Roles And Mechanism Of Polysaccharides From Stichopus Japonicus In The Migration Of Neural Stem Cells

Neural stem cells (NSCs) are the multipotent stem cells with the capacity for unlimited self-renewal and the ability to differentiate and form the three terminal functional cells of nervous system, which are neuorns, astocrytes and oligodendoreytes. NSCs transplantation is one of the most potential treatment of neurodegenerative diseases such as Parkinson’s disease, Huntington’s disease, nerve injury, stroke and so on. Therefore, many studies have been performed to find the potential drug to induce the directional migration, differentiation and proliferation of NSCs and therefore to improve the efficacy of NSCs transplantation. Recently, Stichopus japonicus polysaccharide and glycoconjugate have been reported about many pharmacological effects, such as anticancer and anticoagulant activity, etc. But there is few report about the effect of Stichopus japonicus glycoconjugate on NSCs, especially about the directional migration and differentiation.In this work, we used polysaccharide extracted from fresh Stichopus japonicus as materials and thoroughly studied the effects and mechanisms of Stichopus juponiciis polysaccharide (SJP) on the differentiation and migration in vitro of embryonic rat neural stem cells. The main methods and results are as follows:1.Extraction and purification of SJPand determination of the propertiesFirst, we isolated the crude polysaccharides from the wall of the fresh Stichopus japonicus. The crude polysaccharides was isolated by DEAE Sepharose Fast Flow ion-exchange column chromatography to get four fractions including SJP1. SJP2, SJP3, SJP4. The neurosphere migration assay showed that SJP3was effective, so SJP3was further separated to two fractions SJP3-1and SJP3-2by chromatography on a Sephacryl S-200HR column. We found that only the SJP3-1(short as SJP) could promote the migration of neurospheres. Then, the chemical composition and nature of the polysaccharide was analyzed. The specific rotation was determined at20±1C with an automatic polarimeler. Total carbohydrate was determined by the phenol sulfuric acid colorimetric method with D-glucose as a standard at490nm. Uronic acid content was performed by Sulfuric acid-Carbazole with D-galacturonic acid as the standard. Sulfate was measured by the BaCl2/gelation method. Protein was analyzed by the method of Bradford. The chemical composition was as follows:polysaccharide content57.18%, uronic acid content12.39%, sulfate groups content29.75%. It had a negative response to the Bradford test and no absorption at280or260nm in the UV spectrum, indicating the free of protein and nucleic acid components. IR spectra showed that SJP has a typical infrared absorption characteristics of sulfuric acid mucopolysaccharide. The glycosaminoglycan consists of a homogenous fraction with an average molecular weight of1.79×105Da, as determined by High Performance Size Exclusion Chromatography (HPSEC). The specific rotation of the polysaccharide was [a]-48.62(ca.0.1, H2O).2. Effects of SJP on the proliferation, migration and differentiation of NSCsTo study neural stem cells in vitro, a neurosphere cell culture system is convenient. First, We obtain cells from the cerebral cortex of14.5-day-embryonic Wistar rats and cultured in the basal medium supplemented with20ng/mL EGF and20. ng/mL bFGF. Next, the two major characteristics of neural stem cells, known as self-renewing capacity and multipotency, were identified. Nestin(NSCs marker) positive cells could be found in the colony neurosphere. To test the ability of NSCs differentiation into neuronal and glial cells, neurospheres cultured were induced to undergo differentiation for7days after growth factor withdrawal. Then, immunostaining was carried out using antibodies against Fuj1(neurons marker), GFAP (astrocvtes marker). We found that the cells differentiated into Tuj1-positive neurons and GFAP-positive astrocytes, which indicated that the cells had multipotency capacity. Together, these results proved the cells we cultured are neural stem cells.To investigate the effect of SJP on the neural cell migration. in vitro migration assay using neurosphere cells, free-floating spherical clusters generated by neural stem cells, was performed. The result demonstrated that SJP itself could promote the neurosphere adhere to the surface and cells migration. The migration of NSCs significantly increased in a dose-dependent manner in the presence of SJP for48h. compared with the control. Proliferation analysis in vitro was performed using BrdU incorporation assay and Hoechst33342/PI staining was performed to investigate the apoptosis of NSCs after SJP treatment. These assays revealed that that SJP could maintain the survival ability of NSCs. Immunofluorescence staining revealed that SJP can promote the differentiation of NSCs into neurons and astrocytes. Moreover, the expressions of GFAP and Tuj1were up regulated at mRNA level after the cells were treated with SJP, while nestin was decreased.3. Potential molecular mechanisms of SJP in NSCs migration in vitroRT-PCR analysis revealed the elevation of N-cadherin mRNA expression in SJP-treated NSCs. Meanwhile, Western blot analysis showed that N-cadherin levels obviously increased following SJP treatment. Next, we analyzed neurosphere migration in the presence of specific inhibitors for the ERK, BMP and PI3K/Akt pathways. SJP-induced migration of neurospheres and the expression of p-Akt was effectively abolished by the PI3K. kinase inhibitor. LY294002. However, they were not affected by the BMP inhibitor. Noggin and ERK inhibitor, PD98059. Next. we investigated the possible involvement of the PI3kinase-Akt pathway in mediating SJP-induced changes in N-cadherin expression. Western blot analysis revealed that SJP-stimulated induction of N-cadherin was completely blocked by LY294002. Thus, SJP-induced up-regulation of N-cadherin mediated neurosphere adhesion migration and differentiation via the PI3K/Akt signaling pathway.All the analysis showed that Stichopus japonicus polysaccharide, SJP, could promote the migration of NSCs as well as the differentiation of these cells into neurons and astrocytes. These novel findings suggest the possibility that SJP may be useful in NSCs-mediated therapy to treat CNS diseases.