Studies On The Preparation Of A Sulfated Polysaccharide Purified From The Sea Cucumber Stichopus Japonicus And Its Proliferative Activity On Neural Stem/Progenitor Cells

Sea cucumber, a kind of marine invertebrate, belongs to Holothurioide genus, Stichopodidae family. As a precious food supplement, it has been used as Chinese folk medicine from time immemorial. The sulfated polysaccharides isolated from sea cucumber displayed various biological activities including anticoagulative effects, inhibition of osteoclastogenesis, modulation of angiogenesis, inhibition of tumor metastasis, inflammatory reactions and so on. In recent years, sulfated polysaccharides (SPS) have obtained much attention in nervous system development and in area of glycobiology. Proteoglycans consisting of SPS are prominent components of the ECM in the CNS and are assumed to play an important role in controlling proliferation, differentiation and migration of NSPCs. However, the study of SPS, especially SPS isolated from marine mollusca, effect on neural stem/progenitor cells (NSPCs) is rather rare.We isolated the polysaccharide from body wall of the fresh sea cucumber Stichopus japonicus by enzymolysis extraction, anion-exchange and gel-permeation chromatography. The homogeneous fraction, a kind of fucoidan named as HS, was obtained and a series of physicochemical properties of HS and its structure were analyzed. The effects on survival, proliferation, aggregation and apoptosis of NSPCs were studied for the first time, indicating HS promotes the proliferation of NSPCs and neurosphere formation, acting synergistically with FGF-2 but not EGF. Moreover, we investigated the possible mechanism leading to neurosphere formation and the related signaling pathway. All the results of HS were summarized as follows.1 Extraction and purification of Stichopus japonicus polysaccharide (HS)Hydrolyzed by double-enzyme, the crude extract (CHS) was precipitated by ethanol. After decolored by macroporous adsorptive resins, the polysaccharide (FHS) was first separated by DEAE-Sepharose column with monitoring of 210nm,280nm ultraviolet light assisted phenol-sulphuric acid colorimetric method. The results have shown that four main fractions named A, B, C and D were obtained, but only D fraction has proliferative effects on NSPCs based on the NSPC proliferative assay. Therefore, the D fraction eluted from DEAE column was further fractionated on a SuperdexTM 200 column which was eluted with 0.15 M sodium chloride at a flow rate of 30 mL/h. The main fraction (D1) was collected, desalted by Sephadex G25 gel column and lyophilized to obtain a purified polysaccharide (D1 fraction, named as HS). The rate of CHS and FHS was 0.23% and 0.053% respectively.2 Analysis of physicochemical properties of HS and its structureThe homogeneity of HS was verified initially by UV spectrum scan and Superdex 200 column chromatography. HS had a weight-average molecular weight of about 4.23×105 Da in reference to standard T-series Dextran. HS appeared as a white powder. It produced a negative response in the Bradford test and no absorption was found at 280 or 260 nm in the ultraviolet spectrum, indicating the absence of proteins and nucleic acid in the fraction. The total sugar content of HS was determined to be 38.12% and uronic acid content was 16.52%. It is noteworthy that the sulfate content of HS was as high as 32.64%, much higher than that of heparin. The monosaccharide composition analysis from TLC showed that HS was mainly composed of fucose and a trace amount of galactose. These results were further substantiated by HPLC. The molar ratio of fucose to galactose in HS was equivalent to 14.29:1 based on the conversion of the peak area of the two monosaccharides. The FT-IR spectrum showed that HS had the characteristic absorption peak of SPS, and that the pyranose residues of HS were linked inβ-configuration. The 1H and 13C NMR chemical shifts of HS were confirmed that HS were linked inβ-configuration.3 Proliferative effects on NSPCs of HS in vitroFirstly, the culture systems for the in vitro expansion of NSPCs using neurosphere in suspension had been established. NSPCs were obtained from the cerebral cortex of 14-day-embryonic Wistar rats and incubated in the free-serum growth medium containing DMEM/F-12 nutrient and B27 supplement. The sternness and the multi-lineage potential of neurospheres formed were identified by immunochemistry assay. Secondly, the effects of HS on the viability and proliferation of NSPCs in vitro were examined by MTT assay, BrdU labelling and neurosphere formation assay respectively. Our results showed that HS alone increased NSPC viability in a dose-dependent manner. Moreover, HS acted synergistically with basic fibroblast growth factor (FGF-2) but not epidermal growth factor (EGF) to enhance the proliferation of NSPCs. At a higher concentration ranging from 2～8μg/mL, HS significantly promoted neurosphere formation in a dose-dependent manner,also acted synergistically with FGF-2. The most effective dose of HS to promote the formation of neurospheres was between 4 and 