坏死性凋亡和p38MAPK通路的相互作用介导高糖引起的H9c2心肌细胞损伤

Interaction between necroptosis and p38MAPK pathway mediates high glucose-induced injury in H9c2 cardiac cells

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。

Abstract：Aim To investigate the role of the interactionbetween necroptosis （ Nec ） andp38mitogn-activatedprotein kinase （ M A P K ） pathway in the high glucose（HG）-induced H9c2 cardiac cells injury. Metli-ods The cell viability was measured by cell counterkit-8 assay. The intraceeular level of reactive oxygnspecies （ ROS ） was tested by DCFH- DA stating followedby photofluorography. Mitochondral membranepotential （ MMP ） was detected by Rhodamine 123 stainingfollowed by photofluorography. The expressionlevels of receptor interaction protein 3 （ RIP3 , an indicatorof Nec） and p38MAPK protein were tested byWestern blot assay. Results The treatment of H9c2cardiac cells with 35 mmol·L^-1 glucose （ high glucose, HG ） for 24 h induced considerable injures , includinga decrease in cell viability , increases in RO Sgeneration as well as MMP loss. The co-treatment of the cells with 100 μmol·L^-1 necrostatin -1（Nec-1 , aspecific inhibitor of Nec ） and H G for of the cells with 3 μmol·L^-1 SB203580 （ aninhibitor of p38MAPK ） for 60 min before HG exposureatenuated the above injuries induced by HG. Moreover,the treatment of the cells with HG for 1,3,6,9,12,24,36 and 48 h significantly increased the expressionlevels of R I P 3,peaking at 24 h. The co-treatmentof the cells with 100 μmol·L^-1 Nec-1 or the pretreatmentof the cells with 3 μmol·L^-1 SB203580considerably blocked the up-regulation of RIP3 expressioninduced by HG. On the other hand,the co-treat- ment of the cells with 100 （μmol·L^-1 Nec-1 alleviatedthe HG-induced up-regulation of the expression of pp38MAPIK Conclusion The interaction between Necand p38MAPK pathway mediates the HG-induced injuryin H9c2 cardiac cells.