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目的:在中枢神经系统,损伤神经元内通常出现c-jun和nNOS基因的高表达,然而两者之间的关系还不清楚。本实验探讨PC12细胞JNK/c-jun信号通路对nNOS基因的调控作用。方法:体外培养分化的PC12细胞,免疫组化检测PC12细胞内JNK/c-jun,p-c-jun和nNOS的定位表达;应用SP600125抑制JNK/c-jun信号通路,MTT法检测不同浓度SP600125对分化的PC12细胞活力的影响,Western blot检测SP600125对JNK/c-jun,p-c-jun和nNOS基因表达的影响,验证c-jun和nNOS之间的关系。结果:全部分化的PC12的细胞核都表达c-jun基因,部分细胞表达p-c-jun,全部分化的PC12细胞均表达nNOS蛋白。与对照组相比,50μmol/L和100μmol/L的SP600125可明显降低细胞活力(P<0.05),50μmol/L的SP600125能有效抑制c-jun的磷酸化,即p-c-jun的表达(P<0.05),而对c-jun的表达无影响(P>0.05),同时引起nNOS蛋白表达的上调(P<0.05)。结论:c-jun,p-cjun和nNOS蛋白共表达于分化的PC12细胞中,SP600125有效抑制了c-jun的磷酸化激活,JNK/c-jun信号激活的抑制同时上调了nNOS的表达。这些结果表明在分化的PC12细胞内c-jun与nNOS之间的功能密切相关。
OBJECTIVES: High expression of c-jun and nNOS genes usually occurs in the central nervous system and injured neurons, but the relationship between the two is unclear. This study was to investigate the regulatory effect of JNK / c-jun signaling pathway on nNOS gene in PC12 cells. Methods: Differentiated PC12 cells were cultured in vitro. The expression of JNK / c-jun, pc-jun and nNOS in PC12 cells was detected by immunohistochemistry. SP600125 was used to inhibit the JNK / c-jun signaling pathway. MTT assay was used to detect the differentiation of PC12 cells The effect of SP600125 on the expression of JNK / c-jun, pc-jun and nNOS gene was examined by Western blot, and the relationship between c-jun and nNOS was verified. RESULTS: The c-jun gene was expressed in all differentiated PC12 nuclei and some cells expressed p-c-jun. All differentiated PC12 cells expressed nNOS protein. Compared with the control group, SP600125 at 50μmol / L and 100μmol / L could significantly reduce the cell viability (P <0.05), and 50μmol / L SP600125 could effectively inhibit the phosphorylation of c-jun, 0.05), but had no effect on the expression of c-jun (P> 0.05), and at the same time caused the up-regulation of nNOS protein expression (P <0.05). CONCLUSION: The c-jun, p-cjun and nNOS proteins are co-expressed in differentiated PC12 cells. SP600125 effectively inhibits the phosphorylation of c-jun and inhibits the activation of JNK / c-jun and up-regulates nNOS expression. These results indicate that the function of c-jun and nNOS in differentiated PC12 cells is closely related.