The Effects Of RhIGF-â…  On The Recovery Of Megakaryocytopoietic Hematopoiesis In Radiation Injuried Mice And Its Related Mechanism

It is widely accepted that the ionizing radiation-induced injury is so common afternuclear weapon explosions and nuclear terrorist attacks, as well as nuclear accidents causedby natural disasters, for example, thousands of people were suffered from radiation-inducedinjury in the atomic bombings events in Hiroshima and Nagasaki, and the ChernobylLeenuclear accident in former Soviet Union.Bone marrow is highly sensitive to irradiation. High doses of ionizing irradiation caninduce serious dysfunction of multiple hematopoietic lineages in bone marrow, which ischaracterized by the decrease of hematopoietic cells such as erythrocytes, leukocytes andplatelets, etc. Therefore, experimental animals and human beings inflicted with large doses ofgamma-ray radiation often suffer from haemorrhage, infection, metabolic disorder caused byhematopoiesis dysfunction. Fortunately, people now have a profound understanding about themechanism of bone marrow hematopoietic dysfunction caused by irradiation after research foryears. Besides the damage of mononuclear cells in bone marrow could be caused byirradiation directly, it is believed that other factors could also induce a further decrease ofhematopoitic cells. Cheng et al. had found that mekaryocytes were phagocytized byneutrophilic cells in rats and dogs with radiation, burns or burn-blast combined injury, whichwas named as "megakaryocytophagia". Due to the fact that the number of megakaryocytes inbone marrow and the level of peripheral platelets decreased significantly after irradiation, therecovery of megakaryocytopoietic hematopoiesis is always a hot topic for the treatment ofradiation injury.Insulin-like growth factor (IGF) was firstly obtained from the Cohn components inhuman serum by Rinderkencth in1976. Two types of IGFs, IGF-Ⅰ and IGF-ⅠI, have beendiscovered until now. IGF-Ⅰ is recognized as a polypeptide composed by70amino acids withthree disulfide bonds and a molecular weight of7.5kD, and acts as an important mediator forgrowth hormone(GH). The generation of IGF-Ⅰ is mainly regulated by GH. In the body, IGF-Ⅰ is mainly secreted by liver, renal and spleen and bone marrow stromal cells. Therefore,rhIGF-Ⅰ has the ability to promote the proliferation of many cell lines and protect them fromapoptosis induced by a variety of factors. It was reported that recombinant humanIGF-Ⅰ(rhIGF-Ⅰ) could significantly inhibit the apoptosis of salivary gland and intestinalepithelial cell after ionizing radiation.As known, rhIGF-Ⅰ also plays an important role in hematopoiesis. Previous studiesindicated that rhIGF-Ⅰ could not only promote the proliferation of CD34+hematopoietic stemcells from umbilical cord blood, but also could enhance erythropoiesis, granulopoiesis andlymphopoiesis in vitro, etc. However, it is not clear whether rhIGF-Ⅰ could affectthrombopoiesis and hematopoietic recovery after irradiation. In addition, it has been a hotspot in clinical research how to treat hemopoietic dysfunction caused by radiotherapy andchemotherapy. Therefore, the study on the effect of rhIGF-Ⅰ in the regulation ofthrombopoiesis and hematopoietic recovery after irradiation will not only help us to betterunderatand about the biological functions of IGF-Ⅰ, but also provide us with new ideas andbasis for the development of new drugs for promoting hematopoiesis.In this study, we aimed to investigate the protective effect of rhIGF-Ⅰ on the recovery ofmegakaryocytic hematopoiesis in irradiated BALB/c mice and its possible mechanism. Firstly,the peripheral blood cell counts and pathological changes of bone marrow in irradiated micewere analyzed to see the radioprotective effect of rhIGF-Ⅰ on megakaryocytopoiesis afterrhIGF-Ⅰ treatment. In order to determine the role of rhIGF-Ⅰ in the regulation of HSCsdifferentiation towards megakaryocytes, the changes of megakaryocyte markers on isolatedmononucler cells cultured with rhIGF-Ⅰ were detected by flow cytometry in vitro. Then, theeffects of rhIGF-Ⅰ on megakaryocyte proliferation and apoptosis induced by irradiation invitro were analyzed by CCK-8assay and flow cytometry respectively. At last, the distributionand rearrangement of actin and β1-tubulin cytoskeleton after rhIGF-Ⅰ treatment wasinvestigated in Meg-01cells.The main results and conclusions of this study are as follows:Results:1. Compared with saline control group, the probability of survival over40days ofBALB/c mice inflicted with a single dose of7.5Gy γ-ray with treatment of100μg/kg rhIGF-Ⅰincreased by more than20%, which indicated that rhIGF-Ⅰ has a significant radioprotective effect against radiation-induced injury.2. Peripheral blood cell counts showed that the platelet level in rhIGF-Ⅰ-treated groupwere significantly higher than those in the saline control group from day10to17in additionto the increase of erythrocyte and leucocyte counts, which was performed in BALB/c miceinflicted with a single dose of5.0Gy γ-ray. This result indicated that rhIGF-Ⅰ could enhancethe recovery of peripheral platelet counts.3. Histological analyses demonstrated that the number of mononuclear cells includingmegakaryocytes significantly increased in bone marrow with rhIGF-Ⅰ treatment after radiation.Moreover, mononuclear cells from rhIGF-Ⅰ-treated mice had a statistically significant highernumbers of CFU-GM and BFU-E and CFU-GEMM compared with the saline control, whichcan further demonstrate that rhIGF-Ⅰ could promote megakaryocytopietic hematopoiesisrecovery after radiation.4. After MACS isolation, flow cytometric analysis showed that Lin~-c-kit~+Sca~+-Ⅰ cellsincreased significantly in rhIGF-Ⅰ-treated group. Moreover, Sca~+-Ⅰ cells from normal micedisplayed a higher expression of CD41with treatment of rhIGF-Ⅰ, which indicated thatrhIGF-Ⅰ could facilitate HSCs proliferation and enhance the differentiation of HSCs towardsmegakryocytes.5. As detected by CCK-8asssy, rhIGF-Ⅰ could not directly promote the proliferation ofMO7e cell, a factor-dependent megakaryoblast cell line, which indicated that rhIGF-Ⅰ mayenhance the proliferation of megakaryocytes in an indirect way.6. The frequency of apoptosis in bone marrow mononuclear cells treated with rhIGF-Ⅰdecreased significantly, compared with that in control group with no treatment afterirradiation. Equivalently, treatment with rhIGF-Ⅰ also resulted in a lower frequency ofapoptosis in megakaryocytes induced by irradiation. These results further indicated that rhIGFhas a radioprotective effect on megakaryocytes against irradiation-induced injury.7. As detected by laser scanning confocal microscope, rhIGF-Ⅰ could induce theaggregation of actin and β1-tubulin cytoskeleton in megakaryocytes, which suggested thatrhIGF-Ⅰ might facilitate proplatelet formation by inducing cytoskeleton polymerization ofmature megakaryocytes.