论文部分内容阅读
【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) can inhibit H9C2 cardiomyocyte injury and inflammation induced by high glucose by inhibiting the interaction between p38 MAPK pathway and necrotic apoptosis. 【Methods】 The survival rate of cardiomyocytes was detected by using cell counting kit 8 (CCK-8). The levels of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF- Protein levels of p38MAPK and RIP3 (indicators of necrotic apoptosis) were determined by Western Blot. Intracellular reactive oxygen species (ROS) levels were measured by fluorescence microscopy with diclofenin. Rhodamine 123 staining fluorescence microscopy Photographic determination of mitochondrial membrane potential (MMP). 【Results】 The results showed that the protein level of intracellular RIP3 in H9C2 cardiomyocytes was significantly increased after 35 mmol / L glucose (HG) for 24 h. Pretreatment of cardiomyocytes with 3 μmol / L SB203580 (p38 MAPK inhibitor) HG can up-regulate phosphorylated (p) p38 MAPK protein level, and 100μmol / L necrostatin-1 (Nec-1, a specific inhibitor of necrotic apoptosis ) Co-treated cardiomyocytes for 24 h blocked the up-regulation of p-p38 MAPK protein by HG. However, pretreatment of cardiomyocytes with 400 μmol / L Na HS for 30 min could inhibit the effect of HG on RIP3 and p- Upregulation of p38 MAPK protein levels. In addition, co-treatment of cardiomyocytes with 400μmol / L Na HS, 3μmol / L SB203580 or 100μmol / L Nec-1 all inhibited the cytotoxicity induced by HG, oxidative stress, mitochondrial damage and inflammatory response, High ROS production, loss of MMP, and decreased secretion of IL-1β and TNF-ɑ. 【Conclusion】 Inhibition of the interaction between p38 MAPK pathway and necrotic apoptosis may be one of the important mechanisms of exogenous H2S against H9c2 cardiomyocyte injury and inflammation induced by high glucose.