Mechanism Of Resveratrol Regulating PI3K/AKT And SIRT2 Signaling Pathway To Alleviate Non-alcoholic Fatty Liver Disease

Background:Non-alcoholic fatty liver disease(NAFLD)is a kind of clinical syndrome with diffuse hepatic bullous steatosis as the main pathological feature caused by non-alcoholic and other definite liver damage factors.NAFLD has a broad spectrum of diseases,including simple fatty liver,nonalcoholic steatohepatitis,hepatic fibrosis,and hepatocirrhosis,which may eventually develop into hepatocellular carcinoma.NAFLD has always been one of the common causes of chronic liver disease in western developed countries,and it is expected that it may become a common indication for liver transplantation in the future.Resveratrol,as a natural polyphenol,has biological activities such as anti-oxidative stress.The PI3K/AKT signaling pathway is involved in lipid metabolism and has anti-inflammatory and anti-oxidative stress functions.Sirtuin2(SIRT2)is a NAD~+-dependent deacetylase that participates in various biological functions such as aging and energy metabolism through protein deacetylation.Objective:The purpose of this experiment is to investigate whether resveratrol can alleviate NAFLD,and to explore its mechanism of regulating PI3K/AKT and SIRT2signaling pathways,so as to provide an experimental basis for the development of resveratrol-based hepatic protective drugs.Methods:1.8-week-old male C57BL/6J mice(18-22g)were randomly divided into control(ctr)group,high-fat diet(HFD)group and high-fat diet+resveratrol(HFD+Res)group(n=10).The control group was only fed with conventional feed.The other two groups were fed with 60%high-calorie diet to induce NAFLD.After feeding for 6 weeks,the HFD+Res group continued to be fed with resveratrol(150mg/kg/d)for 4 weeks,while the HFD group was given the same volume of normal saline as control.The body weight of mice in each group was recorded.Enzyme-linked immunosorbent assay(ELISA)was used to measure the changes of serum ALT,AST,TG and TC in each group.Quantitative Real-time PCR(q PCR)was used to detect the levels of inflammatory cytokines(IL-1α,IL-1β,IL-6,TNF-α)and chemokines(CCL3,CCL4,CCL5,CCL11).The hepatic tissues were stained with HE and Oil Red O for pathological examination,and NAFLD activity scores(NAS)were performed.Transcriptome sequencing was used to determine the differential genes between HFD group and HFD+Res group,and further GO enrichment and KEGG pathway enrichment were carried out.The expressions of JNK,PI3K,AKT and other proteins in each group were detected by Western blotting.2.Primary mouse hepatocytes were cultured and divided into four groups:oleic acid(0.25mmol/L)group;resveratrol 1μmol/L+oleic acid group;resveratrol 4μmol/L+oleic acid group;resveratrol 8μmol/L+oleic acid group.The viability of hepatocytes in each group was detected by CCK8.The expressions of BCL2,BAX,PI3K,AKT and other proteins in each group were detected by Western blotting.The oxidative stress products in each group were detected by ROS staining.3.In order to observe the effect of transfection of PI3K siRNA on primary hepatocytes,primary hepatocytes were divided into four groups:oleic acid(0.25mmol/L)group;resveratrol(8μmol/L)+oleic acid group;PI3K siRNA+resveratrol+oleic acid group;PI3K siRNA+oleic acid group.The viability of hepatocytes in each group was detected by CCK8.The expressions of BCL2,BAX,PI3K,AKT and mitochondrial proteins SDH and COX were detected by Western blotting.The oxidative stress products in each group were detected by ROS staining.The levels of antioxidant enzymes SOD,CAT and GSH were measured by ELISA.4.The expression of SIRT2 protein in each group was detected by Western blotting.And then,sirtinol,an inhibitor of SIRT2,was further used to inhibit the SIRT2 signaling pathway in mice.120 mice(40 mice for 3 cycles)were randomly divided into 4 groups:each group was fed HFD diet for 10 weeks.From the 6th week,the first