Resveratrol Promotes Hepatic Lipid Hydrolysis By SIRT1-PLIN2 Mechanism

BackgroundNonalcoholic fatty liver disease(NAFLD)is a clinicopathologic syndrome characterized by diffuse fatty degeneration of hepatocytes with no history of excessive alcohol consumption.It includes simple steatosis,non-alcoholic steatohepatitis,fibrosis,cirrhosis and hepatocellular carcinoma.The increasing prevalence of NAFLD seriously endangered people’s health and aggravated our country’s medical burden.One of the important mechanisms fot the occurrence and progression of NAFLD is the abnormal accumulationg of triglycerides(TG)riched lipid droplets and consequently changes in liver incluing the accumulation of fat in hepatocytes,liver inflammation and metabolic disorders.Therefore,inhibition of lipid droplet accumulation,including reduction of lipid droplet formation and promotion of lipolysis or lipophagy,is one of the important intervention targets for the prevention and treatment of NAFLD.Lipolysis and lipophagy are two main pathways of hepatic lipid hydrolysis.During lipolysis,adipose triglyceride lipase(ATGL)are activated by the binding with its coactivators,the comparative gene identifiers 58(CGI58),then hormone sensitive lipase(HSL)acts on diacylglycerol and releases two FFAs and one glycerol molecule.Lipohagy is a process in which lipid droplets are wrapped in double membranes and transported to lysosomes to form autolysosomes and degrade excess lipid droplets deposited in cells.Ras superfamily small Gprotein or small GTPase7(Rab7)/microtubule associated protein 1 light chain 3 β(LC3β)are involved in the process of lipophagy.There are five kinds of perilipins(PLINs)covering the surface of lipid droplets.PLIN1 is mainly expressed in mammalian adipose tissue,while PLIN2 is mainly expressed in liver tissue.Different peridodroplet proteins can recognize lipid droplets of different sizes or mature states to physically block the approach of lipolysis related enzymes to TG and cholesterol-rich ester cores,thus regulating lipolysis and lipophagy.Phytochemicals such as polyphenols in plant foods can improve the occurrence and development of NAFLD to some extent.Resveratrol(RSV)is one of the typical polyphenolic compounds,which has anti-inflammatory,antioxidant,anti-platelet generation,regulation of lipid metabolism and many other effects.Studies have shown that RSV intervention can reduce lipid accumulation in the liver of animals induced by high fat diet,and the possible mechanism is RSV regulates silent information 1(silent information 1,SIRT1)/activating transcription factor-6(ATF6).RSV can reduce lipid accumulation in steatosis Hep G2 cells by activating SIRT1 and then increasing PLIN2.However,the mechanism of RSV regulating lipolysis and lipophagy through PLIN2 remains unclear.Based on the above previous research results of our laboratory and the pathway of hepatic lipid hydrolysis(lipolysis and liphagy),we proposed the following research hypothesis: RSV may promote the expression and interaction of lipolysis related molecules ATGL/CGI58 and lipophagy related molecules Rab7/LC3β through promoting SIRT1-PLIN2 pathway and improve hepatic lipid hydrolysis to reduce lipid accumulation in liver.In this study,we constructed NAFLD mouse model and the steatosis model of human hepatocyte line5(HHL-5)to clarify the improvement effect of RSV and explore the mechanism of PLIN2 in NAFLD.This study will provide new evidence of scientific experiment and new intervention targets for the treatment of NAFLD by RSV.ObjectiveTo clarify the effect of RSV in promoting hydrolysis in liver and hepatocytes,and to clarify the mechanism of RSV in promoting hepatic lipid hydrolysis by up-regulating the expression of lipolysis and lipophagy related molecules through SIRT1-PLIN2 pathway.Methods1.C57BL/6 male mice aged 6-8 weeks were randomly divided into the control group(CON group,n=8),high-fat group(HFD group,n=8),high-fat with low-dose resveratrol group(FLR group,n=8)and high-fat with high-dose resveratrol group(FHR group,n=8).The control group was fed with ordinary diet for 6 weeks,and the other three groups were fed with high-fat diet for 6weeks to establish NAFLD mouse model.In addition to feeding the same diet,each group was then given gavage for 6 weeks to observe the therapeutic effect of RSV on NAFLD.The intervention doses of RSV in FLR and FHR groups were 100mg/(kg·bw)and 200mg/(kg·bw)per day respectively.The body weight and liver weight of mice in each group were recorded.Blood lipids and liver function indicators were detected by the fully automatic biochemical analyser,and the blood glucose tolerance of 2h after meal was tested by blood glucose test paper.The degree of lipid accumulation in liver tissue was evaluated by oil red O staining and HE staining.Western blot and tissue immunofluorescence staining were used respectively to detect the protein expression levels of hepatic lipid hydrolysis related molecules SIRT1,PKA,phosphorylation protein kinase A(p-PKA),PLIN2,ATGL,CGI58,hormone sensitive lipase(HSL),Rab7,LC3β and co-location situation of lipolysis and lipolysis pathway related genes(ATGL/CGI58,Rab7/LC3β)in mouse liver.2.Palmitic acid(PA)was used to induce HHL-5 cells to establish the model of cell steatosis.HHL-5 cells with steatosis were treated with different concentrations of RSV(5μM,10μM,20μM,40μM)for 24 hours.CCK-8 was used to detect cell viability,and bodipy staining and TG kit was used to detect intracellular lipid accumulation.rt-q PCR and Western blot were used to detect the effect of RSV on the m RNA and protein expression levels of HHL-5hepatocyte lipidolytic