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目的探讨姜黄素对H9c2心肌细胞氧化应激损伤的保护作用及其机制。方法首先用5～15μmol.L-1姜黄素处理H9c2心肌细胞12 h,双波长法检测血红素加氧酶-1(HO-1)活性。其次用400μmol.L-1过氧化氢(H2O2)处理H9c2心肌细胞3 h,以建立细胞氧化应激损伤模型,给予15μmol.L-1姜黄素预处理12 h,或在姜黄素预处理1 h前给予HO-1抑制剂Znpp-Ⅸ10μmol.L-1共孵育。四甲基偶氮唑蓝(MTT)法检测细胞存活率;采用流式细胞术Annexin-V FITC/PI染色和Caspase-3活性检测评估细胞凋亡;硫代巴比妥酸显色法检测细胞丙二醛(MDA)水平,氮蓝四唑显色法检测细胞总超氧化物歧化酶(SOD)活性;采用Western blot法检测细胞核因子-κB(NF-κB)蛋白表达水平。结果 HO-1活性检测显示,与对照组比较,5～15μmol.L-1姜黄素能够呈浓度依赖性地诱导HO-1活性增加。MTT和细胞凋亡检测显示,与H2O2组比较,15μmol.L-1姜黄素能够显著上调细胞存活率,下调细胞凋亡率和Caspase-3活性。细胞MDA水平和总SOD活性检测显示,15μmol.L-1姜黄素能够显著降低细胞MDA水平,提高总SOD活性。此外,与H2O2组相比,15μmol.L-1姜黄素能显著下调细胞NF-κB蛋白表达水平。HO-1抑制剂Znpp-Ⅸ能够部分逆转姜黄素的上述作用。结论姜黄素可能通过上调HO-1活性,抑制NF-κB激活以及维持细胞氧化还原平衡状态,对抗H2O2诱导的H9c2心肌细胞氧化应激损伤。
Objective To investigate the protective effect of curcumin on oxidative stress injury of H9c2 cardiomyocytes and its mechanism. Methods H9c2 cardiomyocytes were treated with 5 ~ 15μmol.L-1 curcumin for 12 hours, and the activity of heme oxygenase-1 (HO-1) was detected by dual-wavelength method. H9c2 cardiomyocytes were treated with 400μmol.L-1 hydrogen peroxide (H2O2) for 3 hours to establish a model of cellular oxidative stress injury, pretreated with 15μmol.L-1 curcumin for 12 hours, or treated with curcumin for 1 hour Pre-given HO-1 inhibitor Znpp-Ⅸ 10μmol.L-1 co-incubation. Cell viability was detected by MTT assay. Cell apoptosis was assessed by flow cytometry with Annexin-V FITC / PI staining and Caspase-3 activity assay. Cell viability was measured by thiobarbituric acid assay (MDA) and the activity of superoxide dismutase (SOD) in the cells were detected by the method of nitrogen blue tetrazolium. The expression of nuclear factor-κB (NF-κB) protein was detected by Western blot. Results HO-1 activity assay showed that 5 ~ 15μmol.L-1 curcumin induced HO-1 activity in a concentration-dependent manner compared with the control group. MTT and apoptosis assay showed that compared with H2O2 group, 15μmol.L-1 curcumin significantly increased cell viability and down-regulated the apoptosis rate and Caspase-3 activity. Cell MDA levels and total SOD activity test showed that 15μmol.L-1 curcumin can significantly reduce cell MDA levels, increase the total SOD activity. In addition, compared with H2O2 group, 15μmol.L-1 curcumin significantly downregulated the expression of NF-κB protein. The HO-1 inhibitor Znpp-IX was able to partially reverse the above effects of curcumin. Conclusion Curcumin may antagonize H2O2-induced oxidative stress injury in H9c2 cardiomyocytes by up-regulating HO-1 activity, inhibiting NF-κB activation and maintaining cellular redox balance.