The Study Of Valproic Acid-induced Neuronal Differenation And Neurite Outgrowth In Mouse Embryonic Neural Stem Cells

ObjectiveMultipotent neural stem cell(NSC) is considered to be the most potential candidate for the regeneration of central nervous system(CNS) injury. In NSCs-based therapy, neuronal differentiation is a key step for regeneration and replacement of lost neurons and neural networks, therefore. Valproic acid(VPA), as a neurotrophic compound, enhances neurogenesis, protects neurons, promotes differentiation, and increases neurite outgrowth. However, the underlying molecular mechanism of VPA-induced neuronal differentiation and neurite outgrowth of NSCs is a complex process that remains to be elucidated. This study investigated the effects of VPA on the NSCs' differentiation and neurite outgrowth of NSC-derived neurons. NSCs were isolated from the cortex of C57BL/6J mice on embryonic day 13.5 and cultured with VPA. Furthermore, SP600125, as specific inhibitors of JNK, were applied to study the molecular mechanism. MethodsNSCs were isolated from the cortex of C57BL/6J mice on embryonic day 13.5. The purified NSCs were observed under light microscope, identified by immuostaination with nestin, ?-III-tubulin?GFAP and CNPase to demonstrate its capacity in multilinage differentiation. The third purified NSCs were cultured in plates coated with Poly-L-lysine(PLL) and cultured with V. By using Western blot, immunocytochemistry, and reverse-transcription polymerase chain reaction(RT-PCR), the neuronal differentiation and neurite outgrowth were analyzed with Image-J, Image-Pro Plus 6.0 and LighterCycler?SW1.1 software. ResultsNSCs were successfully isolated from the cortex of C57BL/6J mice on embryonic day 13.5, and identified with morphology observation, Nestin immuostaining, ?-III-tubulin?GFAP and CNPase immunostaining. Immunostaining showed a significant increase in the percentages of ?-?-tubulin-positive cells and microtubule associated protein-2(MAP2)-positive cells(P < 0.001; P < 0.001), while significant decrease were observed in the percentage of glial fibrillary acidic protein(GFAP)-positive cells, a marker of astrocytes(P < 0.001; P < 0.001). It was further confirmed by Western blot and RT-PCR results showing the significant improvement in the ??-tubulin(P < 0.001;P < 0.001) and decrease in the GFAP expression(P < 0.05); ?-?-tubulin immunostaining results showed that VPA group had longer total neurite length?longest neurite length, mean neurite length and more branch points of NSCs-derived neurons; The phosphorylation of JNK and c-Jun were demonstrated to be up-regulated in the VPA group, and their expression decreased when the JNK specific inhibitor SP600125 was applied, meanwhile VPA-induced neuronal differentiation of mouse embryonic NSCs and neurite outgrowth of NSCs-derived neurons were inhibited by the SP600125. ConclusionVPA promoted neuronal differentiation of NSCs isolated from the cortex of C57BL/6J mice on embryonic day 13.5, and improve the neurite outgrowth of NSCs-derived neurons; JNK involved in the VPA-induced neuronal differentiation of mouse NSCs and neurite outgrowth of NSCs-derived neurons.