Uncovering the mechanism of Huanglian-Wuzhuyu herb pair in treating nonalcoholic steatohepatitis based on network pharmacology and experimental validation

The Huanglian-Wuzhuyu herb pair (HWHP) is a classic Chinese herbal formula consisting of the root of Coptis chinensis Franch and dried, nearly mature scented fruit of Tetradium ruticarpum (A.Juss.) T.G.Hartley. It is widely utilized to treat gastrointestinal and liver diseases such as diarrhea, dysentery, cholestasis, hepatocellular carcinoma, and nonalcoholic steatohepatitis (NASH). However, the mechanism of HWHP in treating NASH remains poorly understood.This study investigated the mechanisms of HWHP in NASH treatment via network pharmacology and validated the results through in vivo experiment using mouse models.The compounds and targets corresponding to the active ingredients of HWHP were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database. The genes associated with NASH were obtained from the DisGeNET database. Cytoscape software was employed to construct a "drug-ingredient-target-disease" network. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were employed to analyze the related signaling pathways affected by HWHP. Moreover, AutoDock software was used to assess the potential binding affinity between the key targets of the hub pathway and the bioactive compounds. Subsequently, in vivo experiment was conducted to verify the findings of network pharmacology.A total of 41 active compounds and 198 targets of HWHP were screened, of which 51 common targets were related to NASH. GO functional enrichment analysis revealed that HWHP may affect NASH by modulating inflammatory response. KEGG pathway enrichment suggested that the NOD-like receptor (NLR) signaling pathway may play an important role in treating NASH. Molecular docking results demonstrated that most HWHP components were successfully docked to NLRP3 with good binding energy. In vivo experiments revealed that HWHP alleviated liver inflammation, improved liver steatosis, reduced TC, TG, LDL-C, ALT, and AST, decreased mRNA expressions of IL-6, IL-18, and TNF-α in the liver, and lowered the expressions of NLRP3, pro-IL-1β, and ASC protein. Also, immunohistochemical findings presented downregulation of caspase-1 and IL-1β by HWHP.The results disclosed that HWHP ameliorates NASH in mice by reducing inflammation and liver steatosis via inhibition of NLRP3 inflammasome. This study revealed the mechanism of HWHP in treating NASH through experiments.