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

发布时间：2018-05-11 15:31

本文选题：坏死性凋亡 + 化学性缺氧　；参考：《中国药理学通报》2017年04期

【摘要】：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(CoCl_2)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~(-1)β)和肿瘤坏死因子-α(TNF-α)的水平。结果 600μmol·L~(-1)CoCl_2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,CoCl_2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~(-1)β和TNF-α)分泌均增多。40~100μmol·L~(-1)坏死性凋亡抑制剂necrostatin-1(Nec-1)共处理可抑制CoCl_2引起的HT22细胞存活率降低,其中80μmol·L~(-1)时对细胞毒性的抑制作用最明显。同时,80μmol·L~(-1)Nec-1可对抗CoCl_2可引起HT22细胞的上述多种损伤和炎症。此外,CoCl_2处理HT22细胞6~48 h可促进RIP3的表达水平,Nec-1可明显抑制CoCl_2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。
[Abstract]:Aim to investigate whether necrotic apoptosis is involved in the injury and inflammation of HT22 hippocampal cells induced by chemical hypoxia. Methods HT22 cells were treated with chemical anoxic analogue cobalt chloride CoCl2) to establish the cell model of chemical hypoxia injury. Western blotting was used to detect the level of receptor interaction protein 3(receptor interacting protein 3, cell counting kit-8 CCK-8 was used to determine the survival rate of hippocampal cells, and lactate dehydrogenase LDH kit was used to detect the activity of LDH in cell culture medium. Detection of mitochondrial membrane potential by Rhodamine 123 staining fluorescence microscopy and dichlorofluorescein staining fluorescence microscopy for intracellular reactive oxygen species oxygen speciesrosimetry The level of tumor necrosis factor- 伪 (TNF- 伪). Results after treated with 600 渭 mol L~(-1)CoCl_2 for 36 h, HT22 cells had obvious cytotoxic effects, and the cell survival rate was reduced to 52.0 卤2.65g / L, and the model of hippocampal cells injured by chemical hypoxia was successfully established, in addition, CoCl2 could induce many kinds of damage and inflammation of HT22 cells. The results showed that the increase of LDH activity and the loss of Ros excessive production of MMP and the increase of 尾 and TNF- 伪 secretion of CoCl_2 induced HT22 cell survival were inhibited by co-treatment with necrostatin-1Nec-1), a necrostatin-1Nec-1) inhibitor of necrostatin-1Nec-1). The inhibitory effect of 80 渭 mol L ~ (-1) on cytotoxicity was the most obvious. At the same time, 80 渭 mol L~(-1)Nec-1 could antagonize the damage and inflammation of HT22 cells induced by CoCl_2. In addition, HT22 cells treated with CoCl2 for 6h for 48 h could promote the expression of RIP3. Nec-1 could significantly inhibit the up-regulation of RIP3 expression by CoCl_2. Conclusion Necrosis apoptosis mediates the injury and inflammation of HT22 hippocampal neurons induced by chemical hypoxia.
【作者单位】：南华大学附属第一医院麻醉科;南华大学附属第一医院疼痛科;南华大学医学院生理教研室;
【基金】：国家自然科学基金资助项目(No 8140050613) 湖南省教育厅资助课题(No 15C1208)
【分类号】：R741

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