| Background:Diabetes mellitus(DM)patients are in conditions of multiple risk factors such as hyperglycemia,dyslipidemia and hypertension which affect each other and cause the occurrence and development of atherosclerosis(AS).Endothelial dysfunction(ED)has been recognized as one of initial processes and the promotion factors in the progression of AS.However,the pathogenesis of ED is extremely complicated,which has not been fully elucidated yet.Autophagy is a protective mechanism against pathophysiological stimuli in vascular endothelial cells.Recent studies have found that autophagy is closely related to the occurrence and development of both ED and AS.The activation of autophagy suppresses the endothelial inflammation and oxidative stress in the present of oxidized-low density lipoprotein(ox-LDL)and advanced glycosylation end products(AGEs)stimulation,while the loss of autophagy may trigger ED.Autophagy is a complex process involving multiple factors and is negatively regulated by the mammalian target protein of rapamycin(mTOR)pathway.Selenoprotein S(SelS)is a transmembrane glucose-regulating protein,which protects endothelium against oxidative stress,inflammation,apoptosis,et al.However,the mechanism by which SelS protects the endothelium from high glucose and ox-LDL-induced injury through regulating the Akt/mTOR autophagy pathway is not fully clarified.This study intends to explore the protective effect of SelS on endothelium and its possible mechanism by observing the effects of SelS on the high glucose and/or ox-LDL-induced Akt/mTOR autophagy pathway and endothelium injury,so as to experimentally and theoretically support SelS to become a new and effective intervention target for the prevention and treatment of AS.Methods:To construct a human aortic endothelial cells(HAECs)injury model,HAECs were treated with different concentrations of glucose and/or ox-LDL and cell survival rates were measured by MTT.To explore the association between SelS and the injury of HAECs caused by high glucose and/or ox-LDL,Western Blot was employed to detect the expression of endothelial nitric oxide synthase(eNOS),endothelin-1(ET-1),intercellular adhesion factor-1(ICAM-1),vascular cell adhesion factor-1(VCAM-1)and SelS.To explore the relationship between high glucose and/or ox-LDL-induced vascular endothelial injury and Akt/mTOR autophagy pathways,transmission electron microscope was used to observe autophagosomes of HAECs after high glucose and/or ox-LDL intervention;the tandem fluorescent mRFP-GFP-LC3 expression construct was employed for monitoring the fluorescent LC3 dots indicative of autophagosomes by using fluorescent microscope;Western Blot was used to detect the expression of LC3Ⅱ/Ⅰ,P62,Beclin-1 and Akt/mTOR changes in phosphorylation level.To investigate the effects of SelS on high glucose and/or ox-LDL-induced Akt/mTOR autophagy pathway and vascular endothelial injury,SelS was overexpressed and knocked down on HAECs in lentiviral infection.In high glucose and/or ox-LDL-intervened HAECs with overexpressed and knocked down of SelS,Western Blot was used to detect eNOS,ET-1 and ICAM-1 expression;mRFP-GFP-LC3 expression construct was employed for monitoring the fluorescent LC3 dots indicative of autophagosomes by using fluorescent microscope;Western Blot was used to detect the expression of LC3 Ⅱ/Ⅰ,P62,Beclin-1 proteins and the phosphorylation of Akt/mTOR autophagy pathway.Results:The high glucose and/or ox-LDL-induced HAECs injury model was successfully constructed as below:the survival rate of HAECs was reduced to(76.57 ±4.47)%,(72.74 ± 6.59)%and(55.84 ± 5.35)%under the stimulation of high glucose(Glu 35 mmol/L),high ox-LDL(ox-LDL 50mg/L)as well as high glucose combined with ox-LDL(35mmol/L Glu+50mg/L ox-LDL),respectively.Under the stimulation of high glucose and/or ox-LDL,eNOS protein levels decreased in a time-dependent manner(P<0.05),while ET-1 and ICAM-1 protein levels increased in a time-dependent manner(P<0.05),indicating