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目的:探讨诱导型一氧化氮合酶/一氧化氮(iNOS/NO)是否介导了氯化钴(CoCl2)引起的PC12细胞损伤及p38 MAPK对其调节作用。方法:应用化学性低氧模拟剂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(CCK-8)比色法检测细胞存活率;Hochest33258核染色法观察细胞凋亡的形态学改变;Western blotting法检测iNOS蛋白的表达水平;Griess试剂盒检测细胞培养基中亚硝酸盐(NO的代谢物)的浓度。结果:应用600μmol/L CoCl2处理PC12细胞24 h可使iNOS表达明显增多;应用600μmol/L CoCl2处理PC12细胞24 h和48 h可使细胞培养基里NO增多;在CoCl2损伤PC12细胞前60 min应用iNOS抑制剂L-canavanine(10μmol/L)预处理能保护PC12细胞对抗600μmol/L CoCl2引起的损伤,使细胞存活率升高,凋亡细胞数目减少;SB203580(p38 MAPK选择性抑制剂)预处理60 min可下调CoCl2引起的iNOS高表达。结论:p38 MAPK-iNOS-NO通路介导CoCl2引起PC12细胞的损伤作用。
AIM: To investigate whether inducible nitric oxide synthase / nitric oxide (iNOS / NO) mediates the damage of PC12 cells induced by cobalt chloride (CoCl 2) and the regulation by p38 MAPK. Methods: Chemical hypoxia injury model was established by PC12 cells treated with chemical hypoxic simulator CoCl2. The cell viability was detected by CCK-8 colorimetric assay; Morphological changes of apoptosis were observed by Hochest33258 nuclear staining; The expression of iNOS protein was detected by Western blotting; Griess kit was used to detect cell viability Concentration of nitrite (NO metabolite). Results: PC12 cells were treated with 600μmol / L CoCl2 for 24 h, the expression of iNOS was significantly increased. Treatment of PC12 cells with 600 μmol / L CoCl2 for 24 h and 48 h resulted in an increase of NO in the cell culture medium; Pretreatment with iNOS inhibitor L-canavanine (10μmol / L) could protect PC12 cells against 600μmol / L CoCl 2 -induced cell injury and increase the cell viability and the number of apoptotic cells. Pretreatment with SB203580 (selective inhibitor of p38 MAPK) 60 min down-regulated CoCl2-induced iNOS overexpression. Conclusion: p38 MAPK-iNOS-NO pathway mediates the injury induced by CoCl2 in PC12 cells.