Study On Salidroside Induces Mouse Bone Marrow Mesenchymal Stem Cells Differentiate Into Neural Cells Directionally Via ERK1/2and PI₃K/AKT/mTOR Signaling Pathways

Objectives1. To elaborate the manner and molecular mechanism of the bone marrow mesenchymal stem cells (BMSCs) induced into neural cells by the traditional Chinese medicine-Salidroside in the research.2. To investigate the role of the extracellular signal-regulated kinase (ERK1/2) and PI3K/AKT/mTOR signaling pathways inducing bone marrow mesenchymal stem cells (BMSCs) differentiation into neural cells.Methods1. Mouse bone marrow-derived mesenchymal stem cell lines (D1cells) were used as research objects. We established the model outside the body and the experiment was divided into control group and SD groups. SD groups (100μg·mL-1)were induced12h,24h,48h and72h respectively. We used the immunofluorescence staining chemical technology to detect the positive rates of β-Tubulin-Ⅲ, NSE, MAP2, NES and GFAP. Then, we used Western blotting and Real-time PCR to detect the protein and mRNA expression levels of β-Tubulin-Ⅲ, NSE and GFAP.2. The model was established outside the body. Different concentrations (Sμg·mL-1,25μg·mL-1,50μg·mL-1,100μg·mL-1and200μg·mL-1) of SD were used to induce24h and the same concentrations of SD (100μg·mL-1) were used to induce at different times (3h,6h,12h and24h). The protein expression levels of NSE, β-Tubulin-Ⅲ, ERK1/2, P-ERK1/2, AKT and P-AKT were detected by Western blotting. Besides, the mRNA expression levels of MAPK3, AKT, and Ps6k were detected by Real-time PCR. Then, we used Western blotting to detect the protein expression levels of ERK1/2, NSE and AKT when signal pathways were blocked. Real-time PCR was used to detect the mRNA expression levels of MAPK3, AKT, Ps6k, NSE, Nr4a2and NES when signal pathways were blocked by PD98059, LY294002and Rapamycin. Results1. The cellular morphology changed from round shape to spindle shape under the microscope. The results of MTT showed that cell viabilities were the highest at48h (P<0.05). The positive expressions of β-Tubulin-Ⅲ, NSE, MAP2, NES and GFAP had expressed with time increased gradually. The positive rates of β-Tubulin-Ⅲ were highest at24h while NSE and MAP2were at48h. Beside, the positive rates of NES were highest at72h while GFAP were at12h. The differences had statistical significance compared with control group (P<0.01). The results of Western blotting showed that the protein expression levels of β-Tubulin-Ⅲ and NSE expressed higher with time increased besides the highest expression was at48h (P<0.05), both of them had time dependence to some extent. The results of Real-time PCR showed that they had expressed at all time besides the highest was at24h.2. When we investigated ERK1/2and PI3K/AKT/mTOR signaling pathways, the results of MTT showed cell viabilities expressed higher first and then depressed with concentrations increased. In first SD group, the protein expression levels of β-Tubulin-Ⅲ, NSE, ERK1/2, P-ERK1/2and AKT expressed higher with concentrations increased, the differences had statistical significance compared with control group (P<0.05). In second SD group, the protein expression levels of ERK1/2and P-ERK1/2expressed higher with time increased while AKT expressed higher first and then depressed. After we blocked the signaling pathways, the protein expression levels of ERK1/2, NSE and AKT decreased significantly (P<0.01). The Real-time PCR showed that the mRNA expression levels of MAPK3, AKT and Ps6k all had expressed in first SD group. Besides, the mRNA expression levels of MAPK3and AKT expressed higher than control group at25μg·mL-1,100μg·mL-1and200μg·mL-1(P<0.05), they expressed higher with concentrations increased. In second SD group, the mRNA expression levels of MAPK3expressed higher than control group at3h and24h while AKT expressed at6h and24h (P<0.05), the mRNA expression levels of Ps6k expressed higher with time increased (P<0.05). When we blocked ERK1/2and PI3K/AKT/mTOR signal pathways, we found the mRNA expression levels of MAPK3, AKT and Ps6k decreased (P<0.05) and the genes of downstream (NSE, Nr4a2and NES) also decreased significantly (P<0.01).Conclusions1. SD can induce BMSCs differentiation into neural cells under some certain conditions.2. It had relationships that SD induced BMSCs differentiation into neural cells with ERK1/2and PI3K/AKT/mTOR signaling pathways on molecular mechanism. They promoted BMSCs differentiation into neural cells by activating coherent signaling pathways.