Study Of The Effect Of Sulfated Polysaccharide Purified From The Sea Cucumber Stichopus Japonicus On Neural Cells

Background:Nervous system disease is the first global debilitating disease, and the third major cause of death, such as the deletion between neurons and neuronal connections in the damaged region caused by stoke, which results in long-term neurological dysfunction.. Drug intervention is largely confined to the acute phase of brain injury, for example, recombinant tissue-type plasminogen activator (t-PA) was justly used to recovering the cerebral circulation in the acute phase. There is no effective treatment for long-term neurological dysfunction and recovery caused by stroke. Therefore, it is significant to search drug or health food for the treatment in subacute and chronic phase.In China, sea cucumber has long been regarded as medicinal and edible seafood to share. It is also widely used in the elderly and infirm, memory loss, stroke patients, as well as neurodegenerative diseases, health and recovery. The sea cucumber played an active role in preventing neurodegeneration. enhancing and restoring the function of nerve tissues. Moreover, the edible sea cucumber has no side effects. Therefore, the in-depth study of pharmacological effects of sea cucumber ingredients plays significant role in maintaining people's health, and promotes the sea cucumber industry developing. In this study, we isolate the active polysaccharides from sea cucumber body wall, and investigate its effect and mechanism on neural cells.Methods:1. Extraction,purification and physicochemical properties of Slichopus japonicus polysaccharideHydroyzedy double-enzyme. the crude extract was precipitated by ethanol. After decolored by macroporous adsorptive resins, the pohsaccharide was first separated by DEAE-sepharose column with monitoring of210nm.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. The isolated components were then screened by neurospheres migration assay (HS-3is the best) and astrocytes reactive assay(HS-4is the best). Activity single polysaccharides were conducted to characterize the physicochemical properties. Purity was checked by HPLC method; optical rotation was detected by automatic polarimeter; phenol sulfuric acid assay detected the total sugar content; sulfuric acid carbazole detected uronic acids content; gelatin-BaCl2assay detected sulfate content; the Bradford assay checked protein content; gel chromatography detected the polysaccharide molecular weight.2. HS-3promoted neurospheres migration.In this study, neurospheres were obtained from the cerebral cortex of14.5-day-embryonic Wistar rats and incubated in the free-serum growth medium containing EGF and FGF-2. Phase contrast microscope measured the distance of HS which induced neural stem cells migration in the basal medium (no EGF/FGF-2). Hoechst/PI double staining detected the cell survival. Cell proliferation detection took the method of BrdU labeling assay. Using immunofluorescence staining observed the state of neural differentiation induced by HS-3medium. Possible mechanism of induction of cell adhesion and migration was detected through signaling pathway inhibitor (PI3K. inhibitor LY294002, ERK inhibitor PD98059, the BMP inhibitor of noggin), Western blotting and RT-PCR techniques.3. HS-4plus FGF-2promoted astrocyte morphological transformation and reactive.Astrocyte reactive contains three main aspects:1) Cellular morphological transformation;2) Resting cells entering into cell division recycle, cell proliferation:3) Cell migration. In this study, Primary astrocyte cultures were prepared from neonatal(day1) Wistar rat. Purified astrocyte cultures from the cerebral cortex were digested by trypsin and separated by mechanical oscillation. Cell viability was assessed by MTT assay to determine the HS-4treatment concentration and duration in following tests. Eosin staining and immunofluorescence staining were used to observe astrocytes morphological transformation that induced by HS/FGF-2. BrdU incorparation assay detected cell proliferation. Flow cytometry detected cell cycle. Western blotting detected the GFAP and cell cycle regulator), proteins. Cyc D1expression levels. Transwell assay measured cell migration effect. The possible cellular morphological transformation mechanism was detected through signaling pathway inhibitor (Rho pathway inhibitor Y27632. P13K inhibitor LY294002. ERK inhibitor PD98059of P38inhibitor SB203580..INK inhibitor of SP600125). immunofluorescence and Western blotting assay. Results:1. Extraction,purification and physicochemical properties of Stichopus japonicus polysaccharideIn this study, we obtained four polysaccharides by ion exchange chromatography. Cell activity assay determined that HS-3promoted neurospheres migration along with differentiation and HS-4induced astrocytes reactive. The physicochemical properties of HS-3were as following: the homogeneous was determined by HPLC detection. HS-3had a weight-average molecular weight of about1.79×105Da in reference to standard T-series Dextrans. The optical rotation was [α]D20-48.62°(c0.1, H2O); HS-3contain57.18%total sugar,12.39%acidic polysaccharide and of29.75%sulfate. The physicochemical properties of HS-3were as following:molecular weight was4.23×105of Da. The total sugar content was38.12%. Acidic