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

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。

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

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(CoCl2),a well-known chemical hypoxia mimetic agent,to establish a chemical hypoxia-induced cellular injury model.Cell viability was detected by cell counter kit(CCK-8).Apoptotic cells were analysed by Flow cytometry(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 CoCl2 for 60 min markedly enhanced the expression of phosphorylated(p)ERK1/2.Pretreatment of PC12 cells with 500 μmol·L-1N-acetyl-L-cystein(NAC),a reactive oxygen species(ROS) scavenger,for 1h before exposure to CoCl2 inhibited the upregulation of p-ERK1/2 expression induced by CoCl2 treatment.Pretreatment of PC12 cells with 10 μmol·L-1U0126(an inhibitor of ERK1/2) for 2h prior to exposure to 600 μmol·L-1CoCl2 protected against the CoCl2-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-1U0126 also attenuated the CoCl2-induced upregulation of p-p38MAPK expression.Furthermore,preteatment with 20 μmol·L-1 SB203580,an inhibitor of p38MAPK,blocked the CoCl2-induced upregulation of p-ERK1/2 expression.Conclusions 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 PC12 cells.