Protective Effects Of Quercetin On High Glucose-induced Diastolic Function Injury In Cultured Human Umbilical Vein Endothelial Cells

Objective: Diabetes(diabetes mellitus, DM) is a common multipleendocrine diseases, The various acute and chronic complications of DM threathuman health seriously. Among them, the large vascular lesions is the mostimportant complication of DM, It is the basis of the other chroniccomplications of DM.Its pathogenesis is associated with the injury of vascularendothelial cell, platelet activation, abnormal blood coagulation andfibrinolysis. The main pathological change is atherosclerosis (atherosclerosis,AS),The injury of endothelial cell is the early important performance ofdiabetic macroangiopathy, it is also the initiating factor for the development ofAS. The vascular endothelial cells play an important role in vasomotor toneregulation,maintain blood flow,prevention of thrombosis and so on,throughthe synthesis and release of a variety of bioactive substances,among them,nitric oxide (NitricOxide, NO) and endothelin (Endothelin-1,ET-1) are theimportant vasodilator and vasoconstrictor, play an important role inmaintaining the homeostasis.NO can relax vascular smooth muscles, inhibitplatelet aggregation, can reduce vascular endothelial dysfunction caused byoxygen free radicals. The elevated levels for ET-1can make vasospasm, theproliferation of vascular smooth muscle cells, platelet adhesion, aggregationand promote thrombosis, induce the AS.In physiological conditions, NO andET-1exist in equilibrium, can maintain some vascular tension. In highglucose concentration, the vascular endothelial barrier function is damaged,from the physiological state that mainly antithrombotic into the pathologicalstate that based on promoting thrombosis. Therefore, protect endothelial cellsfrom injury, enhance the resistance and tolerance for vascular endothelial cellto harmful factors, have important implications for preventing large vascular lesions of diabetes. The Chinese medicine causes more and more attentionfrom scholars. According to researches, that flavonoids have resistance toatherosclerosis, antihypertension, anti oxidative stress, inhibiting aldosereductase, restraining the enzyme glycosylation and other pharmacologicaleffects. Quercetin (Qu) is one of the strongest material in antioxidation. Thedata shows: Qu can effectively play the role of resistance to atherosclerosis,but the mechanism is not yet clear.This experiment is to utilize differentconcentrations of Qu to act on the in vitro cultured human umbilical vein end-othelial cells (HUVECs), observe the morphology,energy of HUVECs,thesecretion of NO and ET-1,and the change of protein expressions ofendothelial nitric oxide synthase (eNOS) and ET-1, investigate the protectionof Qu to the damage of HUVECs endothelial diastolic function induced byhigh glucose. and explore the possible mechanisms of the drug to treat diabeticmacroangiopathy further.Methods:The HUVECs were cultured in vitro,and the logarithmicphase-growth cells were randomly divided into six groups：①normal controlgroup (glucose concentration is5.5mmo1/L);②high glucose group (glucoseconcentration is30mmo1/L);③mannitol control group (5.5mmol/L glucose+24.5mmol/L mannitol)；④low doses of Qu group (25μmol/LQu+30mmol/L glucose)；⑤medium doses of Qu group(50μmol/LQu+30mmol/Lglucose);⑥high doses of Qu group (100μmol/LQu+30mmol/L glucose).Themedicaments are added to each drug group respectively2hours beforetreatment by high glucose, continue to culture for12,24,48hours, to observethe morphology of HUVECs, determin cell survival viability by MTT,determine the NO produced in cell supernatant by means of nitrate reductasemethod，determine the ET-l by means of radio-immune method，determineprotein levels of ET-l,eNOS and its phosphorylation in HUVECs culturedcells by means of Western blot method.Results:1At the same time period, cell activity of the high glucose group wasobviously lower than that of normal control group, the difference was statistically significant (P＜0.05).There was no significant difference in cellviability between mannitol control group and normal control group,The cellactivity in high glucose group declined gradually within48hours as the timewent on(P＜0.05).The cell activities of Qu with different concentration groupswere higher than the high glucose group, the differences had statisticssignificance (P＜0.05),and it showed time and dose dependence.2Compared with normal control group, the production of NO in cellsupernatant and the expression in cells of p-eNOS were decreased obviouslyin the high glucose group, and there were statistically significant (p＜0.05),but the expression of total eNOS had no significant change (p＞0.05), andthe secretion and the expression of ET-1had a significant rise, and therewere statistically significant (p＜0.05); Compared with the high glucose group,after Qu intervention, the production of NO in cell supernatant and theexpression in cells of p-eNOS were apparent rebounded, there were significantdifferences (p＜0.05), the secretion and the expression of ET-1were declinedsignificantly, there were statistically significant (p＜0.05), it showed dosedependence.Conclusion:1High glucose can induce the decrease of HUVECs activity, reductionsin eNOS phosphorylation、secretion of NO, ET-1expression and secretion ofET-1.2A certain concerntration of Qu can enhance the activity of cell underhigh glucose condition, promote eNOS phosphorylation of HUVECs underhigh glucose condition, increase the generation of NO derived from eNOS,inhibit ET-1increase, and it shows dose dependence within a certain range,Qu may improve endothelial diastolic function induced by high glucose, byadjusting the balance of NO and ET-1.This may be one of the protectionmechanisms of Qu to diabetes large vessels.