Effect Of Sitagliptin On Autophagy And PI3K/Akt/mTOR Signaling Pathway In Human Umbilical Vein Endothelial Cells Induced By High Glucose Through Up-regulating HMGB1 Expression

Objective: 1.To establish an in vitro human umbilical vein endothelial cell(HUVEC)hyperglycemic injury model.The expression of HMGB1(High mobility group box 1),PI3K(Phosphatide 3-kinases),Akt(Protein kinase B),mTOR(Mammalian target of rapamycin),P62(Sequestosome 1),LC3-II(Microtubuleassoeiated protein 1 light hain 3-II)and other proteins were observed after intervention with different concentrations of sitagliptin.2.To investigate the relationship between HMGB1 and autophagy and PI3K/Akt/mTOR signaling pathway in human umbilical vein endothelial cells.Methods: HUVEC in logarithmic growth period was selected as experimental object1.Effects of hyperglycemia on HUVEC injury: cultured cells were randomly divided into normal control group(5.5mmol/L),mannitol group(25mmol/L),and hyperglycemia group(30mmol/L).Observation indexes: 1.Cytological morphology;2.Secretion of NO;3.Western Blot was used to detect the expressions of HMGB1,mTOR,LC3-II and P62 proteins in cells of each group.2.Effects of different concentrations of sitagliptin on HMGB1 expression and autophagy of HUVEC induced by high glucose: experimental group of high sugar group and high sugar + sitagliptin low concentration group(0.1?mol/L),high sugar + concentration in sitagliptin group(1?mol/L),high sugar + sitagliptin high concentration group(10?mol/L),Observation indexes: 1.Cytological morphology;2.Secretion of NO;3.Western Blot was used to detect the expressions of HMGB1,PI3 K,Akt,mTOR,LC3-II and P62 proteins in cells of each group.3.The effect of HMGB1 expression on autophagy and PI3K/Akt/mTOR signaling pathway: the experimental group was divided into the hyperglycemia group,the hyperglycemia + sitagliptin optimal concentration group(10?mol/L),the hyperglycemia + sitagliptin optimal concentration group +HMGB1 inhibitor group Glycyrrhizic-acid(100?mol/L),and the hyperglycemia + sitagliptin optimal concentration group +PI3K inhibitor group LY294002(20?mol/L).Observation indexes: 1.Cytological morphology;2.Secretion of NO;3.Western Blot was used to detect the expressions of HMGB1,PI3 K,Akt,mTOR,LC3-II and P62 proteins in cells of each group.Results: 1.The cells in the normal control group were uniform in size and clear in boundary,and the cells were closely connected with each other like paving stones.In the high-glucose group,the cells were shriveled,the cell spaces were widened,and the boundaries were unclear.Compared with the normal control group,the secretion of NO in the high glucose group was significantly reduced,the difference was statistically significant(P<0.05);the expression of HMGB1 and LC3-II protein was decreased,the difference was statistically significant(P<0.05);while the expression of mTOR and P62 protein was increased,and the difference was statistically significant(P<0.05).However,the cell morphology of the mannitol group was similar to that of the normal control group,and there were no statistically significant differences in HMGB1,mTOR,LC3-II and P62 protein expressions(P >0.05).2.Compared with the high-sugar group,the morphology of HUVEC was improved in the high sugar + low/medium/high concentration sitagliptin group.With the increase of the concentration of sitagliptin,the secretion of NO increased,and the difference was statistically significant(P<0.05);HMGB1 and LC3-II protein increased,and the difference was statistically significant(P<0.05);while the expression of PI3 K,Akt,mTOR and P62 protein decreased,and the difference was statistically significant(P<0.05).The effect was most obvious in the high-sugar + sitagliptin group(10?mol/L).3.Compared with the high-sugar + sitagliptin optimal concentration(10?mol/L)+ HMGB1 inhibitor Glycyrrhizic-acid group(100?mol/L)and the high-sugar + sitagliptin optimal concentration group(10?mol/L),HUVEC showed more obvious shrinkage,wider intercellular space and unclear boundaries.NO secretion decreased,and the difference was statistically significant(P<0.05);The protein expressions of HMGB1 and LC3-II were decreased,and the difference was statistically significant(P<0.05);PI3K,Akt,mTOR,P62 protein expressions were increased,and the difference was statistically significant(P<0.05).However,High sugar + sitagliptin optimal concentration(10?mol/L)+ PI3 K inhibitors LY294002 group(20?mol/L)with high sugar + sitagliptin optimal concentration group(10?mol/L),the morphology of HUVEC was improved,and HMGB1 expression was not significantly changed(P>0.05).NO amount of secretion increased,and the difference was statistically significant(P<0.05);LC3-II protein expression increased,and the difference was statistically significant(P<0.05);PI3K,Akt,mTOR and P62 protein expressions were decreased,and the difference was statistically significant(P<0.05).Conclusion:1.High glucose can induce the down-regulation of HMGB1 expression in human umbilical vein endothelial cells and inhibit autophagy.2.Sitagliptin may inhibit the PI3K/Akt/mTOR signaling pathway and promote autophagy by up-regulating the expression of HMGB1,thus playing a role in vascular protection.