Effect Of Dapagliflozin On Autophagy In Human Umbilical Vein Endothelial Cells Induced By High Glucose And Its Mechanism

Objective:To investigate the effect of Dapagliflozin（DAPA）on high glucose（HG）-induced autophagy in human umbilical vein endothelial cells（HUVEC）and the mechanism.Materials and testing methods:after human umbilical vein endothelial cells were resuscitated,cultured and passaged,the following investigations were carried out with the human umbilical vein endothelial cells in logarithmic growth period:1.To investigate the effect of DAPA on the viability of human umbilical vein endothelial cells with different concentrations and treating time.There were applied two different treating time:24h and 48h.For each treating time,the following groups were carried out:normal glucose group（5.5mM）,high glucose group（30mM）and high glucose with different concentrations of DAPA（10μM,20μM,30μM,50μM,80μM and 120μM）.Method MTT technique was used to observe the optimal concentration（OC）and action time of DAPA.2.To investigate the effects of DAPA on HG-induced autophagy in human umbilical vein endothelial cells and the molecular mechanisms.This part of experiment was divided into five groups:group of normal glucose（group A）,group of high glucose（group B）,group of high glucose with DAPA_OC（group C）,group of high glucose with Rapamycin（group D）,group of high glucose with DAPA_OCC and Rapamycin（group E）.The relative protein expression levels of the autophagy signaling factors,LC3-II/LC3-I,P62,AMPK,and mTOR were measured by Western Blot;the relative mRNA expression of Beclin-1,AMPK and mTOR were examined by RT-qPCR.Results:1.The effects on cellular viability of HG-induced human umbilical vein endothelial cells with various concentrations of DAPA and with different treating time:（1）After 24h,compared with the control group,the viability of HG-induced human umbilical vein endothelial cells had been conspicuously suppressed（P<0.05）.It was obviously improved that the cell viability of the groups treating with DAPA 50μM and DAPA 80μM（P<0.05）;（2）After 48h,the viability of the HG-induced human umbilical vein endothelial cells was largely reduced compared with the control group（P<0.05）.There was a great improvement of cellular viability in all groups with DAPA except the group of DAPA 10μM,and the most significant improvement was found in the group of DAPA 50μM（P<0.05）.2.To explore the effect and possible mechanism of DAPA on HG-induced autophagy in human umbilical vein endothelial cells:（1）The results of Western Blot:group B had a higher expression of LC3-II/LC3-I with lower expressions of P62,mTOR and AMPK than those in group A（P<0.05）;the expression of LC3-II/LC3-I decreased while the expressions of P62,mTOR and AMPK significantly increased in group C compared with group B（P<0.05）;the expression of LC3-II/LC3-I was enhanced while the expressions of P62,mTOR and AMPK were reduced in group E compared with group C（P<0.05）;（2）The results of RT-qPCR:compared with group A,the relative mRNA expression of Beclin-1 increased while the relative mRNA expressions of AMPK and mTOR decreased were found in group B（P<0.05）;the relative mRNA expression of Beclin-1 in group C treated with DAPA was significantly lower than that in group B,and the relative mRNA expressions of AMPK and mTOR were significantly higher than those in group B（P<0.05）;when added autophagy inducer Rapamycin,the relative mRNA expression of Beclin-1 declined and the relative mRNA expressions of AMPK and mTOR enhanced in group E compared with group C（P<0.05）;Conclusions:1.DAPA can protect HG-induced human umbilical vein endothelial cells by inhibiting autophagy;2.DAPA can up-regulate the expression of mTOR and down-regulate the expression of Beclin-1 to inhibit the over-activated autophagy in endothelial cells.