Objective:To develop a method of employing flow cytometry to directly detect the γ-H2AX expression levels in peripheral blood lymphocytes of mice through fixation and lysis and to evaluate the feasibility of applying this method to research on the radiation-related biological effects and the efficacy evaluation of radioprotective drugs.Methods:A total of 41 male C57BL/6J mice were used. First, 21 mice were randomly divided into 7 groups according to different radiation doses (0, 1, 2, 4, 6, 8, and 10 Gy) with 3 mice in each group. Blood samples were collected from the tail vein of mice at 1, 4, 8, and 24 h after irradiation and immediately fixed with formaldehyde. Red blood cells (RBC) were lysed with Triton X-100, and γ-H2AX was labeled with specific antibodies. DRAQ5 dye was used to further exclude debris and anucleate cells. The mean fluorescence intensity of γ-H2AX in lymphocyte populations was directly analyzed by flow cytometry through forward and side scatter, and dose-effect curves after irradiation were established. Then, the other 20 mice were divided into radiation alone groups and radiation combined with WR-2721 administration groups at 4 and 6 Gy, respectively, with 5 mice in each group. Blood samples were collected from the tail vein of mice at 1, 4, 8, and 24 h after irradiation to detect the average fluorescence intensity of γ-H2AX in lymphocytes, which was used to evaluate the degree of DNA damage in mice and the therapeutic effect of WR-2721.Results:The expression of γ-H2AX in peripheral blood lymphocytes of mice significantly increased with the increase of radiation doses, and reached a peak at 1-2 h and then decreased. The dose-effect relationship was significant ( R2 = 0.9914). At 24 h after 4 and 6 Gy irradiation, compared with the radiation alone groups, the average fluorescence intensity of γ-H2AX in the radiation combined with WR-2721 administration groups was lower (144.8 ± 8.0 and 109.5 ± 9.7, vs. 178.0 ± 18.5 and 136.6 ± 5.4), with statistically significant difference ( t = 3.78, 5.48, P < 0.05). The average fluorescence intensity of γ-H2AX at 24 h after irradiation was consistent with the lowest values of the three blood cell lines at 7 or 14 d after irradiation. Conclusions:The application of flow cytometry with a fixation/dissolution protocol to directly detect the mean fluorescence intensity of γ-H2AX in peripheral blood lymphocytes of mice has significant application value in radiation biology effect research, radiation protection drug screening, and efficacy evaluation.

Objective:To develop a method of employing flow cytometry to directly detect the γ-H2AX expression levels in peripheral blood lymphocytes of mice through fixation and lysis and to evaluate the feasibility of applying this method to research on the radiation-related biological effects and the efficacy evaluation of radioprotective drugs.Methods:A total of 41 male C57BL/6J mice were used. First, 21 mice were randomly divided into 7 groups according to different radiation doses (0, 1, 2, 4, 6, 8, and 10 Gy) with 3 mice in each group. Blood samples were collected from the tail vein of mice at 1, 4, 8, and 24 h after irradiation and immediately fixed with formaldehyde. Red blood cells (RBC) were lysed with Triton X-100, and γ-H2AX was labeled with specific antibodies. DRAQ5 dye was used to further exclude debris and anucleate cells. The mean fluorescence intensity of γ-H2AX in lymphocyte populations was directly analyzed by flow cytometry through forward and side scatter, and dose-effect curves after irradiation were established. Then, the other 20 mice were divided into radiation alone groups and radiation combined with WR-2721 administration groups at 4 and 6 Gy, respectively, with 5 mice in each group. Blood samples were collected from the tail vein of mice at 1, 4, 8, and 24 h after irradiation to detect the average fluorescence intensity of γ-H2AX in lymphocytes, which was used to evaluate the degree of DNA damage in mice and the therapeutic effect of WR-2721.Results:The expression of γ-H2AX in peripheral blood lymphocytes of mice significantly increased with the increase of radiation doses, and reached a peak at 1-2 h and then decreased. The dose-effect relationship was significant ( R2 = 0.9914). At 24 h after 4 and 6 Gy irradiation, compared with the radiation alone groups, the average fluorescence intensity of γ-H2AX in the radiation combined with WR-2721 administration groups was lower (144.8 ± 8.0 and 109.5 ± 9.7, vs. 178.0 ± 18.5 and 136.6 ± 5.4), with statistically significant difference ( t = 3.78, 5.48, P < 0.05). The average fluorescence intensity of γ-H2AX at 24 h after irradiation was consistent with the lowest values of the three blood cell lines at 7 or 14 d after irradiation. Conclusions:The application of flow cytometry with a fixation/dissolution protocol to directly detect the mean fluorescence intensity of γ-H2AX in peripheral blood lymphocytes of mice has significant application value in radiation biology effect research, radiation protection drug screening, and efficacy evaluation.

Exposure to ionizing radiation intervenes in genomic stability and gene expression,resulting in the disruption of normal metabolic processes in cells and organs by causing complex biolog-ical responses.Altered genomic variations,gene expression and metabolite concentrations in blood or tissue samples reflect systemic radiation damage.With the application of new techniques and exten-sive study on the mechanisms for ionizing radiation damage,related indicators such as chromosomal variation,gene expression,lipid and metabolism are being recognized and promise to be the markers for early diagnosis and prognosis of radiation exposure.Therefore,this article reviews recent progress in and potential applications of biomarkers related to ionizing radiation injury.

