Jin-Gui-Shen-Qi Wan alleviates fibrosis in mouse diabetic nephropathy via MHC class II

Ethnopharmacological relevance: Jin-Gui-Shen-Qi Wan (JGSQW) is a traditional Chinese medicine formula that has been traditionally used to alleviate urinary system ailments such as frequent urination and polyuria. Clinical studies have indicated that when combined with hypoglycaemic drugs, JGSQW exhibits a synergistic effect and can improve diabetic nephropathy (DN), yet its underlying mechanism and targets remain unclear. Aim of the study: This study aims to investigate the therapeutic efficacy of JGSQW and its underlying mechanisms using a DN db/db mouse model. Materials and methods: Ultrahigh-performance liquid chromatography coupled with mass spectrometry was utilized to analyse the primary active compounds, blood levels, and pharmacokinetics of JGSQW. Additionally, the therapeutic effects of JGSQW and metformin on blood glucose levels, lipid levels, renal function, and renal pathology in diabetic nephropathy mice were investigated using a db/db mouse model. Proteomic analysis was carried out to identify the primary target of JGSQW in treating DN. The mechanism of action was verified by western blotting, immunohistochemistry, and immunofluorescence. Then, molecular docking and molecular dynamics, transfection, drug affinity responsive target stability (DARTS) assay and cell thermal migration assay (CETSA) further validated the targeted binding effect. Results: JGSQW combined with metformin significantly improved the blood glucose levels, blood lipids, renal function, and renal pathology of DN mice. JGSQW mainly exerted its therapeutic effect on DN by targeting major histocompatibility complex class II (MHC class II) molecules. Immunohistochemistry results showed that JGSQW inhibited the expression of collagen I, fibronectin, and alpha smooth muscle actin (alpha-SMA) expression. Immunofluorescence and Western blot results showed that JGSQW inhibited the expression of H2-Ab1 and H2-Aa, which are MHC class II molecules, thereby suppressing CD4+ T-cell infiltration and improving diabetic kidney fibrosis. The binding ability of paeoniflorin to H2-Aa was predicted and verified by molecular, DARTS, and CETSA assays. Treatment with 80 mu M paeoniflorin effectively alleviated high glucose-induced injury in the MPC5 injury model. H2-Aa was overexpressed at this model concentration, and Western blotting further confirmed that paeoniflorin reduced glomerular podocyte fibrosis by regulating H2-Aa. Conclusions: JGSQW combined with metformin may have a synergistic effect to alleviates renal fibrosis in diabetic nephropathy by downregulating immune complex MHC class II molecules and attenuating the antigen presentation effect of MHC class II on CD4.