PKC/ERK信号通路在百草枯致人胚胎神经干细胞增殖过程中的作用
Effect of paraquat on proliferation of human embryonic neural stem cells via regulation of PKC/ERK signaling pathway
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摘要:
目的 探讨蛋白激酶C（PKC）/细胞外调节蛋白激酶（ERK）信号通路在百草枯（PQ）调控人胚胎神经干细胞增殖中的作用。
方法 以永生化的人胚胎神经干细胞系（ReNcell CX）为细胞模型，以终浓度为0、5、25、50、100 μmol/L的百草枯处理细胞24 h，采用间接免疫荧光法检测巢蛋白（Nestin）、β-微管蛋白Ⅲ（β-tubulin Ⅲ）和胶质纤维酸性蛋白（GFAP）；采用四甲基偶氮唑盐（MTT）法检测细胞存活率；分光光度法检测培养上清中乳酸脱氢酶（LDH）活性；5-溴-2-脱氧尿嘧啶（BrdU）掺入法检测细胞增殖能力；流式细胞仪检测荧光强度，以此反映细胞内钙离子浓度，免疫印迹法检测细胞膜PKCα和细胞pERK1/2蛋白表达。
结果 与对照组比较，25、50、100 μmol/L百草枯染毒组的细胞活力出现明显抑制作用，且细胞存活率与百草枯浓度对数值呈负相关（r=-0.96，P < 0.05）；50、100 μmol/L百草枯染毒组的细胞上清中LDH水平明显升高，差异有统计学意义（P < 0.05）；免疫荧光染色显示，随着百草枯染毒浓度的增加，BrdU阳性细胞数逐渐减少。其中25、50、100 μmol/L百草枯染毒能明显抑制神经干细胞的增殖（P < 0.05）；流式细胞仪检测细胞内Ca2+荧光强度，25、50、100 μmol/L百草枯染毒组细胞的峰值钙离子浓度明显降低，随染毒浓度增加，Ca2+荧光强度降低（r=-0.97，P < 0.05）；各浓度百草枯染毒均能够下调人神经干细胞的PKCα、pERK1/2蛋白表达，差异有统计学意义（P < 0.05）。
结论 百草枯通过降低细胞内钙离子浓度，进而抑制Ca2+依赖的PKCα活性，减少细胞ERK1/2的磷酸化。Ca2+/PKCα/ERK1/2信号通路可能是介导百草枯抑制神经干细胞增殖的途径之一。
Abstract:Objective To evaluate the role of protein kinase C/extracellular signal-regulated kinase (PKC/ERK) signaling pathway in the proliferation of human embryonic neural stem cells regulated by paraquat.
Methods ReNcell CX immortalized cells cultured in vitro were exposed to paraquat at designed concentrations of 0, 5, 25, 50, and 100 μmol/L, respectively, for 24 h. Nestin, β-tubulin Ⅲ, and glial fibrillary acidic protein (GFAP) were detected by indirect immunofluorescence assay. Cell viability was determined by MTT assay. Lactate dehydrogenase (LDH) activity in supernatant was determined by spectrophotometry assay. Proliferation rate was evaluated by BrdU incorporation assay. Intracellular Ca2+ concentration was determined with flow cytometer. The protein expressions of PKCα and pERK1/2 were measured by Western blot.
Results Compared with the control group, cell viability decreased with increasing log-translated paraquat concentrations (25, 50, and 100 μmol/L) (r=-0.96, P < 0.05). The level of LDH significantly increased after the paraquat treatment at 50 and 100 μmol/L (P < 0.05). The results of immunofluorescent staining showed the BrdU positive cell counts decreased with the increasing paraquat concentrations; especially the paraquat treatment at 25, 50, and 100 μmol/L significantly inhibited ReNcell CX cell proliferation (P < 0.05). By flow cytometry, the paraquat treatment at 25, 50, and 100 μmol/L induced reduction in peak intracellular Ca2+ concentrations, and with the treatment concentration increasing, the Ca2+ fluorescence intensity was decreased (r=-0.97, P < 0.05). Meanwhile, the protein expressions of PKCα and pERK1/2 significantly decreased after treatment with various concentrations of paraquat (P < 0.05).
Conclusion Paraquat could decrease intracellular Ca2+ concentration and further reduce Ca2+-dependent PKCα activity and ERK1/2 phosphorylation. Ca2+/PKCα/ERK signaling pathway may be one of mechanisms that paraquat inhibits the proliferation of human embryonic neural stem cells.
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