The Mechanism Of The Amelioration Of D-chiro Inositol On Vascular Endothelial Dysfunction-based On AMPK Activity

Endothelium is monolayer cell which direct contact with blood,and is the first barrier of human in contact with the stimuli in blood.Endothelial dysfunction is the initial feature of cardiovascular disease and diabetes,and it is throughout the whole pathological process and is the pathological basis of these diseases.More importantly,endothelial dysfunction is a reason for the development of the long-term sub-health state.Endothelial cells are vulnerable to oxidative stress which induced by excess free fatty acids.Oxidative stress is crucial event for endothelial dysfunction.Mitochondria are the main sites for ROS production.While,NADPH oxidase(NOX)are dedicated for the generation of cellular ROS in blood vessels.In order to prevent cardiovascular disease and diabetes,the inhibition of endothelial dysfunction might be one of the effective strategies.Meanwhile,the supplement of natural plant extracts or their biological activity compounds for long time is useful to inhibit oxidative stress and endothelial dysfunction.D-chiro inositol(DCI),which belongs to inositols,a small molecule exist in human had the function of the suppression of oxidative stress,hypotensive,hyperglycemic and anti-aging.In addition,studies had been found that the significantly reduction of DCI content in diseases with endothelial dysfunction.The supplement of DCI improved endothelial function in patients with high blood pressure and diabetes.However,the functional and the mechanism of DCI in the amelioration of endothelial dysfunction in sub-health state is unclear.In this study,the protective effect of DCI on oxidative stress and endothelial function,the mechanisms underlying the prevention of endothelial dysfunction with emphasis on oxidative stress and mitochondrial fission were researched in vivo and in vitro.And all above things were assayed using HPLC,Elisa,Western blot,immunohistochemistry,immunofluorescence and tissue perfusion methods.The results of this study will provide a theoretical basis for the application of DCI in the prevention and treatment of diseases with endothelial dysfunction.The main results of this study were as follows.1.The regulation of D-chiro inositol extract on endothelial dysfunction(1)In the primary rat aortic endothelial cells(RAECs),DCI extract significantly inhibited NOX4 activity and prevented oxidative stress(P<0.05).Consistent with the inhibitory effect on NOX4 activity in cells,DCI extract also prevented NOX4 in the aorta of rats and in the aorta of high fed mice(P<0.05).Thus provided valuable clues for the prevention and treatment of buckwheat in endothelial dysfunction.(2)DCI extract prevented Drp1 activity in RAECs.DCI extract also prevented Drp1 activity in in the aorta of rats and in the aorta of high fed mice.Thus DCI extract protected mitochondrial morphology and.function.In the aorta of rat,PA stimulation led to vasodilation impairment,whereas treatment with DCI extract effectively improved endothelium-dependent vasodilation(P<0.05).TheNOX inhibitor and mitochondrial fission inhibitor reduced the promotion of vasodilation,suggesting that the inhibitory effect on NOX4 and Drp1 might be atherapeutic target in the amelioration of endothelial dysfunction.All aboved results showed that D-chiro inositol extract can ameliorate endothelial dysfunction by inhibiting oxidative stress and preventing mitochondrial dysfunction.2.Mechanisms of D-chiro inositol on regulating oxidative stress and endothelial dysfunction(1)DCI was found to prevent ROS production,inhibit the induction of NOX4 in RAECs.It also enhanceNrf2 activity in PA-stimulated cells,showing that DCI prevents oxidative stress.DCI suppressed Ser616 phosphorylation and increased Ser637 phosphorylation of Drp1,and inhibited PA-induced mitochondrial fission(P<0.05).Knockdown of Drp1 attenuated NOX4 activity and increased the inhibitory effect of DCI.In addition,DCI enhanced AMPK activity through the LKB1-dependent pathway.AMPK knockdown diminished the inhibitory effect of DCI on Drp1/NOX4 induction,indicating that AMPK is essential for Drp1 and NOX4 suppression by DCI.(2)Mitochondrial fission lead to mitochondrial dysfunction and apoptosis.DCI effectively restored ??m and inhibited cytochrome c release from mitochondria,suggesting the protective effect of DCI on mitochondrial function.DCI significantly prevented caspase-3activity and cell apoptosis;DCI promoted the phosphorylation of eNOS Ser1177 and the production of NO,demonstrating that DCI could inhibit endothelial dysfunction through preventing cell apoptosis.(3)In the aorta of rat,PA stimulation led to vasodilation impairment,whereas treatment with DCI effectively improved endothelium-dependent vasodilation(P<0.05).The AMPK inhibitor reduced the promotion of vasodilation induced-by DCI,suggesting that the improvement effect of DCI on endothelial function was dependent on AMPK.(4)Oral administration of DCI reduced the high level of FFAs in high fat-fed mice.DCI inhibited ROS production and prevented NOX4 and Drp1 activity in the aorta of high fed mice.It also inhibited caspase-3 activity and promoted NO production,showing that DCI prevented oxidative stress and mitochondrial fission in the aorta of high fed mice and confirming its protective role in endothelial function in vivo.The above results showed that D-chiro inositol inhibits endothelial cell oxidative stress through AMPK pathway,subsequent the protection of mitochondrial function,inhibition of the mitochondrial rely on cell apoptosis,and the function of protecting endothelial function.This study also provided that it is feasible that buckwheat and soybean,which contains high amount DCI,had direct effect against several chronic diseases in vivo.