Effect Of Sitagliptin On Autophagy And AMPK/mTOR Signaling Pathway In Human Umbilical Vein Endothelial Cells Induced By High Glucose

Objectives:1.To observe the effect of sitagliptin on autophagy,a high glucose-induced injury model of human umbilical vein endothelial cells（HUVEC）was established and treated with different concentrations of sitagliptin;2.To investigate the relationship between the regulation of autophagy by sitagliptin and AMPK/mTOR signaling pathway.Materials and methods:The resuscitated HUVEC was inoculated into a cell culture flask with complete medium,and cultured in a37℃,5%CO₂ incubator.The HUVEC in logarithmic growth period was selected as the experimental research object.1.Effects of different doses of sitagliptin on autophagy induced by high glucose in HUVEC:The experiment was divided into the following6 groups:group A:normal control group（5.5 mmol/L）,group B:mannitol group（25 mmol/L）,group C:high glucose group（30 mmol/L）,groupD:high glucose+sitagliptin 0.1μmol/L group,group E:high glucose+sitagliptin 1μmol/L group,group F:high sugar+Sitagliptin 10μmol/L group.Observed indicators:（1）cell morphology changes;（2）determination of NO content in cell culture supernatant by nitrate reductase method;（3）Western Blot detection of each group of AMPK,p-AMPK,p70S6K,p-p70S6K,LC3-II,p62 protein expression;2.To investigate the role of AMPK/mTOR signaling pathway in the induction of HUVEC autophagy by sitagliptin:the experiment was divided into the follow ing three groups:group A:high glucose group（30mmol/L）,group B:high glucose+sitagliptin 10μmol/L group,group C:high glucose+sitagliptin 10μmol/L+AMPK inhibitor Compound C group.Observed indicators:（1）Determination of NO content in cell culture supernatant by nitrate reductase method;（2）Western Blot detection of AMPK,p-AMPK,p70S6K,p-p70S6K,LC3-II,p62 protein expression.Results:1.Effects of different doses of sitagliptin on autophagy induced by high glucose in HUVEC:（1）Morphological observation of cells in each group:group A was characterized by dense cells,small intercellular space,clear contour,consistent size,full cytoplasm,spindle-shaped,and good growth state.Compared with group A,the cells in group C were shriveled and sparse,with obviously wider intercellular spaces and irregular shapes;Compared with the group C,cell morphology of groups D,E,and F was improved to different degrees and gradually returned to normal with the increase of sitagliptin concentration,and the improvement in group F was the most significant;Compared with the group A,group B show no significant difference in cell morphology;（2）Change of NO content in each group:compared with group A,the NO content of group C decreased significantly,and the difference was statistically significant（P<0.05）;Compared with group C,NO content in group D,E and F increased gradually with the increase of sitagliptin concentration,and the change in group F was the most significant,and the difference was statistically significant（P<0.05）;There was no significant difference in NO content between group B and group A;（3）Western blot results of each group showed that compared with group A,the expression of LC3-II and p-AMPK in group C decreased,while the expression of p62 and p-p70S6K increased,and the differences were statistically significant（P<0.05）;compared with group C,With the increase of sitagliptin concentration,the expression of LC3-II and p-AMPK increased gradually,and the expression of p62 and p-p70S6K decreased gradually,and the changes was most significant in group F,and the differences were statistically significant（P<0.05）;there was no significant differences in the expression levels of LC3-II,p-AMPK,p62and p-p70S6K between group B and group A.2.To investigate the role of AMPK/mTOR signaling pathway in the induction of HUVEC autophagy by sitagliptin:（1）Changes of NO content in each group:compared with group A,the content of NO in group B increased,and the difference was statistically significant（P<0.05）;Compared with group B,the content of NO in group C decreased,the difference was statistically significant（P<0.05）;（2）Western blot results of each group showed that compared with group A,the expression of p-AMPK and LC3-II in group B increased,while the expression of p-p70S6K and p62 decreased,and the differences were statistically significant（P<0.05）;Compared with group B,the expression of p-AMPK and LC3-II in C group decreased,while the expression of p-p70S6K and p62 increased,and the differences were statistically significant（P<0.05）.Conclusions:1.Sitagliptin can alleviate the HUVEC injury induced by high glucose through promoting autophagy;2.Sitagliptin may activate AMPK by phosphorylation and inhibit the activity of mTORC1 to promote autophagy.