ObjectiveTo study the inhibitory effect of polyphyllin Ⅰ （PPI） on the growth of colorectal cancer cells and its molecular mechanism. MethodRKO cells were cultured and divided into a blank group and PPI treatment groups with concentrations of 0.6， 0.8， 1.0 μmol·L^-1， respectively. HRT18 cells were cultured and divided into a blank group and PPI treatment groups with concentrations of 1.2， 1.4， 1.6 μmol·L^-1， respectively. The effects of PPI on the proliferation and morphology of colorectal cancer were detected by cell proliferation toxicity assay， trypan blue exclusion assay， plate clone formation assay， and confocal high-intension cell imaging analysis system. Flow cytometry was used to detect the apoptosis rate of colorectal cancer cells. The pQCXIP-GFP-LC3 plasmid transfection assay was used to detect the formation of autophagosomes in colorectal cancer cells after PPI treatment. Western blot was used to detect the expression of apoptosis-related proteins Caspase-3， Caspase-8， and poly ADP ribose polymerase （PARP）， the expression of autophagy related protein LC3Ⅱ， and the expression and phosphorylation of Hippo signaling pathway proteins LATS1 and YAP. In the plvx-Flag-YAP plasmid transfection assay， YAP was overexpressed and treated with PPI， and the proliferation of colorectal cancer cells was detected by cytotoxicity assay. The expression of LC3Ⅱ and PARP in colorectal cancer cells was detected by Western blot. SwissADME predicted pharmacokinetic parameters of PPI. ResultAs compared with the blank group， the survival rate and clone formation ability of colorectal cancer cells in the PPI group were significantly decreased （P<0.01）， the cell area of colorectal cancer cells in the PPI group was significantly decreased， and the roundness of colorectal cancer cells was significantly increased （P<0.01）. As compared with the blank group， the apoptosis rate of colorectal cancer cells in PPI treatment groupw was significantly increased （P<0.01）， the expression of apoptotic proteins Caspase-3 and Caspase-8 protein precursor in PPI treatment groups was decreased， and the cleavage of PARP was increased （P<0.01）. As compared with the blank group， the expression level of autophagy-related protein LC3Ⅱ in colorectal cancer cells in PPI treatment groups was significantly increased， and the formation of autophagosomes was promoted （P<0.01）. As compared with the blank group， the expression of YAP protein in colorectal cancer cells in PPI treatment groups was significantly decreased， and the expressions of phosphorylated LATS1 and YAP were significantly increased （P<0.01）. As compared with the blank group， overexpression of YAP could significantly antagonize the effect of PPI on apoptosis， autophagy activation， and proliferation inhibition of colorectal cancer cells. SwissADME simulation results showed that PPI had good drug like activity. ConclusionPPI can induce apoptosis and autophagy of colorectal cancer cells through targeted activation of Hippo signaling pathway， thereby inhibiting their proliferation.

Objective To observe the early changes of related indexes after high dose of 60Co γ-ray irradiation on rhesus monkey hematopoietic system.Methods A total of 33 rhesus monkeys were randomly divided into normal control and different irradiation control group,rhesus monkeys in irradiation control group were given different doses(4,8,12 Gy) irradiation to establish acute radiation sickness(ARS) models.XE-2100 automatic blood cell analyzer detected the peripheral blood before and after the irradiation of 3,6,9,12,24,48,80 h.The rhesus monkeys were sacrificed to have a observation of sternum pathological changes at 6,48 and 80 h after 4,8,12 Gy 60Co γ-ray irradiation.Results The number of white blood cell in peripheral blood of the rhesus monkeys after 4 and 8 Gy 60Co γ-ray irradiation were lower than that before irradiation at 3 h after irradiation,as was significant increased at 6 h after irradiation,the highest values were 136.04%.and 221.38% after 9 h(with before irradiation values was 100.00%,the same below),become obviously drooped from 12 h after irradiation,show clearly temporary peak.But the number of white blood cell after 12 Gy 60Co γ-ray irradiation was significant increased at 6 h after irradiation,at the highest of 9 h,become obviously drooped from 12 h after irradiation.Peripheral blood neutrophile count was significant increased at 6 h after irradiation,at the highest of 9 h,become obviously drooped from 12 h after irradiation.Peripheral blood lymphocyte count fell sharply after irradiation,3 h detection value was only 12.02%-25.04% of before irradiation.Sternal bone marrow nucleated cell number decreased sharply after irradiation,the more irradiation dose,the less residual hematopoietic cells.Conclusion In the early stage of BM-ARS,temporary peaktime node of the white blood cell and neutrophil count could be regarded as the best delivery time of hematopoietic cytokine therapy.

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.

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.

Objective To explore the clotting mechanism in beagles irradiated by 4.5 Gy γ-rays after treatment with supportive care,or supportive care and combined cytokines.Methods Sixteen beagles were divided into irradiation control group,Supportive care group and combined cytokines treatment group.Platelet aggregation test,thrombelagtography (TEG) and the time measurement were analyzed in vitro.Results In irradiation group and supportive care group,the platelet aggregation rates in beagles were decreased markedly and the k value of TEG was increased 7 d post-irradiation,while those indexes in combined cytokines treatment group changed little.At 14 d post-irradiation,each parameter of TEG in irradiated group changed obviously.The values of r,k,r+k and M were elevated significantly,clotting time and the maximum coagulation time of thrombus delayed,the Ma value was decreased markedly,and the maximum elasticity amplitude of thrombus was diminished.All parameters in combined cytokines treatment group were better than those in supportive care group.The thrombin time was prolonged obviously in irradiated group 14 d post-irradiation,while the thrombin time was the longest at 2-3 weeks post irradiation in supportive care group and combined cytokines treatment group(P>0.05).Conclusions Cytokines could improve the platelet aggregation and the blood clotting functions of beagles suffering from acute radiation sickness.

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.

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.