Mechanism Of Pax9 Regulating Autophagy In Oxidative Stressinduced Vascular Endothelial Injury

Background:Cardiovascular disease(CVD)is still one of the diseases with high mortality worldwide.Most of the pathophysiological processes of CVD are related to oxidative stress injury.The pathophysiological processes of atherosclerosis,hypertension,myocardial infarction and other common cardiovascular diseases are involved in oxidative stress injury of myocardial cells and vascular endothelial cells.Oxidative stress injury is considered to be the main cause of vascular endothelial dysfunction,which is an imbalance caused by increased production of reactive oxygen species(ROS)and decreased of endogenetic antioxidant defense systems.The rise of ROS can cause inflammatory reaction,apoptosis and cell proliferation,which was important in the progression of diseases.Therefore,reducing oxidative stress injury is helpful to provide a new treatment for cardiovascular and cerebrovascular diseases.Pax9 is a transcription factor,a member of Pax family,with specific DNA binding sites.Previous studies have shown that Pax9 plays an important role in embryonic development and tumorigenesis.Two recent studies have shown that Pax9 is involved in cardiovascular development.Previous studies of our team illustrated that Pax9 can promote the proliferation,migration and phenotypic transformation of vascular smooth muscle cells.We suspect that Pax9 may function in the pathophysiology of cardiovascular disease.However,few studies have reported the relationship between Pax9 and cardiovascular disease.Cell death is a complex and precisely controlled process.Apoptosis is a kind of cell death process which was programmed by genes.In the past decades,apoptosis has been considered to be vital in cell death.With the deepening of the research on apoptosis,recent studies have found that the fate of cells is not only determined by the process of apoptosis,cell death can be roughly divided into three forms:apoptosis,autophagy and necrosis.There are complex interactions between autophagy and apoptosis.The initiation of apoptosis may be due to autophagy,but the terminal stage will converge in apoptosis.Autophagy is usually induced under various stress conditions,such as organelle dysfunction,atrophy,chemotherapy intervention,starvation and so on.If the pressure is controlled,cells can maintain homeostasis through autophagy and return to the initial state.However,if stress persists and autophagy fails to support cell survival,apoptosis will be activated to reduce the damage of surrounding cells and tissues.There are many direct or indirect interactions between apoptosis and autophagy.Their interaction forms a balance between cell survival and cell death.This study will explore the expression of Pax9 in vascular endothelial oxidative stress injury,whether Pax9 can participate in the disease process of vascular endothelial oxidative stress injury through the regulation of autophagy and apoptosis,and the specific mechanism of Pax9 reducing or aggravating vascular endothelial oxidative stress injury.Objective:To clarify the expression of Pax9 in vascular endothelial oxidative stress injury and explore the mechanism of how Pax9 participated in vascular endothelial oxidative stress injuryMaterials and methods:Part One:To observe the effect of oxidative stress on Pax9expression in rats'vascular tissue.Male SD rats of 4-6 weeks were cultured in normal rat food at room temperature for 3 days.The left common carotid artery of the rat was the operation group,and the right common carotid artery of the same rat was the control group.The left carotid artery was stimulated by oxidative stress.After 48 hours,the bilateral carotid artery tissues were taken.The expression of Pax9 was examined by Western blot and immunohistochemistry.Part two:To explore the effect of oxidative stress on Pax9 and the effect of Pax9 on apoptosis of HUVEC.Firstly,hydrogen peroxide(H₂O₂)with increasing concentration gradient(0,100,200,400umol/L)was used to stimulate HUVEC for 12 hours.The outcome of H₂O₂ on Pax9 expression and apoptosis related proteins were examined by Western blot.The accumulation of ROS was observed by ROS fluorescence assay.The cell viability was observed by CCK-8 assay.The apoptosis rate of HUVEC was measured by flow cytometry.Next,H₂O₂ was stimulated by Pax9 overexpression and Pax9knockdown,respectively The outcome of Pax9 overexpression or knockdown on ROS accumulation level were observed by ROS fluorescence assay,the effects of Pax9overexpression or knockdown on HUVEC cell viability were detected by CCK-8 assay,and the effects of Pax9 overexpression or knockdown on HUVEC apoptosis rate were measured by flow cytometry.Part three:In human umbilical vein endothelial cells(HUVEC),to explore the effect and specific mechanism of Pax9 on autophagy in oxidative stress injury,and to explore the regulation relationship between apoptosis and autophagy in HUVEC under oxidative stress injury.First of all,the