The Phytochemical Polydatin Ameliorates Non-alcoholic Steatohepatitis By Restoring Lysosomal Function And Autophagic Flux

Research Background and PurposePolygonum cuspidatum is a kind of traditional Chinese medicine which is usually used to relieve pain and diuresis.It has the effects of eliminating dampness and removing jaundice,heat-clearing and detoxification,dispersing blood stasis and relieving pain,relieving cough and reducing sputum.Polydatin is a single crystal compound extracted from Polygonum cuspidatum,and is the main active ingredient of Polygonum cuspidatum.The benefits of polydatin have been widely reported,including antitussive,lipid-lowering,cholesterol-lowering,anti-shock and treatment of liver damage.Recently,it has been reported that polydatin can alleviate non-alcoholic fatty liver disease(NAFLD)by promoting autophagy in mice,but its therapeutic mechanism has not been fully elucidated.Studies have shown that autophagic flow is inhibited in non-alcoholic steatohepatitis(NASH)mouse models.However,the molecular mechanism by which autophagic flow is inhibited remains unclear.According to our preliminary data,autophagy damage in NAFLD is caused by the acidification inhibition of lysosomes.Transcription factor EB(TFEB)is a transcription factor that activates the autophagy lysosome function.In this study,we tested whether polycyrine can play a protective role in NASH mouse model and fatty liver cell model by upregulation of TFEB to repair the damage of autophagy lysosome.Research Methods and Contents(1)Liver tissue of NASH mice was stained with H&E staining method,and then NASH score was performed.The contents of triglyceride,cholesterol and ALT in liver and serum of mice were measured.LO2 cells of fatty liver cell model and liver tissues of NASH mouse model were stained with oil red O staining method,and the lipid content was observed and calculated(2)Western Blot and immunohistochemistry were used to detect the protein levels of P62,LC3B-I and LC3B-Ⅱ in mouse liver and L02 cells.(3)The activity of lysosomal enzymes in the liver of mice was detected.Western Blot was used to detect the levels of precursor and mature cathepsin D in liver and L02 cells of mice.(4)LO2 cells were transfected with mCherry-GFP-LC3 plasmid.The colour of autophage-lysosome was observed under confocal microscope and the number of autophage-lysosome was calculated(5)Western Blot was used to detect the level of TFEB protein in mouse liver and L02 cells.The cytoplasm and nucleus of L02 cells were separated and the levels of TFEB protein in cytoplasm and nucleus were detected respectively.The activity of TFEB luciferase in L02 cells was detected to understand the changes of TFEB transcriptional activity.The expression levels of mRNA of TFEB,TFE3,MITF and TFEB target genes in mouse liver and L02 cells were detected by reverse transcription quantitative PCR.Research Results:(1)After polydatin treatment,the NASH score of liver decreased,the ALT of serum decreased,and the lipid content of liver,serum and L02 cells also decreased.(2)After polydatin treatment,the accumulation of proteins of P62 and LC3B-II in liver and L02 cells decreased.(3)After polydatin treatment,the lysosomal enzyme activity of liver recovered and the precursor cathepsin D was transformed into mature catheps:in D.(4)After polydatin treatment,the number of acid autophages-lysosomes in L02 cells increased.(5)After polydatin treatment,TFEB protein levels in liver and L02 cells increased,and the expression levels of mRNA of TFEB,TFE3,MITF and TFEB target genes were increased.The transcriptional activity of TFEB in LO2 cells was increased and translocated into the nucleus.Conclusion:Polydatin can improve NASH by upregulating TFEB of liver cells to restore lysosomal function and autophagic flow.