Study On The Mechanism And Protection Of Diabetes-Induced Vascular Endothelial Dysfunction

ObjectiveTo explore the intracellular signal transduction mechanism about the occurrence and melatonin protection of diabetic vascular endothelial dysfunction.MethodsAt the animal level,the type 2 diabetic rat model was first constructed,and the rats were randomly divided into 3 groups,including normal group,type 2 diabetes group and type 2 diabetes+melatonin group.In the experiment,the status of rat aortic endothelial injury in each group was first evaluated by the aortic endothelium-dependent diastolic function experiment.Then the status of glucose and lipid metabolism disorder of rats in each group was evaluated with the blood biochemical test to observe its correlation with arterial endothelial injury.Finally,the activation status of the Nrf2 signaling pathway and ROS/NLRP3 inflammation pathway in the arterial tissues of each group were evaluated with the oxidative stress detection,western blot,RT-PCR and ELISA methods to observe their correlation with arterial endothelial injury.At the cellular level,in experimental design,we first used high glucose(30 mM)to injury vascular endothelial cells,and then applied small molecule inhibitors and siRNA interference technique to explore whether relevant signaling pathways are involved in high glucose-induced vascular endothelial cell dysfunction and the molecular mechanism of melatonin protection.In the detection method,DCFH-DA fluorescent probes,western blot,RT-PCR,ELISA,LDH release experiment and Hoechst/PI staining were used to evaluate the activation of Nrf2 signaling pathway and ROS/NLRP3 pyroptosis pathway in endothelial cells;Besides,CCK-8,ELISA,cell scratching test and transwell test were used to evaluate the functional status of endothelial cells.ResultsAt the animal level,after 12 weeks of type 2 diabetes in rats,arterial endothelial function was significantly impaired,glucose and lipid metabolism was significantly abnormal,the activation of the Nrf2 antioxidant pathway in arterial tissue was significantly inhibited,the oxidative stress in arterial tissue was significantly increased,and the NLRP3 inflammasome pathway in arterial tissue was significantly activated.Melatonin treatment for 12 weeks can significantly reduce these injured changes caused by type 2 diabetes.At the cellular level,1)The high glucose intervention on vascular endothelial cells for 72 hours significantly aggravated cell pyroptosis and cell dysfunction.After specific inhibition of the ROS/NLRP3 pyroptosis pathway by N-acetylcysteine(500μM)and MCC950(50μM),the high glucose-induced vascular endothelial cell dysfunction was significantly relieved;2)With the prolongation of high glucose intervention time,the expression of Nrf2 nucleoprotein,total Nrf2 protein and Nrf2 mRNA in vascular endothelial cells showed an expression trend of increasing first and then decreasing,and all showed a peak expression at 6-12h.After 72 hours of high glucose intervention,their expression was significantly lower than that of the non-intervention group;3)High glucose intervention for 72 hours on vascular endothelial cells can lead to the significant suppression of compensatory activation of the Nrf2 signaling pathway,the significant activation of the ROS/NLRP3 pyroptosis pathway,and the significant aggravation of cell dysfunction.Melatonin treatment can significantly improve the above injury caused by high glucose.However,after knocking down the expression of Nrf2 gene with siRNA,the injury caused by high glucose was even worse,and the protective effect of melatonin also decreased significantly.In addition,after applying Luzindole to block the MT1/MT2 melatonin membrane receptors,the protective effect of melatonin on the activation of the Nrf2 signaling pathway also decreased significantly.ConclusionsInhibition of compensatory activation of Nrf2 antioxidant pathway and excessive activation of ROS/NLRP3 pyroptosis pathway are involved in vascular endothelial dysfunction caused by diabetes;Melatonin can activate the Nrf2 signaling pathway through its MT1/MT2 receptor-dependent pathway,promote the expression of HO-1,NQO1,SOD2 antioxidant enzymes,inhibit the excessive activation of the ROS/NLRP3 pyroptosis pathway of vascular endothelial cells caused by diabetes,and finally improve endothelium function.This study reveals a new mechanism of diabetic vascular endothelial dysfunction and provides a basic theoretical basis and potential therapeutic reference for the prevention and treatment of diabetic angiopathy.