Rapamycin Protects Against Radiation-induced Hematopoietic Damage In Mice

Objects:To explore the effects of pretreatment of Rapamycin against radiation-induced hematopoietic damage in mice.Methods:(1) C57BL/6 mice were randomly divided into untreatment group and radiation-treatment group, and each group includes vehicle- and rapamycin-treatment settings. Mice received rapamycin by i.p. 4mg/kg, for 5 times every each other day before 5 Gy TBI, vehicle was given to mice at the same time. The change of WBC, LYM, RBC, PLT and HGB were measured with peripheral blood from untreatment and radiation-treatment group at day 0.5, 1, 2, 3, 5, 7, 15, 30, 40, 70 and 100.(2) Bone marrow cells were obtained from femur and tibia of mice in 6 and 12 hour after TBI to measure the accumulation of γ-H2 A.X by FACS and WB, and to measure key proteins in DNA damage repair pathway by WB.(3) Chromosomes were obtained from bone marrow cells at day 30 after TBI by hypotonic treatment to analyse genomic stability attributed to rapamycin pretreatment.(4) Sternum from untreatment and radiation-treatment groups at day 1, 5, 15 and 30 were stained by HE to analyse the distribution of nucleated cells, the ratio of nucleated cells and mature red blood cells as well as the changes of vessels.(5) At day 15 after TBI, liver and spleen from each group were obtained. Livers were stained by HE to analyse the change of tissue structure. Spleen was immersed into Tellyesniczky solution to count the clot which indicates endogenous colony formation ability.(6) Bone marrow cells from vehicle- and rapamycin-treated mice in 4 hour after 5 Gy TBI were injected to lethally irradiated mice by i.v. to analyse the difference of median survival time.(7) Mice were treated with vehicle and rapamycin in different dose(1 mg/kg, 4 mg/kg, 8 mg/kg) by i.p. then were given 8.5 Gy TBI to analyse the difference of median survival time.Results:(1) Before radiation: Rapamycin treatment decreased the amount of WBC and LYM, increased RBC number and HGB level, and have no effects on PLT. After radiation: Rapamycin treatment slows down the decrease of RBC and PLT, enhance the recovery of WBC and LYM in number.(2) Before radiation: γ-H2 A.X was undetectable in both groups, while the expression of Rad50 and Lig4 was the same. After radiation: The DNA damage maker γ-H2 A.X was lower and DNA damage repair proteins Rad50 and DNA ligase 4 were higher in rapamycin-treatment group than the control group.(3) In vehicle-treatment group, Chromosomes from bone marrow cells obtained 30 days after TBI were found abnormality, such as chromosome nick and break, however, no abnormality was found in rapamycin-treatment group.(4) Before radiation: Rapamycin-treatment did not have any effects on the distribution of nucleated cells and red blood cells, the ratio of nucleated cells and red blood cells as well as the structure of vessel. After radiation: nucleated cells decreased, but red blood cells increased, the distribution of red blood cells was in a mess due to the destruction of vessels. At day 15 after TBI, nucleated cells were recovered, red blood cells were decreased, and vessels were reconstructed; however, the recovery of nucleated cells in vehicle-treatment group was slower than the rapamycin-treatment group, and what is more, malignant erythropoiesis was found in the vehicle-treatment group.(5) Before radiation: There was no difference between two groups. After radiation: 15 days later, severe hepatocyte hydropic degeneration was detected in vehicle-treatment group, and the colony formation ability of spleen was lower in vehicle-treatment group by counting the white spots of spleen.(6) Lethally-radiated mice received bone marrow transplantation. The bone marrows were immediately obtained from the vehicle-treatment or rapamycin-treatment mice after 5 Gy TBI. Contrast to vehicle-treatment group, the bone marrow from rapamycin-treatment group slightly prolonged the median survival time.(7) Rapamycin treatment slightly prolonged the median survival time of 8.5Gy TBI, and the most adaptive dose was 4 mg/kg.Conclusions:(1) Treatment of rapamycin before radiation effectively reduces the hematopoietic damage, and enhances the recovery of peripheral blood and bone marrow.(2) Treatment of rapamycin before radiation reduces suppression of DNA damage repair pathway proteins and reduces radiation-induced genomic instability.(3) Treatment of rapamycin before radiation may protect extramedullary hematopoiesis possibly by reducing hepatocyte hydropic degeneration and enhancing the endogenous colony formation ability.(4) Treatment of rapamycin before radiation indirectly protects hematopoietic stem/progenitor cells, and prolongs survival time after 8.5 Gy TBI.