Phb In AngⅡ H9C2 Cardiac Muscle Cells Induced By Oxidative Stress And Apoptosis Of The Role Of Research

At present,about 550,000 people die of sudden cardiac death in China every year,and thousands of people die of sudden cardiac death on average every day.Sudden death is happening more frequently and at younger ages.Patients after acute myocardial infarction face a higher risk of sudden cardiac death,and the risk of sudden cardiac death is highest within 1 month after myocardial infarction.Insufficiency of blood supply to the left ventricle caused by coronary artery closure leads to severe and persistent hypoxia and ischemia of cardiomyocytes,and finally leads to myocardial infarction.The apoptosis of cardiomyocytes plays an important role in the development of cardiovascular diseases.A large number of studies have shown that the production of reactive oxygen species(ROS)induced by the renin-angiotensin system(RAS)is related to this pathological process.Increased activity of the RAS system is common in chronic heart failure and can act as a marker for further disease progression.Angiotensin 2(AngⅡ)has been shown to increase intracellular ROS and oxidative stress as well as the activity and expression of NADPH oxidase,leading to an increase in H9C2 cell apoptosis index.Human eukaryotic represtin(PHB for short)is a membrane protein that is very specific for different cell localization.It is involved in a variety of cellular functions,including energy metabolism,proliferation,Overexpression of PHB induces cell resistance to various stimuli through the mitochondrial apoptotic pathway,while knockdown of PHB increases the sensitivity to apoptotic stimuli.Mitochondria are the main source of ROS,and the PHB complex is involved in the regulation of ROS.The overexpression of PHB in cardiomyocytes can protect cells from the effects of oxidative stress-induced mitochondrial apoptosis.apoptosis,and aging.Objective: In this study,the expression of AT1 R and AT2 R in oxidative stress and apoptosis induced by AngⅡ was investigated,and whether PHB could play a protective role in oxidative stress and apoptosis induced by AngⅡ and whether it could stimulate the beneficial effect of AT2RMethods: this study used AngⅡ induced apoptosis and oxidative stress cell model is established,and by observation of cut on PHB gene H9C2 cardiomyocytes apoptosis protein,ROS levels and cell apoptosis.First determined by CCK 8 experimental AngⅡmodeling concentration for 10 μmol/L.Validation with 10 μmol/L AngⅡ processing cell apoptosis protein and oxidation index has difference compared with control subjects.Proved by AngⅡ oxidatie stress and apoptosis model was established successfully.In the transient transfection method,Lipofectamin2000 was used as the transfection reagent to transfect the constructed overexpressed p HB plasmid into H9C2 cardiomyocytes.The real-time quantitative reverse transcription PCR assay was used to detect whether the target gene was successfully transferred and transcripted.Lipofectamin2000 was also used as the transfection reagent to transfer si RNA into H9C2 cardiomyocytes.Real-time quantitative reverse transcription PCR assay was used to detect whether si RNA was transfected into cells and could inhibit the expression of target genes,Detected by western blot experiments AngⅡ induced group AT1 R expression is higher,and higher AT2 R expression is not very obvious,explain AngⅡ of induced myocardial cells mainly activated the AT1 R receptor pathway,trigger apoptosis.However,the expression of AT1 R was decreased and the expression of AT2 R was increased in myocardial cells treated with upregulated PHB gene,while the expression of AT2 R was increased while the expression of AT2 R was increased while the expression of AT2 R was decreased while the expression of AT2 R was increased while the expression of AT2 R was increased while the expression of AT2 R was increased while the expression of AT2 R was decreased with the downregulation of PHB gene,which proved that upregulated PHB gene could activate the AT2 R receptor pathway and inhibit the activation of AT1 R,and the down-regulation of PHB gene enhanced the activation of AT1 R receptor and reduced the expression of AT2 R.Then it was divided into up-regulated PHB gene and down-regulated PHB gene for subsequent experiments.Increase group is divided into control group,add AngⅡ group,raised the PHB gene AngⅡ group,raised the PHB genome.The transfected cells were screened out for further culture;The expressions of Nrf2 and Nox2,as well as the expressions of apoptotic proteins Caspase9,Caspase3,Bax and anti-apoptotic protein Bcl2 were detected by