Nrf2-ARE通路在缺氧/吡那地尔后处理减轻大鼠心肌细胞缺氧复氧损伤中的作用

 目的：探讨核因子E2相关因子2（Nrf2）-抗氧化反应元件（ARE）通路在缺氧后处理和吡那地尔后处理减轻大鼠心肌细胞缺氧复氧损伤中的作用。方法：建立成年大鼠心肌细胞缺氧复氧模型，分离、培养心肌细胞，随机分为6组（n=8）：正常组（N组）、模型组（M组）、缺氧后处理组（IPO组）和不同浓度吡那地尔后处理组（P25组、P50组和P100组）。分离后的心肌细胞培养20 h后，除N组继续培养外，其余各组均缺氧45 min，复氧60 min。IPO组缺氧45 min后复氧、缺氧循环3次，每次各5 min，在继续复氧60 min；P25组、P50组和P100组缺氧45 min后分别以25、50和100 μmol/L吡那地尔处理5 min，复氧60 min。采用real-time PCR及Western blotting技术检测复氧末心肌细胞中Nrf2、血红素加氧酶1（HO-1）、NAD(P)H:醌氧化还原酶1（NQO1）和超氧化物歧化酶1（SOD1） mRNA及蛋白表达水平。结果：与N组比较，其它各组与Nrf2、NQO1、SOD1及HO-1的mRNA及蛋白质表达均降低（P＜0.05）；与M组比较，其它各组mRNA及蛋白质表达均显著增高（P＜0.05）；其中IPO组和P50组mRNA转录量显著高于P25组和P100组（P＜0.05）；P25组SOD1 mRNA显著高于P100组（P＜0.05）。IPO组和P50组Nrf2、SOD1和NQO1蛋白质表达显著高于P25和P100组（P＜0.05）；P50组HO-1蛋白质表达量显著高于IPO组、P25组和P100组（P＜0.05）；P25组HO-1和NQO1蛋白质表达显著高于P100组（P＜0.05）。结论：缺氧后处理和吡那地尔后处理可能通过激活Nrf2-ARE通路减轻大鼠心肌细胞缺氧复氧损伤。

Role of Nrf2-ARE signaling pathway in protective effect of hypoxia or pinacidil postconditioning against hypoxia-reoxygenation injury in adult rat cardiomyocytes

AIM：To investigate whether nuclear factor E2-related factor 2 (Nrf2)-antioxidant response element (ARE) signaling pathway is involved in the protective effect of hypoxia or pinacidil postconditioning against hypoxia-reoxygenation injury in adult rat cardiomyocytes. METHODS：Cardiomyocytes were isolated from the left ventricle of male adult Sprague-Dawley rats (250~300 g) by Langendorff perfusion and collagenase II digestion. The cells were randomly divided into six groups (n=8 in each group). The cells in N group were cultured for 22 h without any treatment. The cells in the other five groups were cultured for 20 h and then underwent 45 min of hypoxia followed by 60 min of reoxygenation (M group), three 5-min cycles of reoxygenation/hypoxia plus 60 min of reoxygenation (IPO group), or treatment with pinacidil (25, 50 and 100 μmol/L) for 5 min plus 60 min of reoxygenation (P25, P50 and P100 groups, respectively). The expression of Nrf2, NAD(P)H:quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO-1) and superoxide dismutase 1 (SOD1) at mRNA and protein levels was detected by real-time PCR and Western blotting, respectively. RESULTS：The mRNA and protein levels of Nrf2, HO-1, NQO1 and SOD1 in M, IPO, P25, P50 and P100 groups were significantly decreased compared with N group (P＜0.05), and those in IPO, P25, P50 and P100 groups were obviously higher than those in M group (P＜0.05). The mRNA levels of Nrf2, HO-1, NQO1 and SOD1 in IPO and P50 groups were obviously higher than those in P25 and P100 groups (P＜0.05). The mRNA expression of SOD1 in P25 group was obviously higher than that in P100 group (P＜0.05). The protein levels of Nrf2, NQO1 and SOD1 in IPO and P50 groups were obviously higher than those in P25 and P100 groups (P＜0.05). The protein expression of HO-1 in P50 group was obviously higher than that in IPO, P25 and P100 groups (P＜0.05). The protein levels of HO-1 and NQO1 in P25 group were obviously higher than those in P100 group (P<0.05). CONCLUSION：Hypoxia or pinacidil postconditioning may attenuate hypoxia-reoxygenation injury in adult rat cardiomyocytes via activation of Nrf2-ARE signaling pathway.