H2O2预处理对大鼠心肌细胞缺氧/复氧损伤的影响

摘要 目的 探讨H2O2预处理对大鼠心肌细胞缺氧/复氧引起H9C2细胞凋亡的影响。方法 大鼠心肌细胞系（H9C2）经体外培养扩增，使用对数生长期细胞做实验处理。分别使用0、5、10、20、50、100μmol/L浓度 H2O2处理H9C2细胞，流式细胞仪（FCM）、MTT法和免疫印迹法（Western blot法）检测不同浓度H2O2处理对H9C2细胞凋亡率、增殖活性和STAT3磷酸化水平的影响，以确定最佳H2O2预处理浓度。观察低浓度H2O2预处理对缺氧/复氧所引起细胞损伤的影响，细胞随机分为4组，①对照组：常规培养；②缺氧／复氧组：预先在 5％CO2，95%N2培养箱中培养 120min，复氧30min ；③H2O2预处理组：给予H2O2处理90min换液，24h后缺氧/复氧处理；④AG490+ H202组：在H2O2预处理前10min给予10μmol/L AG490处理，其余处理同H2O2预处理组。AnnexinV-FITC/PI双染法及流式细胞仪（FCM）检测细胞凋亡，MTT法检测H9C2细胞增殖活性，免疫印迹法（Western blot法）检测STAT3磷酸化水平。结果20μmol/L组的STAT3磷酸化水平明显高于其他各浓度组（P<0.01），心肌细胞凋亡率亦明显低于50μmol/L及100μmol/L浓度组（P<0.01），而其心肌细胞增殖活性及细胞凋亡率与5μmol/L及10μmol/L浓度组无明显差异； H2O2预处理能降低大鼠心肌细胞凋亡率（P<0.05）,增加心肌细胞活性（P<0.05）上调P- STAT3水平（P<0.05）。缺氧损伤后给予AG490处理可使H2O2预处理保护作用消失。结论20μmol/L H2O2预处理对心肌细胞缺氧/复氧损伤具有适应性保护作用。可能机制是激活 JAK2-STAT3通路发挥抑制细胞凋亡作用。

The effect of H2O2 pretreatment on mouse myocardial hypoxia/reoxygenation injury

Abstract Objective To investigate the H2O2 pretreatment on mouse myocardial hypoxia/reoxygenation caused apoptosis of H9C2 cells. Methods Mice cardiac cells (H9C2) cultured in vitro amplification, using the logarithmic growth phase cells to do the experimental treatment. Use respectively 0, 5, 10, 20, 50, 100 μ mol/L concentration H2O2 treatment H9C2 cells, flow cytometry ( FCM ), MTT and immunoblotting method ( Western blot ) detection of H2O2 with different concentration on H9C2 cell apoptosis rate, Proliferation activity and the STAT3 phosphorylation level effects, in order to determining the optimal concentration of H2O2 pretreatment. Watch the effects of low concentrations of H2O2 pretreatment on hypoxia/reoxygenation caused cell injury. Cells were divided into four groups namely control group, hypoxia/reoxygenation group, H2O2 pretreatment group, AG490 group.① Control group: conventional cultivation; ② hypoxia/reoxygenation group: pre in 5% CO2, 95%N2 incubator 120min, reoxygenation 30min; ③H2O2 pretreatment groups: pre-treatment given to determine the concentration of H2O2 90min for liquid 24h after hypoxia/ reoxygenation; ④AG490 group: give 10min before the H2O2 pretreatment 10μmol/L AG490 treatment, after treatment with H2O2 pretreatment group.AnnexinV-FITC / PI double staining method and flow cytometry (FCM) detection of apoptosis, MTT assay H9C2 cell proliferation activity, immunoblot (Western blot method) to detect STAT3 phosphorylation level. Results 20μmol/L group of STAT3 phosphorylation significantly higher than all other level of concentration group (P < 0.01), myocardial cell apoptosis rates were also significantly lower than 50 μ mol/L and 100 μ mol/L concentration group (P < 0.01), and the myocardial cell proliferation activity and cell apoptosis rate and 5 μ mol/L and 10 μ mol/L concentration group there were no obvious difference ； H2O2 pretreatment can reduce the small rat myocardial cells apoptosis (P <0.05), increased myocardial cell viability (P<0.05) increased P-STAT3 expression (P<0.05). Oxygen injury to AG490 after treatment can make the H2O2 pretreatment protection disappear.Conclusions 20μmol/L H2O2 pretreatment on myocardial hypoxia/reoxygenation injury adaptive protection. The possible mechanism is activated JAK2-STAT3 pathways play to the role of inhibiting cell apoptosis.