| BackgroundNonalcoholic fatty liver disease(NAFLD)is a clinicopathological syndrome characterized by excessive lipid deposition in hepatocytes except for alcohol and other definite factors.Currently,there is no specific drug to treat NAFLD.Dapagliflozin,a new class of hypoglycemic agents,achieves hypoglycemic effects by inhibiting SGLT-2 and reducing renal glucose reabsorption.Dapagliflozin has been reported to improve hepatic steatosis in patients with T2DM and NAFLD,but the exact mechanism is still unclear.Therefore,this study is to explore whether Dapagliflozin improves hepatic steatosis and the possible molecular mechanism,so as to provide experimental basis for the prevention and treatment of NAFLD.ObjectiveSimple hepatic steatosis model was established by high fat diet and PA induction respectively in vivo and in vitro to study the effect of dapagliflozin on lipid deposition in steatotic hepatocytes and its possible mechanism.Methods1.Eight-week-old ZDF rats were selected and induced by high fat for 4 weeks to establish an obese diabetic rat model.The experiment was divided into three groups:normal group,model group and dapagliflozin treatment group.All rats were fed with high fat diet for 9 weeks and treated accordingly.Randomized blood glucose and weight changes were monitored weekly.After the rats were killed,blood lipid levels were detected.Lipid accumulation was observed by HE and oil red O staining.The expressions of LC3,Beclinl and P62 and the expressions of ACC 1 and ACOX1 were determined by Western blot and immunohistochemistry.2.In vitro,human normal liver cell lines(LO2)and human hepatocellular carcinoma cell lines(HepG2)were used to establish the hepatic steatosis model induced by palmitic acid.Cell viability was detected to determine the optimal intervention concentration of dapagliflozin.The groups are as follows:normal group,model group,dapagliflozin treatment group,dapagliflozin +chloroquine group,dapagliflozin+Compound C group.Oil red O and BODIPY 493/503 staining were used to assess the degree of lipid accumulation in each group.The expressions of LC3,Beclin1,p62,ACC1 and ACOX1 were evaluated by immunofluorescence and Western blot.The expression of SGLT-2 in liver tissue,LO2 and HepG2 cells was detected by immunohistochemistry,Western blot and immunofluorescence.Finally,the AMPK-mTOR pathway was detected to probe the possible mechanism of autophagy.Results1.After 9-week intervention,HE and oil red O staining results showed that dapagliflozin improved liver lipid accumulation;Western blot results showed that dapagliflozin increased ACC1 phosphorylation and ACOX1 expression.Compared with the model group,dapagliflozin increased the expression of LC3B and Beclinl,while decreased the expression of P62.2.CCK-8 was used to detect the activity of LO2 and HepG2 cells,and the intervention concentration of dapagliflozin was determined to be 20 μM.Oil red O and BODIPY 493/503 staining results showed that dapagliflozin improved lipid accumulation in hepatocytes.Western blot results showed that dapagliflozin increased ACC1 phosphorylation and ACOX1 expression compared with model group.Western blot and immunofluorescence results revealed that in comparison with model group,dapagliflozin increased the expression of LC3B and Beclinl in liver cells,while decreased the expression of p62.The results of oil red O showed that CQ could inhibit the effect of dapagliflozin on reducing lipid deposition.3.Western blot,immunohistochemistry and immunofluorescence results showed that SGLT-2 was expressed in LO2 and HepG2 cells.The results in Western blot showed that dapagliflozin increased AMPK expression and decreased mTOR phosphorylation.The results of oil red O showed that Compound C could inhibit the effect of dapagliflozin.Conclusion1.Dapagliflozin can reduce lipid deposition in steatotic hepatocytes;2.Dapagliflozin may improve lipid deposition in steatotic hepatocytes by enhancing autophagy;3.AMPK-mTOR signaling pathway may be involved in the autophagy of dapagliflozin in hepatic cells. |
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