The Signal Transduction Pathways Of Neural Differentiation From Mesenchymal Stem Cells Induced By Tetramethylpyrazine

Mouse bone marrow-derived mesenchymal stem cells(BMSCs) were induced by the tetramethylpyrazine(TMP) to investigate the effect of TMP on the neural differentiation from BMSCs in vitro, and study the signaling transduction pathways involved in BMSC neural differentiation induced by TMP.The experiments were divided into 3 groups: the TMP induction group(2.4 g·L-1 TMP),β-mercaptoethanol(BME) induction group as positive control and the BMSCs group as negative control. Synchronization experiments were performed in each group. RT-PCR was used to detect the expression of several neural genes including Nestin,β-Tubulin III, NSE(neuron-specific enolase), Nurrl(nuclear receptor relatedfactor 1) and GFAP(glial fibrillary acidic protein). Flourescence immunohistochemistry and Western blot methods were used to detect several neural specific proteins, such as nestin and NSE.The blocking agents, such as EGTA(Ca²⁺ chelator), Nifedipine(L-type Ca²⁺ channel blocker) and LY294002(PI3K inhibitor), were applied to block the cellular Ca²⁺ signal pathways. MEK-ERK and p38MAPK pathways were inhibited by PD98059 and SB203580 respectively. We also inhibited the MEK-ERK and PI3K or p38MAPK and PI3K signal pathways simultaneously. TMP was used to induce BMSCs after different signaling pathways inhibited. RT-PCR and Western blot methods were performed respectively to detect the expression of NSE, Nurr1 mRNA and NSE protein to study the effect of Ca²⁺ signal, MEK-ERK and p38MAPK signal transduction pathways on the neural differentiation of BMSCs induced by TMP.The results showed that the induced BMSCs by TMP gradually exhibited neuronal morphological characteristics. The mortality rate of BMSCs caused by TMP is much lower than BME; The expression of several neural specific proteins, such as nestin and NSE were detected by immunocytochemistry and Western blot methods. The levels of these proteins in TMP group were much higher than positive group. RT-PCR analysis revealed that an expression of several neural genes including Nestin,β-Tubulin III, NSE and Nurrl were found in the induced BMSCs. These results suggest that the mouse BMSCs could be differentiated into neuron-like cells in vitro with the induction of TMP and TMP was a good neural inducer. The study also found that a significant increase of nueral cell differentiation was observed after the ectocytic Ca²⁺, L-type Ca²⁺ channel and PI3K blocked respectively or simultaneously compared with the control group; The excess ectocytic Ca²⁺ could inhibit neural differentiation from BMSCs induced by TMP; The inhibition of MEK–ERK and p38MAPK signaling pathways could impairs BMSC neural differentiation induced by TMP; A further reduction in cell differention was detected by blockading MEK-ERK and PI3K or p38MAPK and PI3K pathways simultaneously.These results suggest that TMP regulate the neural differentiation of BMSCs via different Ca²⁺ signaling pathways, MEK-ERK and p38MAPK signaling transduction pathways.