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Objective To understand the radiation protection effect of pre-irradiation administrations of nilestriol on the mice with bone marrow type of acute radiation syndrome after irradiation with 60Co γ-rays,along with its mechanisms for improvement of hematopoiesis.Methods The nilestriol administration protocols were prepared by analysis of peripheral blood cell counts and survival rate experiment on mice.The mechanisms by which the pre-irradiation twice administrations improved the post-irradiation recovery of bone marrow hematopoiesis were studied by the analysis of the surface marker of bone marrow hematopoietic stem/progenitor cells of mice and by the inspection of hematopoietic progenitor cell colony and by using histopathological assessment of bone marrow.Results Pre-irradiation administration of nilestriol at two-or three-day intervals had been shown to increase survival rates up to 100％ in mice exposed to 9.0 Gy γ-rays,which was superior to a single administration (20％,x2 =21.66,21.66,P ＜0.05).The pre-irradiation administration both at one-day or two-day intervals were capable of improving the recovery of peripheral blood counts,including white blood cell (WBC),red blood cell (RBC),and platelet in mice exposed to 6.5 Gy (F =21.33,100.9,49.34,19.19,P ＜ 0.05),showing the better effects than a single administration (F =17.11,63.38,21.89,14.37,P ＜ 0.05).The two-day-interval administration of nilestriol could significantly increase the numbers of bone marrow hematopoietic stem/progenitor cell counts (t =8.58,2.80,P ＜ 0.05) in mice on day 10 after 6.5 Gy irradiation.This also could be capable to significantly improve colony formation,with there being statistical difference compared with single administration(t =4.29,6.34,P ＜ 0.05).Also the administration at two-day-interval were also usefull in reconstruction of hematopoietic cell hyperplasia of bone marrow of irradiated mice.Conclusions As compared with conventional single admination,the pre-irradiation multiple administrations of nilestriol showed significantly improved radiation protection effects.Considering a nuclear medical emergency rescue,it is recommended to follow the pre-irradiation administration of nilestriol at two-day interval,which could obtain the best protection effects at minimum administration frequency.
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Objective To investigate the effect of ionizing radiation on the invasion of the pulmonary adenocarcinoma cell line A549.Methods The invasiveness of A549 cells irradiated with 2 and 4 Gy doses of γ-rays was detected by using transwell invasion assay.The expression levels of matrix metalloproteinase (MMP)-2 mRNA and protein and phosphorylated signal transducers and activators of transcription 3 ( STAT3 ) protein were detected by reverse transcription PCR and Western blot.Results After irradiation with 2 or 4 Gy, the invasiveness of A549 cells increased by 200.0% ( F = 111.7, P ＜ 0.01 ) and 390.9% ( F = 593.7, P ＜ 0.01 ), respectively, compared with that in untreated A549 cells.Furthermore, the transcription and protein expression of MMP-2 24 h after irradiation and the phosphorylation of STAT3 12 h after irradiation were promoted.The irradiation-induced elevation of MMP-2 protein expression was suppressed using STAT3 phosphorylation specific inhibitor (AG490).Moreover,compared with 4 Gy of irradiation alone, treatment with 4 Gy of irradiation plus AG490 decreased the number of invasive cells by 76.1% ( F = 555.9, P ＜ 0.01 ), and the number of invasive cells in 4 Gy of irradiation plus AG490 group made up only 117.8% of that in untreated group ( F = 3.6, P ＞ 0.05 ).Conclusions Ionizing radiation could activate STAT3, which triggers the transcription of MMP-2, and then promote the invasiveness of A549 cells.
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Objective To investigate the mechanism of treatment of granulocyte colony-stimulating factor(rhG-CSF),recombinant human interleukin-11(rhIL-11)and recombinant human interleukin-2 (rhIL-2)on hematopoietic injuries induced by 4.5 Gy60 Coγ-ray irradiation in beagles,and to provide experimental evidence for the clinical treatment of extremely severe myeloid acute radiation sickness (ARS).Methods Sixteen beagle dogs were given 4.5 Gy60 Co γ-ray total body irradiation(TBI),then randomly assigned into irradiation control group,supportive care group or cytokines+supportive care (abbreviated as cytokines)group.In addition to supportive care,rhG-CSF,rhlL-11 and rhIL-2 were administered subcutaneously to treat dogs in cytokines group.The percentage of CD34+cells,cell cycle and apoptosis of nucleated cells in peripheral blood were examined by Flow cytometry.Results After 4.5 Gy 60 Co γ-ray irradiation,the CD34+cells in peripheral blood declined obviously(61.3%and 52.1% of baseline for irradiation control and supportive care group separately).The cell proportion of nucleated cells in Go/G1 phase was increased notably(99.27% and 99.49% respectively).The rate of apoptosis(26.93% and 21.29% separately)and necrosis(3.27% and 4.14%,respectively)of nucleated cells were elevated significantly when compared with values before irradiation(P<0.05) 1 d post irradiation.When beagles were treated with cytokines and supportive care,the CD34+cells in peripheral blood were markedly increased(135.6% of baseline).The effect of G0/G1 phase blockage of nucleated cells became more serious(99.71%).The rate of apoptosis(5.66%)and necrosis(1.60%)of nucleated cells were significantly lower than that of irradiation control and supportive care groups 1 d after exposure.Conclusions Cytokines maybe mobilize CD34+cells in bone marrow to peripheral blood,indce cell cycle block at G0/G1 phase and reduce apoptosis,and eventually cure hematopoieticinjuries induced by irradiation.
