血红素氧和酶-1在原代心肌细胞缺氧/复氧损伤中的表达及意义

The expression and significance of heme oxygenase-1 in primary myocardial cells during hypoxia/reoxygenation injury

目的 探讨血红素氧和酶-1（HO-1）在姜黄素对原代心肌细胞缺氧/复氧（H/R）损伤后处理中的表达及意义.方法 实验分为5组：正常对照组、姜黄素组（10μmol/L）、H/R组、姜黄素（10μmol/L）＋ H/R组、姜黄素＋ZnPP-Ⅸ（HO-1抑制剂）＋H/R组.检测细胞活性及培养液中乳酸脱氢酶（LDH）活性、丙二醛（MDA）浓度和超氧化物歧化酶（SOD）活性,评估细胞损伤情况;检测半胱氨酰天冬氨酸特异性蛋白酶（Caspase）-3凋亡酶活性;JC-1探针检测细胞膜电位;通过Western blot检测各组细胞HO-1、B细胞淋巴瘤/白血病-2相关X蛋白（bax）、B细胞淋巴瘤/白血病-2（bcl-2）表达.结果 与H/R组比较,姜黄素可以诱导HO-1和bcl-2的表达,抑制bax的表达,加入ZnPP-Ⅸ后,HO-1和bcl-2表达减弱,bax表达上调（P=0.001、0.006、0.007）.与对照组比较,H/R组线粒体膜电位显著降低（31±7）%比100%（P=0.004）,姜黄素后处理可以提高线粒体膜电位（75±16）%比（31±7）%（P=0.011）,而加入ZnPP-Ⅸ阻断了姜黄素对心肌细胞的保护作用（19±4）%比（75±16）%（P=0.001）.姜黄素同时具有增加心肌细胞活性和SOD活性,降低LDH、MDA浓度,及降低Caspase-3活性,降低细胞损伤、氧化应激损伤和凋亡.结论 姜黄素可通过诱导HO-1表达,减轻氧化应激及抗凋亡而具有心肌细胞保护作用,而HO-1抑制剂ZnPP-Ⅸ可以阻断姜黄素的心肌保护作用.

Objective To explore the expression and significance of heme oxygenase-1 （HO-1） on primary myocardial cells during hypoxia/reoxygenation （H/R） injury with curcumin post-treatment.Methods Five groups were set up in this experiment： normal control group;curcumin group （10 μmol/L）;H/R group;curcumin （10 μmol/L）＋H/R group;curcumin＋ZnPP-Ⅸ inhibitors （HO-1）＋H/R group.Cell viability,lactate dehydrogenase （LDH） and superoxide dismutase （SOD） activity,and malondialdehyde （MDA） concentration were measured to evaluate cell damage.Cysteinyl aspartate-specific protease （Caspase）-3 was detected.JC-1 probe was used to test cell membrane potential.Western blotting was used to detect the expression of B cell lymphoma/leukemia-2 associated X protein （bax）,HO-1 and B-cell lymphoma/leukemia-2 （bcl-2） proteins.Results As compared with H/R group,curcumin could induce the expression of HO-1 and bcl-2,inhibit the expression of bax,but after addition of ZnPP-Ⅸ,the expression of HO-1 and bcl-2 was decreased and the bax expression was up-regulated （P=0.001,0.006,0.007）.As compared with control group,the mitochondrial membrane potential in H/R group was decreased significantly （31±7）% vs.100% （P=0.004）,curcumin post-treatment could increase the mitochondrial membrane potential （75±16）% vs.（31±7）% （P=0.011）,and the addition of ZnPP-Ⅸ blocked the protective effects of curcumin on myocardial cells （19±4）% vs.（75±16）% （P=0.001）.Curcumin could increase the viability of myocardial cells and SOD activity,reduce LDH and MDA concentrations,and decline the Caspase-3 activity,which could alleviate cell injury,oxidative stress injury and apoptosis.Conclusion Curcumin can reduce oxidative stress and apoptosis by inducing by the HO-1 expression,which can protect the myocardial cells,and HO-1 inhibitor ZnPP-Ⅸ can block the myocardial protective effect of curcumin.