Zexie Decoction alleviates diet induced nonalcoholic fatty liver disease via regulating AMPK mediated lipid metabolism

Objective Nonalcoholic fatty liver disease (NAFLD) is a continuum of liver abnormalities from nonalcoholic fatty (NAFL) to nonalcoholic steatohepatitis (NASH), which has become a major public health problem worldwide. Zexie Decoction (ZXD), a traditional Chinese formula derived from Synopsis of the Golden Chamber , has been reported to have the effects of lowing hyperlipidemia and ameliorating NAFLD. Nevertheless, the underlying mechanism by which ZXD alleviates NAFL and NASH remains elusive. This study aims to explore the therapeutic role of ZXD in the process of NAFLD and elucidate the mechanism by which ZXD maintains lipid homeostasis in vivo and in vitro so as to provide a potential candidate for clinical treatment of NAFLD. Methods NAFL and NASH mice models introduced by high fat and high cholesterol diet (HFHCD) or methionine and choline deficient diet (MCD) were established and used to investigate the benefits of ZXD on the progress of NAFLD in vivo . Human hepatocellular carcinoma cell line (HepG2) and mouse alpha liver 12 cell line (AML12) were stimulated by oleic acid and palmitic acid (OPA) to induce lipid accumulation model in vitro . The levels of total cholesterol (TC), triglyceride (TG), nonesterified free fatty acids (NEFA), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) were measured by commercial kits. Hematoxylin and eosin (HE) staining, Oil Red O staining, Sirius red staining and immunofluorescence staining were applied to observe the liver lesions such as steatosis, inflammation, and fibrosis. Levels of interleukin-1 β (IL‑1 β ), IL-6 and tumor necrosis factor- α (TNF‑ α ) were detected by ELISA. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting analysis were conducted to detect the related mRNA and protein levels after ZXD intervention in both mice liver and hepatocytes. Results In vivo, ZXD ameliorated hepatocellular steatosis and hepatocellular ballooning in NAFL mice. Additionally, ZXD affected lipid accumulation, inflammation and fibrosis in mice with NASH. Compared with the control group, HFHCD and MCD diets can downregulate phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK), phosphorylated acetyl CoA carboxylase (p-ACC) and carnitine palmitoyltransferase 1 (CPT1) protein levels, upregulate sterol regulatory element-binding protein 1c (SREBP-1c) and cluster of differentiation 36 (CD36) protein levels, and also increase TC, TG, AST, ALT, ALP levels in mice liver. ZXD could reverse these changes significantly. Besides, ZXD could significantly reduce mRNA levels of Srebp-1c , Fasn , Acc1 , Cd36 and increase the levels of peroxisome proliferator activated receptor alpha ( Pparα ) and Cpt1 after HFHCD and MCD diet stimulation. In vitro , ZXD could increase mitochondrial membrane potential, ameliorate lipid accumulation by inhibiting the synthesis and uptake of fatty acid, while promoting fatty acid oxidation. These results suggest that the therapeutic effect of ZXD on NAFLD may be related to the phosphorylation of AMPK, activation of target protein such as ACC and CPT1, and inhibition of SREBP-1c and CD36. Conclusion ZXD could be a promising natural medicine for the treatment of NAFL/NASH and related diseases.