【摘要】 目的探讨叔丁基对苯二酚(t BHQ)对化学性低氧模型剂氯化钴(Co Cl2)诱发H9c2心肌细胞氧化应激损伤的影响及其作用机制。方法将细胞分成4组:正常对照组、Co Cl2组(500μmol/L Co Cl2)、DMSO(二甲基亚砜)+Co Cl2(0.005%DMSO+500μmol/L Co Cl2)组、t BHQ+Co Cl2(50μmol/L t BHQ+500μmol/L Co Cl2)组;MTT法检测细胞生长活性;DCFH-DA探针测定细胞内ROS含量;Western Blot检测细胞内cleaved-caspase-3和Nrf2及其调控的II相解毒酶NAD(P)H:醌氧化还原酶1(NQO1)和抗氧化酶γ-谷氨酰半胱氨酸连接酶(GCLC)蛋白的表达;应用Real-time PCR检测NQO1和GCLC m RNA表达。结果 Co Cl2组与正常对照组相比,细胞活性明显降低,细胞内的cleaved-caspase-3蛋白表达显著升高,ROS含量增高;DMSO+Co Cl2组与Co Cl2组无差异;t BHQ预处理能够显著降低Co Cl2对H9c2心肌细胞氧化应激损伤。与Co Cl2组相比,t BHQ预处理能够显著增加细胞内的Nrf2蛋白水平,且细胞内NQO1和GCLC的m RNA及蛋白表达均明显高于Co Cl2组。结论 t BHQ能够诱导H9c2心肌细胞内Nrf2的蛋白表达,进而上调其下游II相解毒酶NQO1和抗氧化酶GCLC的转录活性及蛋白表达,拮抗Co Cl2诱发的化学性缺氧对H9c2心肌细胞的氧化应激损伤。更多还原

【Abstract】 Objective To investigate the effect of tert-butylhydroquinone(t BHQ) on the oxidative stress damages induced by chemical hypoxia-mimetic agent(cobalt chloride, Co Cl2) in H9c2 myocardial cells as well as its mechanisms. Methods The H9c2 cells were divided into 4 groups: normal control group, Co Cl2 group(induced by 500 μmol/L Co Cl2), DMSO+Co Cl2 group(induced by 0.005%DMSO + 500 μmol/L Co Cl2), t BHQ+Co Cl2 group(induced by 50 mol/L t BHQ +500 mol/L Co Cl2). The cell growth viability was detected by MTT assay, and the content of ROS in cells was measured by the DCFH-DA probe. The protein expression of cleavedcaspase-3, Nrf2 and its regulation of phase II detoxifying enzyme NAD(P) H: quinone oxidoreductase 1(NQO1) and antioxidant enzymes γ-glutamate cysteine ligase catalytic subunit(GCLC) were determined with Western Blot. The m RNA expression of NQO1 and GCLC were measured by real-time polymerase chain reaction(PCR). Results In comparison with those in the normal control group, the cell viability decreased markedly, and the expression of cleaved-caspase-3 protein and ROS content increased significantly in the Co Cl2 group. There were no significant differences between DMSO+Co Cl2 group and Co Cl2 group. Pretreatment with t BHQ could markedly inhibit Co Cl2 induced-oxidative stress damages in H9c2 cells. As compared with Co Cl2 group, t BHQ pretreatment remarkably increased the level of Nrf2 protein, and the expression levels of NQO1 and GCLC m RNA and protein were significantly elevated in the Co Cl2 group. Conclusion TBHQ may protect H9c2 myocardial cells against Co Cl2-induced oxidative stress injuries by inducing the expression of Nrf2 protein expression, activating the transcription of the downstream phase II detoxifying enzymes NQO1 and antioxidant enzymes GCLC and up-regulating the protein expression of NQO1 and GCLC in H9c2 cells.更多还原