Experimental Study Of High Glucose Aggravates Injury Of Cerebral Microvascular Endothelial Cells Induced By Oxygen Glucose Deprivation / Reoxygenation And Its Relationship With Autophagy

Objective To investigate the role of autophagy in high glucose aggravated cerebral ischemia-reperfusion injury and its related mechanism.Methods In this study,brain microvascular endothelial cells were cultured in vitro and divided into normal media control group(Control group),normal medium oxygen glucose deprivation group(NG group),high glucose medium oxygen glucose deprivation group(HG group),high glucose medium oxygen glucose deprivation group plus 3-MA group(HG +3-MA group)and high glucose medium oxygen glucose deprivation group plus RAPA group(HG + RAPA group).In NG group,the cells were cultured in normal medium for 24 hours,then subjected to hypoxia and glucose deprivation for 6 hours and reoxygenation for24 hours.In HG group,the cells were cultured in high glucose medium for 24 hours,then subjected to hypoxia and glucose deprivation for 6 hours and reoxygenation for 24 hours.In HG + 3-MA group,the cells were cultured in high glucose medium with 2 mmol/L3-methyladenine for 24 hours,followed by hypoxia and glucose deprivation for 6 hours and reoxygenation for 24 hours.In HG + RAPA group,the cells were cultured in high glucose medium with 100 nmol / L rapamycin for 24 hours,then subjected to hypoxia and glucose deprivation for 6 hours and reoxygenation for 24 hours.Cell morphology was observed by invert microscope,cell survival rate was detected by CCK-8 assay,cell apoptosis was detected by flow cytometry,and cell damage was detected by LDH assay.The autophagosomes of cerebral microvascular endothelial cells were labeled by MDC kit.Immunofluorescence and Western blot were used to detect the expression of autophagy related factors LC3-Ⅱ/Ⅰ,Beclin1 and P62.Results CCK-8 results showed that with the increase of glucose concentration,the activity of cultured cerebral microvascular endothelial cells gradually decreased,and concentration of 50 mmol/L glucose was selected as the high sugar treatment concentration in this experiment.CCK-8 and Western blot were used to determine that the concent ration of3-methyladenine was 2 mmol/L and rapamycin was 100 nmol/L.Cell morphology s howed that the cells in the Control group were full in shape,well adherent,dense in arrangement and spindle shape.In normal glucose OGD/R group,the intercellular space widened and some cells shrunk.In the high glucose OGD/R group,the cells were spars e and some of them fell off and floated,and the adherent cells showed different degrees of shrinkage,rounding and smaller;after 3-methyladenine intervention,the adherent cells improved and the number of exfoliated cells decreased;on the contrary,after rapamycin intervention,a large number of cells exfoliated.CCK-8 results showed that the cell survival rate of NG group was lower than that of control group,and that of HG group was significantly lower than that of NG group.Compared with HG group,the survival rate of HG+3-MA group was decreased,the survival rate of HG+RAPA group was increased.LDH results showed that compared with the Control group,the cell injury rate of NG group was significantly higher,and that of HG group was apparently higher than that of NG group.Compared with HG group,the cell damage rate was decreased after treatment with autophagy inhibitor,the cell damage rate was increased after treatment with autophagy agonists.The results of flow cytometry showed that the apoptosis rate of NG group was higher than that of Control group,and that of HG group was significantly higher than that of NG group.Compared with HG group,the apoptosis rate of HG+3-MA group was decreased,and the apoptosis rate of HG+RAPA group was markedly increased.The results of MDC showed that the formation of autophagy in NG group was more than that in Control group,and the formation of autophagy in HG group was more tha n that in NG group.Compared with HG group,the formation of autophagy decreased after3-MA intervention,but increased after RAPA intervention.Western blot results showed that compared with the Control group,the positive autophagy related factors LC3-Ⅱ/I and Beclin1 protein expression increased,and the negative autophagy related factor P62 protein expression decreased in NG group.Compared with NG group,the expression of LC3-Ⅱ/Ⅰ and Beclin1 in HG group was significantly increased,while the expression of P62 in HG group was significantly decreased.Compared with HG group,the expression of LC3-Ⅱ/I and Beclin1 protein decreased and P62 protein increased in HG+3-MA group,while the expression of LC3-Ⅱ/I and Beclin1 protein increased and P62 protein decreased in HG+RAPA group.Immunofluorescence was used to label the autophagy related fact ors LC3,Beclin1 and P62.The results were consistent with Western blot’s result.Conclusion Oxygen glucose deprivation / reoxygenation can cause the brain microvascular endothelial cell injury,and high glucose can aggravate the brain microenvironment endothelial cell injury caused by oxygen glucose deprivation /reoxygenation.Oxygen glucose deprivation / reoxygenation can activate the autophagy of brain microvascular endothelial cells.High glucose aggravates the damage of brain microvascular endothelial cells induced by oxygen glucose deprivation / reoxygenation,which is related to the increase of autophagy.