体外培养神经元内JNK/c-jun信号通路对nNOS表达的影响

The effect of JNK/c-jun signaling pathway on the expression of nNOS in culture cells in vitro

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。

Objective： In central nervous system, both c-inn and nNOS genes are strongly expressed inside injured neu- rons ; however, the relation of these two genes in neuronal diseases remains uncertain. In the present study, we investiga- ted the role of JNK/c-jun signaling pathway on nNOS gene in neuron-like differentiated PC12 cells. Methods： Differenti- ated PC12 ceils were cultured in vitro, the expression of JNK/c-jun, p-c-inn and nNOS were detected by immunohisto- chemistry. An inhibitor-SP600125 was used to inhibit JNK/c-jun signaling pathway, the cell viability of differentiated PC12 were detected by MTT assay after incubating with different concentrations of SP600125. The effect of SP600125 on JNK/c-jun, p-c-jun and nNOS gene expression were investigated by Western blot, which were used to further verify the re- lationship between c-jun and nNOS. Results： c-inn gene was expressed in the nuclei of all differentiated PC12 cells, while p-c-jun was expressed in part of them, nNOS-immunoreactivity was expressed in the cytoplasm of all diffeentiated PC12 cells. 50 Ixmol/L and 100 txmol/L of SP600125 significantly reduced cell viability （ P 〈 0.05 ）, 50 txmol/L SP600125 inhibited the c-jun phosphorylation （P 〈 0.05 ）, while there was no effect on c-inn expression （P 〉0.05 ）.Moreover, the nNOS protein levels were also upregulated in differentiated PC12 cells following 50 p.mol/L SP600125 in- cubation （P 〈0.05）. Conclusion： The c-jun, p-c-jun and nNOS proteins were co-expressed in the differentiated PC12 cells, the inhibition of JNK/c-jun phosphorylation upregulated the nNOS protein expression in differentiated PC12 cells. These resuhs indicate that there is a functional relationship between c-jun and nNOS in differentiated PC12 ceils.