NADPH Oxidase 2 Mediates Necroptosis In H9C2 Cells And Its Underlying Mechanism

Cardiovascular disease(CVD)is a serious threat to human health,and its incidence rate and mortality are increasing year by year.Heart failure(HF)is the terminal stage of cardiovascular disease.Oxidative stress and myocardial cell necrosis apoptosis coexist in the development of heart failure.However,whether NADPH oxidase 2(Nox2)can mediate drug and hypoxia induced necroptosis of cardiomyocytes and its mechanism have not been fully clarified.Therefore,the purpose of this study is to investigate the role and mechanism of Nox2 in the necroptosis of myocardial cells induced by drugs and hypoxia.Firstly,we cultured H9C2 cells in vitro,and established a myocardial cell injury model with the intervention of tumor necrosis factor-α(TNF-α),Norepinephrine(NE)and Hypoxia/reoxygenation(H/R),drugs and hypoxia can induce oxidative stress and necroptosis of H9C2 cells;Secondly,investigate whether silencing the Nox2 gene can affect the level of necroptosis in normal H9C2 cells;Finally,in TNF-α,NE and H/R induced myocardial cell injury model,the role and mechanism of silencing Nox2 gene in H9C2 cell necroptosis were observed.This paper consists of three parts:Part I: Effects of TNF-α,NE and H/R Intervention on oxidative stress and necroptosis in H9C2 cardiomyocytes Objective:Exploring TNF-α,NE and H/R induce oxidative stress and necroptosis in H9C2 cells.The isolated rat H9C2 myocardial cells were resuscitated,frozen and subcultured,and then TNF was applied to the cells in 6-well plates TNF-α、 NE drug intervention and grouping: control group(Control group): adding 2000 ul of complete culture medium;TNF-α Group(10 ng/ml TNF-α):Join TNF-α Working fluid 20ul+1980ul complete culture medium;The NE 50 group(50umol/L NE)was added with 16.9ul NE+1983.1ul complete culture medium;NE 100 group(100 umol/L NE): Add 33.8ul NE+1966.2ul complete culture medium.Construct a 1% hypoxia/reoxygenation(H/R)model of H9C2 cells and group them into control group(Control group): add 2000 ul of complete culture medium;H/R 1h group: reoxygenation after 1 hour of hypoxia treatment;H/R 6h group:reoxygenation after 6 hours of hypoxia treatment.After the establishment of myocardial cell injury model,the activity of H9C2 cells was detected by CCK-8 method,the level of LDH in cells was detected by LDH kit,the expression of Nox2,RIP1 and RIP3 m RNA was detected by RT-PCR,and the expression of Nox2 and RIP3 protein was detected by Western blot.Results:(1)Compared with the control group,TNF-α,Intervention with NE and H/R for 24 hours can reduce cell survival rate in H9C2 cells;The survival rate of cells in the NE 100 group decreased compared to the NE 50 group,and the results showed that an increase in NE concentration had adverse effects on cells;At H/R,the cell survival rate decreased after 6 hours of hypoxia compared to 1 hour.The results showed that the longer the cell hypoxia time,the lower the cell survival rate;(2)Compared with the control group,TNF-α,Intervention with NE and H/R can significantly increase intracellular LDH levels in H9C2 cells.The LDH levels in the NE100 group were higher than those in the NE 50 group,and the results showed that an increase in NE concentration would exacerbate cell damage;The LDH level of cells increased after 6 hours of hypoxia at H/R compared to 1 hour,indicating that prolonged cell hypoxia time would exacerbate cell damage;Methods:(3)Compared with the control group,TNF-α,NE and H/R intervention can significantly upregulate the expression of Nox2 m RNA and Nox2 protein in H9C2 cells,indicating TNF-α,NE and H/R intervention can activate NADPH oxidase and induce oxidative stress in H9C2 cells;(4)TNF-α,NE and H/R intervention in H9C2 cells can upregulate the expression of necroptosis related RIP1 m RNA,RIP3 m RNA,and RIP3 protein,indicating TNF-α、NE and H/R induced the increase of myocardial necrosis apoptosis mediated by RIP1-RIP3.Part 2: Effect of NADPH oxidase 2 gene silencing on necroptosis of H9C2 cells Objective:Effect of NADPH oxidase 2 gene silencing on necrotizing apoptosis of H9C2 cells.Methods:The efficiency of Nox2 gene silencing was verified by Real time PCR and Western blot methods,respectively,to detect the expression of Nox2,RIP3 m RNA and Nox2,RIP3 protein.Results:Silencing the NADPH oxidase 2 gene can reduce the m RNA and protein expression of H9C2 RIP3,suggesting that silencing the Nox2 gene may interfere with RIP3 expression,thereby reducing the level of necroptosis.Part III: Effect of NADPH oxidase 2 gene silencing on necroptosis of myocardial cells induced by TNF-α,NE and H/R.Objective:Based on the model of myocardial cell injury intervened by TNF-α,NE and H/R,the role and mechanism of silencing Nox2 gene in necrotizing apoptosis of H9C2 cells were observed.Methods:In vitro rat H9C2 myocardial cells were used for cell resuscitation,cryopreservation,subculture,after the Nox2 gene was silenced by si Nox2,the cells were intervened by TNF-α and NE drugs,and H/R models were constructed and grouped.The experimental groups were as follows:(1)Control group;(2)NE 50 group;(3)NE 100 group;(4)TNF-αGroup;(5)H/R 1h group;(6)H/R 6h group(7)si Nox2 group;(8)NE 50+si Nox2 group;(9)NE 100+si Nox2 group;(10)TNF-α+ Si Nox2 group;(11)H/R 1h+si Nox2 group;(12)H/R 6h+si Nox2 group;Nox2 si RNA(1)～(6)directly using TNF-α,NE and H/R intervened in H9C2 cells,and Nox2 si RNA was transfected into H9C2 cells in groups(7)to(12),followed by TNF-α,NE and H/R intervened in H9C2 cells,and the expression of Nox2 m RNA,RIP3 m RNA,and RIP3 protein in each group were detected by RT-PCR and Western blot.Results:The results of this study indicate that silencing the Nox2 gene can effectively inhibit TNF-α,Upregulation of Nox2,RIP3 m RNA,and RIP3 protein expression after NE and H/R intervention in H9C2 cells,reducing TNF-α.The injury of H9C2 cells caused by NE and H/R intervention suggests that Nox2 is involved in the necroptosis of myocardial cells and reduces the level of necroptosis,and this process is mediated by RIP1-RIP3 pathway.Therefore,silencing the Nox2 gene can to some extent inhibit the upregulation of RIP3 and protein expression,a marker of necroptosis in H9C2 cells,and reduce the level of necroptosis.Nox2 gene silencing plays a certain protective role in drug and hypoxia induced necroptosis in H9C2 cells.Oxidative stress is closely related to necroptosis.Nox2 mediates necroptosis of cardiomyocytes through RIP1-RIP3 pathway.Conclusion:1.TNF-α,NE and H/R intervention,H9C2 cells undergo oxidative stress and RIP1-RIP3 mediated necroptosis levels increase,and the oxidative stress and RIP1-RIP3 signaling pathways are activated;2.Silencing the Nox2 gene can interfere with the expression of RIP3,and Nox2 is correlated with RIP1-RIP3 mediated necroptosis;3.Silencing the Nox2 gene significantly inhibits TNF-α,NE and H/R intervened in the expression of RIP3,a marker of necroptosis of H9C2 cells,and Nox2,a key enzyme of oxidative stress,mediated necroptosis of cardiomyocytes through RIP1-RIP3 pathway.