8μg/mL. Besides, HS significantly promoted neurosphere formation even though incubating in the the medium containing 1% fetal bovine serum, but neurite branch was less than the control, and showed more elaborate networks of neurites. Finally, HS did not induce apoptosis of NSPCs among the treatment of HS and/or FGF-2.4 Possible mechanism leading to neurosphere formation and the related signaling pathwayIn the present study, we observed a significantly faster formation of neurosphere units on the same day of plating. And a larger free space between cells and/or neurosphere units appeared in HS cultures, as compared to the control group. Meanwhile, HS significantly promoted neurosphere formation even though incubating in the the medium containing 1% fetal bovine serum. Therefore, we thought that proliferation of NSPCs induced by HS was not the only reasons for the faster formation of neurosphere units. Thus we attempted to explore the mechanisms of the faster formation of neurosphere units from these two aspects:aggregation and proliferation of NSPCs. The results showed that several NSPCs dispersed on the plate begin to aggregate induced by HS at the early culture stage, and then 3-5 cells will aggregate to form neurosphere units. The cell aggregates formed might provide a favorable environment for the proliferation of NSPCs. However, the aggregation was not caused by chembtactic migration of NSPCs, as evidenced by the transwell chamber assay. On the other hand, cell cycle analysis showed that HS increased the percentage of cells in S phase by 2.8-fold, as compared with the control. Thus, we demonstrated that HS was able to promote cell proliferation and aggregation of NSPCs which could lead to the formation of neurospheres, and suggested that HS can serve as an adjuvant for promoting proliferation of NSPCs and formation of neurospheres. 5 NF-κB activation and the effects of HS on NSPCsNF-κB is ubiquitously expressed throughout the nervous system, and the pathway is activated by cell surface receptors that signal to degrade its inhibitor IκB, leading to NF-κB nuclear translocation. The NF-κB signaling pathway plays a central role in neuronal integrity, synaptic plasticity, neuroprotection and neurogenesis. Since NF-κB is known to control the proliferation and aggregation of NSPCs stimulated by TNF-α, we hypothesized that there was a relationship between the effects of HS on NSPCs and the activation of nuclear factor NF-κB. We used an ELISA kit to measure the amount of p65 in the nucleus of NSPCs that indicated the extent of activation of NF-κB. The results showed that HS significantly activated the translocation of NF-κB and the effects were dose-dependent. HS (50μg/mL) increased NF-κB nuclear translocation nearly 1.5-fold greater than the controls. These findings suggested that HS stimulation was related to the activation of NF-κB signaling pathway.6 Stemness and multi-lineage potential of NSPCsHS originated from marine invertebrate which is less likely to contain infectious agents, such as viruses or prions. More importantly, the anticoagulant activity of fucoidan from sea cucumber is lower than heparin, so that the hemorrhage risk is lowered for clinical use of transplanting NSPCs into the CNS if contaminated with fucoidan.Due to its safety, we suggested that HS can serve as an adjuvant for promoting the proliferation of NSPCs. Therefore, we examined the sternness and multi-lineage potential of neurospheres formed by HS. The results showed that HS-stimulated formation of neurospheres did not alter the lineage of after differentiation. Neurospheres were remained positive for intermediate filament protein (Nestin), a NSC marker, and the neurosphere formed with HS had the ability of multi-lineage potential, the markers of three neural lineages, such as oligodendrocytes marker O4, astrocytes marker GFAP and neuronal marker MAP2 did not change after HS treatment. These findings suggest that HS stimulation might not influence sternness of neurospheres and their multi-lineage potential. Originality of the article are the following:(1) A homogeneous sulfated polysaccharide named as HS was obtained from Stichopus japonicus. HS had a weight-average molecular weight of about 4.23×105 Da. The total sugar content and uronic acid content of HS was 38.12% and 16.52% respectively. The sulfate content of HS was as high as 32.64%.(2) The effects on survival, proliferation, aggregation and apoptosis of NSPCs were studied for the first time. The results indicated HS promotes the proliferation of NSPCs and neurosphere formation, acting synergistically with FGF-2. HS did not induce apoptosis of NSPCs.(3) The aggregation effects on NSPCs of HS were studied for the first time. At the early culture stage, HS induced a significantly rapid aggregation of NSPCs, resulting in formation of 3-5 cells aggregates. Meanwhile, HS increased the percentage of cells in S phase and promoted proliferation of NSPCs. These two roles of HS leaded to a rapid formation of neurospheres.(4) The effects on NSPCs of HS were related to the activation of NF-κB signaling pathway.