Mechanism Of Salidroside Induces Mouse Bone Marrow Mesenchymal Stem Cells Differentiate Into Neuronal Cells Mediated By Calcium

ABSTRACTObjective: To study the change of cell morphology and expression of neural cell markers,discuss effects of salidroside on calcium signal related proteins and genes, reveal role and mechanism of Ca2+ signaling pathway in the process of salidroside inducing D1 cells to differentiate into neuronal cells, in order to provide the basis of molecular biology experiment for Traditional Chinese Medicine theory of Kidney producing marrow.Method: D1 cells were cultured untill 80% are conjugate, divided them into two groups:the one was control group and the other was salidroside group. Control group was cultured with complete culture solution D/F12, while salidroside inducing groups were induced with100mg·L-1salidroside for different time(12, 24, 48 and 72 hours): ①Cells proliferation rate that before and after induction was detected by MTT method; ②Cells morphology was observed by immunohistochemistry method that marked with cytoskeletal protein β-Tubulin;③ Neuronal cell markers such as neuron-specific enolase(NSE), microtubule associated protein 2(MAP2) and β-Tubulin Ⅲ were detected before and after induction by fluorescence immunocytochemistry staining method.D1 cells were divided into control groups and induction groups, the first induction groups were cultured with different concentrations of SD(5, 10, 20, 50 and 100 mg·L-1) for24 hours, and the second groups were induced with 100 mg·L-1 salidroside for different time(12, 24, 48 and 72 hours): ①MAP2 was decected by western blot before and after induction;②Intracellular Ca2+ were assessed in Fluo-3/AM, decected the change of calcium fluorescent intensity;③Key portein of Calmodulin(Ca M) and Calmodulin dependent protein kinaseⅡ(Ca MKⅡ) in Ca2+/Ca M singaling pathway were decected by western blot.D1 cells were blocked with Ca2+ specific blockers(EGTA, Nifedipine and LY294002)respectively, detected the protein expression of Ca M and NSE by western blot.D1 cells were induced with salidroside for 24, 48 and 72 hours, PCR-array assay was used to dectct expression of 84 calcium related genes, chose significant difference genes to analysis, the up-regulated gene of Ptgs-2 protein was detected by western blot.RESULTS:(1) Cytoskeletal staining results: control group cells were polygonal or fusiform shape,D1 cells were induced with 24 hours coud find that apart of cells formed projections and cells body were larger than before, when they were induced with 48 hours, they had typical nerve cells characters and cells body were smaller, synapses were slender, a part of them connected into the network.(2)The results of MTT: after D1 cells were induced for 12, 24, 48 and 72 hours, compared with the control group, the Cell proliferation rate was significantly decreased in 48 and 72 hours groups(P< 0.05).(3)The results of immunohistochemistry: after D1 cells were induced for 12, 24, 48 and72 hours, compared with the control group, positive rate of NSE, MAP2 and β-Tubulin Ⅲwas significantly increased in salidrosid inducing groups(P < 0.01, P<0.05).(4) The results of confocal laser scanning microscopy: BMSCs were induced with different concentration of salidrosid, the calcium fluorescence intensity was different, 100mg·L-1 salidrosid induction groups was significantly higher than other groups(P<0.01); 100mg·L-1 salidrosid induced BMSCs with different time, the fluorescence intensity was highest than other groups(P<0.01).(5)The results of western blot: compared with the control group, the expression of MAP2 was upregulated with the increase of salidrosid inducing dosage(P<0.01, P<0.05), the same concentration of salidrosid induced D1 cells for 24 and 48 hours, the expression of MAP2 were significantly increased, and it had dose-dependent and time-dependent effect. Different concentrations of salidrosid induced D1 cells for 24 hours, compared with the control group,the protein expression of Ca M was significantly downregulated(P<0.01, P <0.05); Compared with the control group, the expressions of Ca MK II in 5, 10 and 20 mg·L-1 salidrosid inducing groups were upregulated(P<0.01), 100 mg/L salidrosid inducing group was downregulated(P<0.05). For the 100 mg·L-1 salidrosid was effect on cells for 12, 24, 48 and 72 hours, the expression of Ca M was upregulated in 12 and 24-hour groups, but downregulated in 48 and72-hour groups, compared with the control group(P<0.01); the expressions of Ca MKII were significantly upregulated in 48-hour group(P<0.01), and downregulated in other inducinggroups(P<0.05, P<0.01), compared with the control group.(6)The results of blocking groups:After blocking with Ca2+ blockers, compared with the control group, the expressions of Ca M was downregulated in salidroside inducing groups(P<0.01); Compared with salidroside inducing group, the expressions of Ca M was upregulated in LY29402 inducing group(P<0.01), the other block groups were downregulated(P<0.01). The expressions of neuron-specific enolase salidrosid group and blocker groups were upregulated than control group(P<0.01, P<0.05); Compared with salidrosid group, LY294002 added with salidrosid group,three kind of blockers added with salidroside group and EGTA group significantly upregulated(P<0.01), EGTA added with salidroside group, LY294002 group and three kind of blockers group were downregulated(P<0.01, P<0.05).(7)The results of PCR-array:100 mg·L-1 salidrosid induced D1 cells for 24, 48 and 72 hours respectively, compared with control group, 38 genes got significant differential expression, which 23 up-regulated and 15 down-regulated, take 5 folds as standard and chose significantly up-regulated genes are DNA damage-inducible transcript3(DDIT3),Heat shock proteins(HSPA5), Protein phosphatase 1 regulatory subunit 15a(Ppp1r15a), Prostaglandin-endoperoxide synthase 2(Ptgs-2),Significantly down-regulated genes are Glucagon(Gcg), Interleukin-2(Il-2),Tumor necrosis factor(TNF)and Somatostatin(Sst).(8)The results of western blot: compared with control group, the expression of STAT3 was downregulated in 24 and 48 hour groups,but up-regulated in 72 hour group(P<0.01), the expression of Ptgs-2 was up-regulated in 72 hour group(P<0.01).Conclusion:(1) Salidroside can induce D1 cells differentiate into neuronal cells.(2) Saliroside may play a important role of L-type calcium channel blocker in the process of induction, and Ca2+/Ca M signaling pathway probably has down-regulation effects.(3)The up-regulation of DDIT3, HSPA5, Ppp1r15 a, Ptgs-2 and down-regulation of Gcg、Il-2、TNF、Sst in the c AMP/Ca2+ signaling pathway may related to the process of salidroside induced D1 cells differentiate into neuronal cells.