Investigation On Potential Pharmacological Mechanisms Of Colquhounia Root Tablets Acting On The Membranous Nephropathy Based On TNFα/IL6-JAK2/STAT3-MMP9 Signal Pathway

Objective: Membranous nephropathy(MN)is the most common cause of non-diabetic idiopathic nephrotic syndrome in adults worldwide.In addition to drug tolerance in some patients,the current main clinical treatment drugs also have side effects such as infection,liver and kidney damage,resulting in a poor prognosis.Clinical studies have found that Colquhounia Root Tablets(CRT)has good efficacy in MN,but its pharmacological basis and mechanism of action are unclear.This study aims to investigate the therapeutic effect and mechanism of action of CRT on MN,and further evaluate the "benefit-risk" of CRT key pharmacodynamic substances in vitro to guide the rational use of clinical drugs.Methods:(1)In this part of the study,we combined wet and dry experiments,firstly,we performed network analysis to predict the targets of CRT intervention in MN,searched the literature for the main chemical components of CRT,collected the corresponding component targets using TCMIP database and Swiss Prediction website,and collected MN disease targets using TCMIP database.Based on the STRING database,we constructed a "disease-drug" interaction network,calculated and screened key network targets,and then performed GO and KEGG pathway enrichment analysis.Subsequently,in vitro and in vivo MN models were established to observe the therapeutic effects of CRT on cationized bovine serum albumin MN mice and C5b-9 damaged mouse foot cells,and the effects of CRT on pathway proteins were further validated in in vitro experiments with the TNF-α inhibitor infliximab.The 24-hour urinary protein levels and the levels of albumin,cholesterol,creatinine,TNF-α,IL-6,MMP-9 and TNF-α,IL-6 and MMP-9 in the supernatant of MPC-5 cells were measured dynamically by enzyme-linked immunosorbent assay.The expression of Nephrin and Podocin in mouse glomeruli was detected by immunohistochemistry,and the expression levels of Nephrin and Podocin in MPC-5 cells,JAK2,STAT3,p-JAK2 and p-STAT3 in MPC-5 cells and mouse kidneys were detected by immunoblotting.protein expression levels in MPC-5 cells and mouse kidney.(2)After clarifying the therapeutic effects of CRT on MN,the evaluation of CRT key pharmacodynamic substances was further evaluated to examine its effects on the activity of human normal hepatocytes L-02,human rheumatoid fibroblast-like synoviocytes MH7 A,human renal tubular epithelial cells HK-2 and mouse foot cells MPC-5.The levels of inflammatory factors in the inflammation-induced MH7 A and glucose-induced HK-2 cell models were investigated by enzyme-linked immunosorbent assay,and the effects of drugs on the abnormal expression of Nephrin and Podocin in the MPC-5 cell model were examined by protein immunoblotting,and the regulation of the expression of MPC-5 cytoskeletal proteins by immunofluorescence assay.The TCMIP v2.0 database was used to obtain the candidate targets of CRT blood components,and the GEO database was used to collect the targets of rheumatoid arthritis,diabetic nephropathy,and MN disease.Finally,we used Auto Dock Vina molecular docking method to investigate the binding ability of rhodopsin and rhodopsin to the corresponding targets in the key pathways.Results:(1)CRT can act on MN through four functional modules: immune,metabolic,oxidative and cell cycle,among which TNFα/IL6-JAK2/STAT3-MMP9 signaling pathway is more relevant.The results showed that CRT reduced 24-hour urinary microalbumin,serum creatinine and total cholesterol levels,increased serum albumin,reduced immune deposits in glomerular capillary loops,increased the expression of podocyte-specific proteins,and regulated the expression of pathway-related proteins in MN mice,thereby maintaining podocyte structure,restoring glomerular filtration rate,and improving renal function.Positive validation experiments further clarified the regulatory effect of CRT on the TNFα/IL6-JAK2/STAT3-MMP9 signaling pathway,indicating that CRT can treat MN through this pathway.(2)Celastrol,epicatechin and triptonide all reduced the supernatant inflammatory factor concentration in MH7 A and HK-2 cells and increased the signature protein content in MPC-5 cells,among which epicatechin had no significant toxic effects on cells.The results of "disease-gene-drug target" association network mining suggest that CRT may interfere with rheumatoid arthritis,MN,and diabetic nephropathy by regulating PI3K/HIF1α/NOS2 signaling pathway.Conclusions:(1)CRT has better efficacy in MN,and can improve renal function,alleviate renal and podocyte injury,and intervene in the occurrence and development of MN through regulating the TNFα/IL6-JAK2/STAT3-MMP9 signaling pathway.The results of this study provide an experimental basis for the expansion of the clinical indications of CRT and expand the clinical therapeutic scope of T.rhamnosus.(2)As the key pharmacodynamic substances of CRT,epicatechin has higher benefit but less risk,and tretinoin and tretinoin lactone have better benefit but higher risk.The results of web mining suggest that PI3 K / HIF1α/ NOS2 signaling pathway may be a key link and pathway for CRT intervention in rheumatoid arthritis,diabetic nephropathy and MN,and the results of this study provide an experimental reference for quality standard improvement and further development of CRT.