that the injury of HAECs aggravated gradually over time.Under the stimulation of high glucose and ox-LDL,the SelS protein level increased in a time-dependent manner(P<0.05),suggesting that SelS is related to the endothelial injury induced by high glucose and/or ox-LDL.Under the stimulation of high glucose and/or ox-LDL,HAECs autophagy evaluated firstly and then decreased,and the characteristics of autophagy achieved to the peak at 6h.HAECs were injured by high glucose and/or ox-LDL at 6h as well.Meanwhile,the eNOS expression decreased but the ET-1 and ICAM-1 expression increased.It was found that the most significant changes were in high glucose combined with ox-LDL group(P<0.05).Accordingly,the expression of SelS increased most significantly in high glucose combined with ox-LDL group and ox-LDL group(P<0.05).Also,the autophagic flux was induced in HAECs with the increase in autophagosomes and fluorescent LC3 dots,LC3Ⅱ/Ⅰ ratio,Beclin-1 and P62 expression,and with the relatively insufficient autophagy product degradation.It was noticed that the most significant changes were in high glucose combined with ox-LDL group(P<0.05).The decreased ratios of p-Akt/Akt and p-mTOR/mTOR in 6h stimulation of high glucose and/or ox-LDL suggested that Akt/mTOR pathway was inhibited.It was also found that the most significant changes were in high glucose combined with ox-LDL group(P<0.05).SelS expression increased significantly in 6h stimulation of high glucose and/or ox-LDL.It was observed that and the increase was more significant in high glucose combined with ox-LDL group and high ox-LDL group(P<0.05),suggesting that SelS may relate to autophagy and Akt/mTOR pathway in HAECs after high glucose and/or high ox-LDL stimulation.The evidences that SelS overexpression relieved HAECs injury induced by high glucose and/or ox-LDL stimulation are as follows:the increased eNOS expression,the significant reduced ET-1 and ICAM-1 expressions(P<0.05);the inhibited autophagic flux with the reduction of autophagosomes and fluorescent LC3 dots,LC3 Ⅱ/Ⅰ ratio,Beclin-1 expression and the increase of P62 expression(P<0.05).However,SelS knockdown showed the opposite results in HAECs.The evidences that SelS knockdown aggravated HAECs injury further in high glucose and/or ox-LDL stimulation are as follows:the reduced eNOS expression;the significant increased ET-1 and ICAM-1 expressions(P<0.05);the increased autophagosomes indicated by mRFP-GFP-LC3 fluorescent spot aggregation,the increased LC3 Ⅱ/Ⅰ ratio and Beclin-1 expression,and the reduced P62 expression(P<0.05).In addition,p-Akt/Akt and p-mTOR/mTOR ratios were increased by SelS overexpression in HAECs with high glucose and/or ox-LDL stimulation(P<0.05),indicating that Akt/mTOR pathway was activated by SelS overexpression.On the contrary,SelS knockdown reduced p-Akt/Akt and p-mTOR/mTOR ratios(P<0.05),indicating that Akt/mTOR pathway was inhibited by SelS knockdown.Conclusion:1.The injury of endothelial cells caused by high glucose and/or ox-LDL stimulation in a time-dependent manner is also accompanied by the change of SelS level.It suggests that SelS expression is related to the endothelial injury and autophagy induced by high glucose and/or ox-LDL.2.In high glucose and/or ox-LDL-intervened HAECs,the characteristics of autophagy evaluate firstly and then decreases;the ratios of p-Akt/Akt and p-mTOR/mTOR rise significantly and the expression of SelS increases.It suggests the association between Akt/mTOR-dependent autophagy pathway and endothelial cell injury induced by high glucose and/or ox-LDL.3.SelS overexpression relieves endothelial cell injury by activating Akt/mTOR pathway and inhibiting autophagy in high glucose and/or ox-LDL-stimulated HAECs,whereas SelS knockdown shows the opposite effect.It suggests SelS protects endothelial cells from high glucose and/or ox-LDL-induced injury through Akt/mTOR-dependent autophagy pathway. |
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