风轮菜总黄酮联合miR-702-5p抑制物对缺氧/复氧诱导的心肌细胞损伤的影响

Effects of total flavonoids of Clinopodium chinense combined with miR-702-5p inhibitor on myocardial cell injury induced by hypoxia/reoxygenation

目的研究风轮菜总黄酮联合miR-702-5p抑制物对缺氧/复氧诱导的心肌细胞损伤的影响。方法实验设置对照组(Con,不作任何处理)、缺氧/复氧组(H/R,缺氧6 h,复氧3 h),H/R+药物-1组、H/R+药物-2组、H/R+药物-3组(6.25μg/ml、12.5μg/ml、25μg/ml的风轮菜总黄酮+缺氧/复氧处理)、H/R+anti-miR-NC组(转染anti-miR-NC+缺氧/复氧处理)、H/R+anti-miR-702-5p组(转染anti-miR-702-5p+缺氧/复氧处理)、H/R+药物-3+anti-miR-NC组(转染anti-miR-NC+25μg/ml风轮菜总黄酮+缺氧/复氧处理)、H/R+药物-3+anti-miR-702-5p组(转染anti-miR-702-5p+25μg/ml风轮菜总黄酮+缺氧/复氧处理)。蛋白质印迹(Western blot)法检测细胞周期蛋白依赖性激酶抑制剂1A(P21)、半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)蛋白表达;细胞计数试剂盒8(CCK-8)检测细胞存活率;流式细胞术检测细胞凋亡;试剂盒检测丙二醛(MDA)含量及乳酸脱氢酶(LDH)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)活性;实时荧光定量PCR(RT-qPCR)检测miR-702-5p表达水平。结果缺氧/复氧诱导的心肌细胞中P21、Caspase-3表达水平升高,细胞存活率降低,细胞凋亡率升高,MDA含量升高,LDH活性升高,CAT、SOD活性降低。不同浓度风轮菜总黄酮处理后P21、Caspase-3表达水平降低,细胞存活率升高,细胞凋亡率降低,MDA含量降低,LDH活性降低,CAT、SOD活性升高。抑制miR-702-5p表达可抑制缺氧/复氧诱导的心肌细胞损伤。风轮菜总黄酮联合miR-702-5p抑制剂可显著提高细胞存活率,抑制细胞凋亡和氧化应激反应。结论风轮菜总黄酮联合miR-702-5p抑制物可保护心肌细胞免受缺氧/复氧引起的损伤。

Objective To investigate the effects of total flavonoids of Clinopodium chinense combined with miR-702-5p inhibitor on myocardial cell injury induced by hypoxia/reoxygenation.Methods The experiment consisted of the control(Con)group(without any treatment),the hypoxia/reoxygenation(H/R)group(hypoxia for 6 h and reoxygenation for 3 h),the H/R+drug-1 group,the H/R+drug-2 group,H/R+Drug-3 group(6.25μg/ml,12.5μg/ml,25μg/ml total flavonoids of Clinopodium chinense+hypoxia/reoxygenation treatment),H/R+anti-miR-NC group(transfecting anti-miR-NC+hypoxia/reoxygenation treatment),H/R+anti-miR-702-5p group(transfecting anti-miR-702-5p+hypoxia/reoxygenation treatment),H/R+drug-3+anti-miR-NC group(transfecting anti-miR-NC+25μg/ml total flavonoids of Clinopodium chinense+hypoxia/reoxygenation treatment),H/R+drug-3+anti-miR-702-5p group(transfecting anti-miR-702-5p+25μg/ml total flavonoids of Clinopodium chinense+hypoxia/Reoxygenation).Western blot was used to detect the expression of cyclin-dependent kinase inhibitor 1A(P21),cysteine aspartase-3(Caspase-3)protein expression;cell counting kit 8(CCK-8)was used to detect cell survival rate;flow cytometry to detect apoptosis;kit was used to detect malondialdehyde(MDA)content and activity of lactate dehydrogenase(LDH),catalase(CAT),superoxide dismutase(SOD);real-time quantitative PCR(RT-qPCR)was used to detect miR-702-5p expression.Results In cardiomyocytes induced by hypoxia/reoxygenation,the expressions of P21 and Caspase-3 were increased,cell survival rate was decreased,cell apoptosis rate was increased,MDA content was increased,LDH activity was increased,and CAT and SOD activity were decreased.After treatment with different concentrations of total flavonoids of Clinopodium chinense,the expressions of P21 and Caspase-3 decreased,the cell survival rate was increased,the cell apoptosis rate was decreased,the MDA content was decreased,LDH activity was decreased,and CAT and SOD activity was increased.Inhibition of miR-702-5p expression can inhibit myocardial cell injury induced by hypoxia/reoxygenation.The total flavonoids of Clinopodium chinense combined with miR-702-5p inhibitor can significantly improve cell survival rate,inhibit apoptosis and oxidative stress response.Conclusion The total flavonoids of Clinopodium chinense combined with miR-702-5p inhibitor can protect myocardial cells from damage caused by hypoxia/reoxygenation.