Unraveling the Molecular Mechanisms of Fufangduzhong Formula in Alleviating High-Fat Diet-Induced Non-alcoholic Fatty Liver Disease in Mice

Yu Mou ¹ Yao Tang ¹ Xiuyan Zheng ² Xiang Liu ¹ Xuemei Wu ¹ Hongji Wang ¹ Jie Zeng ¹ Qing Rao ² Yaacov Ben-David ^2* Yanmei Li ^2* Lei Huang ^2*

• ¹ Guizhou Medical University, Guiyang, China
• ² Key Laboratory of Chemistry for Natural Products of Guizhou Province (CAS), Guizhou, China

Background: Non-alcoholic fatty liver disease (NAFLD) is a common chronic liver disease, characterized by hepatic lipid accumulation. The Fufangduzhong formula (FFDZ) is a traditional Chinese medicine (TCM) formulation composed of Eucommia ulmoides Oliv., Leonurus artemisia (Lour.) S. Y. Hu, Prunella vulgaris Linn, Uncariarhynchophylla (Miq.) Miq. ex Havil., and Scutellaria baicalensis Georgi. It has demonstrated hepatoprotective effects and the ability to reduce lipid accumulation. However, its mechanisms against NAFLD remain unclear.Methods: UPLC-MS/MS was used to identify FFDZ metabolites. C57BL/6J mice were fed a high-fat diet (HFD) supplemented with or without FFDZ (HFD+L, 0.45 g/kg/d; HFD+H, 0.9 g/kg/d) for 12 weeks. Biochemical indicators and histopathological observations were utilized to assess the extent of metabolic homeostasis disorder and hepatic steatosis. An analysis of differentially expressed genes and regulated signaling pathways was conducted using hepatic transcriptomics. Metabolomics analysis was performed to investigate the significantly changed endogenous metabolites associated with NAFLD in mice serum using UPLC-Q-TOF/MS. Western blot was employed to detect proteins involved in the lipid metabolism-related signaling pathways. Oleic acid-induced hepatic steatosis was used to examine the lipid-lowering effect of FFDZ-containing serum in vitro.Results: A total of eight active metabolites were identified from the FFDZ formula and FFDZ-containing serum through UPLC-MS/MS analysis. FFDZ reduced body weight, liver weight, and levels of inflammatory cytokines, and it ameliorated hepatic steatosis, serum lipid profiles, insulin sensitivity, and glucose tolerance in mice with HFD-induced NAFLD. Transcriptomics revealed that FFDZ modulated the lipid metabolism-related pathways, including the PPAR signaling pathway, Fatty acid metabolism, and AMPK signaling pathway. Meanwhile, western blot analysis indicated that FFDZ down-regulated the expression of lipid synthesis-related proteins (Srebp-1c, Acly, Scd-1, Fasn, Acaca, and Cd36) and up-regulated the fatty acid oxidation-related proteins (p-Ampk, Ppar-α, and Cpt-1). Furthermore, metabolomics identified FFDZ-mediated reversal of phospholipid dysregulation (PC, PE, LPC, LPE). Additionally, FFDZ-containing serum remarkedly reduced OA-induced lipid accumulation in HepG2 cells.Conclusion: The present results demonstrate that FFDZ exerts anti-NAFLD effects by enhancing glucose tolerance and insulin sensitivity, as well as regulating the Ampk signaling pathway to ameliorate lipid metabolism disorder, lipotoxicity, hepatic steatosis, and inflammatory responses.