The Mechanism Of IL-17a Induced Endothelial Cell Senescence Via NOX4 /ROS/p53 Signaling Pathway

Background:Age-related cardiovascular disease is a kind of chronic and common disease that seriously threatens the health of middle-aged and elderly people.With the increase of age,endothelial cells gradually become senile,and their morphological changes and abnormal functions will lead to dysfunction of normal vascular regulation.Currently,there are two main hypotheses about cell aging:one is replicative aging characterized by telomere shortening,and the other is premature cell senescence induced by DNA damage,oxidative stress and oncogene activation.In normal adults,vascular endothelial cells are generally in a static state,rarely dividing,and only under pathological conditions(such as wound healing and tumor growth)can the signaling of endothelial cell division and proliferation be activated.Premature senescence is characterized by cell metabolism disorder,structural damage and activation of cell senescence signaling pathway when exposed to a variety of physical and chemical stimulus(such as blood flow disorders,high concentration of glucose,low density lipoprotein,homocysteine,inflammatory factors or radiation,chemical drugs and other causes of increased oxidative metabolites)that lead to telomerase inactivation,proteins and DNA and other cell components damage and abnormal gene expression.Doubtlessly,endothelial senescence is involved in the occurrence of vascular dysfunction and related diseases,and is an important pathological basis for the occurrence of cardiovascular diseases.The oxidative stress of aging organism leads to chronic inflammation.Aging endothelial cells are often in a state of chronic inflammatory and secrete pro-inflammatory factors that promote the development of atherosclerotic plaque.Interleukin-17A(IL-17A)is an inflammatory cytokine mainly produced by Th17 cells(a subgroup of helper T cells),which can induce inflammatory cell infiltration and tissue damage by promoting cells of several types to synthesize and secrete a large number of other inflammatory cytokines(such as IL-6,granulocyte colony-stimulating factor,chemokines,etc).It is reported that the plasma level of IL-17 in patients with acute coronary syndrome is also significantly higher than that in healthy people.In addition,Th17 cell promotes the instability of atherosclerotic plaque and the occurrence and development of AS through the pro-inflammatory effect of IL-17.Our previous study found that IL-17 is closely related to the aging of vascular endothelial cells,but the specific mechanism remains unclear.Reactive oxygen species(ROS)is a family of electronic reduction products of molecular oxygen.The free radical theory of aging suggests that aging is caused by the harmful attack of free radicals on cellular components,and in this case,mainly oxygen free radicals.Studies have proved that NADPH oxidase family(NOx)and ROS are involved in the pathogenesis of hypertension,atherosclerosis and other vascular diseases.The main member of NADPH oxidase family in vascular endothelial cells is NOX4.A study worked by Jula et al showed that IL-17A disrupts the blood-brain barrier by inducing NADPH oxidase or xanthine oxidase-dependent ROS.Additionally,IL-17 induces the cell stress microenvironment to promote apoptosis of melanocytes or proliferation of human mesenchymal stem cells.In this study,we first detected the senescence indexes of primary mouse aortic endothelial cells after IL-17A intervention,then explored the effects of IL-17A on endothelial cell senescence-related signaling pathway proteins,reactive oxygen species levels and NOX4 expression,and finally observed whether inhibition of NOX4 activity and ROS production could change the aging phenotype of endothelial cells stimulated by IL-17A,and preliminarily elucidate the role and mechanism of IL-17A/NOX4/ROS signaling pathway in endothelial cell aging..Objective:To explore whether IL-17A can induce oxidative stress in endothelial cells through NOX-dependent reactive oxygen species generation,which activates the aging signaling pathway and lead to endothelial cell senescence.This investigation may provide a new therapeutic target for the prevention of aging related cardiovascular diseases.Method:(1)In this study,primary vascular endothelial cells were cultured from the aorta of eight-week-old C57BL/6J mice,and the ratio of CD31 and CD105 double positive primary cells was detected by flow cytometry to identify the purity of the cultured mouse aortic endothelial cells(MAEC).(2)After MAEC was treated with different concentrations of IL-17A(0,10,20,30,40,50 ng/m L)and different time(12h and 24h),CCK-8 was used to detect cell proliferation and Western blot was used to detect the expression levels of age-related proteins p53,p21,and p16.(3)After determining the optimal concentration and time of IL-17 A intervention,?-galactoside staining was used to observe the degree of cell senescence,cell cycle and intracellular reactive oxygen species(ROS)levels were measured by flow cytometry,the expression levels of NOX4 and p22phox were detected by Western blot,and last,whether the addition of ROS scavenging agent NAC could reverse IL-17A-induced aging phenotype was observed in the following experiment.Results:(1)A simple and effective primary culture method of mouse aortic vascular endothelial cells was successfully established.The endothelial markers CD31 and CD105 were detected in96.5%cells by flow cytometry.(2)The result of CCK-8 showed that endothelial cell proliferation was inhibited by the intervention of IL-17A,and the inhibitory effect was increased with the increase of concentration and time.The expression levels of p53 and p21 pathway proteins associated with cell senescence significantly increased after the treatment of MAEC with 30ng/ml IL-17A for24h(P<0.01,P<0.05),and there was no significant change in p16 protein.(3)The data of senescence?-gal enzyme staining in the 30ng/ml IL-17A intervention group showed a significant increase in the proportion of positive cells compared to the control group(P<0.05,P<0.01),as well as the proportion of cells blocked in G0/G1 phase(P<0.05).The expression level of NOX4 protein in endothelial cells increased after IL-17A treatment(P<0.05),while there was no significant change in p22^phoxexpression.The addition of reactive oxygen scavenger NAC successfully inhibited the expression of IL-17A-induced oxidative stress and aging proteins p53 and p21 in MAEC.(4)The addition of NOX4 inhibitor GKT137831 decreased the expression levels of p53 and p21 induced by IL-17A(P<0.05),and successfully decreased IL-17-induced increased senescence galactosidase activity(P<0.01).Conclusion:High purity endothelial cell lines were obtained by primary culture technique in this study,which could reflect the physiological and pathological conditions in vivo.As a cytokine,IL-17A can induce the increase of?-galactosidase activity,cell cycle arrest and cell proliferation inhibition in mouse aortic endothelial cells,suggesting that IL-17A can induce endothelial cell senescence.The specific mechanism of IL-17A induced endothelial cell senescence may be the activation of NOX4/ROS/p53/p21 senescence signaling pathway in cells.