摘要
目的探讨硫化氢对血管内皮细胞缺氧性损伤的保护作用及机制。方法将人脐静脉内皮细胞于2%O_2、5%CO_2、93%N_2培养箱中培养24h,复制缺氧模型。以硫氢化钠(NaHS)作为硫化氢供体,采用DCFH-DA和激光共聚焦系统检测细胞内活性氧含量;用Fura-2/AM和显微荧光成像系统检测细胞质内钙离子([Ca^(2+)]_i)浓度;使用Rh123和激光共聚焦系统检测细胞线粒体膜电位(mitochondria membrane potential,MMP);用Hoechst33342染色观察细胞核形态,计算细胞凋亡率。结果缺氧使细胞ROS含量显著升高,硫氢化钠预处理显著降低缺氧所致ROS含量的升高;缺氧使细胞[Ca^(2+)]_i显著升高,而NaHS、活性氧清除剂NAC、IP_3受体抑制剂Xesto C均抑制缺氧诱导[Ca^(2+)]_i的升高;缺氧显著降低细胞MMP,NaHS、NAC预处理缺氧细胞则抑制。MMP降低;NaHS、Xesto C预处理可显著降低缺氧所致细胞凋亡。结论硫化氢可能一方面通过减轻缺氧时ROS增多引起的线粒体膜电位下降,保护线粒体功能;另一方面抑制缺氧时ROS增多诱导的钙超载,从而减少缺氧时人脐静脉内皮细胞的凋亡。
Objective To investigate the protective effect of H2S on the hypoxia-induced apoptosis of human umbilical vein endothelial cells （HUYECs） and its possible mechanism. Methods HUVECs were cultured in hypoxic conditions （2% 02, 5% CO2, and 93% N2 at 37℃） for 24h to establish the hypoxic models. Sodium hydrosulfide （NariS） served as the donor of exogenous hydrogen sulfide （H2S）. DCFH-DA and laser scanning confocal microscopy （LSCM） were used to detect the ROS content in the cells. Fura- 2/AM and fluorescence microscopic imaging technology were employed to detect the calcium concentration. MMP was measured by Rh123 staining and detcted by LSCM. Cell nuclear morphology was observed and apoptosis rates calculated by Hoechst33342 staining. Results Hypoxia significantly increased ROS content, and this effect was greatly reduced by NariS pretreatment. Hypoxia significantly increased intracellnlar [ Ca^2 ＋ ] i, and this was inhibited by NariS, NAC （ a ROS scavenger） or Xesto C （ an inhibitor of IP3 receptor）. Hypoxia significantly reduced MMP, and this effect was attenuated by Naris or NAC pretreatment. Naris or Xesto C pretreatment noticeably attenuated hypoxia-induced apoptosis. Conclusion H2S may attenuate the decrease of MMP by inhibiting the ROS increase caused by hypoxia to protect mitochondrial functions. On the other hand, it may prevent the calcium overload induced by ROS, thereby reducing the hypoxia-induced apoptosis of HUVECs.
作者
毛慕华
张竹铭
罗友根
冯毅婷
方洪霞
刘名纲
李志强
甘海丽
魏楚蓉
Mao Muhua Zhang Zhuming Luo Yougen Feng Yiting Fang Hongxia Liu Minggang Li Zhiqiang Gan Haili Wei Churong(The Research Center of Neurodegenerative Diseases and Aging, Medical College of Jinggangshan University, Ji＇an, 343009 The infirmary of Ji＇an Career Technical College, Ji＇an, 343000)
出处
《中国组织化学与细胞化学杂志》
CAS
CSCD
2016年第6期482-486,共5页
Chinese Journal of Histochemistry and Cytochemistry
基金
江西省自然科学基金(No.20142BAB205030
2015ZBAB205008)
井冈山大学博士科研启动基金
2015年国家级大学生创新创业训练计划(201510419016)
