坏死性凋亡与活性氧的相互作用介导高糖引起的H9c2心肌细胞损伤  

The Interaction Between Necroptosis and Reactive Oxygen Species Mediates High Glucose-induced Injury in H9c2 Cardiac Cells

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

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,广东省广州市511400 [3]中山大学附属第一医院黄埔院区心血管内科CCU,广东省广州市510700 [4]中山大学附属第一医院心血管内科,广东省广州市510080

出　　处：《中国动脉硬化杂志》2016年第8期781-787,共7页Chinese Journal of Arteriosclerosis

基　　金：国家自然科学基金资助项目(81270296);广东省财政科技项目(2014SC107)

摘　　要：目的探讨坏死性凋亡（Nec）与活性氧（ROS）之间的相互作用能否介导高糖引起的H9c2心肌细胞损伤。方法应用蛋白质免疫印迹试验（Western blot）检测心肌细胞RIP3蛋白的表达水平;细胞计数盒测定心肌细胞存活率;罗丹明123（Rh123）染色荧光显微镜摄片测定线粒体膜电位（MMP）;2’,7’-二氢二氯荧光素二乙酸酯（DCFH-DA）染色荧光显微镜摄片检测心肌细胞内ROS水平;Hoechst 33258核染色荧光显微镜摄片测定凋亡细胞的数量。结果应用高糖（HG,35 mmol/L葡萄糖）分别处理H9c2细胞0~24 h,其中3、6、9、12和24 h均能明显地上调RIP3蛋白的表达水平,24 h时RIP3蛋白表达上调最为明显。应用100μmol/L Nec的特异性阻断剂necrostatin-1（Nec-1）共处理心肌细胞可对抗高糖引起的RIP3蛋白表达的上调作用。此外,100μmol/L Nec-1能显著地抑制高糖引起的细胞毒性、线粒体损伤及氧化应激,使细胞存活率升高,MMP丢失及ROS生成减少。1 000μmol/L ROS清除剂N-乙酰-L-半胱氨酸（NAC）预处理心肌细胞60 min,可明显地抑制高糖对心肌细胞RIP3蛋白表达的上调作用;此外,高糖诱导的细胞凋亡和细胞毒性作用,经NAC预处理后均能得到显著的抑制。结论 Nec与ROS之间的相互作用介导高糖对心肌细胞的损伤。Aim To investigate whether the interaction between necroptosis（ Nec） and reactive oxygen species（ ROS） mediates high glucose（ HG）-induced injury in H9c2 cardiac cells. Methods The expression level of RIP3protein（ an important index that reflects Nec） was determined by Western blot assay. The cell viability was measured by cell counting kit-8（ CCK-8） assay. Mitochondrial membrane potential（ MMP） was examined by Rhodamine 123（ Rh123） staining followed by photofluorography. The intracellular levels of ROS were detected by 2＇,7＇-dichlorfluoresceindiacetate（ DCFH-DA） staining followed by photofluorography. Apoptotic cells were evaluated by the nuclear morphology observed with Hoechst 33258 staining followed by photofluorography. Results After the H9c2 cells were treated with35 mmol / L glucose（ HG） for 0 ~ 24 h,respectively,the expression levels of RIP3 protein were significantly increased at 3h,6 h,9 h,12 h and 24 h,reaching the maximum level at 24 h. Cotreatment of the cells with necrostatin-1（ Nec-1,a specific inhibitor of Nec） considerably blocked the up-regulation of RIP3 expression level induced by HG. Moreover,cotreatment with Nec-1 obviously inhibited HG-induced injuries（ including cytotoxicity,mitochondrial damage and oxidative stress）,leading to an increase in the cell viability,decreases in a loss of MMP and ROS generation. On the other hand,pretreatment of the cells with 1000 μm N-acetyl-L-cysteine（ NAC,a scavenger of ROS） for 60 min before HG exposure obviously reduced the HG-induced increase in the expression of RIP3. In addition,pretreatment of the cells with NAC dramatically alleviated the HG-induced apoptosis and cytotoxicity. Conclusion The interaction between Nec and ROS mediates HG-induced injury in H9c2 cardiac cells.

关 键 词：坏死性凋亡 活性氧 高血糖 心肌细胞 

分 类 号：R363[医药卫生—病理学]