姜黄素下调miR-21-5p减轻缺氧/复氧诱导的H9C2心肌细胞凋亡

Curcumin Down-regulated miR-21-5p to Reduce Apoptosis of H9C2 Cardiomyocytes Induced by Hypoxia/Reoxygenation

【目的】探讨姜黄素通过调控miR-21-5p对缺氧/复氧诱导的H9C2心肌细胞凋亡的影响。【方法】观察姜黄素对缺血/复氧心肌细胞的影响:四甲基偶氮唑盐(MTT)法检测细胞增殖活力;乳酸脱氢酶(LDH)试剂盒检测LDH活性;Hoechst 33258染色法检测细胞凋亡;蛋白免疫印迹(Western Blot)法检测Bcl-2、Bax和caspase-3蛋白表达水平;逆转录—聚合酶链反应(RT-PCR)检测miR-21-5p、Bcl-2 mRNA水平。荧光素酶报告实验检测miR-21-5p与Bcl-2靶向关系。采用RT-PCR法和Hoechst 33258染色法观察姜黄素对miR-21-5p mimic转染心肌细胞miR-21-5p、Bcl-2 mRNA水平及凋亡的影响。采用Western Blot法和Hoechst 33258染色法观察姜黄素对si-Bcl-2转染心肌细胞Bcl-2蛋白表达及凋亡的影响。【结果】与空白对照组比较,缺血/复氧组细胞活力显著降低,LDH活性,细胞凋亡率,caspase-3、Bax蛋白表达水平,miR-21-5p mRNA表达水平显著升高,Bcl-2蛋白和mRNA表达水平显著降低(均P<0.01)。与缺血/复氧组比较,姜黄素5、10μmol/L组细胞活力显著升高,LDH活性,细胞凋亡率,caspase-3及Bax蛋白表达水平,miR-21-5p mRNA表达水平显著降低,Bcl-2蛋白和mRNA表达水平显著升高(均P<0.01)。荧光素酶报告实验表明miR-21-5p和Bcl-2存在直接靶向作用关系。姜黄素10μmol/L可抑制缺血/复氧条件下miR-21-5p mimic和si-Bcl-2转染的H9C2细胞凋亡。【结论】姜黄素可通过下调miR-21-5p减轻缺氧/复氧诱导的H9C2心肌细胞凋亡。

Objective To explore the effect of curcumin on ischemia/reoxygenation-induced H9C2 cardiomyocytes by regulating miR-21-5p.Methods The effects of curcumin on ischemia/reoxygenation-induced H9C2 cardiomyocytes were observed as follows:The cell viability was detected by-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide(MTT)assay,lactate dehydrogenase(LDH)activity was detected with LDH kit,cell apoptosis was determined by Hoechst 33258 staining,protein expression of Bcl-2,Bax and caspase-3 were detected by Western blotting assay,mRNA expression of miR-21-5p and Bcl-2 was detected by reverse transcription-polymerase chain reaction(RT-PCR).The targeting relationship between miR-21-5p and Bcl-2 was detected by phototinase reporter assay.The effects of curcumin on the mRNA expression of miR-21-5p and Bcl-2 in miR-21-5p mimic transfected ischemia/reoxygenation-induced H9C2 cardiomyocytes and cell apoptosis were observed by RT-PCR and Hoechst 33258 staining method,respectively.The effects of curcumin on protein expression of Bcl-2 in si-Bcl-2 transfected ischemia/reoxygenation-induced H9C2 cardiomyocytes and cell apoptosis were observed by Western blotting assay and Hoechst 33258 staining method,respectively.Results Compared with the blank control group,the cell vitality was reduced,LDH activity,apoptosis rate,caspase-3 and Bax protein expression levels,miR-21-5p mRNA expression level were increased,and Bcl-2 protein expression level was decreased in the ischemic/reoxygenation group(all P<0.01). Compared with the ischemic/reoxygenation group,the cell vitality was increased,LDH activity,apoptosis rate,caspase-3 and Bax proteinexpression levels,miR-21-5p mRNA expression level were decreased,and Bcl-2 protein expression level wasincreased in curcumin 5,10 μmol/L group(all P<0.01). There were direct targeting relationship between miR-21-5p and Bcl-2. The apoptosis of miR-21-5p mimic or si-Bcl-2 transfected ischemia/reoxygenation-inducedH9C2 cardiomyocytes was inhibited by 10 μmol/L of curcumin. Conclusion Curcumin reduces H9C2cardiomyocytes apoptosis induced by hypoxia/reoxygenation through down-regulating miR-21-5p.