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目的:研究苯妥英(diphenylhydantoin,DPH)(100μmol/L)对体外培养的小脑颗粒神经元(CGN)毒性的细胞内信号机制。方法:比较DPH对体外培养的大脑皮质神经元(CN)和小脑颗粒神经元的作用;考查信号通路抑制剂对苯妥英诱导的小脑颗粒神经元存活的作用;采用相关信号通路中蛋白分子的抗体及其活性部位磷酸化特异性抗体蛋白质印迹法检测信号分子c-Jun,p44/42,JNK,p38的活性及表达;RT-PCR检测c-jun mRNA的表达。结果:DPH 100μmol/L可选择性诱导CGN凋亡,而对CN无明显毒性;JNK/p38抑制剂SB203580 10μmol/L和MLK抑制剂CEP-110041μmol/L可明显抑制苯妥英诱导的小脑颗粒神经元的凋亡;DPH可诱导CGN中c-Jun的活性及表达,c-jun mRNA亦随之上调;SB203580 10μmol/L和CEP-11004 1μmol/L可抑制DPH引起的c-Jun活性的上调;DPH不影响JNK和p38的活性表达量;DPH不影响p44/42的表达,但可明显抑制p44/42的活 性。结论:DPH对体外培养的CGN的神经毒性源于其介导的CGN的凋亡,在此过程中c-Jun的表达及活性被上调,p44/42活性被抑制。
OBJECTIVE: To study the intracellular signaling mechanism of the toxicity of diphenylhydantoin (DPH) (100 μmol / L) to cultured cerebellar granule neurons (CGN) in vitro. Methods: The effects of DPH on neurons (CN) and cerebellar granule neurons cultured in vitro were compared. The effects of inhibitor of signal pathway on the survival of granule neurons induced by phenytoin were examined. Antibody of protein molecule in relevant signal pathway and The activity and expression of c-Jun, p44 / 42, JNK and p38 were detected by Western blotting of the active site phosphorylation-specific antibody. The expression of c-jun mRNA was detected by RT-PCR. RESULTS: DPH at 100μmol / L selectively induced apoptosis of CGN without obvious cytotoxicity to CN; the inhibitory effect of phenytoin-induced granule neurons was inhibited by 10μmol / L JNK / p38 inhibitor SB203580 and CEP-110041μmol / L MLK inhibitor DPH induced c-Jun activity and expression in cGN, and c-jun mRNA was up-regulated. DP20-induced c-Jun activity was inhibited by SB203580 10μmol / L and CEP-11004 1μmol / Affect the activity of JNK and p38 expression; DPH does not affect the expression of p44 / 42, but can significantly inhibit p44 / 42 activity. CONCLUSION: The neurotoxicity of DPH on CGN cultured in vitro derives from the apoptosis of CGN induced by DPH. The expression and activity of c-Jun are up-regulated during this process, and p44 / 42 activity is inhibited.