) Long-Term Exposure to Air Pollution and Increased Risk of Membranous Nephropathy in China. J Am Soc Nephrol 27(12):3739\u0026ndash;3746\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDisease GK (2021) Improving Global Outcomes Glomerular Diseases Work, KDIGO 2021 Clinical Practice Guideline for the Management of Glomerular Diseases. Kidney Int 100(4S):S1\u0026ndash;S276\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHofstra JM, Wetzels JF (2010) Alkylating agents in membranous nephropathy: efficacy proven beyond doubt. Nephrol Dial Transplant 25(6):1760\u0026ndash;1766\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Y, Deng Y, Ni Z, Chen N, Chen X, Shi W, Zhan Y, Yuan F, Deng W, Zhong Y (2013) Efficacy and safety of traditional chinese medicine (Shenqi particle) for patients with idiopathic membranous nephropathy: a multicenter randomized controlled clinical trial. Am J Kidney Dis 62(6):1068\u0026ndash;1076\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHopkins AL (2008) Network pharmacology: the next paradigm in drug discovery. Nat Chem Biol 4(11):682\u0026ndash;690\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi S, Zhang B (2013) Traditional Chinese medicine network pharmacology: theory, methodology and application. Chin J Nat Med 11(2):110\u0026ndash;120\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi S (2011) Network target: a starting point for traditional Chinese medicine network pharmacology. China J Chin Med 36(15):2017\u0026ndash;2020\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang J, Huang K, Zhong G, Huang Y, Li S, Qu S, Zhang J (2016) Acupuncture Decreases NF-κB p65, miR-155, and miR-21 and Increases miR-146a Expression in Chronic Atrophic Gastritis Rats, Evid Based Complement Alternat Med (2016) 9404629\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeng X, Zhang H, Mezei M, Cui M (2011) Molecular docking: a powerful approach for structure-based drug discovery. Curr Comput Aided Drug Des 7(2):146\u0026ndash;157\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei L, Yong J, Zhang X, Ling C, Wu Y, Xu Z, Zhang H, Cao X, Sheng L, Zhang Q, Chen Y, Wang L (2023) Shenqi granule upregulates CD2AP and alpha-actinin4 and activates autophagy through regulation of mTOR/ULK1 pathway in MPC5 cells. J Ethnopharmacol 303:115942\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePetrosyan A, Cravedi P, Villani V, Angeletti A, Manrique J, Renieri A, De Filippo RE, Perin L, Da Sacco S (2019) A glomerulus-on-a-chip to recapitulate the human glomerular filtration barrier. Nat Commun 10(1):3656\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiaofan H, Jing X, Chenni G, Yifan W, Xialian Y, Li L, Hong R, Wen Z, Weiming W, Xiaoxia P, Jingyuan X, Nan C (2019) New risk score for predicting progression of membranous nephropathy. J Transl Med 17(1):41\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu L, Lai J, Ling Y, Weng Y, Zhou S, Wu S, Jiang S, Ding X, Jin X, Yu K, Chen Y (2021) A Review of the Current Practice of Diagnosis and Treatment of Idiopathic Membranous Nephropathy in China. Med Sci Monit 27:e930097\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRonco P, Beck L, Debiec H, Fervenza FC, Hou FF, Jha V, Sethi S, Tong A, Vivarelli M, Wetzels J (2021) Membranous nephropathy. Nat Rev Dis Primers 7(1):69\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGlassock RJ (2010) The pathogenesis of idiopathic membranous nephropathy: a 50-year odyssey. Am J Kidney Dis 56(1):157\u0026ndash;167\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGarg P (2018) A Review of Podocyte Biology. Am J Nephrol 47:1\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNagata M (2016) Podocyte injury and its consequences. Kidney Int 89(6):1221\u0026ndash;1230\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlaine J, Dylewski J (2020) Regulation of the Actin Cytoskeleton in Podocytes. Cells 9(7)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBlanchoin L, Boujemaa-Paterski R, Sykes C, Plastino J (2014) Actin dynamics, architecture, and mechanics in cell motility. Physiol Rev 94(1):235\u0026ndash;263\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLing C, Zhang X, Wang L (2018) Chen\u0026rsquo;s Yiqi Huoxue Huashi Recipe protected PAN cultured podocyte by regulating autophagy. Chin J Integr Nephrol 19(09):760\u0026ndash;763\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYong J, Ling C, Zhang X, Wang L (2019) Regulation of Yiqi Huoxue Huashi formula on podocyte slit membrane protein α-actinin-4 and CD2AP in passive Heymann nephritis model rats. Shanghai J Traditional Chin Med 53(05):69\u0026ndash;75\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMargaria JP, Campa CC, De Santis MC, Hirsch E, Franco I (2020) The PI3K/Akt/mTOR pathway in polycystic kidney disease: A complex interaction with polycystins and primary cilium. Cell Signal 66:109468\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFruman DA, Chiu H, Hopkins BD, Bagrodia S, Cantley LC, Abraham RT (2017) The PI3K Pathway in Human Disease. Cell 170(4):605\u0026ndash;635\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoncalves MD, Hopkins BD, Cantley LC (2018) Phosphatidylinositol 3-Kinase, Growth Disorders, and Cancer. N Engl J Med 379(21):2052\u0026ndash;2062\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu Z, Han X, Ou D, Liu T, Li Z, Jiang G, Liu J, Zhang J (2020) Targeting PI3K/AKT/mTOR-mediated autophagy for tumor therapy. Appl Microbiol Biotechnol 104(2):575\u0026ndash;587\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang L, Li X, Yang Z, Pan X, Liu X, Zhu M, Xie J (2017) Crotonaldehyde induces autophagy-mediated cytotoxicity in human bronchial epithelial cells via PI3K, AMPK and MAPK pathways. Environ Pollut 228:287\u0026ndash;296\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaxton RA, Sabatini DM (2017) mTOR Signaling in Growth, Metabolism, and Disease. Cell 168(6):960\u0026ndash;976\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD'Arcy MS (2019) Cell death: a review of the major forms of apoptosis, necrosis and autophagy. Cell Biol Int 43(6):582\u0026ndash;592\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohan S, Abdelwahab SI, Kamalidehghan B, Syam S, May KS, Harmal NS, Shafifiyaz N, Hadi AH, Hashim NM, Rahmani M, Taha MM, Cheah SC, Zajmi A (2012) Involvement of NF-kappaB and Bcl2/Bax signaling pathways in the apoptosis of MCF7 cells induced by a xanthone compound Pyranocycloartobiloxanthone A. Phytomedicine 19(11):1007\u0026ndash;1015\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK.Vucicevic V, Jakovljevic N, Colovic N, Tosic T, Kostic I, Glumac S, Pavlovic T, Karan-Djurasevic M, Colovic (2016) Association of Bax Expression and Bcl2/Bax Ratio with Clinical and Molecular Prognostic Markers in Chronic Lymphocytic Leukemia. J Med Biochem 35(2):150\u0026ndash;157\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYip KW, Reed JC (2008) Bcl-2 family proteins and cancer. Oncogene 27(50):6398\u0026ndash;6406\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBorkan SC (2016) The Role of BCL-2 Family Members in Acute Kidney Injury. Semin Nephrol 36(3):237\u0026ndash;250\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFuchs Y, Steller H (2011) Programmed cell death in animal development and disease. Cell 147(4):742\u0026ndash;758\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAsadi M, Taghizadeh S, Kaviani E, Vakili O, Taheri-Anganeh M, Tahamtan M, Savardashtaki A (2022) Caspase-3: Structure, function, and biotechnological aspects. Biotechnol Appl Biochem 69(4):1633\u0026ndash;1645\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWaterhouse N, Kumar S, Song Q, Strike P, Sparrow L, Dreyfuss G, Alnemri ES, Litwack G, Lavin M, Watters D (1996) Heteronuclear ribonucleoproteins C1 and C2, components of the spliceosome, are specific targets of interleukin 1beta-converting enzyme-like proteases in apoptosis. J Biol Chem 271(46):29335\u0026ndash;29341\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChang H, Yang X (2000) Proteases for cell suicide: functions and regulation of caspases. Microbiol Mol Biol Rev 64(4):821\u0026ndash;846\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElmore S (2007) Apoptosis: a review of programmed cell death. Toxicol Pathol 35(4):495\u0026ndash;516\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoreba M, Str\u0026oacute;zyk A, Salvesen GS, Drag M (2013) Caspase substrates and inhibitors. Cold Spring Harb Perspect Biol 5(8):a008680\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":"Shenqi granule(SQ), Membranous nephropathy(MN), Network pharmacology, Molecular docking, MPC5","lastPublishedDoi":"10.21203/rs.3.rs-3800699/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3800699/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIdiopathic membranous nephropathy (IMN), a common pathological type of nephrotic syndrome. Shenqi granule(SQ) is a traditional Chinese medical formula that has been used for decades to treat IMN, and there is a large amount of clinical data confirming its effectiveness,but the mechanism is unclear. This study explores the potential mechanisms and targets of action of SQ through network pharmacology and validates them through in vitro experiments and molecular docking techniques. Network pharmacology is a method that can determine how TCM works through pharmacokinetic evaluation, allowing us to study its molecular mechanisms. Through in vitro experiments, MPC5 cells are used to establish puromycin aminonucleoside (PAN)-induced podocytes damage models to extract cell protein, western blot detection signal pathway protein and related target proteins, molecular docking was performed for the validation. The network pharmacology study results indicate that SQ has 106 compounds, and 195 shared targets with MN. The treatment of IMN with SQ is mainly related to the apoptosis, PI3K/AKT/mTOR signaling pathway and other significant signaling pathways. In vitro experiments showed that SQ could effectively inhibit the activity of the PI3K/AKT/mTOR signaling pathway, increase the expression of Bcl2, and suppress the expression levels of apoptosis-related proteins such as Calaspase-3 and Bax in MPC5 cells. This study initially investigated the pharmacological effects of SQ, which effectively ameliorates IMN by potentially regulating the phosphorylation level of the AKT/mTOR pathway, inhibiting apoptotic activity, and restoring skeletal proteins.\u003c/p\u003e","manuscriptTitle":"Analysis of the regulating PI3K/AKT/mTOR signaling pathway and anti-apoptosis activity of Shenqi granule through Network Pharmacology and in vitro experiments","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-03 08:13:11","doi":"10.21203/rs.3.rs-3800699/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":"368be509-6e3f-4df0-9758-9fe657303666","owner":[],"postedDate":"January 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-01-07T07:59:16+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-03 08:13:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3800699","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3800699","identity":"rs-3800699","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}