ATP敏感性钾通道-Akt通路在硫化氢对抗高糖损伤H9c2心肌细胞中的作用  

Role of ATP-sensitive potassium channels-Akt pathway in hydrogen sulfide inhibiting high glucose-induced injury in H9c2 cardiac cells

作　　者：梁伟杰 陈景福[3] 何洁仪 宋明才 余盛龙 张稳柱 郑东诞[3] 廖新学[4] 

机构地区：[1]广州市番禺区中心医院心血管内科 [2]广州市番禺区心血管疾病研究所,广东广州511400 [3]中山大学附属第一医院黄埔院区心血管内科CCU,广东广州510070 [4]中山大学附属第一医院心血管内科,广东广州510080

出　　处：《中国药理学通报》2016年第4期530-536,共7页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No81270296);广东省财政科技项目(No2014SC107)

摘　　要：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。Aim To investigate the role of ATP-sensitive potassium channels-Akt pathway in exogenous hydrogen sulfide( H_2S) inhibiting the high glucose( HG)-induced injury in H9c2 cardiac cells. Methods The expression level of Akt protein was tested by Western blot assay. The cell viability was measured by cell counter kit-8( CCK-8 assay). The number of apoptotic cells was tested by Hoechst 33258 nuclear staining followed by photofluorography. The intracellular levels of reactive oxygen species( ROS) were detected by DCFH-DA staining followed by photofluorography. Mitochondrial membrane potential( MMP) was examined by JC-1 staining followed by photofluorography. Results H9c2 cells were treated with 35 mmol·L^(-1) glucose( high glucose,HG) for 0 ~ 24 h respectively. After treating for 3 h,the expression level of phosphorated( p)-Akt protein began to be obviously reduced,the maximum reduced expression level was observed after the cells were exposed to HG for 24 h. Pretreatment of the cells with 50 μmol · L^(-1) pinacidil( Pin,a KATP channel opener) or 400 μmol·L^(-1) NaHS( a donor of H_2S) prior to exposure to HG considerably blocked the down regulation of p-Akt expression level induced by HG. However,pretreatment with 1 mmol · L^(-1) KATP channel blocker glibenclamide( Gli) obviously attenuated the inhibitory effect of NaHS on HG-induced downregulation of p-Akt expression level. On the other hand,the protective effects of NaHS against the HGinduced cardiomyocyte injury were markedly blocked by 30 μmol·L^(-1) Ly294002( an inhibitor of Akt),as indicated by the decrease in cell viability and MMP dissipation as well as the increases in the number of apoptotic cells and ROS generation. Conclution KATP channels-Akt pathway mediates the protective effect of H_2S against the HG-induced cardiac injury.

关 键 词：ATP敏感性钾通道 AKT通路 硫化氢 高血糖 损伤 心肌细胞 

分 类 号：R322.11[医药卫生—人体解剖和组织胚胎学] R329.24[医药卫生—基础医学]