| Objective To investigate the potential lipophagy related mechanisms of ajugol on nonalcoholic fatty liver disease(NAFLD)by using a high-fat diet(HFD)-induced NAFLD in mice and palmitate-induced lipid accumulation in AML 12 cells and primary mouse hepatocytes.Methods 1 Male C57BL/6 mice(6 weeks old;weight 20-23 g)were fed with HFD for the preparation of NAFLD model.Treatment started concomitantly with the administration of HFD and consisted of daily oral introduction with ajugol(50 mg/kg/d body weight)in mice with a safe dose for 12 weeks;GTT was performed at week 10 and ITT was performed and food intake was measured at week 11;At week 12,mice were sacrificed for serum and livers tissue collection;serum concentrations of triglyceride(TG),low-density lipoprotein cholesterol(LDL-C),alanine aminotransferase(ALT),high-density lipoprotein cholesterol(HDL-C),total cholesterol(TC)and aspartate aminotransferase(AST)were determined using a biochemical auto-analyser;liver sections were stained with hematoxylin and eosin(H&E)to assess liver morphology and oil red O(ORO)for lipid assessments.2 To explore whether ajugol can treat hepatic steatosis in vitro,primary mouse hepatocytes and AML12 cells were treated with palmitate(0.3 m M)to mimic lipid overload conditions;the cellular TG and TC contents were analysed with a commercial kit;meanwhile,m Cherry-GFP-LC3 was transfected into AML12 cells to evaluate autophagic flux,Bodipy 493/503 and Lyso Tracker double staining to evaluate lipophagy and LC3 and LAMP2 double staining to detected the colocalisation of lysosomes and lipid droplets;LAMP2,MCOLN,UVRAG,PGC1α,and ATP6V1 A m RNA expression were amplified with real-time quantitative PCR;western blotting assay were used to detect the expression levels of TFEB,P62,LC3,PGC1α,ATP6V1 A,m TOR,p-m TOR,ERK,p-ERK,GSK-3β,cathepsin B,p-GSK-3β,p-S6 K,S6K,p-4E-BP1 and4E-BP1.Results 1 In our study,it was found for the first time that ajugol could significantly decreased body weight,liver-to-body weight ratio,and TG content of liver in HFD-fed mice,as well as improved insulin resistance;also protected against HFD-induced liver injury and decreased serum levels of TG,LDL-C,and TC,as well as LDL-C/HDL-C;meanwhile,H&E and ORO staining found that the liver of HFD-fed mice with histological changes such as inflammation,hepatocellular ballooning and/or steatosis.However,ajugol significantly decrease hepatic inflammation,hepatocellular ballooning and/or steatosis induced by HFD-fed mice.2 Ajugol significantly improved hepatic steatosis in AML 12 cells and primary mouse hepatocytes induced by palmitate;meanwhile,ajugol promoted the TFEB nuclear translocation,enhanced the m RNA expressions of LAMP2,MCOLN,UVRAG,ATP6V1 A and PGC1α;upregulated the LAMP2,TEFB,PGC1α and ATP6V1 A protein expressions;decreased the ratio of pm TOR/ m TOR,p-S6K/ S6 K and p-4E-BP1/4E-BP1;increased the LC3-II expression and p62 degradation,promoted TFEB mediated lysosomes biogenesis to restore autophagosome and lysosome fusion and enhance lipophagy to reduce lipid accumulation.Conclusion Ajugol ameliorated hepatic steatosis via m TOR-TFEB-mediated lysosomal biogenesis and subsequent improved autophagy-lysosomal fusion,lipophagy and reduced lipid accumulation.This study provides evidence for the potential application of ajugol as a promising therapeutic agent for NAFLD and supports exploration of a potential therapeutic role for lipophagy and lysosomal modulation in the prevention of NAFLD.Figure 7;Table 5;Reference 135... |
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