| ObjectiveBone marrow is a spongy tissue found in the mesenchymal mesh of long,flat and irregular bones,which has hematopoietic,immune and defense functions and is extremely sensitive to ionizing radiation [1].After acute radiation exposure,the body may suffer from hematopoietic abnormalities or dysfunction,and is prone to hemorrhage,infection,and other symptoms,which can lead to death in severe cases [2].In this study,we investigated the protective effect of Brusatol(Bru),a bitter lactone compound of Crowberry,on 60 Co γ-ray-induced Bone Marrow form of Acute Radiation Sickness(BM-ARS)in mice.To investigate the mechanisms involved,and to provide a new means of prevention and treatment for radiation-induced hematopoietic damage and experimental basis.Methods1、To observe the survival rate of lethally irradiated mice at different dose concentrations: 40 male C57B6/J mice were randomly divided into four groups: IR group,Bru low,medium and high concentrations(0.5 mg/kg,1.0 mg/kg and 1.5 mg/kg),and were simultaneously irradiated with 8.5 Gy 60 Co γ-ray radiation lethal dose.The mice in the Bru group were injected intraperitoneally with different concentrations of Bru at two time points,24 h before irradiation and 3 h before irradiation,and the IR group was given the same volume of excipient as Bru 1.0 mg/kg.The survival of mice was observed 30 days after irradiation.2、Detection of peripheral blood cell changes in 6.5 Gy 60 CO γ-ray mice under different Bru administration concentrations: 24 C57 male mice were divided into 4 groups,IR group,Bru low,medium and high concentration(0.5 mg/kg,1.0 mg/kg,1.5 mg/kg)group,and received 6.5 Gy 60 Co γ-ray irradiation at the same time.The mice in the Bru group were given intraperitoneal injections of different concentrations of Bru at two time points,24 h before irradiation and 3 h before irradiation,and the IR group was given the same volume of excipient as Bru 1.0 mg/kg.Observe the peripheral blood cell changes of mice 30 days after irradiation to determine the optimal administration concentration.3、To detect the changes of peripheral blood cells in 6.5 Gy 60 Co γ-ray mice at different administration time points: mice were divided into IR group,double time point administration group(administered 24 h + 3h before irradiation),and single time point administration group(administered 3h before irradiation.1.0 mg/kg Bru was injected intraperitoneally in the Bru group,and the IR group was given the same volume of excipient as Bru1.0 mg/kg.The mice were also irradiated with 6.5 Gy 60 Co γ-rays,and the peripheral blood cell changes were observed for 30 days to determine the optimal dosing time.4、Bone marrow radiation protection effect of Bru under the optimal dosing regimen: Mice were divided into two groups,IR(same volume of excipient as Bru 1.0 mg/kg)and IR + Bru(24 h before irradiation + 3 h before irradiation,Bru 1.0 mg/kg),and received 6.5 Gy 60 Co γ-irradiation at the same time.(1)The changes in the bone marrow lumen were observed by HE staining at day 14 after irradiation;(2)The changes of bone marrow nucleated cells were measured by cell counter at day 14 after control.(3)Bone marrow nucleated cells(BMNCs)were counted using a cell counter at day 14,and hematopoietic progenitor cells(Lin-Sca-1-c-Kit +,LK),hematopoietic stem cells(Lin-Sca-1 + c-Kit +,LSK),and long-term stem cells in hematopoietic stem cells(CD135-CD34-Lin-Sca-1 + c-Kit +,LSK)were detected using flow cytometry.(CD135-CD34-Lin-Sca-1+ c-Kit+,LT-HSC),short-term stem cells(CD135-CD34+ Lin-Sca-1+ c-Kit+,ST-HSC),multipotent progenitor cells(CD135+ CD34+ Lin-Sca-1+ c-Kit+,MPP)as a proportion of BMNCs and hematopoietic stem and progenitor cells(Hematopoietic stem progenitor cells(HSPCs)to evaluate the change in the number of bone marrow cells;(4)colony formation assay to evaluate the proliferation and differentiation ability of HSPCs 14 days after irradiation;(5)competitive transplantation assay on day 14 after irradiation and peripheral blood at weeks 4,8,12,and 16 after transplantation.Chimerism rate assay;Tlymphocyte,B-lymphocyte and granulocyte assay at 12 weeks;LK and LSK ratios of mouse donor sources were examined at week 16,and all results of competitive