Mechanism Of HDAC9 In The Injury Of Vascular Endothelial Cell Mediated By P38 MAPK Pathway

Background and Aim:Cerebral infarction is a common disease which seriously endangers human health and life.Its morbidity rate,mortality rate and disability rate are increasing year by year.Cerebral infarction is often associated with the formation of atherosclerotic plaque in the intracranial artery.Atherosclerosis(AS)plays an important role in the occurence and development of cerebral infarction.Previous studies have shown that the injury and dysfunction of vascular endothelial cells are the earliest pathological changes of AS.The damaged vascular endothelial cells often release a large number of cellular inflammatory factors which then promote the accumulation of inflammatory cells in the vascular lesion,and further expand the extent of damage.It can be seen that effectively inhibiting the inflammatory damage of vascular endothelial cells is beneficial to reducing the formation of atherosclerotic plaque.In recent years,genome-wide association studies have found that the gene sequence of Histone Deacetylase 9(HDAC9)located on chromosome 7p21.1 is closely related to the diseases caused by AS.Existing researches suggest that HDAC9 is likely to incur AS by mediating inflammatory damage in vascular endothelial cells.At present,the molecular mechanism of HDAC9-mediated vascular endothelial injury is undefined.However,there were studies proving that HDAC9 can mediate nerve cell injury induced by cerebral ischemia via P38 mitogen-activated protein kinase(P38 MAPK)in mouse glial cells.It could thus be inferred that HDAC9 in vascular endothelial cells is likely to mediate inflammatory injury through the P38 MAPK pathway.Sodium valproate(SVA)is a routine anti-epileptic drug with its recognized clinical safety and stability.The fact that SVA inhibits HDAC9 activity and anti-inflammatory effects has been confirmed,which may antagonize vascular endothelium inflammatory damage of cells,and therefore reduce the formation of atherosclerotic plaque,becoming a potential drug for the prevention and treatment of cerebral infarction.To sum up,this study aims to discuss the impact and molecular regulation mechanism of HDAC9 on vascular endothelial cells inflammatory injury induced by Oxidized low-density lipoprotein(ox-LDL).Meanwhile,the effect of SVA on ox-LDL-induced vascular endothelial cell injury is also verified,thus providing theoretical basis for SVA clinical prevention and treatment of cerebral infarction.Methods:1.ox-LDL-induced injury of vascular endothelial cell CCK-8 assay was adopted and screened to construct the optimum concentration for EA.HY926 cell injury model.EA.HY926 cells were divided into two groups,that is,control group and ox-LDL group.The early and late apoptosis of the two groups were tested by Flow cytometry.The expression levels of HDAC9,P38 MAPK,p-P38 MAPK,TNF-a and MCP-1 of the two groups were compared by Western blot.2.The effect of HDAC9 on the injury of vascular endothelial cell The expression levels of HDAC9 in the EA.HY926 cells which were treated with different concentrations of ox-LDL,were detected by Western blot.EA.HY926 cells were divided into six groups,including control group,sh Vector group,shHDAC9group(HDAC9 knockdown group),ox-LDL group,sh Vector+ox-LDL group and shHDAC9+ox-LDL group.The viability of the above six groups were tested by CCK-8 assay.The early and late apoptosis of the above six groups were detected by Flow cytometry.The expression levels of HDAC9,P38 MAPK,p-P38 MAPK,TNF-a and MCP-1 of the above six groups were detected by Western blot.3.HDAC9 mediating the injury of vascular endothelial cell by P38 MAPK pathway The optimum concentration of SB203580 antagonizing ox-LDL was screened by CCK-8 assay.EA.HY926 cells were divided into four groups,namely,control group,SB203580 group,ox-LDL group and SB203580+ox-LDL group.The detection method was the same as Method 1.4.SVA antagonizing P38 MAPK pathway-mediated injury of vascular endothelial cell The optimum concentration of SVA antagonizing ox-LDL was screened by CCK-8 assay.EA.HY926 cells were divided into 4 groups,covering control group,SVA group,ox-LDL group and SVA+ox-LDL group.The detection method was the same as Method 1.Results:1.ox-LDL-induced vascular endothelial cell injury EA.HY926 cells were treated with different ox-LDL concentrations for 24 hours.CCK-8 assay were used to test the cell survival rate.Results showed that the activity of EA.HY926 cells declined with the increase of ox-LDL concentration.When ox-LDL concentration reached 150μg/ml,the cell survival rate decreased by 50%.The difference in cell survival rate between the 150μg/ml group and the control group was statistically significant(P <0.01).Therefore,this concentration was used to construct model of vascular endothelial cell injury in follow-up experiments.The results of the Flow cytometry indicated that the apoptosis rate of ox-LDL group was significantly higher than that of the control group,showing statistical differences(P<0.01).Meanwhile,The comparative results of the Western blot revealed that the expression level of HDAC9,p-P38 MAPK,TNF-a and MCP-1 of the ox-LDL group was higher than that of the Control group,and the above four proteins expression of the two groups was statistically significant(P<0.01).2.The effect of HDAC9 on the injury of vascular endothelial cell EA.HY926 cells were treated with different ox-LDL concentrations for 24 hours.According to the result of Western blot,the expression of HDAC9 in EA.HY926 cells increased with the rise of ox-LDL concentration.A dual comparison among various groups with different concentrations was conducted,results were statistically significant(P <0.01).The results of CCK-8 assay demonstrated that knockdown of HDAC9 can increase the viability of EA.HY926 cells,and the cell survival rate of shHDAC9 group was higher than that of control group,which showed statistical difference between the two groups(P <0.05).The cell survival rate of the shHDAC9+ox-LDL group improved significantly when comparing with the ox-LDL group,showing statistical difference(P <0.01).According to the results of Flow cytometry,the apoptosis rate of shHDAC9+ox-LDL group was significantly lower than that of ox-LDL group,showing statistical difference(P <0.01).Meanwhile,the results of the Western blot revealed that knockdown of HDAC9 had not effect on the expression of P38 MAPK.There was no significant statistical difference in the expression of P38 MAPK when comparing the above six groups(P=0.998).The knockdown of HDAC9 only affected the expression of p-P38 MAPK and the effectors(TNF-a and MCP-1)of the P38 MAPK pathway.The expression level of the above four proteins(HDAC9,p-P38 MAPK,TNF-a and MCP-1)of the shHDAC9+ox-LDL group were lower than that of the ox-LDL group,showing statistical difference(P <0.01).3.HDAC9 mediating the injury of vascular endothelial cell by P38 MAPK pathway CCK-8 assay was adopted to screen the optimal concentration of SB203580 to antagonize ox-LDL,which was 6μM.According to the results of cell apoptosis by Flow cytometry,SB203580 could reduce the apoptosis induced with ox-LDL and the apoptosis rate of SB203580+ox-LDL group was lower than that of ox-LDL group,which revealed statistical difference(P <0.01).The results of the Western blot showed that the existence of SB203580 did not affect the expression of HDAC9 in the cells.The inhibitor’s antagonism to ox-LDL was achieved by decreasing the phosphorylation level of P38 MAPK.The difference in the expression of HDAC9 between SB203580+ox-LDL group and ox-LDL group was statistically insignificant(P =0.912).The expression levels of p-P38 MAPK,TNF-a and MCP-1 of SB203580+ox-LDL group were lower than those of the ox-LDL group,showing statistical differences between the two groups(P <0.01).4.4.SVA antagonizing P38 MAPK pathway-mediated injury of vascular endothelial cell CCK-8 assay was adopted to screen the optimal concentration of SVA to antagonize ox-LDL,which was 4μM.According to the results of cell apoptosis by Flow cytometry,SVA could reduce the apoptosis induced with ox-LDL and the apoptosis rate of SVA+ox-LDL group was lower than that of ox-LDL group,which revealed statistical difference between the two groups(P <0.01).The results of the Western blot showed that SVA did not affect the expression of HDAC9 in the cells.The inhibitor’s antagonism to ox-LDL was related to reducing the deacetylation activity of HDAC9,which led to the elevated level of histone H3K9 acetylation.There was no statistical difference in the HDAC9 expression between SVA+ox-LDL group and ox-LDL group(P =1).The expression levels of p-P38 MAPK,TNF-a and MCP-1 of the SVA+ox-LDL group were lower than those of ox-LDL group,and the difference between the two groups was statistically significant(P <0.01).The expression of acetylated histone H3K9 in control group was higher than that of ox-LDL group(P <0.01)while the the expression level of acetylated histone H3K9 of SVA+ox-LDL group was higher than that of ox-LDL group,and the difference between groups was statistically significant(P <0.01).Conclusion:1.HDAC9 activates P38 MAPK signaling pathway by P38 MAPK phosphorylation,which mediates inflammatory injury of vascular endothelial cells.2.SVA can antagonize ox-LDL-induced vascular endothelial cell inflammatory injury by inhibiting the activity of HDAC9,which makes it a potential drug of the prevention and treatment of cerebral infarction.