Western blot.To prove that the up-regulation of PHB gene can reduce the oxidative stress response of cardiomyocytes by enhancing the activity of antioxidant response elements and inhibiting the NADPH oxidase NOX2,reduce the apoptosis of cardiomyocytes,and protect cardiomyocytes by inhibiting apoptosis proteins and activating antiapoptotic proteins.ROS content in each group was detected by reactive oxygen detection kit.To prove up PHB gene indeed reduce the active oxygen content in the myocardial cells,increase the AngⅡ induced myocardial cell’s ability to fight ROS damage.TUNEL apoptosis detection kit each cell apoptosis,rise from cellular level directly proved that PHB gene can reduce AngⅡ induction of apoptosis;According to statistical figure analysis experimental results raise PHB genes on AngⅡ induced myocardial cell apoptosis and the effects of oxidative stress.Cut set is divided into control group,add AngⅡ group,cut PHB gene AngⅡ group,the lower the PHB genome.The expressions of Nrf2 and Nox2,as well as the apoptotic proteins Caspase9,Caspase3,Bax and anti-apoptotic protein Bcl2 were detected by Western blot.To prove that the downregulation of PHB gene increased the expression of NADPH oxidase Nox2 and inhibited the activity of antioxidant reaction element Nrf2,thus increasing the oxidative stress level of cardiomyocytes.By reducing the activity of anti-apoptotic protein Bcl2,the expression of apoptotic proteins Caspase9,Caspase3 and Bax increased the apoptosis of cells.ROS content in each group was detected by reactive oxygen detection kit.This indicated that the down-regulation of PHB gene triggered the production of ROS and increased the damage of cells.The TUNEL apoptosis detection kit was used to detect the apoptosis of cells in each group.Also proved that cut from a cellular level of PHB gene increased cell apoptosis,and cut PHB in gene and AngⅡ induced under the dual role of cell apoptosis necrosis number more faster.To make statistical figure cut PHB gene analysis of AngⅡinduced myocardial cell apoptosis and the effects of oxidative stress.Results: the CCK 8 experiments AngⅡ concentration for 10 μmol/L when the cell survival rate is about 50%;In the real-time quantitative reverse transcription PCR experiment,the m RNA level of the transfected overexpressed p HB plasmid group was significantly higher than that of the control group,and the m RNA expression was the highest when the ratio of plasmid to transfection reagent was 6:6.Compared with the control group,the m RNA level of PHB gene in the si RNA transfected group was significantly decreased,and the expression of the number 787 group was the lowest.Western blot experiments AngⅡ induced myocardial cells AT1 R expression is significantly higher than the control group,the AT2 R slightly higher than the control group,raised the PHB genome AT1 R myocardial cells significantly below add AngⅡgroup,significantly better than AT2 R add AngⅡ group.Cut PHB AT1 R genome myocardial cells were significantly higher than those with AngⅡ group,AT2 R also significantly lower than the added AngⅡ group;The myocardial cell apoptosis proteins induced by AngⅡ Caspase9,Caspase3,Bax is significantly higher than the control group,Bcl2 antiapoptotic proteins is lower than the control group.The oxidative stress index NOX2 was significantly higher than that of the control group,and the anti-oxidation reaction element NRF2 was decreased.And raised the PHB gene after add AngⅡ group of myocardial cell apoptosis protein Caspase9,Caspase3,Bax below AngⅡ group,Bcl2 above plus AngⅡ group,Nox2 expression below add AngⅡ group,significantly better than Nrf2 add AngⅡ group.The expression trend of down-regulation group was opposite to up-regulation group.ROS active oxygen detection and AngⅡ group was obviously higher than that of control group,raised the PHB gene Ang add after the content of ROSⅡ group was obviously lower and AngⅡ group.Cut PHB group and ROS levels higher than the control group,after cut PHB gene add AngⅡ group of ROS has the highest level;TUNEL experiment china-canada Ang apoptosis number Ⅱ group was obviously higher than that of control group,raised after the PHB gene plus AngⅡ group cells apoptosis than AngⅡ group.Cut the PHB genome apoptosis quantity will increase a lot,and the cut after the PHB gene add AngⅡ induction,cell apoptosis necrosis number more Conclusion: At1 R plays a major role in AngⅡ-induced cell apoptosis and oxidative stress.Upregulation of PHB gene can reduce AngⅡ-induced oxidative stress and apoptosis of cardiomyocytes,which can protect the myocardium,and stimulate the beneficial effect of At2 R to reduce cell apoptosis.