硫化氢通过抑制坏死性凋亡对抗高糖引起的H9 c2心肌细胞损伤

摘要

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 le-vels of reactive oxygen species (ROS) were detected by 2’, 7’-dichlorfluorescein diacetate staining followed by photofluo-rography.Mitochondrial membrane potential (MMP) was examined by rhodamine 123 staining followed by photofluorogra-phy.The number of apoptotic cells was observed by Hoechst 33258 nuclear staining followed by photofluorography .RE-SULTS: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.Pretreat-ment of the cells with 400μmol/L NaHS (a donor of H2S) or co-treatment of the cells with necrostatin-1 (Nec-1;a speci-fic inhibitor of necroptosis) considerably blocked the up-regulation of RIP3 protein induced by HG.Moreover, pretreatment with NaHS 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 NaHS also reduced the num-ber of apoptotic cells and the protein level of cleaved caspase-3 in the HG-treated H9c2 cardiomyocytes .CONCLUSION:H2 S protects H9c2 cardiomyocytes against HG-induced injury by inhibiting necroptosis .%目的：探讨硫化氢（hydrogen sulfide，H2S）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（ NaHS；为H2 S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， NaHS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 NaHS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：H2 S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。