Protective Effect of Polysaccharides Isolated from Tremella fuciformis against Radiation-induced Damage in Mice

Wenqing XU Xiu SHEN Fujun YANG Ying HAN Ruifeng LI Dan XUE Chengfeng JIANG Radioprotection/Tremella fuciformis/Hematopoietic system/Micronuclei/Chromosome aberration. WTF-B, a type of water-soluble homogeneous polysaccharide, was isolated and purified from Tremella Fuciformis. To investigate the radioprotective effect of WTF-B, we employed a 30-day survival assay. Mice were treated with WTF-B once per day for three consecutive days before 8-Gy gamma irradiation. The treatment groups receiving 54 and 72 mg/kg body weight (b.w.) of WTF-B showed 50% survival post-irradiation. The hematological parameters of the peripheral blood indicated that WTF-B, when administered at doses of 72 mg/kg b.w., significantly restored hemoglobin, white blood cell counts and red blood cell counts by the 14th day and 18th day. In addition, spleen colony forming units (CFU-S), the number of nucleated cells in bone marrow (BMNC) and spleen index were used to investigate the radioprotective effect of WTF-B on the hematopoietic system. The treatment groups receiving WTF-B at 18, 54 and 72 mg/kg b.w. doses presented significantly higher BMNC compared to radiation-only group. The group administered 72 mg/kg b.w. WTF-B presented a significant change in CFU-S compared to the radiation-only group. We also completed micronucleus and chromosome aberration assays to explore genotoxicity. The results of those assays indicated that the number of micronuclei induced by 2-Gy irradiation in a group treated with 72 mg/kg b.w. WTF-B decreased from 30.30 to 11.32. The chromosomal aberration produced by 3-Gy irradiation in the group receiving 72 mg/kg b.w. WTF-B decreased from 56.01% to 28.13%. The results of the present study indicate a potential use for WTF-B as a radioprotector. - Wide varieties of people are exposed to ionizing radiation and are potentially at increased risk for adverse health effects. Radioprotective agents are of significant importance in medical, industrial, environmental, military and space applications. Radioprotectors might reduce the cancer risk in a population exposed to radiation directly or indirectly through industrial or military applications. Over the past 50 years, the possible radioprotective effects of many synthetic or natural agents have been investigated. Earlier studies were centered on thiols, aminothiols and their derivatives. Through these studies, the gold standard, the radioprotective drug amifostine, also known as Ethyol or WR2721 was discovered and applied in clinical therapy. Some of the other agents also had the effects of protecting cells, membranes and biomolecules, such as DNA and proteins in vitro, and demonstrated promising results in laboratory studies, but were of limited utility for human use due to several factors including toxicity at radiation protective doses and availability.1) Plant extracts, such as those from Emblica officinalis Gaertnd,2) Phyllanthus Amarus,3,4) Amaranthus Paniculatus,5) Tinospora cordifolia,6) Myristica fragrans7) and Ganoderma lucidu,8) have been reported to have radioprotective effects in animal model systems. Tremella fuciformis Berk belongs to the Tremellaceae family of the class Heterobasidiomycetes. The fungus is known as a nutritious mushroom and an important medicine for people in China. Polysaccharides are one of the main bioactive components of Tremella fuciformis Berk. Great advances have been made in chemical and bioactive studies of Tremella fuciformis polysaccharides in the last twenty years.911) Experimental results indicate that the fungus has immunomodulatory,1214) anti-cancer15) and anti-inflammatory activity.16) In our study, a homogeneous polysaccharide, WTF-B, was obtained and identified from crude extracts of Tremella fuciformis Berk. The chemical and physical characteristics of WTF-B were determined. In addition, its protective effects in mice exposed to radiation were explored in terms of whole body survival, protection of the hematopoietic system, genotoxicity and hematological parameters. MATERIALS AND METHODS Preparation polysaccharide of tremella fuciformis berk Fungal spores (150 g) were extracted three times with boiling water. The extract was concentrated in a rotary evaporator under reduced pressure, precipitated by ethanol at 4C for 24 h, and then centrifuged (5000 r, 10 min). The precipitate was dissolved in water and then deproteinated with chloroform and n-butanol eight times. The resulting aqueous fraction was extensively dialyzed against running water for 3 d and then against distilled water for 1 d. The retentate was concentrated under reduced pressure to a small volume, and 4 volumes of ethanol were added stepwise with stirring at 4C. The mixture was stored overnight at 4C. The resulting precipitate obtained by centrifugation consisted of crude polysaccharide. To purify the crude polysaccharide, sequential column filtration was used. First, the extract was applied to a DEAESephadex A-25 column and eluted with 0.1 M NaCl. No carbohydrates were detected via a phenol-sulfuric acid color reaction in the fractions prior to linear gradient elution at 3.9 M and 0.1 M NaCl. The corresponding fractions, WTF-A and WTF-B were pooled, dialyzed, and lyophilized. WTFB, with the highest activity, was further fractionated on a Sephadex G-200 column and eluted with 0.1 M NaCl, resulting in one fraction. This fraction was purified by rechromatography on the same exclusion column three times. The resulting homogeneous polysaccharide obtained was designated WTF-B (yield: 2.5 g). The sugar composition of WTF-B was analyzed by gas chromatography (GC), paper chromatography (PC) an infrared spectrum (IR). The carbohydrate and protein contents were measured using the phenol-sulfuric acid method and Lowry method, respectively. The glucuronic acid content was measured using the sulfuric acid-carbazole method. Homogeneity and molecular weight measurements were made with high power liquid chromatography (HPLC). Animals ICR mice (68 weeks old) with an average body weight of 22 2 g were obtained from the Animal Center of the Chinese Academy of Medical Sciences. They were maintained under controlled laboratory condition at a temperature of 25 2 C with a controlled light cycle (14 hours of light and 10 hours of darkness). The mice were fed standard animal food pellets and tap water ad libitum. All animal experiments were conducted according to the guidelines of the institutional ethics committee. Irradiation of animals Total body gamma irradiation (TBI) was accomplished at room temperature using a 137Cs Gamma Tissue Irradiator at a dose rate of 0.78 Gy/min (Theratron 780E, Canada) during the experimental period. Each mouse was kept in a perforated plastic container. The mice being irradiated were placed on a rotating platform to ensure even dose delivery to all tissues. Administration of WTF-B WTF-B was dissolved in normal saline for administration at the desired concentrations, and the dose was expresse (...truncated)