The Carboxyl Terminus Of Heat Shock Protein 70 Interacting Protein (CHIP) Participates In High Glucose-induced Vascular Endothelial Cell Injury

Background:With the aging of the world’s population,the number of people suffering from diabetes is increasing year by year.According to the data of the International Diabetes alliance 2019,the number of diabetes patients aged 20 to 79 in the world has reached463 million,with an average of 1 person in 11 suffering from diabetes,and it is expected to exceed 700 million in 2045.Long term hyperglycemia in the body will lead to pathological changes of peripheral blood vessels,and eventually lead to organ failure,such as diabetic nephropathy,diabetic heart disease and diabetic eye disease.Among the deaths of diabetic patients,1 / 2 of them died of cardiovascular complications.Diabetic cardiovascular diseases mainly include diabetic cardiomyopathy and diabetic atherosclerosis.Although the pathogenesis of cardiovascular disease caused by long-term hyperglycemia has not been fully elucidated,a large number of studies have confirmed that the inflammatory response,oxidative stress and apoptosis of cardiovascular cells play an important role in the pathogenesis of diabetic cardiovascular disease.Studies have shown that MAPK and NF-κ B are activated in a large amount in the cardiovascular tissue of diabetic patients,while reducing MAPK and NF-κ B activity can improve the inflammatory response and cells Apoptosis reduces cardiovascular cell damage and improves cardiac and vascular function.The carboxyl terminus of heat shock protein 70 interactiNG protein(CHIP)is a member of E3 ubiquitin ligase.Professor Ballinger first found it in human cardiac cDNA library,which is highly expressed in human cardiovascular system.Its N-terminal TPR domain can interact with heat shock protein 70 / 90,while its C-terminal U-box domain has E3 ubiquitin ligase activity.The unique structure of CHIP determines that it can mediate the ubiquitination and degradation of disease-related proteins and misfolded proteins,so as to achieve the stability of theintracellular environment.The results showed that in the mouse model of myocardial remodeling induced by angiotension II,the down-regulation of CHIP gene aggravates the inflammatory response and apoptosis of mouse cardiomyocytes,suggesting that CHIP participates in the process of inflammation and apoptosis.However,there is no report on whether CHIP is involved in hyperglycemic mediated endothelial cell injury.Objective:1.Whether CHIP is involved in high glucose mediated vascular endothelial cell injury.2.Suppose CHIP is involved in vascular endothelial cell injury,and explore its molecular mechanism.Methods:The primary umbilical vein endothelial cells of healthy pregnant women were extracted and used in the experiment for 2-6 generations.Before the experiment,mannitol was used to eliminate the influence of osmotic pressure,The recombinant lentivirus LV siRNA NC and LV siRNA CHIP were transfected into the primary umbilical vein endothelial cells.The umbilical vein endothelial cells were treated with 5.5mmol/l(normal glucose: NG)and 25.5mmol/l(high glucose: Hg)glucose concentration medium for 48 hours.The experiment was divided into four groups:NG + siRNA NC,NG + siRNA CHIP,Hg + siRNA NC,Hg + siRNA CHIP.1.MTT and TUNEL methods were used to detect cell death rate and apoptosis rate,2.Endothelin-1(ET-1)was detected by ELISA;3.The activity of SOD and the content of NO were detected by the kit;4.ROS level was detected by DHE;5.qRT-PCR was used to detect the relative expression of IL-8 and MCP-1mRNA;6.Western blot was used to detect the protein levels of CHIP,iNOS,eNOS,NOX2,NOX4,p38,p65,p-p38,p-p65,BAX and BCL-2.Results:1.Compared with the 5.5mmol/l glucose concentration group,there was no significant difference in cell survival rate of the 5.5mml/l glucose + 20 mmol / L mannitol concentration group.2.Compared with NG + siRNA GFP group,the cell survival rate,apoptosis rate and the mRNA expression of IL-8 and MCP-1 were increased,the level of ROS was increased,while the antioxidant capacity of SOD was decreased,the expression of ET-1 were increased,while NO was decreased.the protein expression of NOX2,NOX4,iINOS,p-p38,p-p65 and BAX were increased,while eNOS,BCL-2was decreased in HG + siRNA NC group.3.Compared with HG + siRNA NC group,the cell survival rate,apoptosis rate and the mRNA expression of IL-8 and MCP-1 were increased,the level of ROS was increased,while the antioxidant capacity of SOD was decreased,the expression of ET-1 were increased,while NO was decreased.the protein expression of NOX2,NOX4,p-p38,p-p65 and BAX were increased,while BCL-2 was decreased in HG + siRNA CHIP group.But there was no significant difference of the expression of eNOS and iNOS between two groups.Conclusion:1.High glucose can mediate the inflammatory response,oxidative stress and apoptosis of primary umbilical vein endothelial cells.2.Down regulation of CHIP expression aggravates high glucose-induced HUVECs injury,which may be related to the regulation of MAPK / p38 and NF-κ B/ p65 activation.