Diazoxide对缺氧大鼠肺动脉平滑肌细胞内氧自由基的变化及细胞增殖的作用

目的： 研究线粒体膜上ATP敏感钾通道（MitoK_ATP）开放剂diazoxide和线粒体膜电位（ΔΨm）在缺氧导致的大鼠肺动脉平滑肌细胞内氧自由基的变化及细胞增殖/凋亡失衡中的作用，探索缺氧性肺动脉重建和肺动脉高压形成的发生机制。方法： 取大鼠正常肺组织，分离出肺动脉平滑肌细胞（PASMCs）进行常氧或慢性缺氧培养。将样本分为6组：① 正常对照组；② 线粒体膜上ATP敏感钾通道（MitoK_ATP）开放剂diazoxide组；③ MitoK_ATP 阻断剂5-HD组；④ 慢性缺氧(CH)组；⑤ CH+diazoxide组；⑥ CH+5-HD组。利用激光共焦显微镜（Leica SP-1 USA）成像检测线粒体膜电位，荧光染色检测细胞内氧自由基含量，流式细胞仪检测细胞周期和MTT法检测细胞增殖情况。结果： ① Diazoxide作用24 h后，R-123荧光强度明显强于正常对照组，线粒体膜电位去极化，细胞内氧自由基含量明显高于正常对照组，细胞增殖明显多于正常对照组、凋亡少于正常对照组，P<0.05；而5-HD作用24 h后，上述指标与正常对照组相比较，均无显著差异,P>0.05；②慢性缺氧24 h，结果与diazoxide组相似，R-123荧光强度明显强于正常对照组，线粒体膜电位去极化，细胞内氧自由基含量明显高于正常对照组，细胞增殖明显多于正常对照组、凋亡少于正常对照组，P<0.05；CH+diazoxide组，R-123荧光强度和线粒体膜电位去极化明显强于缺氧组，细胞内氧自由基含量明显高于缺氧组，细胞增殖明显多于缺氧组、凋亡少于缺氧组，P<0.05；CH+5-HD组，R-123荧光强度和线粒体膜电位去极化明显弱于缺氧组，细胞内氧自由基含量明显低于缺氧组，细胞增殖明显少于缺氧组、凋亡多于缺氧组，P<0.05;R-123荧光强度、MTT的A值与细胞内氧自由基含量均呈显著正相关；凋亡细胞百分比与细胞内氧自由基含量呈显著负相关。结论： 本实验结果显示，diazoxide能够通过开放线粒体膜上ATP敏感的钾通道，引起ΔΨm去极化，ΔΨm的变化影响细胞内氧自由基的含量，氧自由基可能作为信号分子通过影响肺动脉平滑肌细胞的增殖/凋亡的平衡，参与了缺氧性肺动脉重建和肺动脉高压的发生和发展过程。

Effect of diazoxide on change of H2O2 in rat pulmonary artery smooth muscle cells and proliferation of hypoxic rat pulmonary artery smooth muscle cells

AIM: To investigate the contribution of diazoxide,an opener of mitochondrial ATP-sensitive K+ channel (MitoK_ATP),and mitochondrial membrane potential (ΔΨm) to change of H₂O₂ in rat pulmonary artery smooth muscle cells (PASMCs) and to unbalance between cell proliferation and apoptosis of PASMCs induced by hypoxia.METHODS: The rat PASMCs were isolated from fresh normal lung tissues and cultured,which were divided into 6 groups,as follows: ① control group;② diazoxide group;③ 5-HD group;④chronic hypoxia group;⑤ chronic hypoxia+diazoxide group;⑥ chronic hypoxia +5-HD group.The relative change in mitochondrial potential was detected with rhodamine fluorescence (R-123) technique.The level of H₂O₂ in rat PASMCs was detected with chemiluminescence method.The proliferation of rat PASMCs was examined by cell cycle analysis and MTT colorimetric assay.RESULTS: After exposed to diazoxide for 24 h,the intensity of R-123 fluorescence,the level of H₂O₂ and the A value in normoxic rat PASMCs were significantly increased,and the apoptosis of rat PASMCs was significantly decreased as compared with control group (P<0.05).However,there were no significant changes in these tests after the rat PASMCs had been exposed to 5-HD for 24 h.Chronic hypoxia or chronic hypoxia+diazoxide markedly increased the intensity of R-123 fluorescence,the level of H₂O₂ and the A value in rat PASMCs,and also markedly decreased the apoptosis of rat PASMCs as compared with control group (P<0.05),and these changes were more significant in chronic hypoxia +diazoxide group than those in chronic hypoxia group (P<0.05).5-HD partly weakened the effect of hypoxia on the intensity of R-123 fluorescence,the level of H₂O₂,the A value and the apoptosis of rat PASMCs (P<0.05).Significant and positive correlations were found between the intracellular H₂O₂ and the R-123 fluorescence or the A value.Significant and negative correlation was found between the intracellular H₂O₂ and the apoptosis of rat PASMCs.CONCLUSION: The results suggest that the opening of MitoK_ATP followed by a depolarization of ΔΨm can contribute to the increase in the level of H₂O₂ in rat PASMCs and to the proliferation of rat PASMCs induced by hypoxia.This might be a mechanism of the development of hypoxic pulmonary hypertension.