Abstract: Objective To investigate the mechanism of Fuzheng Huayu Formula (FZHY) in treating metabolic dysfunction-associated steatohepatitis (MASH). Methods The study utilized TCMSP to systematically analyze FZHY′s active ingredients and target proteins, while the potential MASH treatment targets were acquired by integrating data from GeneCards and OMIM resources.Intersecting targets were analyzed via protein-protein interaction (PPI) networks to identify core proteins, followed by KEGG enrichment analysis to determine key signaling pathways. A methionine-choline-deficient (MCD) diet-induced MASH mouse model was established. Mice were randomly divided into a model control group, an obeticholic acid group (10 mg/kg/day), a FZHY group (20.57 g crude drug/kg/day), and a normal control group (n=10/group). The MCD diet was administered to model and treatment groups for 8 weeks, while the normal control group received standard chow diet. Drug interventions began at week 3 and lasted for 6 weeks. Serum and liver tissues were collected 12 hours after the final dose. Serum liver function markers, hepatic total cholesterol (TC), triglycerides (TG), and hydroxyproline levels were measured using biochemical kits. Histopathological changes were assessed via hematoxylin-eosin (HE), Sirius red, and Oil Red O staining. Hepatic IL-6, IL-1β, and TNF-α levels were quantified by ELISA. Key network pharmacology-predicted signaling molecules were validated via qRT-PCR and Western blotting. Results The analysis yielded 136 shared targets between the drug′s action targets and disease pathogenesis targets. Both PPI network and KEGG pathway enrichment analyses demonstrated significant enrichment of the PI3K-Akt signaling transduction pathway (P<0.05), indicating its potential role as a pivotal regulatory pathway for FZHY′s therapeutic effects on MASH.The experimental results demonstrated that, compared with the control group, the model group exhibited elevated serum AST、ALT 、hepatic TC and TG levels, (79.95 ± 28.69) IU/L、(147.60 ± 27.11) IU/L and (0.25 ± 0.04) mmol/L、 (0.51 ± 0.09) mmol/L,FZHY treatment group showed significantly reduced serum AST、ALT、hepatic TC and TG levels, (29.67 ± 11.10) IU/L、(62.41 ± 20.13) IU/L and (0.14 ± 0.01) mmol/L、 (0.20 ± 0.06) mmol/L(P<0.05).Additionally, FZHY treatment alleviated hepatic IL-6, IL-1β, TNF-α levels, liver fibrosis, and liver lipid accumulation. It decreased mRNA expression of AKT1, NF-κB, HSP90, CASP9, BCL2, and TP53, and decreased protein levels of AKT, phosphorylated AKT (p-AKT), NF-κB, phosphorylated NF-κB (p-NF-κB), and TP53. Conclusion FZHY exerts therapeutic effects on MASH in mice, potentially through modulation of the PI3K-Akt signaling pathway.

Key words: Fuzheng Huayu Formula, Metabolic dysfunction-associated steatohepatitis, Network pharmacology, Mechanism