Effect Of Early Intervention Of Recombinant Human Thrombopoietin On Radiation-induced Long-term Myelosuppression

Background:Acute radiation sickness occurs in people exposed to nuclear radiation due to high doses of radiation,and bone marrow acute radiation sickness with hematopoietic injury as the basic injury is more common.The hematopoietic damage caused by ionising radiation includes acute haematopoietic damage and long-term bone marrow suppression.Therefore,re-establishing haematopoietic function and promoting hematopoietic recovery is the key to the treatment of myeloid acute radiculopathy.The therapeutic effect of hematopoietic growth factors on radiation induced acute hematopoietic damage has been recognised by national and international scholars.For example,recombinant human granulocyte colony-stimulating factor has been approved for clinical use in the treatment of myeloid acute radiculopathy.However,the effects of hematopoietic factors on radiation-induced long-term myelosuppression have been less well studied.In particular,it has been suggested that the early use of hematopoietic growth factors after irradiation may lead to depletion of haematopoietic stem cells and exacerbate long-term myelosuppression,but this view is controversial.Previous studies have shown that the early post-irradiation administration of recombinant human thrombopoietin（rh TPO）,a hematopoietic growth factor,significantly promotes the recovery of acute haematopoietic damage after radiation by regulating haematopoietic stem cells,and has been recommended for the treatment of acute radiation sickness.However,whether rh TPO has an effect on radiation-induced long-term myelosuppression is unclear and has not been reported.Based on this,in order to investigate the effect of rh TPO on radiation-induced long-term myelosuppression and to provide an experimental basis for the safe and rational use of rh TPO in the treatment of bone marrow acute radiation sickness,this project carried out a study on the effect and mechanism of early intervention of rh TPO on radiation-induced long-term myelosuppression.The results of this study will also provide reference data to elucidate whether haematopoietic factors treating acute hematopoietic injury while exacerbating long-term bone marrow suppression.Methods:C57BL/6J mice were irradiated with 9.0 Gy ⁶⁰Co gamma rays as an acute radiation disease survival model.The experimental animals were divided into irradiated control group,subcutaneous injection of rh TPO 100μg/kg group,intramuscular injection of rh TPO 25μg/kg,50μg/kg and 100μg/kg group,eight animals in each group.On the basis of determining the optimal administration protocol for rh TPO,C57BL/6J mice were irradiated with 6.5 Gy⁶⁰Co gamma rays in whole body as an experimental model of myeloid acute radiation sickness.The experimental animals were divided into normal control group,irradiated control group and rh TPO group.The effect of rh TPO on the long-term hematopoietic injury effect in irradiated mice was investigated by observing the peripheral blood picture at 0 d,10 d,14 d,18 d,21 d,30 d,60 d,90 d,120 d and 180 d after irradiation.And the changes of peripheral blood T,B,G,Treg and NK immune cell populations in each group of mice at one,two,three and six months after irradiation.In addition,the bone marrow structure,the number of nucleated cells,the number of in vitro bone marrow cell colonies,the proportion of hematopoietic stem cells,the chimerism rate of peripheral blood cells and the proportion of differentiated cells of each lineage in recipient mice were analyzed in one,two and six months post-irradiation competitive transplantation experiments.Based on the above experiments,flow cytometry was used to detect the changes of one,two and six months HSC cycles and apoptosis after irradiation in mice.Flow sorting technique,real-time fluorescence quantification andβ-galactosidase staining were used to detect one,two and six months HSC senescence after irradiation in mice,in order to clarify the cytological mechanism of rh TPO affecting radiation-induced long-term bone marrow suppression.Flow cytometry was used to detect the changes in reactive oxygen species content and DNA damage in HSC one,two and six months after irradiation.The changes in m RNA expression of HSC ROS-related pathways one,two and six months after irradiation were detected by flow sorting technique,magnetic bead sorting technique and real-time fluorescence quantification to reveal the molecular mechanism of rh TPO affecting radiation-induced long-term myelosuppression in mice.Results:1.rh TPO improved peripheral blood cell abnormalities in mice with long-term bone marrow suppression after radiation.Survival experiments