genes(SIRT1,PKA,p-PKA,PLIN2,ATGL,CGI58,HSL,Rab7,LC3β)The co-localization of genes related to lipolysis and lipophagy pathway(ATGL/CGI58,Rab7/LC3β)were observed by immunofluorescence staining.In order to elucidate that RSV promotes hepatic lipid decomposition by regulating ATGL/CGI58 and Rab7/LC3β through PLIN2.PLIN2 si RNA was further used to transfect into HHL-5 cells to knockdown PLIN2,which is for detecting the effect changes of RSV regulation on the above indicators.ResultsAnimal experimental results1.RSV intervention can improve the lipid accumulation in the liver of NAFLD mice.Compared with the HFD group,the relative area of lipid droplets in the livers of mice treated with RSV was significantly decreased,and the liver HE staining showed the recovery of pathological damage to some extent and the reduction of NAS score.2.RSV intervention could inhibit the decrease of SIRT1,p-PKA and PLIN2 molecular protein expression levels in the liver tissues of NAFLD mice,but did not affect the PKA protein expression level.3.RSV can promote the expression of ATGL,CGI58 and HSL proteins which are related to the lipolysis in NAFLD mice,and also promote the interaction of ATGL/CGI58.The molecular protein expression levels of ATGL,CGI58 and HSL in liver tissue of mice fed with high fat diet were decreased compared with those of mice fed with ordinary diet,and the RSV intervention of high dose could inhibit the decrease of the protein expression levels induced by high fat diet.The degree of co-localization of ATGL/CGI58 decreased in the HFD group,but increased after RSV treatment.4.RSV can promote the expression and interaction of liphagy related molecules Rab7 and LC3β in NAFLD mice.The molecular protein expression levels of Rab7 and LC3βⅡ in HFD group were decreased compared with CON group,while the molecular protein expression levels of Rab7 and LC3β in FHR group were increased compared with HFD group.The co-localization degree of Rab7/LC3β was decreased in the high fat group and increased after RSV treatment.Cell experimental results1.Compared with the control group,the content of TG in 0.2m M,0.3m M,0.4m M and0.5m M PA groups was significantly increased.When PA concentration was 0.3m M,the cell viability was lower than 50%,and the drug concentration was higher than the median lethal dose.Thus,HHL-5 cells were treated with 0.2m M PA for 24 h to establish the cell steatosis model.2.RSV intervention can inhibit PA-induced hepatocyte steatosis.RSV concentrations of40μM and below had little effect on cell viability,but RSV of high concentrations(over 80μM)reduced cell viability.Treatments of 20 and 40μM RSV decreased the content of TG and the average fluorescence intensity of lipid droplets compared with the PA group.3.RSV can increase the expression levels of SIRT1,p-PKA and PLIN2.Compared with PA group,the protein expression levels of SIRT1,p-PKA and PLIN2 increased after 40μM RSV treatment,but the PKA protein expression levels did not change.4.RSV intervention can promote lipolysis and lipophagy of HHL-5 cells with steatosis through ATGL/CGI58,Rab7/LC3β and their interactions,respectively.Compared with PA group,the protein expression levels of ATGL,CGI58 and HSL were increased in PA+RSV40 group.RSV intervention improved the average fluorescence intensity of ATGL and CGI58,and the colocalization degree of ATGL/CGI58 was higher than that in the PA group.The levels of m RMA,protein expression and average fluorescence intensity of Rab7 and LC3β,the key molecules of liphage,were decreased in the PA group,and RSV had a recovery effect on the above reductions.The co-localization degree of Rab7/LC3β after RSV intervention was higher than that in the PA group.5.After PLIN2 knockdown,the inhibitory effect of RSV on HHL-5 cell steatosis was blocked.After transfection of PLIN2 si RNA into HHL-5 cells,TG content in si PLIN2+PA group was higher than that in the si PLIN2 group,but there was no decrease in TG content after RSV intervention.The results of Bodipy staining were as the same as those of TG content,and the inhibitory effect of RSV on hepatocyte steatosis induced by PA disappeared.6.PLIN2 knockdown did not affect the regulation of PA and RSV on SIRT1,PKA and pPKA protein expression.PLIN2 knockdown could inhibit the effect of RSV on promoting lipolysis and liphagy of HHL-5 cells.The regulation of PA and RSV on SIRT1,PKA and p-PKA protein expression was not affected by PLIN2 si RNA treatment.There was no difference in protein expression of ATGL,CGI58 and HSL between si PLIN2 group,si PLIN2+PA group and si PLIN2+PA+RSV40 group,and there was no difference in average fluorescence intensity of ATGL and CGI58.There was no significant difference between si PLIN2+PA+RSV40 group and si PLIN2+PA group in the degree of ATGL/CGI58 colocalization.Compared with si PLIN2+PA+RSV40 group,the protein expression level of Rab7 and LC3β did not change,the average fluorescence intensity of Rab7 and LC3β did not change,and the co-localization degree of Rab7/LC3β was not significantly different.Conclusion1.RSV can improve NAFLD by reducing lipid accumulation in liver tissue of NAFLD mice and HHL-5 hepatocytes of human with steatosis.2.RSV can promote the expression and interaction of ATGL/CGI58 and Rab7/LC3β in liver tissues of NAFLD mice through up-regulation of SIRT1-PLIN2.3.RSV can promote hepatic lipid decomposition by up-regulating PLIN2 to increase the expression and interaction of ATGL/CGI58 and Rab7/LC3β related molecules in lipolysis and liphagy pathway.PLIN2 knockdown can largely eliminate the effect of RSV on lipolysis and lipophagy and consequently the accumulation of lipid in HHL-5 cells with steatosis.