The Effect And Mechanism Of AMPK Regulate Calcium Influx On Endothelial Cell Apoptosis Induced By High Glucose

Background:As the increase of the aging in our country,the health of the elderly has been paid more and more attention.Diabetes mellitus is one of the underlying diseases of the aged,and its vascular complications are the major underlying causes of death and disability,and atherosclerosis is the most common cardiovascular complications of diabetes.But its etiology has not been fully clarified.The excessive apoptosis of endothelial cells is an important form of endothelial dysfunction,while the formation of atherosclerosis is also the initiating factor.Studies have shown that under hyperglycemic conditions,the main characteristics of vascular endothelial cells in the regulation of transcription factors,resulting in a series of related factors,lead to endothelial dysfunction,caused by the occurrence and development of diabetic vascular complications.High glucose plays an crucial role in the apoptosis of endothelial cells.Hyperglycemia induces oxidative stress,which leading to apoptosis of vascular endothelial cells directly or indirectly through a series of complex signaling pathways.AMPK contains a heterotrimeric serine / threonine protein kinase activity,catalytic subunit alpha,beta and gamma subunit through the formation and arrangement of different conformations.Alpha subunit has alpha 1 and alpha 2 two subtypes,AMPK alpha 1 and alpha 2 were expressed in endothelial cells,but mainly to alpha 1.Recent evidence indicates that AMPK is a promising role in the treatment of atherosclerosis and vascular dysfunction,but its specific mechanisms need to be further studied.Objectives:In this study,in vitro pancreatic vascular endothelial cells(MS1)culture and establishment of diabetic atherosclerosis mouse modelas the research object,By using laser confocal microscopy,TUNEL detection of apoptosis of endothelial cells,sought to demonstrate that: 1)AMPK inhibits apoptosis of endothelial cells induced by hyperglycemia;2)high glucose induced endothelial cell apoptosis and calcium ion influx related;3)AMPK inhibited the ROS production stimulated by hyperglycemia;4)AMPK inhibitedthe expression of SOCC proteinand calcium influx induced by high glucose;5)AMPK inhibit the formation of atherosclerotic plaque.Methods:1.The endothelial cell apoptosis was detected by TUNEL method in vitro culture of MS1 endothelial cell line.Cells were divided into 6 groups: high glucose group(30mmol/l glucose stimulated 48h),AMPK activation agent group(1mmol/l AICAR pretreated 2h),high glucose group and AMPK agonist group(glucose 30mmol/l stimulated 48h+1mmol/l AICAR 2h pretreated),SOC channel inhibitor(100umol/l2-APBpretreated 2h)group and high glucose +SOC channel inhibition group(30mmol/l glucose stimulated 48h+100umol/l 2-APB pretreated 2h),while the establishment of the control group(without any intervention).2.The calcium influx was detected by laser confocal microscopy.The endothelial cells were cultured in HEPES buffer solution,the fluorescence intensity of Ca2+ probe Fluo-3AM was detected by laser confocal system.The cells were divided into control group,high glucose group,AICAR group,high glucose +AICAR,SOC channel inhibitor(2-APB)and high glucose +SOC channel inhibitor group.3.The DHE probe was used to observe intracellular ROS formation and would be seeded with endothelial cells in 6-well plates and cultured cells fertility after the probe DHE The ROS could be observe by fluorescence microscope.Cells were divided into control group,high glucose group,AICAR group,high glucose +AICAR group.4.SOCC protein was detected by WB technique.Endothelial cells to 105 inoculum density in 6-well plates,each group of three holes,the cell grouping ditto,to 1 mmol/l AICAR pretreated 2h,to give high glucose stimulation for 48 h.The cells were washed with PBS two times,cells were collected to detect the influence of AMPK on expression of Orai1 and Stim1 protein by applying WB.5.Oilred O?HE staining was used to observe the aortic plaque formation.Experimental groups as the normal group,high fat group,STZ group,STZ + control virus group and STZ +AMPK alpha 1 overexpression group.In the thoracic aorta of mice,the plaque formation was observed by oil red O staining after longitudinal incision.The rest,made into paraffin section,HE staining was used to observe the effect of AMPK on the formation of atherosclerosis.Results:1.Compared to the control group,high glucose treated endothelial cell apoptosis significantly increased(P < 0.05),and compared with the high glucose stimulated group pretreatment AICAR significantly inhibits high glucose induced apoptosis of endothelial cells(P < 0.05).2.After high glucose stimulated,calcium influx of endothelial cells increased significantly,while the 2-APB pretreatment,calcium influx and endothelial cell apoptosis induced by high glucose was significantly inhibited by SOC channel inhibitor(P<0.05).3.High glucose stimulated endothelial cells,the intracellular ROS production increased,after AICAR pretreatment,the high glucose induced ROS production was significantly inhibited by AMPK agonist AICAR(P<0.05).4.High glucose stimulated endothelial cells,the expression of Stim1 and Orai1 protein increased,compared with the high glucose group,high glucose group +AICAR could significantly inhibit the expression of Orai1 ?Stim1and calcium influx(P<0.05).5.Compared with the high fat group,the mice were treated with STZ induced diabetes,aortic atherosclerotic plaque formation was obvious,after the injection of AMPK alpha 1 overexpression of lentivirus,aortic plaque formation was significantly reduced(P<0.05).Conclusion:1.High glucose induced apoptosis of endothelial cells,while AMPK can significantly inhibit the endothelial apoptosis induced by high glucose.2.High glucose induced endothelial apoptosis was related to calcium influx,SOC channel mediated calcium influx induced by high glucose.3.High glucose induced ROS production,while AMPK significantly inhibited the ROS induced by hyperglycemia4.AMPK could significantly inhibit the increase of Stim1 and Orai1 protein expression induced by high glucose,and inhibited the calcium influx mediated by SOC protein.5.AMPK siginificantly inhibit the formation of atherosclerotic plaque...