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目的研究坏死性凋亡(necroptosis,Nec)和p38丝裂原激活蛋白激酶(mitogen-activated protein kinase,MAPK)通路的相互作用在高糖引起H9c2心肌细胞损伤中的作用。方法应用细胞计数盒检测心肌细胞存活率;双氯荧光素染色荧光显微镜照相法检测细胞内活性氧(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);蛋白质免疫印迹法测定RIP3蛋白(反映Nec的指标)和p38MAPK蛋白的表达水平。结果高糖(35 mmol·L~(-1)葡萄糖,HG)作用H9c2心肌细胞24 h可引起明显的细胞损伤,表现为细胞存活率降低,ROS生成及MMP丢失增多;应用100μmol·L~(-1)necrostatin-1(Nec-1,Nec特异性抑制剂)和HG共处理心肌细胞24 h或3μmol·L~(-1)SB203580(p38MAPK抑制剂)预处理心肌细胞60 min,再予HG作用24 h可减轻高糖引起的上述损伤。此外,HG作用心肌细胞1、3、6、9、12、24、36和48 h均能明显增加RIP3蛋白的表达水平,其中24 h时表达水平增加最明显。应用100μmol·L~(-1)Nec-1共处理或3μmol·L~(-1)SB203580预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。另一方面,应用100μmol·L~(-1)Nec-1共处理心肌细胞能阻断HG对磷酸化(p)-p38MAPK表达的上调作用。结论 Nec和p38 MAPK通路的相互作用介导高糖引起H9c2心肌细胞损伤。
Objective To investigate the role of necroptosis (Nec) and p38 mitogen-activated protein kinase (MAPK) pathway in H9c2 cardiomyocyte injury induced by high glucose. Methods The survival rate of cardiomyocytes was detected by using cell counting kit. The level of reactive oxygen species (ROS) in cells was detected by fluorescence microscopy with diclofenin staining. The mitochondrial membrane potential (mitochondrial membrane potential) was measured by rhodamine 123 staining. , MMP). Western blotting was used to detect the expression of RIP3 protein (an indicator of Nec) and p38MAPK protein. Results The H9c2 cardiomyocytes exposed to high glucose (35 mmol·L -1 glucose, HG) for 24 h could cause obvious cell injury, which showed the decrease of cell viability, the increase of ROS production and the loss of MMP. The application of 100 μmol·L ~ 1) Nec-1, Nec-specific inhibitor and HG co-treated cardiomyocytes for 24 h or 3 μmol·L -1 SB203580 (p38MAPK inhibitor) for 60 min, and then HG Action for 24 h can reduce the high glucose caused the above injury. In addition, HG-treated cardiomyocytes at 1, 3, 6, 9, 12, 24, 36 and 48 h significantly increased the expression of RIP3 protein, and the expression of RIP3 protein increased most significantly at 24 h. Pretreatment of cardiomyocytes with 100μmol·L -1 Nec-1 or 3μmol·L -1 SB203580 significantly inhibited the upregulation of RIP3 protein by HG. On the other hand, co-treatment of cardiomyocytes with 100μmol·L -1 Nec-1 blocked the upregulation of phosphorylated (p) -p38MAPK by HG. Conclusion The interaction of Nec and p38 MAPK pathway mediates H9c2 cardiomyocyte injury induced by high glucose.