硫化氢通过抑制p38MAPK通路和坏死性凋亡的相互作用对抗高糖引起的H9C2心肌细胞损伤和炎症  

Hydrogen Sulfide Protects H9C2 Cardiac Cells against High Glucose-Induced Injury and Inflammation by Inhibiting the Interaction between p38 MAPK Pathway and Necroptosis

作　　者：梁伟杰 陈美姬[2] 何洁仪 陈景福[3] 郑东诞[3] 李健豪 张稳柱 廖新学[4] LIANG Wei-jie CHEN Mei-ji HE Jie-yi CHEN Jing-fu ZHENG Dong-dan LI Jian-hao ZHANG Wen-zhu LIAO Xin-xue(Department of Cardiology, Central Hospital of Panyu District//Cardiovascular Institute of Panyu District, Guangzhou 511400, China Department of Paidology Department of Cardiology, Huangpu Division of The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510700, China Department of Cardiology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou510080, China)

机构地区：[1]广州市番禺区中心医院心血管内科、广州市番禺区心血管疾病研究所,广东广州511400 [2]中山大学附属第一医院黄埔院区儿科,广东广州510700 [3]中山大学附属第一医院黄埔院区儿科心血管内科,广东广州510700 [4]中山大学附属第一医院心血管内科,广东广州510080

出　　处：《中山大学学报（医学科学版）》2016年第5期649-656,共8页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金(81270296);番禺区科技计划项目(2015-Z03-57)

摘　　要：【目的】探讨外源性硫化氢(H2S)能否通过抑制p38丝裂原激活蛋白激酶(p38 MAPK)通路和坏死性凋亡的相互作用对抗高糖引起的H9C2心肌细胞损伤和炎症。【方法】采用细胞计数试剂盒8(CCK-8)检测心肌细胞的存活率;ELISA法检测细胞培养液中白介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)的水平;蛋白质免疫印迹法(Western Blot)测定p38MAPK和RIP3蛋白(反映坏死性凋亡的指标)的蛋白水平;双氯荧光素染色荧光显微镜照相测定细胞内活性氧(ROS)水平;罗丹明123染色荧光显微镜照相测定线粒体膜电位(MMP)。【结果】H9C2心肌细胞经35 mmol/L葡萄糖(高糖,HG)作用24 h,胞内RIP3的蛋白水平明显增加,应用3μmol/L SB203580(p38 MAPK抑制剂)预处理心肌细胞60 min能明显地抑制HG对RIP3蛋白水平的上调作用;另一方面,HG可上调磷酸化(p)p38 MAPK的蛋白水平,应用100μmol/L necrostatin-1(Nec-1,坏死性凋亡的特异性抑制剂)共处理心肌细胞24 h能阻断HG对p-p38 MAPK蛋白水平的上调作用;而400μmol/L Na HS(为H2S的供体)预处理心肌细胞30 min能同时抑制HG对RIP3和p-p38 MAPK蛋白水平的上调作用。此外,400μmol/L Na HS、3μmol/L SB203580预处理或100μmol/L Nec-1共处理心肌细胞均可抑制HG引起的心肌细胞毒性、氧化应激、线粒体损伤和炎症反应,使细胞存活率升高,ROS生成、MMP丢失及IL-1β和TNF-ɑ的分泌水平减少。【结论】抑制p38 MAPK通路和坏死性凋亡的相互作用可能是外源性H2S对抗高糖引起的H9c2心肌细胞损伤和炎症的重要作用机制之一。[Objective] To investigate whether exogenous hydrogen sulfide （H2S） protects H9C2 cardiac cells against high glucose-induced injury and inflammation by inhibiting the interaction between p38 mitogen-activated protein kinase （p38 MAPK） pathway and necroptosis. [Methods] The cell viability was measured by cell counter kit-8 （CCK-8） assay. The levels of interleukin- 1β （IL-1β） and tumor necrosis factor-α （TNF-α） were detected by ELISA. The protein levels of p38 MAPK and RIP3 （an indicator of necroptosis） were tested by western blotting. The intracellular levels of reactive oxygen species （ROS） were detected by 2＇, 7＇- dichlorfluorescein-diacetate （DCFH-DA） staining followedbyphotofluorographv. Mitochondrial membrane potential （MMP） was examined by rhodamine？ 123 （Rh 123） staining followed by photofluorography. [Results] After the H9C2 cells were treated with 35 mmol/L glucose （high glucose, HG） for 24 h, the protein level of RIP3 was significantly increased; pre-treatment of the cells with 3 μmol/L SB203580 （an inhibitor of p38 MAPK） for 60 min considerably blocked the up-regulation of RIP3 protein level induced by HG. Moreover, co-treatment of the cells with 100 Ixmol/L necrostatin-1 （Nec-1, a specific inhibitor of necroptosis） attenuated the up- regulation of phosphorated （p）-p38 MAPK protein level induced by HG. In addition, pre-treatment of the cells with 400 txmol/L NariS （a donor of H2S） inhibited the up-regulation of both RIP3 and p-p38 MAPK protein levels induced by HG. On the other hand, pre-treatment of the cells with 400 μmol/L NariS or 3 μmol/L SB203580 or co-treatment of the ceils with 100 μmol/L Nec-1 ameliorated the HG-induced cytotoxicity, oxidative stress, mitochondrial damage and inflammation, evidenced by an increase in cell viability, decreases in the levels of ROS generation, MMP loss as well as the secretion levels of IL-1β and TNF-α. [ Conclusion ] Inhibiting the interaction between p38 MAPK pathway and neeroptosis may

关 键 词：硫化氢 P38丝裂原激活蛋白激酶 坏死性凋亡 相互作用 高糖 心肌细胞 

分 类 号：R363[医药卫生—病理学]