| Objective To investigate the possible mechanism of naringin in improving non-alcoholic fatty liver disease(NAFLD)by in vitro and in vivo experiments.Methods 1 In vivo experiment:C57BL/6 mouse liver lipid deposition model was induced by high-fat diet(HFD).50 mg·kg-1·day-1,100 mg·kg-1·day-1 and 200 mg·kg-1·day-1 of naringin were intervened simultaneously with high-fat feeding.Every week,weigh and measure food consumption,do a glucose tolerance test at 14 weeks,an insulin tolerance test at 15 weeks,and anesthesia sacrificed at 16 weeks,with blood and liver samples obtained.Detect serum low density lipoprotein cholesterol(LDL-C),triglyceride(TG),serum glutamic oxaloacetic transaminase(AST),and total cholesterol(TC)by biochemical analyzer,Serum glutamic pyruvic transaminase(ALT),high density lipoprotein cholesterol(HDL-C);through hematoxylin and eosin(H&E)and oil red O(ORO)Staining to observe liver histological changes and lipid deposition;Western blot analysis to detect autophagy marker LC3-Ⅱ and substrate p62 protein,lysosomal marker nuclear TFEB and downstream LAMP1,ATP6V1A protein levels.2 In vitro experiment:0.3 mM palmitate(PA)was used to induce lipid droplet accumulation in alpha mouse liver-12(AML12)cells and mouse primary hepatocytes,and intervention with naringin at 20 μM,40 μM,and 80 μM respectively.BODIPY493/503 and ORO dyes were used to measure intracellular lipid droplet accumulation;kits were used to measure TG and TC contents in cells;netautophagic flux and mCherry-GFP fluorescently labeled LC3 to detect autophagic flux;LC3 and LAMP1 antibodies labeled autophagosomes and lysosomes,to observe the number of autophagic lysosomes;BODIPY493/503 and Lysotracker dyes label intracellular lipid droplets and lysosomes to observe the number of lipid lysosomes.Western blot analysis detects LC3-II,p62,TFEB,LAMP1,ATP6V1A,p-S6K1,p-AMPK and downstream Beclin 1 proteins Horizontal;electron microscopy to observe the number of autophagosomes and lysosomes in cells.3 Liver-specific knockout TFEB mice(TFEBΔhep)and littermate control(TFEBfl/fl)mice were fed HFD for 16 weeks with or without 200 mg·kg-1 naringin intervention,respectively.Serological indexes and liver pathological changes as well as the expression levels of LC3-Ⅱ and p62 in liver tissue were detected;the ultrastructure of liver was observed by transmission electron microscope.Results In vivo experiments revealed that mice in the naringin group lost weight,blood lipid levels,pathological changes in liver tissue,and lipid droplet deposition when compared to the model control group,implying that naringin alleviates lipid metabolism disorder and reduces liver lipid deposition in mice caused by HFD.In vitro tests revealed that naringin lowers lipid droplet formation as well as TG and TC levels in hepatocytes induced by PA.However,qPCR results showed that naringin treatment had no significant effect on lipid uptake,lipid synthesis and lipolysis gene expression.Furthermore,western blotting revealed that in HFD liver tissue or PA-treated hepatocytes,the expressions of autophagy marker LC3 Ⅱ and autophagy substrate p62 were considerably up-regulated compared to the control group.LC3 Ⅱ net flux and mCherry-GFP viral dual fluorescence suggest autophagic flow blockade in steatotic hepatocytes.In the naringin treatment group,LC3 Ⅱ was up-regulated,p62 was down-regulated,the net flux of LC3 Ⅱ was increased,the red fluorescence of mCherry-GFP was enhanced,and the autophagic flux was improved,suggesting that naringin alleviated liver lipid deposition by improving the autophagic flux.Further research revealed that naringin might counteract HFD or PAinduced downregulation of TFEB,a key regulator of lysosomal biogenesis,and downstream LAMP 1,ATP6V1A protein production.Naringin therapy enhanced the quantity of autophagolysosomes and lipsolysosomes,according to LC3 and LAMP1 colocalization and Bodipy493/503 and Lysotracker co-localization.The effects of naringin in boosting lysosomal biosynthesis,improving autophagic flux,and reducing hepatic lipid accumulation were eliminated when the TFEB gene was silenced at the cell level.According to the results of Compound C inhibition,naringin increased TFEB nucleation via AMPK.At the animal level,liver-specific deletion of the TFEB gene reduces the effects of naringin on dyslipidemia,autophagy,and hepatic lipid accumulation.Conclusion Naringin enhanced lysosomal biosynthesis,improved autophagic flux and alleviated HFD or PA-induced hepatic lipid deposition via AMPK/TFEB pathway.Figure 8;Table 5;Reference 102... |
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