Neuron-like differentiation of bone marrow mesenchymal stem cells induced by Rehmannia decoction withCimicifuga heracleifolia Kom. andAchyranthes bidentata Bl. as guiding drugs

doi:10.3969/j.issn.2095-4344.2015.19.003

Abstract：BACKGROUND:Rehmannia decoction has remarkable effects on the neurologic function recovery of cerebral ischemia-reperfusion model rats undergoing bone marrow mesenchymal stem cel transplantation. OBJECTIVE:To investigate the feasibility of bone marrow mesenchymal stem cels differentiating into neuron-like cels induced by Rehmannia decoction withCimicifuga heracleifolia Kom. andAchyranthes bidentata Bl. as guiding drugs and to further explore the regulatory role. METHODS: Bone marrow mesenchymal stem cels were harvested from 4-week-old Sprague Dawley rats and culturedin vitro using the whole bone marrow adhesion method. Flow cytometry assay was used to detect the expression of CD90 and CD45 of bone marrow mesenchymal stem cels at passage 3. Cels at passage 3 were divided into four groups according to different culture conditions: blank control group (1% fetal bovine serum+DMEM/F-12), Rehmannia decoction group (serum containing Rehmannia decoction+1% fetal bovine serum+DMEM/F-12),Achyranthes bidentata Bl. group (serum containing Rehmannia decoction withAchyranthes bidentataBl. as the guiding drug+1% fetal bovine serum+DMEM/F-12),Cimicifuga heracleifoliaKom. group (serum containing Rehmannia decoction withCimicifuga heracleifolia Kom. as guiding drug+1% fetal bovine serum+DMEM/F-12). Then the celular morphology was observed under inverted phase contrast microscope. At 7 days after induction, immunocytochemistry staining and western blot assay were used to detect expression of neuron-specific enolase and glial fibrilary acidic protein; and RT-PCR was used to detect mRNA expression of glial cel line-derived neurotrophic factor and brain-derived neurotrophic factor. RESULTS AND CONCLUSION: Bone marrow mesenchymal stem cels roughly presented a single long spindle or flat-shaped morphology at passage 3, grew with close whirlpool-like arrangement, and the CD90 expression level was up to 98%, but the expression level of CD45 was only 1.3%. At 7 days after induction, bone marrow mesenchymal stem cels in the groups of Rehmannia decoction, Achyranthes bidentataBl. andCimicifuga heracleifoliaKom. became round, extended multiple distinct processes and had connections among some processes, exhibiting typical neuron-like cel morphology, but in the blank control group, there was no obvious change. Immunocytochemistry staining showed that compared with the Rehmannia decoction group, the protein expression levels of neuron-specific enolase and glial fibrilary acidic protein inAchyranthes bidentataBl. andCimicifuga heracleifoliaKom. groups were obviously increased (P < 0.05); and these protein levels were also higher in the Cimicifuga heracleifolia Kom. group than theAchyranthes bidentataBl. group (P < 0.05). Western blot results were consistent with the findings of immunocytochemistry staining. RT-PCR results showed the mRNA levels of glial cel line-derived neurotrophic factor and brain-derived neurotrophic factor were ranked as folows:Cimicifuga heracleifolia Kom. group <Achyranthes bidentata Bl. < Rehmannia decoction < blank control group, and there were significant differences between different groups (P < 0.05). Taken together, Rehmannia decoction withCimicifuga heracleifolia Kom. andAchyranthes bidentataBl. as guiding drugs can induce the neuronal differentiation of bone marrow mesenchymal stem cels, and Wnt/β-catenin signaling pathway may play an important role in neural differentiation of bone marrow mesenchymal stem cels.