transplantation experiments were combined to examine the ability of mouse bone marrow cells to reconstitute hematopoiesis after illumination to assess the functional changes of bone marrow cells.Clarify the protective effect of Bru on bone marrow tissue based on all the above experiments.5 、 To investigate whether the radiation protection effect of Bru on hematopoietic tissue still exists after Nrf2 gene deletion: 16 Nrf2 wild-type(WT)mice were divided into WT IR group and WT +Bru group,16 Nrf2 knockout(KO)mice were divided into KO IR group and KO IR +Bru group,8 mice in each group,24 h before irradiation and 3 h before irradiation,respectively.The mice in the Bru group were injected intraperitoneally with 1.0 mg/kg Bru,and the IR group was given the same volume of excipient as Bru 1.0 mg/kg,while receiving 8.0 Gy 60 Co γ-irradiation.The survival rate of mice 30 days after irradiation and the changes of peripheral blood cells at the extreme stage were observed.6、Preliminary in vivo and in vitro experiments to investigate the radiation protection mechanism of Bru on bone marrow tissues: the peripheral serum of mice was taken 6h after irradiation and the changes of granulocyte colony stimulating factor(G-CSF)were detected using ELISA kits;RAW 264.7 cell line was selected to investigate the target sites where Bru exerts its radiation protection effect.Different concentrations of Bru(100,200,400 nmol/L)were added into RAW264.7 cell medium for 12 h.400 nmol/L Bru was added into RAW264.7 cell medium for different time points(3,6,12 h).RNA was extracted from RAW264.7 cells,and the relative expression of G-SCF,IL-6 and TNF-a were detected by q RT-PCR;the expression levels of related proteins p50,p65 and p105 were detected by Western blot.Results1、Survival experiment: Bru increased the survival rate of mice irradiated with lethal doses: compared with the IR group,the intraperitoneal injection of Bru 24 h before and 3 h before irradiation significantly increased the survival rate of mice after irradiation,and the effect was most obvious when the concentration of Bru was 1.0 mg/kg compared with the IR group(10% vs.50%,P < 0.01).2、Optimal dosing concentration: Bru can significantly improve the rapid decrease of major blood cells in peripheral blood of mice after 6.5 Gy 60 Co γ-irradiation and accelerate the hematopoietic recovery process,and the effect is most obvious at the dosing concentration of 1.0 mg/kg,which is consistent with the survival experiment.Therefore,the optimal concentration of Bru in this subject is 1.0 mg/kg.3、Optimal administration time: After mice were irradiated with 6.5 Gy 60 Co γ-rays,the dynamic peripheral blood cell changes were monitored for 30 days,and the experimental results showed that the dual time point administration(24h before irradiation + 3h before irradiation)was effective in protecting red blood cell(RBC),white blood cell(WBC),platelet(PLT)and Neutrophils(NE).The protection of platelets(PLT)was more significant.The optimal dosing regimen was determined to be Bru concentration of 1.0 mg/kg at 24 h and 3 h prior to the treatment.4、The effect of Bru on bone marrow radiation protection under the optimal dosing regimen(Bru 1.0 mg/kg,24 h before and 3 h before irradiation).(1)On day 14 after receiving 6.5Gy 60 Co γ-irradiation,HE results observed less vacuolization and higher density of bone marrow cells in the femoral bone marrow cavity of Bru group mice compared with the IR group.(2)The results of bone marrow nucleated cell count on day 14 after irradiation showed that the number of bone marrow nucleated cells in the Bru group was better than that in the IR group(3.0±0.51×107 vs.1.9±0.57×107,P < 0.01);the LK(0.38±0.07% vs.0.19±0.068%,P < 0.01)and LSK(0.04±0.0129% vs.0.0129%,P < 0.01)of mice in the Bru administration group were better than those in the IR group.0.0129% vs 0.018±0.009%,P <0.05),LT-HSC(0.014±0.0049% vs 0.005±0.0028%,P < 0.05),and ST-HSC(0.024±0.008% vs 0.006±0.001%,P < 0.05)in BMNCs were differentially extent than the IR group mice.Flow cytometric detection of apoptosis of HSPCs on day 14 after 6.5 Gy 60 Co γ-irradiation showed that compared with the IR group,the proportion of apoptosis of LK(14.83% ± 1.9% vs.22.57% ± 3.26%,P < 0.01)and LSK(9.7.12% ± 2.01% vs.16.69% ± 2.58%,P < 0.01)in the Bru group was higher than that in the IR group.2.58%,P < 0.01)were lower than those in the IR group.These results suggest that Bru significantly reduced the apoptosis of HSPCs induced by irradiation and the sharp decrease of myeloid cells in the extreme phase after radiation.