group:intraperitoneal injection of PBS(equal to resveratrol);the second group:intraperitoneal injection of resveratrol(150mg/kg/d);the third group:intraperitoneal injection of resveratrol+sirtinol(1mg/kg/d);fourth group:intraperitoneal injection of sirtinol.The expressions of SIRT2,FOXO1,BCL2,BAX,SDH,COX and other proteins in each group were detected by Western blotting.The levels of SOD,CAT,GSH in each group were measured by ELISA.5.In order to observe the effect of SIRT2 siRNA transfection on primary hepatocytes,primary hepatocytes were divided into four groups:oleic acid(0.25mmol/L)group;resveratrol(8μmol/L)+oleic acid group;SIRT2 siRNA+resveratrol+oleic acid group;SIRT2 siRNA+oleic acid group.The viability of hepatocytes in each group was used to detecte by CCK8.The protein expressions of BCL2,BAX,PI3K,AKT,SDH and COX were detected by Western blotting.The levels of antioxidant enzymes SOD,CAT and GSH were measured by ELISA.6.PI3K siRNA and SIRT2 siRNA were transfected respectively to observe the changes of PI3K/AKT and SIRT2 signaling pathway related proteins,and to clarify the relationship between upstream and downstream.Results:1.The weight of mice in HFD+Res group was lower than that in HFD group(P<0.05).The levels of ALT,AST,TG and TC in HFD+Res group were lower than those in HFD group(P<0.05).The results of inflammatory cytokines IL-1α,IL-1β,IL-6 and TNF-αshowed that HFD+Res group was lower than HFD group(P<0.05).Resveratrol could also decrease the expression of chemokines CCL-4 and CCL-11(P<0.05).However,there was no statistical difference in the expression of chemokines CCL-3 and CCL-5.HE staining and Oil Red O staining of pathological sections showed that resveratrol could reduce the lipid accumulation of mouse hepatic tissue induced by HFD,and the difference of NAS score was significant(P<0.05).Through differential gene sequencing,GO enrichment,and KEGG pathway enrichment,and finally focused on PI3K/AKT signaling pathway.Western blotting showed that resveratrol inhibited the expression of p-JNK,but increased the expression of p-PI3K and p-AKT(P<0.05).2.The increase of resveratrol concentration increased the viability of primary hepatocytes(P<0.05).Western blotting showed that resveratrol increased the protein expressions of p-PI3K,p-AKT and BCL2,but decreased the expression of BAX(P<0.05).Resveratrol reduced the release of oxidative stress products.3.The viability of hepatocytes decreased after PI3K siRNA transfection(P<0.05).Western blotting showed that the protein expressions of p-PI3K,p-AKT and BCL2 decreased,while the expression of BAX increased,and the expression of mitochondrial proteins SDH and COX increased(P<0.05).PI3K siRNA increased the release of oxidative stress products and reduced the levels of SOD,CAT,GSH and other antioxidant enzymes(P<0.05).4.Western blotting showed that resveratrol increased the expression of SIRT2 protein(P<0.05).However,sirtinol decreased the protein expression of SIRT2,FOXO1,and BCL2,and increased the protein expression of BAX,SDH,and COX(P<0.05).Sirtinol also reduced the levels of antioxidant enzymes such as SOD,CAT,GSH(P<0.05).5.The viability of hepatocytes decreased by SIRT2 siRNA(P<0.05).Western blotting showed that SIRT2 siRNA decreased the protein expressions of SIRT2,FOXO1 and BCL2,but increased the protein expressions of BAX,SDH and COX(P<0.05).SIRT2 siRNA decreased the levels of SOD,CAT,GSH(P<0.05).6.The protein expressions of p-PI3K,p-AKT,SIRT2,and FOXO1 decreased after transfection of PI3K siRNA(P<0.05).However,after SIRT2 siRNA transfection,the protein expressions of SIRT2 and FOXO1 also decreased(P<0.05),but there was no statistical difference in the expression of p-PI3K and p-AKT.Conclusion:Resveratrol can alleviate NAFLD.Resveratrol can activate the PI3K/AKT signaling pathway,thereby regulate SIRT2 signaling pathway,effectively inhibit hepatocyte apoptosis,reduce oxidative stress injury,protect hepatocytes,and alleviate NAFLD.