摘要
目的:探讨硫化氢(hydrogen sulfide,H_2S)能否通过调控坏死性凋亡(necroptosis)对抗高糖(HG)引起的H9c2心肌细胞损伤。方法:应用Western blot法检测心肌细胞内能反映坏死性凋亡的RIP3蛋白和cleaved caspase-3蛋白的水平;细胞计数盒测定心肌细胞存活率;双氯荧光素染色荧光显微镜照相法检测细胞内活性氧簇(reactive oxygen species,ROS)水平;罗丹明123染色荧光显微镜照相法测定线粒体膜电位(mitochondrial membrane potential,MMP);Hoechst 33258核染色荧光显微镜照相法测定凋亡细胞的数量。结果:应用HG(35 mmol/L葡萄糖)处理H9c2心肌细胞3 h、6 h、9 h、12 h和24 h均能明显地上调RIP3蛋白的表达水平,其中24 h时RIP3蛋白水平增加最明显。400μmol/L硫氢化钠(Na HS;为H_2S的供体)预处理或坏死性凋亡的特异性阻断剂necrostatin-1(Nec-1;100μmol/L)共处理心肌细胞均能明显地抑制HG对RIP3蛋白表达的上调作用。此外,Na HS预处理或Nec-1共处理心肌细胞均显著地抑制HG引起的心肌细胞损伤,使细胞存活率升高,ROS生成及MMP丢失减少。另一方面,400μmol/L Na HS预处理心肌细胞能使凋亡细胞数量及cleaved caspase-3表达明显减少。结论:H_2S可通过抑制坏死性凋亡保护心肌细胞,对抗高糖引起的损伤。
AIM:To study whether hydrogen sulfide( H2S) protects H9c2 cardiomyocytes against high glucose( HG)-induced injury by inhibiting necroptosis.METHODS:The protein levels of RIP3( an indicator of necroptosis) and cleaved caspase-3 were determined by Western blot.The cell viability was measured by CCK-8 assay.The intracellular levels of reactive oxygen species( ROS) were detected by 2',7'-dichlorfluorescein diacetate staining followed by photofluorography.Mitochondrial membrane potential( MMP) was examined by rhodamine 123 staining followed by photofluorography.The number of apoptotic cells was observed by Hoechst 33258 nuclear staining followed by photofluorography.RESULTS:After the H9c2 cells were treated with HG( 35 mmol/L glucose) for 0 - 24 h,the protein expression of RIP3 in the H9c2 cells was significantly increased at 3 h,6 h,9 h,12 h and 24 h,reaching the maximum level at 24 h.Pretreatment of the cells with 400 μmol/L Na HS( a donor of H2S) or co-treatment of the cells with necrostatin-1( Nec-1;a specific inhibitor of necroptosis) considerably blocked the up-regulation of RIP3 protein induced by HG.Moreover,pretreatment with Na HS or co-treatment with Nec-1 obviously inhibited HG-induced injuries,leading to an increase in the cell viability,and decreases in the generation of ROS and MMP loss.On the other hand,pretreatment with Na HS also reduced the number of apoptotic cells and the protein level of cleaved caspase-3 in the HG-treated H9c2 cardiomyocytes.CONCLUSION:H2S protects H9c2 cardiomyocytes against HG-induced injury by inhibiting necroptosis.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2016年第3期385-391,共7页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81270296)
广东省财政科技项目(No.2014SC107)
关键词
坏死性凋亡
硫化氢
高糖
心肌细胞
Necroptosis
Hydrogen sulfide
High glucose
Cardiomyocytes