Integrating WGCNA and network pharmacology to unravel the multi-target mechanisms of Ludangshen Oral liquid against nephrotic syndrome: Involvement of TGF-β1/Smad3 and gut microbiota.

Ludangshen oral liquid (LDSKFY, abbreviated from the pinyin initials of "Ludangshen Koufuye") is a traditional Chinese medicinal product clinically used to treat chronic nephritis. This study aimed to investigate the therapeutic effects of LDSKFY on nephrotic syndrome (NS) and its underlying mechanisms. UPLC-MS was used to analyze the chemical composition to identify the active components of LDSKFY. An integrated approach combining weighted gene co-expression network analysis (WGCNA) and network pharmacology was employed to predict its potential mechanisms against NS. The protective effects of LDSKFY were further evaluated both in vivo and in vitro, using an Adriamycin (ADR)-induced NS model in SD rats and an ADR-induced fibrosis model in HBZY-1 cells, respectively. Renal histopathology was examined to assess morphological changes. Western blot and RT-qPCR were conducted to assess TGF-β1/Smad3 pathway-related proteins and mRNA expression. Additionally, 16S rRNA gene sequencing technology was used to investigate the gut microbiota research to identify microbial changes in rat fecal samples. The interactions between active components and key targets were further validated through molecular docking and molecular dynamics simulations. UPLC-MS analysis identified 20 important active components, and network pharmacology predictions suggested their mechanism of action was associated with the TGF-β signaling pathway. In vivo results indicated that LDSKFY reduced kidney index and histopathological grading in ADR-induced NS. Furthermore, LDSKFY significantly decreased the levels of 24-h urinary protein (24 h-TP), 24-h microalbumin (24 h-mALB), triglyceride (TG), cholesterol (CHO), blood urea nitrogen (BUN), and serum creatinine (CRE), while increasing total protein (TP) and albumin (ALB), indicating improved renal function. In vitro, LDSKFY effectively restored the expression of podocyte-specific markers nephrin and synaptopodin and suppressed desmin expression in HBZY-1 cells. Mechanistically, LDSKFY prevents the activation of the TGF-β1/Smad3 pathway in both renal tissue and cells. In addition, gut microbiota analysis revealed that LDSKFY treatment promoted a more stable gut microbial community structure. Molecular docking and dynamics simulations confirmed that multiple active components of LDSKFY stably bound to key targets including TGF-β1, Smad3, and Smad7. Our findings elucidate the multi-target mechanisms of LDSKFY against NS, involving the TGF-β1/Smad3 pathway and gut microbiota modulation, which provides a theoretical basis for its clinical application in the treatment of chronic nephritis and NS.