Phillygenin Ameliorates Non-alcoholic Fatty Liver By Lipophagy

Objective:To explore the pharmacological effect and mechanism of phillygenin on non-alcoholic fatty liver,and provide scientific basis for its development and application in lipid-lowering.Methods:（1）To establish a cellular model of NAFLD by stimulating hepatocytes with palmitate（PA）,and using CCK8 method to screen the effective modeling concentration and dosing concentration;（2）BODIPY 493/503 and Oil RedОstaining were used to detect neutral lipid content in hepatocytes;（3）NAFLD model was established by feeding mice with high fat diet（HFD）,and liver function indexes were detected by an automatic biochemical analyzer,H&E staining and Oil RedОstaining were used to visualize liver tissue morphology and lipid deposition;（4）The expression levels of autophagy-related proteins were detected by immunofluorescence and western blot;（5）AML12 cells were transfected with m Cherry-GFP-LC3B adenovirus,and the numbers of autophagosomes and lysosomes were counted under a laser confocal microscope;（6）Autophagic flux was evaluated by monitoring turnover of the autophagic marker LC3B-II by western blot analysis in the presence and absence of chloroquine;（7）The protein expression of TFEB and the downstream target genes were tested by western blot analysis;（8）To further verify the role of phillygenin in the treatment of NAFLD,we applied RNA interference techniques to knock down TFEB;（9）Fluo-4/AM was used to detect Ca²⁺changes in cells;（10）The expression levels of inflammation-related factors were detected using western blotting and q RT-PCR.Results:1.The result of CCK-8 measurement showed that 0.4 m M PA had no significant effect on the viability of hepatocytes,and could induce lipid deposition.This concentration was the best condition for the establishment of an in vitro NAFLD model in hepatocyte.Phillygenin（20μM）had no significant effect on the viability of hepatocytes.2.In hepatocytes,phillygenin treatment reduced lipid deposition as determined by Oil red O staining and BODIPY 493/503 staining（P<0.05 or P<0.01）,with concomitant decreases the levels of TG and TC（P<0.05 or P<0.01）.3.The expression level of LC3B-II and p62 were increased after PA treatment（P<0.05or P<0.01）,which represented decreased autophagic flux.After treatment with phillygenin,the expression level of p62 in hepatocytes was decreased（P<0.05）,while the protein accumulation of LC3B-II increased（P<0.05）.4.PA inhibited lysosome biogenesis in hepatocytes,causing decreased lysosome numbers and insufficient autophagic flux.Phillygenin increased the number of lysosomes,facilitated formation of autolysosomes,and improved the impaired autophagic flux.5.PA inhibited the nuclear translocation of TFEB in hepatocytes,thereby disrupting lysosome biogenesis.After treatment with phillygenin,dephosphorylated TFEB translocated to the nucleus,thereby enhancing lysosomal biogenesis.6.Phillygenin improved NAFLD by inducing Ca²⁺release from the endoplasmic reticulum,triggering calcineurin-dependent TFEB nuclear translocation to enhance autophagy.7.PA activated the NLRP3 inflammasome and induced the release of inflammatory factors.Phillygenin inhibited the inflammatory response by negatively regulating autophagy.8.The HFD-fed mice displayed significantly increased body and liver weight as compared to the ND group（P<0.05 or P<0.01）,accompanied by hepatic steatosis.The HFD-fed mice also exhibited obvious liver function damage,dyslipidemia and inflammation.After treatment with phillygenin,the body weight of the HFD mice was reduced（P<0.05）,the liver color was restored to ruddy,the lipid accumulation in the liver was reduced,the liver function was significantly improved,and the inflammation was reduced.The beneficial effects of phillygenin on NAFLD mice were blocked by TFEB knockout.Taken together,phillygenin can restore lipophagy and further inhibit steatosis and inflammation by regulating the Ca²⁺-calcineurin-TFEB axis.