淫羊藿苷对大鼠心肌细胞缺氧再复氧损伤的作用及机制

Effect of icariin on hypoxia/reoxygenation injury in neonatal rat cardiomyocytes

目的探讨淫羊藿苷对心肌细胞缺氧复氧损伤的作用及可能机制。方法原代培养新生SD大鼠心肌细胞，给予不同浓度淫羊藿苷预处理24h后建立缺氧复氧模型，Tunel法检测心肌细胞凋亡，免疫印迹检测凋亡相关蛋白的表达；免疫荧光检测p65核转位，免疫印迹检测p65信号通路的改变。结果缺氧复氧后心肌细胞凋亡率显著增高（对照组：1．5％±0．1％；模型组：23．4％±1．3％，P〈0．05），不同浓度的淫羊藿苷显著减少心肌细胞的凋亡（1μmol／L淫羊藿苷组：7．2％±0．9％；10μmol／L淫羊藿苷组：3．9％±0．8％，均P〈0．05）；免疫印迹结果显示缺氧复氧后心肌细胞促细胞凋亡蛋白Bax表达显著增加（对照组：0．19±0．05；模型组：0．41±0．03，P〈0．05），而抗凋亡蛋白BCL-2的表达明显减少（对照组：0．15±0．02；模型组：0．03±0．01，P〈0．05），淫羊藿苷处理后Bax表达减少（1μmol／L淫羊藿苷组：0．29±0．01；10μmol／L淫羊藿苷组：0．33±0．03，均P〈0．05），而BCL-2蛋白表达增加（1μmol／L淫羊藿苷组：0．10±0．03；10μmol／L淫羊藿苷组：0．11±0．02，均P〈0．05）；免疫荧光结果显示：缺氧复氧后心肌细胞核因子κB（p65）核转位显著增加（对照组：3．6％±0．5％；模型组：89．5％±4．8％，P〈0．05），而淫羊藿苷能减少p65核转位（1μmol／L淫羊藿苷组：32．6％±2．3％；10μmol／L淫羊藿苷组：10．6％±1．0％，均P〈0．05），此外淫羊藿苷能减少缺氧复氧后心肌细胞p65的活化和IKBα的磷酸化（P〈0．05）。结论淫羊藿苷能保护心肌细胞缺氧复氧损伤，其机制可能与阻断p65信号通路有关。

Objective To investigate the effect of icariin on myocardial hypoxia reoxygenation injury and the possible mechanism. Methods Neonatal Sprague-Dawley rat cardiomyocytes in primary culture were treated with different concentrations of icariin for 24 h prior to hypoxia/reoxygenation injury. Cardiomyocyte apoptosis was evaluated with Tunel staining. The expression levels of apoptosis proteins were detected by Western blotting. The nuclear translocation of p65 was evaluated by immunofluorescence. The p65 signaling pathway was also detected by Western blotting. Results Myocardial apoptosis rate significantly increased after hypoxia/reoxygenation （ control： 1.5% ± 0.1% ; Model ： 23.4% ± 1.3% , P 〈 0.05 ）. While icariin significantly reduced cardiomyocyte apoptosis induced by hypoxia./reoxygenation （ 1 Ixmol/L icariin ： 7.2% ± 0.9% ; 10 μmol/L icariin ： 3.9% ±0.8%, both P 〈 0. 05 ）. Western blot showed that the expression levels of pro-apoptotic protein, Bax, increased significantly （control： 0. 19 ± 0. 05; Model： 0.41 ± 0.03, P 〈 0.05）, while the expression of anti-apoptotic protein, B-Cell CLL/ Lymphoma 2 （ BCL-2）, was significantly reduced （ control ： 0.15 ± 0.02 ; Model ： 0.03 ±0.01, P 〈 0.05 ） after hypoxia/reoxygenation. Notably, icariin reduced the expression of Bax （ 1 μmol/L icariin： 0.29 ± 0. 01 ; 10 μmol/L icariin： 0. 33 ±0. 03, both P 〈 0. 05 ） and increased expression of BCL-2 （ 1 μmol/L icariin ： 0. 10 ± 0.03 ; 10 μmoL/L icariin ： 0.11 ± 0.02, both P 〈 0.05 ）. Immunofluorescence showed that NFKB-p65 nuclear translocation in cardiomyocytes was increased after hypoxia/reoxygenation （control： 3.6%± 0.5% ; Model： 89.5% ± 4.8%, P 〈 0.05 ）, while icariin reduced the nuclear translocation of p65 （1 μmol/L icariin： 32.6%± 2.3% ; 10 μmol/L icariin： 10.6% ± 1.0%, both P 〈 0.05 ）. Moreover, icariin reduced the activation of p65 and phosphorylation of IKBα induced by hypoxia/reoxygenation in cardiomyocytes. Conclusion Icariin can protect cardiomyocytes against hypoxia reoxygenation injury, which may be via blocking p65 signaling pathway.