坏死性凋亡介导化学性缺氧引起的HT22海马神经元损伤和炎症

摘要

Aim To investigate whether necroptosis mediates chemical hypoxia-induced HT22 mouse hippocampal cell injury and inflammation.Methods HT22 hippocampal cells were exposed to cobalt chloride (CoCl2) to establish a model of the chemical hypoxia-induced injury and inflammation.The expression level of RIP3 (an index of necroptosis) was determined by Western blot.Cell counter kit-8 (CCK-8) assay was used to test the cell viability.Lactate dehydrogenase (LDH) activity in the culture medium was measured with commercial kits.Mitochondrial membrane potential (MMP) was examined by rhodamine123 staining followed by photofluorography.The intracellular level of reactive oxygen species (ROS) was detected by 2', 7'-dichlorfluorescein-diacetate (DCFH-DA) staining followed by photofluorography.The secretion levels of interleukin-1β (IL-1β) and tumor necrosis factor-a (TNF-α) were measured by ELISA.Results Treatment of HT22 hippocampal cells with 600 μmol·L-1 CoCl2 for 36 h markedly induced cytotoxicity, leading to a decrease in cell viability to (52.0±2.65) % , indicating that chemical hypoxia-induced cellular injury model was successfully set up.Besides, CoCl2 induced considerable injuries and inflammation, evidenced by increases in LDH activity, ROS production, MMP loss, as well as the secretion levels of IL-1β and TNF-α.Co-treatment of the cells with 40~100 μmol·L-1 Nec-1 (a specific inhibitor of necroptosis) and CoCl2 markedly attenuated the decrease in viability induced by CoCl2, reaching the best anti-cytotoxicity inhibitory effect at 80 μmol·L-1.Meanwhile, the co-treatment with 80 μmol·L-1 Nec-1 blocked the above injuries and inflammatory response induced by CoCl2.In addition, treatment of HT22 hippocampal cells for 6~48 h up-regulated the expression of RIP3, and Nec-1 alleviated the up-regulation of RIP3 expression level induced by CoCl2.Conclusion Necroptosis mediates chemical hypoxia-induced HT22 hippocampal cell injury and inflammation.%目的 探讨坏死性凋亡(necroptosis)是否参与化学性缺氧诱导的小鼠HT22海马细胞损伤和炎症.方法 采用化学性缺氧模拟剂氯化钴(CoCl2)作用HT22细胞建立化学性缺氧损伤的细胞模型.蛋白质免疫印迹法测定受体相互作用蛋白3(receptor interacting protein 3,RIP3)的水平;应用细胞计数试剂盒(cell counting kit-8,CCK-8)测定海马细胞的存活率;乳酸脱氢酶(lactate dehydrogenase,LDH)试剂盒检测细胞培养液中的LDH活性;罗丹明123染色荧光显微镜照相法检测线粒体膜电位(mitochondrial membrane potential, MMP);双氯荧光素染色荧光显微镜照相法测定胞内活性氧(reactive oxygen species,ROS)生成水平;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.结果 600 μmol·L-1 CoCl2作用HT22细胞36 h可产生明显的细胞毒性作用,使细胞存活率降至(52.0±2.65)%,成功建立化学性缺氧损伤的海马细胞模型;此外,CoCl2可引起HT22细胞的多种损伤和炎症,表现为培养液中LDH活性升高,ROS过度生成,MMP丢失以及炎症因子(IL-1β和TNF-α)分泌均增多.40~100 μmol·L-1坏死性凋亡抑制剂necrostatin-1(Nec-1)共处理可抑制CoCl2引起的HT22细胞存活率降低,其中80 μmol·L-1时对细胞毒性的抑制作用最明显.同时,80 μmol·L-1 Nec-1可对抗CoCl2可引起HT22细胞的上述多种损伤和炎症.此外,CoCl2处理HT22细胞6~48 h可促进RIP3的表达水平,Nec-1可明显抑制CoCl2对RIP3表达的上调作用.结论 坏死性凋亡介导化学性缺氧诱导的HT22海马神经元损伤和炎症.