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Objectivc To observe the therapeutic effects of combined cytokines on hematopoietic injuries induced by 4.5 Gy60 Co γ-rays irradiation in beagles,and to provide experimental evidences for the clinical treatment of extremely severe myeloid acute radiation sickness(ARS).Methods 16 beagles were given 4.5 Gy60 Co γ-rays total body irradiation,and then randomly assigned into irradiation control group,supportive care group and cytokines group.In addition to supportive care,recombinant human granulocyte colony-stimulating factor (rhG-CSF),recombinant human interleukin-11(rhIL-11)and recombinant human interleukin-2(rhIL-2)were administered subcutaneouly to dogs in cytokines group.Peripheral blood hemogram was examined once every two days.Bone marrow and peripheral blood were collected to proceed colony cultivation 4 d pre-irradiation and 1 and 45 d post-irradiation.Conventional histopathological sections of sternum were prepared to observe the histomorphology changes. Results After irradiation,the population of all kinds of cells in peripheral blood declined sharply.WBC nadir Was elevated(1.04×109/L,but 0.28×109/L and 0.68×109/L for the irradiation control group and the supportive care group separately),the duration of thrombocytopenia was shortened (24 days,but 33 days for the supportive care groug) and red blood cell counts were maintained in the range of normal values after cytokincs treatment in combination.The colony forming efficiency of haemopoietic stem cells(HSCs)in bone marrow and peripheral blood decreased obviously 1 d post irradiation,but recovered to the level of that before irradiation 45 d post irradiation after supportive care and cytokines treatment.Hematopoietic cells disappeared in bone marrow of animals in irradiation control group,but hematopoietic functions were recovered after cytokines were administrated.Conclusions RhG-CSF.rhIL-11 and rhIL-2 used in combination could elevate WBC nadir,accelerate the recovery of leukocytes,platelets and red blood cells and promote the proliferation,differentiation and maturity of HSPCs left in the body after 4.5 Gy γ-rays total body irradiation,eventually restore the hematopoietic function.Hence,combination of rhG-CSF,rhIL-11 and rhIL-2 could serve as better therapeutic strategy to treat extremely severe myeloid ARS.
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Objective To explore the mechanisms of cytokines on acute radiation disease in irradiated beagles.Methods The sera of beagles irradiated with 4.5 Gy γ-rays with cytokines treatment was collected at different time points post irradiation.The two-dimensional gel electrophoresis(2-DE)was used to isolate and compare the differentially expressed proteins in sera.HD-MS was used to analyze the differentially expressed proteins with significance,and the amino acid sequences should be determined. Results High resolution 2-DE gel map was obtained.There were six differentially expressed proteins in sera of irradiated beagles at different time points.Four protein spots were successfully identified by MS.A significant spot was identified as serum amyloid A(SAA)by HD-MS,with relative molecular mass of 13 077 and isoelectfie point of 6.26.Expression of SAA was not found 1 d pre-irradiation and 36 d postirradiation,but increased slightly 1 d(0.2166)and significantly 14 d post-irradiation(0.4577). Conclusions The expression of serum amyloid A was consistent with the process of acute radiation injury,which might indicate the turnover of the disease.
