Study Of The Protective Effect Of Stichopus Japonicus Fucoidan On Cultured PC12Cell Injury Induced By Hypoxia/Reoxygenation

Stichopus japonicas (selenka) is the only edible sea cucumber in the north mailand, it mainly distributed over sea area of Lianyungang City and Bohai Sea estuary. Recent studies reported that Stichopus japonicas polysaccharide possessed many pharmacological effects such as anticoagulant, anticancer, antiviral and immunity enhance activity, etc. But the effect of Stichopus japonicas polysaccharide and glycoconjugate on nerve system disease is few reported. This issue extracted fucoidan from fresh Stichopus japonicus with current technology, studied the hypoxia/reoxygenation injury on cultured PC12cells induced by Na2S2O4and the protective effect of fucoidan extracted from Stichopus japonicas. Main contents of this paper are as follows:1. Extraction and separation of polysaccharide from fresh Stichopus japonicas. On the basis of preliminary laboratory work, the raw material was hydrolyzed by double-enzyme and then precipitated by ethanol, the crude extract was then decolored by macroporous adsorptive resins. Using DEAE-Sepharose column for first separation with phenol-sulphuric acid colorimetric method to monitor each component, we obtained four main components. The active component named SJP-3was collected, Superdex200column for second separation, we obtained SJP-3A、 SJP-3B, then desalted by Sephadex G-25column. According to the preliminary results, we selected SJP-3A for the follow-up study.Determination of physical and chemical properties showed that SJP-3was homogeneous; the molecular weight of it was179000Dal; the sulfate content of SJP-3measured by barium sulfate turbidimetric method was29.75%, significantly higher than that of chondroitin and heparin sulfate; Phenol-sulphuric acid colorimetric method measured its saccharides content to be57.18%.2. Protective effect of fucoidan in Stichopus japonicas on hypoxia/reoxygenation injury in PC12cells. We first studied the influence of SJP-3on cell morphology and viability, found that concerntrations of SJP-3lower than500μg/mL has no obvious toxic effect on PC12cells. After exposed to10mmol/L Na2S2O4for2hours, PC12cells were transferred to reoxygenation environment for24hours. After building the hypoxia/reoxygenation injury model, we studied the protective effect of SJP-3to it. The results showed that,100、300、500μg/mL SJP-3can effectively improve the hypoxia/reoxyganation injury in cell morphological change, improve cell viability, reduce LDH leakage, reduce MDA value and improve SOD activity. Hoechst33342staining and Annexin/PI double staining results through flow cytometry showed that hypoxia/reoxygenation damage cause apoptosis in PC12cells. Pre-treated with SJP-3in could reduce apoptosis.3. Mechanism study about the protective effect of fucoidan in Stichopus japonicas on hypoxia/reoxygenation injury in PC12cells. Detection of mitochondrial membrane potential (MMPT) to the different experimental treatment groups showed that MMPT started to decline in model group, MMPT in SJP-3-treated groups changed a little. Western Blot results showed that hypoxia/reoxygenation injury reduce the expression of Bcl-2, improve protein expression of Bax、caspase-3and caspase-9. With the protective effect of SJP-3, Bcl-2expression in PC12cells was imporoved while the expression of Bax、caspase-3and caspase-9were decreased. All these results suggested that the protective effect of fucoidan in Stichopus japonicas on hypoxia/reoxygenation injury in PC12cells may due to its ability to stabilize the MMPT and to resistance of Cyt-C/mitochondrial apoptotic pathway.In this paper, we used PC12cells to represent nerve cells, researched the protective effect of fucoidan in Stichopus japonicas on hypoxia/reoxygenation injury and studied its possible mechanism. In this experiment, we initially identified the mechanism of SJP-3resistant to cell injury induced by hypoxia/reoxygenation. It provides potential theoretical basis and possible sources of drug for the prevention and treatment of cerebral ischemia/reperfusion-induced nerve damage. It also contributes to the high value-added utilization of marine biological polysaccharide.