effects of Pax9 on autophagy were determined by Western blot.Next,the binding of Pax9 to p62 was examined by co-immunoprecipitation assay(Co IP).H₂O₂stimulation was given to observe whether the binding degree of Pax9 and P62 changed.Finally,since the experiment found that Pax9 knockdown can promote autophagy,3-methyladenine(3-MA)was used to inhibit autophagy,observe the changes of apoptosis level after inhibiting autophagy,and explore the regulation of apoptosis and autophagy in endothelium oxidative stress injury.The gray value of Western blot was measured by Image J(ver 1.8.0)software.ROS and immunohistochemical cell area were measured by Image J,too.The absorbance value of cells in CCK-8 was measured by microplate.Western blot,ROS fluorescence,immunohistochemistry and flow cytometry images were processed by Photoshop 2019.All the bar charts were made by graphpad prism(ver 7.0.4).All statistical analyses were performed by SPSS(IBM)software,and the results were shown as mean±standard error.The statistical methods were independent sample t-test analysis and one-way ANOVA(Bonferroni and s-n-k methods were used for post test).P<0.05 for the difference between the data was statistically significant.Results:1.Part I:compared with the sham operation group,the expression of PAX9 was decreased in the operation group which was stimulated by H₂O₂.At the same time,we found that the expression of LC3II also decreased.This result indicate that oxidative stress injury in blood vessels leads to the increase of autophagy.2.Part2:(1)Along with the increase of H₂O₂ concentration,the expression of Pax9 was gradually decreased,and the apoptosis related protein Caspase3 and Bax/Bcl₂ were gradually increased.The accumulation level of ROS increased when increased the concentration of H₂O₂,and the cell viability of HUVEC decreased with the increase of H₂O₂ concentration.The results of flow cytometry and Western blot were consistent.With the increase of H₂O₂concentration,the apoptosis rate of HUVEC increased gradually.(2)The cells were divided into four groups:con group,con+H₂O₂ group,Pax9overexpression group and Pax9+H₂O₂ overexpression group.Compared with con+H₂O₂group,overexpression of Pax9+H₂O₂ decreased the expression of Caspase3 and Bax/Bcl₂.These results suggest that overexpression of Pax9 can inhibit HUVEC apoptosis induced by oxidative stress.In addition,compared with con+H₂O₂ group,Pax9+H₂O₂overexpression group showed decreased ROS accumulation level,increased cell viability and decreased apoptosis rate of HUVEC.(3)The cells were divided into four groups:con group,con+H₂O₂ group,Pax9 knockout group and Pax9+H₂O₂ knockout group.Compared with con+H₂O₂group,Pax9+H₂O₂ knockdown group showed higher expression of Caspase3 and Bax/Bcl₂.This suggests that Pax9 knockdown can promote HUVEC apoptosis induced by oxidative stress injury of vascular endothelium.3.Part3:(1)In H₂O₂ induced vascular endothelial oxidative stress injury,with the increase of H₂O₂ concentration,autophagy related protein LC3II/LC3I gradually increased,p62gradually decreased.(2)The cells were divided into four groups:con group,con+H₂O₂group,Pax9 overexpression group and Pax9+H₂O₂ overexpression group.Compared with con+H₂O₂ group,Pax9+H₂O₂ overexpression group showed decreased expression of autophagy related protein LC3II/LC3I and increased expression of p62.This suggests that overexpression of Pax9 can inhibit HUVEC autophagy induced by oxidative stress injury.(3)The cells were divided into four groups:con group,con+H₂O₂ group,Pax9 knockout group and Pax9+H₂O₂ knockout group.Compared with con+H₂O₂ group,Pax9+H2O2knockdown group showed higher expression of autophagy related protein LC3II/LC3I and lower expression of p62.(4)Since previous experiments have confirmed that Pax9knockdown can cause autophagy and apoptosis of HUVEC at the same time,we explore the mutual regulation of autophagy and apoptosis in vascular endothelial oxidative stress injury.When the autophagy inhibitor 3-MA was added in the presence of Pax9 knockdown,the increase of apoptosis caused by Pax9 knockdown was reduced.These results indicate that the autophagy induced by Pax9 knockdown is involved in the apoptosis of vascular endothelial cells induced by oxidative stress injury.(5)Immunoprecipitation assay showed that Pax99 could bind to p62,and the binding of Pax9 and p62 decreased with the stimulation of H₂O₂.The results are consistent with the previous experimental results.These results indicate that the inhibition of Pax9 on autophagy is reduced after H₂O₂stimulation,resulting in the increase of apoptosis under oxidative stress.Conclusion:(1)The expression level of Pax9 was decreased in vascular endothelial oxidative stress injury.(2)Pax9 can inhibit HUVEC apoptosis induced by oxidative stress.(3)Pax9 can inhibit HUVEC autophagy in vascular endothelial oxidative stress injury by binding to p62.(4)In vascular endothelial oxidative stress injury,the autophagy induced by Pax9 knockdown is involved in HUVEC apoptosis.