Salidroside induces the differentiation of mouse bone marrow mesenchymal stem cells into neuron-like cells mediated by calcium/calmodulin signaling pathway

doi:10.3969/j.issn.2095-4344.2014.37.021

Abstract

Abstract:

BACKGROUND: Our previous studies have shown that salidroside can induce bone marrow mesenchymal stem cells directly into neuron-like cells, and Ca²⁺ signal is one important way to achieve its biological signal transduction.
OBJECTIVE: To investigate the role and mechanism of the calcium/calmodulin (Ca²⁺/CaM) signaling pathway inducing bone marrow mesenchymal stem cells to differentiate into nerve cells.
METHODS: Bone marrow mesenchymal stem cells were divided into two groups: control groups and salidroside groups. Salidroside groups were induced with different concentrations of salidroside (5, 10, 20, 50 and 100 mg/L) for 24 hours and 100 mg/L salidroside was added to culture cells for different time (12, 24, 48 and 72 hours). Western blot assay was used to detect the expression levels of neural cell marker, microtubule-associated protein 2, and the important protein of Ca²⁺/CaM signaling pathway: CaM and calmodulin dependent kinase II (CaMK II). Then Ca²⁺/CaM signaling pathway specific blockers were applied to cells respectively for 30 minutes, including 500 µmol/L EGTA (Ca²⁺ chelator), 1 mmol/L Nifedipine(L-type Ca²⁺ channel blocker) and 10 mmol/L LY294002 (PI3K inhibitor). Then, 100 mg/L salidroside was added and cultured for 24 hours. Western blot assay was used to detect the expression of neuron-specific enolase and CaM in the Ca²⁺/CaM signaling pathway.
RESULTS AND CONCLUSION: (1) After inducing with salidroside, the expression of microtubule-associated protein 2 were upregulated (P < 0.01), indicating that salidrosid can induce the neuronal differentiation of bone marrow mesenchymal stem cells. (2) After different concentrations of salidrosid induced bone marrow mesenchymal stem cells for 24 hours, the expressions of CaM and CaMK II were significantly upregulated in the 10 mg/L group ( P < 0.01); For the 100 mg/L salidrosid that was added for cell induction for different time, the expressions of CaM and CaMK II were significantly downregulated in 72-hour group (P < 0.01). (3) After blocking extracellular Ca²⁺ and PI3K signaling pathway, the expressions of neuron-specific enolase and CaM were higher than those in salidrosid groups (P < 0.05). These results suggest that salidrosid can induce bone marrow mesenchymal stem cell to directly differentiate into nerve cells by inhibiting the Ca²⁺/CaM signaling pathway.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: bone marrow, mesenchymal stem cells, rhodiola, neurons, calcium signaling

CLC Number:

R394.2

Zhao Ling, Zhao Hong-bin, Pan Qian, Li Gen, Wang Jiu-na, Tang Jun-jie. Salidroside induces the differentiation of mouse bone marrow mesenchymal stem cells into neuron-like cells mediated by calcium/calmodulin signaling pathway[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(37): 6019-6023.

Figures/Tables 2

2.1 红景天苷诱导后神经细胞标志分子的表达不同质量浓度红景天苷(5，10，20，50，100 mg/L)诱导D1细胞24 h后，与空白对照组比较，10-100 mg/L红景天苷诱导组MAP2蛋白表达显著上调，差异有非常显著性意义 (P < 0.01)，具有剂量-效应关系，见图1，表1。100 mg/L红景天苷作用细胞12，24，48，72 h后，与空白对照组比较，红景天苷诱导组MAP2蛋白表达显著上调，差异有非常显著性意义(P < 0.01)，具有时间-效应关系，见图2，表2。 2.2 红景天苷对Ca2+/CaM信号通路中CaM表达的影响不同质量浓度红景天苷作用D1细胞24 h后，与空白对照组比较，除10 mg/L红景天苷诱导组之外其余各组CaM蛋白表达水平均下调，差异有非常显著性意义(P < 0.01)，见图3，表1。100 mg/L红景天苷作用D1细胞24，48，72 h CaM表达水平与空白对照组比较差异均有非常显著性意义 (P < 0.01)，其中24 h组上调，48，72 h组下调，见图4，表2。 2.3 红景天苷对Ca2+/CaM信号通路中CaMKⅡ表达的影响不同质量浓度红景天苷作用细胞24 h后，除100 mg/L红景天苷诱导组之外，其余各组CaMKⅡ表达水平与空白对照组比较均上调，差异有非常显著性意义(P < 0.01)，见图5，表1；同一质量浓度红景天苷作用细胞不同时间后CaMKⅡ蛋白表达水平与空白对照组比较，差异均有非常显著性意义(P < 0.01)，其中48 h组上调，其余各时间点均下调，见图6，表2。 2.4 阻断Ca2+/CaM信号通路后NSE、CaM的表达与空白对照组相比，各阻断剂组CaM表达明显上调(P < 0.01，P < 0.05)；与红景天苷诱导组相比，CaM表达均显著上调，差异有非常显著性意义(P < 0.01)。与空白对照组相比，EGTA阻断剂组、LY294002阻断剂组NSE表达明显上调，差异有非常显著性意义(P < 0.01)；与红景天苷诱导组相比，EGTA阻断剂组、LY294002阻断剂组明显上调，差异有非常显著性意义(P < 0.01)，见图7，表3。"

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