Protective Effect Of Resveratrol On Endothelial Cell Injury Induced By High Glucose And The Related Mechanisms

Objective:To investigate the protective effect of Resveratrol（RSV）on vascular endothelial injury induced by high glucose,and explore its related mechanism.Methods:1.To study the protective effect of RSV on HG induced vascular endothelial injury in animal experiments:three groups were set up,and 8 BKS mice aged 8 weeks were randomly fed with basic diet as the normal control group（BKS group）.In addition,16 db/db male mice aged 8weeks were fed with basal diet,and fasting blood glucose≥11.1mmol/L for two consecutive times was considered as successful modeling of type 2 diabetes.db/db mice were randomly divided into Model group（Model group）and resveratrol group（RSV group）,with 8 mice in each group.RSV was dissolved in 0.5%CMC-Na solution,and RSV group was given400mg/kg/d intragastric administration,and the other two groups were given 0.5% CMC-NA solution intragastric administration for 12 weeks.The body weight and blood glucose of mice were monitored during the intervention.Orbital blood was collected after gavage,and serum was taken to detect the levels of TNF-α,IL-6,IL-1β and VEGF.Aorta specimens of mice were dissected,and vascular tissue sections were taken.Morphological changes of blood vessels were observed by HE staining,and expression levels of Akt and Cleaved caspase-3protein in blood vessels were detected by immunohistochemistry.2.The protective effect of RSV on HG induced HUVECs was studied by cell experiment:Six groups were set up,including control group（5.5mmol/L glucose,Control group）,mannitol group（5.5mmol/L glucose+27.8mmol/L mannitol,Man group）,high glucose group（33.3mmol/L glucose,HG group）,HG+RSV（5μmol/L,10μmol/L,20μmol/L）group,HUVECs were pretreated with RSV at different concentrations for 4h,and then stimulated with 33.3mmol/L glucose for 48 h.Cell viability was detected by CCK-8method;transwell chamber was used to detect cell migration;the lumen formation ability of the cells was detected by Matrigel assay;apoptosis was detected by Tunel assay and Annexin V-FITC;the expression levels of caspase-3,Cleaved caspase-3,Bcl-2,Bax,TNF-α,IL-6,VEGF and Akt were detected by Western blot.Intracellular production of Reactive Oxide Species（ROS）was detected by fluorescence probe DCFH-DA and flow cytometry.Mitochondrial membrane potential was detected by rhodamine 123.Results:1.Animal experiments showed that RSV had a protective effect on HG induced vascular endothelial injury:Compared with the BKS group,fasting blood glucose and random blood glucose were increased in the Model group,but there was no significant difference in body weight gain in the Model group;compared with the Model group,fasting blood glucose and random blood glucose were decreased in the RSV group,and body weight gain was decreased in the RSV group.Compared with BKS group,the serum levels of TNF-α,IL-6 and IL-1βwere increased in Model group,while the secretion levels of VEGF was decreased;Compared with Model group,the secretion levels of TNF-α,IL-6 and IL-1β in RSV group were decreased,while the secretion levels of VEGF was increased.The results of HE experiment showed that the nuclei of aorta in BKS group were arranged neatly,the endothelial cell layer was smooth and smooth,and the endodermis was well continuous;in Model group,the nuclei of aorta were disordered and absent,and the endodermis was fractured and vacuolated;in RSV group,the nuclei of aorta were arranged in order,the rupture of endodermis was improved,and the vacuolar change of endodermis was reduced.Immunohistochemistry showed that compared with the BKS group,the expression of Akt was decreased,but the expression of Cleaved-Caspase-3 was increased in the Model group;Compared with the Model group,the expression of Akt was increased,but the expression of Cleaved-Caspase-3 was decreased in RSV group.2.Cell experiments showed that RSV had a protective effect on HG induced endothelial injury of HUVECs:Compared with the Control group,the proliferation,migration and tube forming ability of HUVECs in RSV group were decreased,but the apoptosis rate of HUVECs were increased;Compared with the HG group,the proliferation,migration and tube forming ability of HUVECs in RSV group（5μmol/L,10μmol/L,20μmol/L）were increased,but the apoptosis rate of HUVECs were decreased.Compared with the Control group,the production of ROS in HG group were increased,and the mitochondrial membrane potential was collapsed;Compared with the HG group,the production of ROS in RSV group（5μmol/L,10μmol/L,20μmol/L）were decreased,and the mitochondrial membrane potential collapsed was improved.Western blot results showed that compared with the Control group,the protein expressions of Akt and VEGF in HG group were decreased,while the protein expressions of TNF-α and IL-6 were increased.Compared with HG group,the protein expressions of Akt and VEGF were increased in RSV（5μmol/L,10μmol/L,20μmol/L）groups,but the protein expressions of TNF-α and IL-6 were decreased in RSV（5μmol/L,10μmol/L,20μmol/L）groups.Conclusion:1.In vivo experiments showed that RSV had a protective effect on HG induced vascular endothelial injury: RSV can reduced cell apoptosis by up-regulating the protein expression of Akt,and reduced the expression of inflammatory factors in db/db mice,thereby reducing the vascular endothelial injury.2.In vitro experiments showed that RSV had protective effects on HG induced HUVECs injury: by reducing cell ROS production,improving mitochondrial membrane potential collapse,promoting HUVECs proliferation,migration and lumen formation,and inhibiting HUVECs apoptosis.These results suggest that RSV may improve mitochondrial function by regulating Akt signaling pathway,thus alleviating HG induced injury in HUVECs.