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目的研究ATP敏感性钾通道(ATP-sensitive K+channel,KATP通道)-Akt通路在外源性硫化氢(hydrogen sulfide,H_2S)对抗高糖引起的H9c2心肌细胞损伤中的作用。方法应用Western blot法检测心肌细胞Akt蛋白的表达水平;细胞计数盒测定心肌细胞存活率;Hoechst 33258核染色荧光显微镜照相测定凋亡细胞数量的变化;双氯荧光素染色荧光显微镜照相法检测胞内活性氧(reactive oxygen species,ROS)水平;JC-1染色荧光显微镜照相法测定线粒体膜电位(mitochondrial membrane potential,MMP)。结果应用高糖(35 mmol·L~(-1),HG)处理H9c2心肌细胞0~24 h,其中3 h起磷酸化(p)-Akt蛋白表达水平开始明显下降,24 h p-Akt表达水平降至最低。在HG处理心肌细胞24 h前,应用50μmol·L~(-1)KATP通道开放剂吡拉地尔(pinacidil,Pin)和400μmol·L~(-1)硫氢化钠(NaHS,为H_2S的供体)预处理30 min均能明显地抑制HG对p-Akt表达的下调作用。应用1mmol·L~(-1)K_(ATP)通道阻断剂格列本脲(glibenclamide,Gli)预处理心肌细胞能阻断NaHS对HG下调p-Akt表达水平的抑制作用。此外,30μmol·L~(-1)Akt的抑制剂LY294002能明显阻断NaHS对抗HG损伤心肌细胞的保护作用,表现为细胞存活率和MMP降低,凋亡细胞数量、ROS生成增多。结论 K_(ATP)通道-Akt通路介导H_2S对抗高糖引起的心肌细胞损伤的保护作用。
Objective To investigate the role of ATP-sensitive K + channel (AKAT) -Akt pathway in H9c2 cardiomyocyte injury induced by high glucose by exogenous hydrogen sulfide (H2S). Methods The expression of Akt protein in cardiomyocytes was detected by Western blot. The survival rate of cardiomyocytes was determined by cell counting kit. The number of apoptotic cells was determined by Hoechst 33258 nuclear staining fluorescence microscopy. Reactive oxygen species (ROS) levels were measured. The mitochondrial membrane potential (MMP) was measured by JC-1 staining and fluorescence microscopy. Results H9c2 cardiomyocytes were treated with high glucose (35 mmol·L -1, HG) for 0-24 h. At 3 h, phosphorylated (p) -Akt protein expression began to decrease significantly, and expression of p-Akt at 24 h To a minimum level. Before HG treatment of cardiomyocytes, pinacidil (Pin) and 400μmol·L -1 sodium hydrosulfide (NaHS), a 50μmol·L -1 KATP channel opener, Body) pretreatment for 30 min significantly inhibited the down-regulation of p-Akt by HG. Pretreatment of cardiomyocytes with 1 mmol·L -1 K (ATP) channel blocker glibenclamide (Gli) blocked the inhibitory effect of NaHS on HG down-regulated p-Akt expression. In addition, LY294002, an inhibitor of 30 μmol·L -1 Akt, could obviously block the protective effect of NaHS against HG-injured myocardial cells. The results showed that the cell survival rate and MMP decreased, the number of apoptotic cells and ROS production increased. Conclusion KA (ATP) channel-Akt pathway mediates the protective effect of H 2 S against cardiomyocyte injury induced by high glucose.