showed that all mice in the irradiated control group died within15 days after irradiation,while the 30 d survival rates of the 100μg/kg dermal,25μg/kg intramuscular,50μg/kg and 100μg/kg groups were 40%,0%,50%and 90%,respectively.Based on this,peripheral blood results showed that rh TPO significantly promoted blood cell recovery in irradiated mice within 30 days post-irradiation and did not inhibit peripheral blood cell counts from one to six months post-irradiation.The results of immune cell detection by flow cytometry showed that the percentage of peripheral blood B cells decreased and the percentage of granulocytes increased at one and two months after 6.5 Gy irradiation,which improved significantly after rh TPO administration.The percentage of peripheral blood CD4⁺T cells increased and the percentage of CD8⁺T cells decreased at one and two months after irradiation,which could be changed by rh TPO administration.The percentage of peripheral blood Treg cells increased at one month after irradiation compared with the normal group and there was significant improvement after rh TPO administration.The percentage of peripheral blood Treg cells in mice was higher than that in the normal group one month after irradiation and rh TPO administration showed significant improvement.The percentage of peripheral blood NK cells in the irradiated control group was still significantly lower than the normal six months after irradiation,and there was no significant difference between rh TPO administration and the normal group.2.rh TPO ameliorates bone marrow hematopoietic cell injury in mice with long-term myelosuppression after radiation.The ratio of bone marrow nuclear cells and hematopoietic stem cells in bone marrow of mice were basically restored one month after irradiation.However,the colony-forming capacity of bone marrow cells remained significantly lower than normal until the sixth month,which was significantly improved after rh TPO administration.The bone marrow reconstructive hematopoietic capacity of the mice was still significantly lower than normal at one,two,and six months after irradiation,and rh TPO administration had a significant effect on its improvement.3.rh TPO ameliorates long-term myelosuppression by mitigating chronic oxidative stress after radiation in mice.The results of HSC cycle and apoptosis assay showed that the proportion of HSC in G0phase decreased significantly and the proportion of cells in G1 and G2/S/M phases increased in the irradiated control group one month after 6.5 Gy irradiation.rh TPO administration significantly improved the HSC cycle disturbance induced by radiation.six months after 6.5 Gy whole body irradiation,the rate of HSC apoptosis increased significantly in the irradiated control group compared with the normal control,while the difference was not statistically significant in the rh TPO group.Detection of hematopoietic stem cell senescence showed a significant increase in p16 m RNA expression and positive senescence staining in mouse bone marrow hematopoietic stem cells from one to six months after irradiation,with a significant improvement after rh TPO administration.The results of molecular mechanism experiments showed that the ROS level in bone marrow hematopoietic stem cells increased significantly at one and two months after 6.5 Gy irradiation,and rh TPO administration significantly reduced the ROS level in HSC,with no significant difference between the groups up to six months after irradiation.TheγH2AX⁺ratio of bone marrow haematopoietic stem cells was significantly increased one month after irradiation and the improvement was significant after rh TPO administration.Further testing of ROS-related pathways revealed that the ameliorative effect of rh TPO administration was achieved primarily through inhibition of the p53-p21 and/or p38 m RNA expression following irradiation.Conclusion:1.The optimal administration regimen for rh TPO is intramuscular injection of rh TPO（100μg/kg）two hours after irradiation,which can effectively promote the recovery of peripheral blood cells and maintain the immune homeostasis of the organism after irradiation.2.rh TPO could effectively improve radiation-induced long-term bone marrow hematopoietic damage by promoting the recovery of bone marrow cell colony formation and hematopoietic reconstitution capacity in mice after irradiation.3.Preliminary mechanistic studies suggest that rh TPO reduce chronic oxidative stress and DNA damage,increase the proportion of resting hematopoietic stem cells in post-irradiation mice,reduce the rate of hematopoietic stem cell senescence,and attenuate radiation-induced long-term myelosuppression by inhibiting the expression of ROS-related pathways after irradiation.