(3)The results of colony formation assay showed that,compared with the IR group,the Bru group showed a significant decrease in megakaryocyte colonyforming units(MK-CFU)(41.3 ± 11.9 vs.16 ± 1.7,P < 0.001),burst erythroid colony-forming units(BFU-E)(33.6 ± 1.2 vs.17 ± 6.9,P < 0.05),and erythroid colony-forming units(CFU-E)(54.7 ± 11.8 vs 34.3 ± 5.1,P < 0.01),granulocyte megakaryocyte colony-forming unit(GM-CFU)(86 ± 3 vs 50.3 ± 2.5,P < 0.0001),mixed colony-forming unit(Mix-CFU)(81 ± 6.6 vs 20.7 ± 5.9,P < 0.0001)colony numbers were significantly higher than those in the IR group,indicating that Bru improved the proliferation capacity of post-irradiation bone marrow cells.(4)The results of the competitive transplantation experiment showed that the chimerism rate of peripheral blood in the IR group showed a decreasing trend from week 4 to week 16 after transplantation: 30.25±2.98%,27.78±8.08%,26.74±13.36% and 20.73±7.13%;the chimerism rate in the IR+Bru group showed a gradual increasing trend: 52.15± 2.82%,57.49±12.04%,58.95±11.89% and 60.95±13.15%.From 4 to 16 weeks after transplantation,the percentage of peripheral blood CD45.2 in the control mice showed a gradual decrease,while the percentage of peripheral blood CD45.2 in the Bru group showed a steady increase and a stable trend.At week 12 post-transplantation,the proportions of T lymphocytes(43.78±1.58% vs.22.35±4.42%,P < 0.01)and B lymphocytes(55.36±20.61% vs.25.67±20.90%,P < 0.01)of donor origin were significantly higher in the Bru group compared with the IR group.The proportions of donorderived LK and LSK in mouse bone marrow cells were detected at week 16 after transplantation,and the proportions of donor-derived LK(81.57±5.39% vs 31.28±15.28%,P < 0.0001)and LSK(84.79±6.31% vs 35.31±15.00%,P < 0.0001)in the Bru group were significantly higher than those in the IR group compared with the IR group.were significantly higher than those of the IR group.Based on the results of competition transplantation experiments,this revealed that Bru could accelerate the reconstruction of hematopoietic multispectrum and improve hematopoietic function in mice after irradiation.5、Radioprotective effect of Bru on hematopoietic tissue after Nrf2 gene deletion: When Nrf2 gene was absent,the protective effect of Bru on the number of important blood cells in peripheral blood remained 14 days after irradiation.6、 In vivo and in vitro experiments explored the radiation protection mechanism of Bru on hematopoietic tissues: ELISA results showed that the level of peripheral blood G-CSF in mice given Bru alone for 6 h was 1050.4±315.4 pg/ml,and the G-CSF increased 50-fold 6 h after administration compared with the normal control group.Radiation itself also caused an increase in G-CSF,with G-CSF reaching 478.9 ± 139.2 pg/ml in the irradiated group alone;the level of G-CSF was 4169.3 ± 1590.905 pg/ml in the pre-irradiated administration group 6 h after irradiation,which could reach as much as 9 times the radiation effect compared to the IR group,P < 0.01.The levels of G-CSF and IL-6 increased significantly with time and Bru concentration after the action of Bru on RAW264.7 cells,and it could activate NF-κB pathway rapidly,which made IκBα phosphorylated and degraded rapidly.p50 / p65 was transferred from cytoplasm to nucleus for transcription of downstream genes after freeing from IκB.ConclusionBru can significantly reduce the reduction of BMNCs,decrease the apoptosis rate of HSPCs,improve the proliferation ability and reconstitute hematopoiesis of HSPCs in irradiated mice,and has good protective effect on acute myeloid radiation disease mice.The mechanism of action may be related to the activation of NF-κB pathway by Bru and the downstream production of cytokines(G-CSF,IL-6,TNF-α)with positive effect on hematopoiesis. |
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