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Taking lipophagy as the breakthrough point, we explored the mechanism of Zexie Decoction(ZXD) in improving lipid metabolism in the hepatocyte model induced by palmitic acid(PA) and in the animal model induced by high-fat diet(HFD) on the basis of protein kinase B(Akt)/transcription factor EB(TFEB) signaling pathway. Co-localization was carried out for the microtubule-associated protein light chain 3(LC3) plasmid labeled with green fluorescent protein(GFP) and lipid droplets(LDs), and immunofluorescence co-localization for liver LC3 of HFD mice and perilipin 2(PLIN2). The results showed that ZXD up-regulated the expression of LC3, reduced lipid accumulation in hepatocytes, and increased the co-localization of LC3 and LDs, thereby activating lipo-phagy. Western blot results confirmed that ZXD increased autophagy-related protein LC3Ⅱ/LC3Ⅰ transformation ratio and lysosome-associated membrane protein 2(LAMP2) in vivo and in vitro and promoted the degradation of sequestosome-1(SQSTM1/p62)(P<0.05). The results above jointly explained that ZXD regulated lipophagy. Furthermore, ZXD activated TFEB expression(P<0.05) and reversed the PA-and HFD-induced decrease of TFEB nuclear localization in hepatocytes(P<0.05). Meanwhile, ZXD activated liver TFEB to up-regulate the expression of the targets Lamp2, Lc3 B, Bcl2, and Atg5(P<0.05). Additionally, ZXD down-regulated the protein level of p-Akt upstream of TFEB in vivo and in vitro. In conclusion, ZXD may promote lipophagy by regulating the Akt/TFEB pathway.

The present study investigated the pharmaceutical effect and underlying mechanism of Zexie Decoction(ZXD) on nonalcoholic fatty liver disease(NAFLD) in vitro and in vivo via the LKB1/AMPK/PGC-1α pathway based on palmitic acid(PA)-induced lipid accumulation model and high-fat diet(HFD)-induced NAFLD model in mice. As revealed by the MTT assay, ZXD had no effect on HepG2 activity, but dose-dependently down-regulated alanine aminotransferase(ALT) and aspartate aminotransferase(AST) in the liver cell medium induced by PA, and decreased the plasma levels of ALT and AST, and total cholesterol(TC) and triglyceride(TG) levels in the liver. Nile red staining showed PA-induced intracellular lipid accumulation, significantly increased lipid accumulation of hepatocytes induced by PA, suggesting that the lipid accumulation model in vitro was properly induced. ZXD could effectively improve the lipid accumulation of hepatocytes induced by PA. Oil red O staining also demonstrated that ZXD improved the lipid accumulation in the liver of HFD mice. JC-1 staining for mitochondrial membrane potential indicated that ZXD effectively reversed the decrease in mitochondrial membrane potential caused by hepatocyte injury induced by PA, activated PGC-1α, and up-regulated the expression of its target genes, such as ACADS, CPT-1α, CPT-1β, UCP-1, ACSL-1, and NRF-1. In addition, as revealed by the Western blot and immunohistochemistry, ZXD up-regulated the protein expression levels of LKB1, p-AMPK, p-ACC, and PGC-1α in vivo and in vitro. In conclusion, ZXD can improve NAFLD and its mechanism may be related to the regulation of the LKB1/AMPK/PGC-1α pathway.