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Objective To evaluate the effects of combined administration of recombinant human interleukin-11(rhIL-11),recombinant human G-CSF(rhG-CSF)and recombinant human interleukin-2 (rhIL-2)on acute radiation sickness(ARS)beagles.Methods Sixteen beagle were irradiated with 4.5 Gy60 Co γ-rays to establish ARS models,and were divided into irradiation control group,supportive care group and combined cytokines treatment group.After irradiation irradiation control group was given no treatment,the dogs in supportive care group received purely symptomatic treatment,while combined cytokines treatment group received rhIL-11 50μg/(kg·d)and rbG-CSF 10μg/(kg·d)subcutaneously(0-14 d)and rhIL-2 1×1 06 U/d(29-43 d)besides symptomatic treatment.Manifestation and characteristics of ARS beagles were observed,and the survival time were recorded.At last,post-mortem examination and histological examination were performed.Results All animals underwent nausea,diarrhea and fever.After irradiation,all animals in irradiation control group died in two weeks,and the mean survival time was 12.7 d,while only one died at 33 d in supportive care group.All dogs in combined cytokine group survived at 45 day after exposure,and their haematopoiesis and gastrointestinal tract were recovered.Conclusions Combination of rhIL-11 + rhG-CSF + rhIL-2 treatment could be significantly effective on ARS beagles irradiated by 4.5 Gy60 Co γ-rays,which could accelerate injured haemotopoiesis and intestinal tract recovery,increase the survival rate and improve the life quality of animals.
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It is therapeutically effective to apply hemopoietic growth factors to treat hemopoietic irradiation injuries, but for other tissues injuries, hemopoietic growth factors nearly do not work. At the same time, they are only suitable for the patients with severe and lower level acute radiation sickness (ARS). Besides, clinical therapeutic efficacy of marrow transplantation is not optimistic due to difficulties of marrow zygosity and severe complications such as graft versus host disease, radiation interstitial pneumonitis. Mesenchymal stem cells (MSCs) could secret hemopoietic growth factors, rebuild hemopoietic microenvironment and be easily transfected by exogenous gene, which also have low immunogenicity. At present, studies on MSCs application in the treatment of radiation injuries are at the early -stage, but the precise mechanism is not clear. However, the specific characteristics can make up the disadvantage of traditional treatment of acute radiation sickness, and the clinical application is wide.
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To evaluate the effects of combined therapy of rhIL 11 and rhG CSF on monkeys irradiated with 8 0 Gy 60 Co ? ray. Animals were divided into control ( n =4) and rhIL 11+rhG CSF treatment group ( n =4). After irradiation the control was given no treatment, while the treatment group was given rhIL 11 50?g/(kg.d)+rhG CSF 10?g/(kg.d) and other symptomatic supportive treatment whenever needed. The results showed that all animals underwent nausea, diarrhea and fever. After irradiation, all blood cells in peripheral blood of the animals declined in quantity rapidly. Animals of the control died of hemorrhage and infection. Their mean surviving time after ? ray exposure was 18 2 days. Aninals of the treatment group all survived on the 45th day after exposure. Their peripheral blood cell counts recovered to near baseline level. Histopathological observation revealed that bone marrow cells of treated animals proliferated actively. The results suggested that a combination of rhIL 11 and rhG CSF treatment was significantly effective in extremely severe hematopoietic acute radiation sickness.
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In order to obtain experimental evidences of therapeutic effect of rhIL 11 for clinical use, we observed the effects of rhIL 11 on colony formation of multilineage human hematopoietic cells in vitro and acceleration of platelet count and bone marrow hematopoietic recovery in 3 0Gy ? ray irradiated rhesus monkeys. The results showed that rhIL 11 significantly increased the nadirs of PLT count and shortened the duration of PLT numbers below 50% of their baseline values. The recovery of PLT in irradiated monkeys was accelerated. Dosage related PLT recovery was not observed. rhIL 11 not only promoted the proliferation and maturation of CFU Mk but also improved the proliferation of CFU GM, CFU e, BFU e and CFU Mix. These data suggest that rhIL 11 may be an effective agent in the treatment of hemopoietic suppression and thrombocytopenia.
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In more than ten years, hematopoietic factors have been approved to be effective in the treatment of radiation sickness. But there are still some problems in the clinical application, such as suitable cases, dosage, administrative timing, and drug combination. Due to those problems, the efficacy was undermined. Our experimental treatment study showed that hematopoietic factors should be administered early after the radiation exposure. The efficacy of early administration group is better than the one treated in acute phase of radiation sickness. There is also a limit of application duration but not the longer the better. RhTPO along can not induce the granulocytopoiesis and rhG-CSF can not improve the recovery of megakaryopoiesis. Hematopoietic factors do not increase the chromosome mutation rate of tumor cells. We did not observe bone marrow stem cell or progenitor cell exhaustion after we treated those animals for a second cycle of hematopoietic factors or exposed those treated animals to radiation again. RhG-CSF is more effective in the recovery of granulocytopoiesis than rhGM-CSF. RhIL-11 can increase the recovery of three cell lines of bone marrow, especially megakaryopoiesis. We recommended the combination of hematopoietic factors in the treatment of acute radiation sickness. RhG-CSF plus rhIL-11 is a preferred combination.