Wenyang Huazhuo Formula Extract Ameliorates Diabetic Kidney Disease in db/db Mice and Is Associated with Modulation of MHC Class II Molecules and Gut Microbiota

Qi Gao¹Xingyao Li¹Yanhong Zhao¹Jiang Tiantian¹Yuetong Wang¹Hailong Zhang²Xili Wu^1*

• ¹The Second Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China
• ²Xi'an Jiaotong University, Xi'An, China

The purpose of this study was to investigate the therapeutic effects and underlying mechanisms of Wenyang Huazhuo Formula (WYHZF) extract in diabetic kidney disease (DKD) in db/db mice. DKD mice were administered WYHZF extract by oral gavage, and glycolipid metabolism, renal function, and renal pathology were assessed. Renal histopathology was evaluated using H&E, PAS, and Masson's trichrome staining. UPLC-QE-Orbitrap-MS, HPLC, and UV-Vis spectroscopy were employed to characterize prototype and potential blood-absorbed metabolites of WYHZF extract. Renal RNA-seq was conducted to identify differentially expressed genes and related pathways. Molecular docking and molecular dynamics simulations were used as supportive in silico analyses to assess the plausibility of interactions between representative metabolites and H2-Aa/H2-Ab1. Western blotting (WB) was performed to validate H2-Aa and H2-Ab1 regulation and to examine fibrosis-associated proteins TGF-β and α-SMA. Gut microbiota alterations were evaluated by 16S rRNA sequencing. WYHZF extract significantly improved hyperglycemia and dyslipidemia, reduced UACR, serum creatinine, and BUN, alleviated glomerular/tubular injury, and restored Nephrin and NGAL expression. Transcriptomic analysis indicated aberrant activation of MHC class II–related pathways in DKD kidneys, which was modulated after WYHZF treatment. Docking and molecular dynamics simulations suggested compatible binding modes and stable interaction patterns between several representative metabolites and H2-Aa/H2-Ab1 under simulated conditions. WB confirmed that H2-Aa and H2-Ab1 were upregulated in DKD and were reversed by WYHZF; notably, TGF-β and α-SMA were also reduced, consistent with attenuation of renal fibrotic features. In addition, WYHZF partially restored gut microbiota diversity and corrected key taxonomic imbalances. Collectively, WYHZF extract may exert multi-level benefits in DKD, accompanied by modulation of MHC class II-related molecules, reduced fibrosis-associated signals, and gut microbiota remodeling.