Regulation And Mechanism Study On The Activity Of Autophagy Of Endothelial Cells In High Glucose Conditions By Spermidine

Purpose:(1)To investigate the possible mechanism of high glucose on autophagy activity of endothelial cells.(2)Aim to investigate the regulation and mechanism of spermidine on autophagy in endothelial cells under high glucose environment,and to explore its theoretical basis for the treatment of microangiopathy such as diabetic retinopathy.Method:(1)Human Umbilical Vein Endothelial Cells were cultured in vitro under suitable environment,including cell resuscitation,cell culture and cell passage.Cells in logarithmic growth phase were taken for experiment.The expression of BECN 1 / Beclin-1 and LC3-II m RNA in human umbilical vein endothelial cells was measured by Western Blot and PCR,to determine the most suitable glucose concentration and the best culture time point for the effect of high glucose on autophagy.(2)After incubating different concentrations of SPD(5?M,10?M,20?M,25?M,50?M,100?M,500?M,1000?M)with HUVECs for 24 hours,detect the absorbance value at 450 nm to observe the toxic effect of SPD on HUVECs,and PCR was chosen to detect the expression level of autophagy related genes in cells to determine the optimal concentration of SPD intervention.(3)HUVECs were stimulated with appropriate high glucose concentration to construct endothelial cell high glucose model.SPD intervened in human umbilical vein endothelial cells induced by high glucose.The m RNA and protein expression levels of autophagy related markers Beclin-1,LC3-II and SQSTM1/p62 were detected by Western blot and RT-PCR.The degree of autophagy of endothelial cells in simple high glucose group and high glucose combined with SPD group was compared.Autophagy flow inhibitor 3-methyladenine was used to observe the effects of high glucose environment and SPD intervention on autophagy flow.(4)The cells were treated with appropriate concentration of drugs,loaded with fluorescent probes according to the reagent instructions,and the level of reactive oxygen species(ROS)in the cells was detected by fluorescent enzyme labeling instrument.Results:(1)Compared with the control group,the expression of LC3-II and BECN1/Beclin-1 m RNA and protein in the high glucose group decreased,as well as the expression of SQSTM1/p62 m RNA and protein increased significantly(P < 0.05).The most significant difference could be found when the glucose concentration in the medium was 33 m M and the culture time was 24 h.(2)Cell viability of HUVECs was not affected when SPD concentration was lower than 500?M(P < 0.05).The most significant effect of inducing autophagy was observed when SPD concentration was 10?M.The expression level of BECN1/Beclin-1 and LC3-II m RNA increased,while the expression level of SQSTM1/p62 m RNA decreased(P < 0.05).(3)After 24 hours of high glucose induction,compared with the intervention without 3-MA,the expression level of BECN1/Beclin-1 and LC3-II m RNA decreased significantly,and the expression level of SQSTM1/p62 m RNA increased significantly(P < 0.05).After 24 hours co culture of high glucose and SPD,the expression level of BECN1/Beclin-1,LC3-II m RNA was significantly higher than those in the simple high glucose induction group,and the level of SQSTM1/p62 m RNA was significantly lower than that(P < 0.05).Meanwhile,compared with no 3-MA intervention,the expression level of BECN1/Beclin-1,LC3-II m RNA decreased significantly,and the level of SQSTM1/p62 m RNA increased significantly(P < 0.05).(4)Compared with the control group,the level of ROS in the simple high glucose intervention group increased significantly,and the level of ROS in the simple SPD intervention group decreased significantly(P < 0.05).After 24 hours of high glucose induction,the ROS level of SPD intervention group was significantly lower than that of simple high glucose intervention group,but still higher than that of the control group.After 24 hours of combined action of SPD and high glucose,the ROS level of 3-MA intervention group was significantly higher than that of nonintervention group,but still lower than that of high glucose combined with 3-m A intervention group(P < 0.05).Conclusions:(1)The autophagy level of human umbilical vein endothelial cells under high glucose environment was significantly inhibited.(2)Spermidine can promote the occurrence of autophagy in HUVECs,reduce the damage of high glucose environment to HUVECs and have a protective effect on cells.(3)High glucose inhibited the autophagic flow of HUVECs,while spermidine can promote the smooth autophagy flow of endothelial cells in high glucose state.