Radioprotective Effect Of Sipunculus Nudus Polysaccharides

With the increasing acknowledge of nuclear and irradiation, people pay more attention to the study of radio-protective agents. Therefor varieties of them were increasing, meanly including thiol, phenols, polysaccharides, cytokines, vitamins, herbal medicine and its extracts, marine materials and so on. While most of them have some disadvantages more or less. For example some of them have good effects but also toxic and side effect, otherwise having little side effects without obvious curative effect. WR2721 is still the most effective radio-protective agents until now. Although it has good effect, it also produces seriously side effects which are bad for people. At present, the most common radio-protective agents in China are estrogens(like “500” and “523”) and plant resource active compounds(like “408”) and so on. Estrogens have obvious effects while they also have side effects such as carcinogenicity and gynecological cancer. The latter has little side effects but the curative effect of it is general and the resource of it is limited. For these reasons, it is necessary and urgent for us to find a radioprotector with more efficiency and less toxicity. This paper isolated polysaccharides from Sipunculus nudus, and characterized its structure, systematically studied the protective effects of it in vivo and in vitro and its primary anti-oxidation mechanism by establishing animal experiment model, cells experiment model and scavenging free radical activity experiment.Sipunculus nudus polysaccharide(SNP) was purified from S. nudus L. via NaOH extraction, trichloroacetic acid deproteination, DEAE-cellulose 52 and Sephacryl S-300 chromatography. The uronic acid content was 9.82% measured by Carbazole-sulfuric acid method. The monosaccharide analysis was detected with PMP-HPLC and SNP was composed of mannose, rhamnose, galacturonic acid, glucose, arabinose and fucose with mole ratio of Man:Rha:Gal A:Glc:Ala:Fuc =1.2: 0.9: 7.5: 88.8: 0.8: 0.9. The molecular weight was 680 kDa tested by HPGPC. FT-IR results showed that SNP had the stretching vibration of the O-H bond, the C-H bond, the C-O-C glycosidic bond vibration and the glucose ring. 1H spectrum and 13 C NMR spectrum showed that α-configuration and C-6 resonance were existed in SNP.To study the radioprotective effects on mice, a total of 60 male BALB/C mice were random Ly divided into six groups(n = 10 per group): normal control group( no administration, no irradiation), irradiated control group( no administration, irradiation), positive control group(administration 300 mg/kg 523, irradiation), 30 mg/kg SNP group(irradiation), 90 mg/kg SNP group(irradiation) and 270 mg/kg SNP group(irradiation). 30 days survival rate was measured by whole body irradiation with 7.5 Gy 137 Cs γ ray. The results showed SNP could improve the survival rates of irradiated mice and prolong the survival time after irradiation. Mice were sacrificed on the 14 th day after whole body irradiation by 4.0 Gy 137 Cs γ ray. RBC, WBC and PLT amounts in peripheral blood were counted and organ indexes were measured including liver index, spleen index, thymus index and testis index. Flow cytometry(FCM)was used to detect the hematopoietic stem cell content, the micronucleus rate in bone marrow cells and the apoptosis rate in bone marrow cells. The results showed that SNP could increase the RBC, WBC and PLT amounts, spleen index and testis index. It also increased the DNA content and hematopoietic stem cell content in bone marrow and significantly decreased the micronucleus rate and apoptosis rate of bone marrow cells. Bone marrow histology pathology was analyzed by HE staining. The results showed that SNP could protect the bone marrow from radiation damage. The LPS, DAO, TNF-α and IL-6 content in blood was measured on 1st and 2nd after irradiation. The results showed that SNP could greatly decrease the contents of LPS and DAO in blood plasma, decreasing the DAO and TNF-α content in serum at the same time. The intestinal tissue pathology and stomach tissue pathology showed that SNP could effectively protect the crypt cell in intestinal and gastric mucosa against irradiation. Besides, it also decreased the apoptosis rate in crypt cells measured by TUNEL staining.To study the radioprotective mechanism of SNP, scavenging free radical activity in rats and in cells were detected. The results suggested that SNP showed significant dose-dependent scavenging activities on hydroxyl radical generated through the Fenton reaction, and significantly increase the activities of SOD and GSH-Px in serum and tesis tissue and CAT in intesdinal and IEC-6 cells. Besides, it decreased the MDA content in serum, tesis intestinal and IEC-6 cells. CCK-8 method tested the protection of SNP on irradiated IEC-6 cells and the results showed that SNP could significantly improve the survival rate and decrease the ROS level of cells after 8.0 Gy 137 Cs γ ray irradiation.SNP showed obvious protection on hematopoietic system and digestive system in irradiated mice and increasing the activity of antioxidant enzyme and decreasing the producing of lipid peroxide would be one of the mechanism. The present study will play an important theoretical and practical role in revealing protective mechanism of radiation and exploiting the natural and efficient raidioprotectors.