活性氧激活的ERK1/2通路在化学性缺氧损伤PC12细胞中的作用  

Role of ROS-activated ERK1/2 pathway in chemical hypoxia-induced injury in PC12 cells

作　　者：徐文明[1] 兰爱平[2] 林建聪[1] 郭润民[2] 沈宁[2] 陈培熹[2] 冯鉴强[2] 

机构地区：[1]中山大学附属第一医院黄埔院区内科,广东广州510080 [2]中山大学中山医学院生理学教研室,广东广州510080

出　　处：《中国药理学通报》2013年第7期985-990,共6页Chinese Pharmacological Bulletin

基　　金：广东省科技计划基金资助项目(No 2012B031800358;2010B080701035)

摘　　要：目的探讨胞外信号调节激酶1/2(ERK1/2)在化学性缺氧损伤PC12细胞中的作用。方法应用化学性低氧模拟剂氯化钴(CoCl2)处理PC12细胞建立化学性缺氧损伤模型。应用细胞计数试剂盒-8(cell counting kit-8,CCK-8)比色法检测细胞存活率;罗丹明123(Rh123)染色荧光显微镜照像检测线粒体膜电位(MMP);流式细胞术检测凋亡细胞百分比;Western blot法测定caspase-3、ERK1/2和p38MAPK蛋白的表达水平。结果应用600μmol.L-1处理PC12细胞60 min可使磷酸化(p)ERK1/2的表达明显升高;在600μmol.L-1CoCl2处理PC12细胞前,应用500μmol.L-1N-乙酰半胱氨酸(NAC,为活性氧的清除剂)预处理1 h可抑制CoCl2对p-ERK1/2表达的上调作用;在600μmol.L-1CoCl2处理PC12细胞前,应用10μmol.L-1U0126(ERK1/2抑制剂)预处理2 h可保护PC12细胞对抗CoCl2引起的损伤,使细胞存活率升高,凋亡细胞数目和cleaved caspase-3表达及线粒体膜电位(MMP)丢失均减少;10μmol.L-1U0126预处理还能抑制CoCl2对p-p38MAPK表达的上调作用。此外,应用20μmol.L-1SB203580(p38MAPK抑制剂)预处理能抑制CoCl2对p-ERK1/2表达的上调作用。结论活性氧激活的ERK1/2通路介导CoCl2对PC12细胞的损伤作用,并与p38MAPK通路存在相互的的激活作用。Aim To explore the role of extracellular signal-regulated kinase 1/2（ERK1/2） in the chemical hypoxia-induced injury in PC12 cells. Methods PC12 cells were treated with cobalt chloride（CoC12）, a well-known chemical hypoxia mimetic agent, to es- tablish a chemical hypoxia-induced cellular injury mod- el. Cell viability was detected by cell counter kit （CCK-8）. Apoptotic cells were analysed by Flow cy- tometry （FCM）. Mitochondrial membrane potential （MMP） was examined by rhodamine 123 （RH123） staining and photoflutograph. The expression levels of caspase-3, ERK1/2 and p38 mitogen-activated protein kinase（MAPK） were tested by Western blot assay. Results Exposure of PC12 cells to 600 μmol·L-1 CoC12 for 60 rain markedly enhanced the expression of phosphorylated （p） ERK1/2. Pretreatment of PC12 cells with 500 μmol·L-1 N-acetyl-L-cystein （ NAC ） , a reactive oxygen species （ROS） scavenger, for l h be- fore exposure to CoCl2 inhibited the upregulation of p- ERK1/2 expression induced by CoC12 treatment. Pre- treatment of PC12 cells with 10 μmol·L-1 U0126（ an inhibitor of ERK1/2 ） for 2h prior to exposure to 600μmol·L-1 CoC12 protected against the CoC12-induced injuries , leading to decreases in the number of apoptotic cells, expression of cleaved caspase-3 as well as MMP loss. Pretreatment with 10 μmol·L-1 U0126 also at- tenuated the CoC1z-induced upregulation of p- pSSMAPK expression. Furthermore, preteatment with 20 μmol·L-1 SB203580, blocked the CoCl2-induced expression. Conclusions an inhibitor of p38MAPK, upregulation of p-ERK1/2 ROS-activated ERK1/2 pathway mediates the CoCl2-induced injury and there is a synergetic interaction between ERK1/2 pathway and p38MAPK in CoCl2 treated PCl2 cells.

关 键 词：ERK1 2 活性氧 氯化钴 凋亡 线粒体膜电位 P38MAPK 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] R329.25[医药卫生—基础医学]