polysaccharide content was16.52%. The sulfate content was32.64%. HS-4was mainly composed of fucose and a trace amount galactose, free of proteins and nucleic acids.2. HS-3promoted neurospheres migration.NSPCs was obtained from the cerebral cortex of14.5-day embryonic Wistar rats. Cells were suspended with basal medium supplemented with basal fibroblast growth factor(bFGF) and epidermal growth factor(EGF). NSPCs proliferated to form neurospheres after7days. The BrdU staining of purified NSPCs was positive and Nestin staining was also positive. NSPCs can differentiate into astrocytes (GFAP positive) and neurons (TUJ1positive) after cultured in2%fetal bovine serum medium for5～7days. HS-3treatment led to neurosphere attachment onto the plate surface and induced cell migration and neurite outgrowth. HS-3promoted cell migration from the neurospheres in a dose-dependent manner. After HS-3treated72h in the concentration of80μg/mL. the migration distance reached to438μm. We also found that HS-3can function as an effective agent for maintaining cell survival following growth factor withdrawal and can sustain viability for at least a week. Neurosphere can differentiate into neuronal and glial cells following3days of exposure to HS-3. The increase in HS-3content was accompanied by a higher proportion of differentiated neurons as determined by RT-PCR. Interestingly. cells expressing mainly TUJI were involved with the initial stages of migration at the forefront of the migratory process, whereas cells stained for GFAP were retained in the neurosphere. SJP-induced attachment and migration of neurospheres were not affected by either an ERK inhibitor (PD98059) or BMP inhibitor (Noggin). However. this process was essentially abolished in the presence of the PI3K inhibitor. LY294002. Pretreatment of neurospheres with LY294002inhibited the Akt-increased serine473phosphory lation and blocked SJP-induced N-cadherin induction. Altogether, these data indicate that HS-3induces N-cadherin expression through PI3K Akt signaling pathway activation. 3. HS-4plus FGF-2promoted astrocyte morphological transformation and reactive.Astrocyte purity was greater than97%as based on GFAP staining. Cells cultured for long periods presented characteristics of senescence with cell body enlargement, and displayed flattened polygonal shapes with actin bundles at the cell edges. As a result of cell body enlargement, cell proliferation stoped and produced cell senescence. Senescence in cells arrested cell growth in G1phase and changed the metabolism. The combination of HS-4and FGF-2changed a number of astrocytic characteristics. Now, normal resting astrocytes were transformed into reactive astrocytes with stellate-shaped, spherical and phase-bright cell somas. There was also a rearrangement of GFAP fibers, an astrocyte marker protein, with changes from their flat polygonal shape into a stellate process-bearing morphology. BrdU-positive astrocytes was significantly increased after culture with HS-4and FGF-2. The percentage of proliferative cells obtained with this assay was13.04%(control), but achieved statistically significant increases of55.92%over controls with the combination of FGF-2and HS-4. The combined treatment of HS-4and FGF-2resulted in an increasing population of cells at the S phase, but a decreasing population in the G1phase accompanied. With respect to cell cycle signaling, immunoblotting assay demonstrated an accumulation of cyclin Dl. The resting state cell could not pass through the transwell membrane, revealing no migration activity. However, reactive astrocytes induced by HS plus FGF-2has the function of migration acityity that could pass through the membrane. Altogether, these data indicate that HS-4could induce the activation of astrocytes.PI3K inhibitor slightly inhibited the HS-4/FGF-2induced astrocytes activation. ERK inhibitor could completely inhibit the activation of astrocytes.JNK inhibitor almost had no inhibition function. On the contrary. P38inhibitor could promote activation induced by HS-4/FGF-2. Medium by adding EGTA could inhibit cell activation through combining of Ca2+An inhibitor of Rho-rock rapidly promoted astrocyte reactivityConclusion:In this study, we obtained two different cell acitvity of intensely sulfated polysaccharides from sea cucumber body wall. HS-3was capable of promoting neurosphere attachment and migration in a dose-dependent manner. Moreover. HS-3effectively maintained cell viability even after being deprived of mitogens. Our current results demonstrated that neurosphere were differentiated into neuronal and glial cells when exposed to HS-3. This HS-3-induced up-regulation of N-cadherin mediated neurosphere adhesion migration and differentiation via the PI3K Akt signaling pathway. Combinational treatment of HS-4and FGF-2synergistically promoted morphological transformation of normal astrocyte into a stella morpholog\(stellation), cell proliferation and cell migration. Probably by activating ERk kinase and inhibiting Rho activity, HS-4induced astrocyte reactive, and led to cytoskeletal protein reorganization, and cell morphological transformation. Activating ERK kinase enhanced the expression of nuclear transcription factors, such as Cyc D1, which promoted cells to change from G0/G1to S phase transition, inducing cell proliferation.