Effect Of SAH On BEnd3 Apoptosis And Its Molecular Mechanism

According to the World Health Organization?WHO?,17.9 million accounting for 31% of the global deaths died of cardiovascular and cerebrovascular diseases in 2016,of which 85% of patients died of stroke and cardiovascular disease,and more than three-quarters occurred in middle-and low-income countries.Studies have shown that the incidence of stroke in China is increasing at a rate of 9% every year.In 2019,an analysis report completed by the Chinese Center for Disease Control and Prevention?CCDC?and the Institute of Health Measurement and Evaluation?IHME?of Washington University also revealed that stroke have become one of the top three chronic diseases causes premature deaths for Chinese,and 54% of recurrent patients will have disability.S-adenosyl homocysteine is the intermediate product of the methionine cycle.In recent years,it has attracted attention as a risk factor for cardiovascular and cerebrovascular diseases that is more sensitive than homocysteine.Studies have found that increasing SAH levels can increase the risk of cerebral venous thrombosis,but its mechanism and its impact on the occurrence of ischemic stroke still need to be proved by research.This study focused on the main initiating link of ischemic stroke—cerebral vascular endothelial cell injury to research.We establish a 3-Deazaadenosine?3-DZA?blockade of SAH to Hcy in the methionine cycle on b End.3,and study the molecular mechanism of its effect.This study is of great significance for further understanding the role of SAH in ischemic stroke,promoting stroke prevention and diagnosis,improving the quality of life of stroke patients and high-risk populations,and reducing the social burden caused by stroke.Part I Establishment of SAH induced apoptosis model of b End.3.Objective: By using 3-DZA to block the conversion of S-adenosyl homocysteine to homocysteine,to establish an apoptosis of b End.3.Method: CCK-8 method was used to detect the viability of b End3 to determine the optimal time and concentration to induce injury.And further determine using flow cytometry to detected cell apoptosis.Result: 3-DZA at concentrations of 0,100,200,400,600,800,1000,1500,and 2000 ?mol/L were used to intervene b End3 cells for 0,24,and 48 hours,respectively.With the increase of treatment time,b End.3 viability gradually decreased?P <0.05?.When treated with 3-DZA at a concentration of 200-400 ?mol/L for 48 h,the cell survival rate caused was moderate.Therefore,100,200,400,and 600 ?mol/L 3-DZA was further selected to perform stability experiments on b End3.The results showed that 3-DZA?200 ?mol/L could reduce the survival rate of b End3 cells?P <0.05?;when 3-DZA ?400 ?mol/L,the survival rate of b End3 cells decreased significantly?P <0.01?.The cell damage stability is good,which is suitable for establishing a damage model and used for subsequent research.Flow cytometry results showed that the apoptosis rate of b End3 was?0.63 ± 0.061?%.After treatment with 3-DZA,the apoptosis rate increased to?1.03 ± 0.067?% ^{3.12 ± 0.040}%.It increased with the increase of 3-DZA concentration,and the difference between any two groups was statistically significant?P <0.01?.Conclusion: The 3-DZA-induced increase in SAH induced b End3 apoptosis model is stable,fast,and simple,which can be further used in subsequent experimental research.Part ? Effects of SAH on b End3 apoptosis and its molecular mechanism.Objective: To evaluate the effect of 3-DZA-induced increase in SAH on apoptosis of b End3 through cell biology methods,and to explore the molecular mechanism of b End3 apoptosis.Method: After the b End3 cells were interfered with 3-DZA at different concentrations for 48 h,the reactive oxygen species?ROS?levels were detected using an active oxygen detection kit;Western Blot was used to detect the expression levels of bcl-2,bax,caspase-9,and caspase-3.Result: Compared with the control group,the ROS level of b End3 in the 3-DZA intervention group was increased with 300 ?mol/L and 400 ?mol/L as the priority?P <0.01?.Western-blot results showed that compared with the control group,400 ?mol/L 3-DZA interfered with the expression of bcl-2 gene in b End3 significantly?P <0.05?,and there was no significant difference between the other intervention groups and the control group.The expression levels of bax,caspase-9 and Cleaved caspase-3 were significantly increased in each intervention group?P <0.01?.Conclusion: The mechanism of 3-DZA-induced b End3 apoptosis is through improving ROS levels of cells,thereby regulating mitochondrial-related apoptosis signaling pathways,down-regulating bcl-2 expression,up-regulating the expression of bax,Cleaved caspase-3,and caspase-9 to promote mitochondria apoptotic pathway,eventually lead to apoptosis of b End3.