The Mechanisms For The Radioprotective Effect Of S.Cerevisiae-?-D-Glucan On Carbon-Ion Irradiation

With the development of astronautical technology,the space radiation,especially the space heavy-ion irradiation also makes it a major source of healthy risk for astronauts.Therefore,it is of great significance to find an effective,low toxicity and orally administrated radioprotector.In the present study,human umbilical endothelial cell line EAhy926 and BALB/c mice were used as in vitro and vivo model system to investigate the radioprotective effect of S.cerevisiae-?-D-glucan on the damage induced by carbon-ion?80MeV/u?irradiation.And the possible mechanism of S.cerevisiae-?-D-glucan exerting the radioprotective effect was studied based on the intracellular signaling triggered by the interaction of S.cerevisiae-?-D-glucan with its receptor.These findings will contribute to the development of S.cerevisiae-?-D-glucan as a potential and safe radioprotective agent.In vitro study:Cytotoxicity of S.cerevisiae-?-D-glucan on human umbilical endothelial cell line?EAhy926?was detected by MTT,and it was nontoxic at the concentration of 0-100?g/mL.The clone formation assay showed that 10?g/mL S.cerevisiae-?-D-glucan pretreatment could increase the survival of carbon-ion irradiated cells.The DNA damage,apoptosis and cell cycle were detected by flow cytometry and the results showed that 10?g/mL S.cerevisiae-?-D-glucan pretreatment could decrease the DNA damage,apoptosis and cell cycle arrest caused by carbon-ion irradiation,and increase the recovery of DNA damage.The intracellular reactive oxygen species?ROS?were detected by flow cytometry,the intracellular malonaldehyde?MDA?and the enzyme activities of SOD,CAT and GPx were detected by spectrophotometer,and the results showed that 10?g/mL S.cerevisiae-?-D-glucan pretreatment could decrease the intracellular ROS and MDA,and increase the antioxidative enzyme activities.The expression of signal related proteins Src,MnSOD,BRCA2,Hsp90?,Bax and Bcl-2 were detected by western blotting,and the nuclear activities of NF-?B subunit p65,p50,RelB,p52 and C-Rel were detected using NF-?B family transcription factor assay kit,and the results showed that the interaction of S.cerevisiae-?-D-glucan with its receptor dectin-1 made the phosphorylation of Src,then triggered the NF-?B activation,especially the p65,p50 and RelB subunit,the activated NF-?B up-regulated the expression of antioxidant protein MnSOD,DNA damage response and repair related protein BRCA2 and Hsp90?,and anti-apoptotic protein Bcl-2.In vitro study demonstrated that S.cerevisiae-?-D-glucan could decrease the damage induced by carbon-ion irradiation and increase the recovery and then enhance the survival through scavenging ROS,increasing intracellular antioxidative enzyme activities,and up-regulating pro-survival signaling triggered by the interaction of S.cerevisiae-?-D-glucan with its receptor.Most of the radioprotective mechanisms of S.cerevisiae-?-D-glucan are focus on its antioxidative and immune regulated activities,our research is the first to detect the mechanism that S.cerevisiae-?-D-glucan could interact with its receptor and regulate the expression of pro-survival related proteins.In vivo study:The DNA double and single strand breaks of bone marrow mononuclear cells in different doses of carbon-ion irradiated mice were detected by two-tailed comet assay at the 1^st,3^rd and 8^th day after irradiation,the results showed that the DNA damage was highest at the 3^rd day post irradiation.Mice were orally administrated by high,medium and low concentration of S.cerevisiae-?-D-glucan for2 weeks before 2Gy carbon-ion irradiation,and the DNA damage of mice bone marrow mononuclear cells were detected at the 3^rd and 8^th day after irradiation,the results showed that the low concentration of S.cerevisiae-?-D-glucan is the most efficient to decrease the DNA damage.Furthe r study indicated that orally administration with low concentration of S.cerevisiae-?-D-glucan significantly decreased the apoptosis,ROS and MDA level,and cell cycle arrests in mice bone marrow mononuclear cells after 2Gy carbon-ion irradiation,and increased the IL-3,IL-6,G-CSF and GM-CSF level in mice plasma,thereby ameliorated the hematopoietic syndrome caused by carbon-ion irradiation.The transcriptomic analysis showed that,S.cerevisiae-?-D-glucan significantly modulated the gene expression in bone marrow mononuclear cells of carbon-ion irradiated mice,not only up-regulated hematopoietic related genes,but also regulated immune system and related genes expressions to maintain the stem cells niche in bone marrow.The results of mice survival indicated that low concentration of S.cerevisiae-?-D-glucan significantly increased the survival rate of 6Gy carbon-ion irradiated mice and decreased the weight loss caused by irradiation.In vivo study demonstrated that orally administration of S.cerevisiae-?-D-glucan could relieve the hematopoietic syndrome and prolong the survival of mice exposed to high-LET carbon-ion irradiation by decreasing oxidative damage and modulating the gene expression in mice bone marrow mononuclear cells,increasing the level of protective cytokines in mice plasma and the recovery of hematopoietic cells,promoting the proliferation of hematopoietic stem cells.These results would be helpful to understand the radioprotection molecular mechanisms of S.cerevisiae-?-D-glucan and be a foundation for the future development of radioprotectors.