Protective Effect And Mechanism Of Puma SiRNA On Damaged Hematopoietic Cells Induced By Radiation

Objective:Radiotherapy is one of the important means of tumor treatment.More than half of the patients receive radiotherapy,and 40%of cancer patients are cured.However,while ionizing radiation kills tumor cells,it also damages normal cells,which leads to acute or chronic radiation damage in the body,such as acute radiation syndrome,cardiotoxicity of chest radiotherapy,anemia,etc.,increasing the disease burden of patients.Therefore,it is necessary to develop a novel approach to ameliorate radiation-induced damage.In this study,we established cellular damage induced by ionizing radiation(IR)to investigate whether the specific silencing of Puma gene by in vivo delivery of small interfering RNA(siRNA)can reduce radiation-induced hematopoietic damage.The present study will provide a new method and strategy for preventing acute damage caused by radiotherapy.Methods:1.Establishment of animal models and interventionsThirty-seven C57BL/6 mice,all 8-10 weeks old male mice,were grouped according to the following random grouping principle:(1)The effects of siPUMA on mice under physiological conditions:C57BL/6mice were divided into 2 groups after sham irradiation(control treatment)and injected with siPUMA plasmid and EGFR control plasmid,respectively.The plasmids(5μg/g)were administered by tail vein injection at 6h before,6h after and 24h after treatment.The corresponding indexes were taken from each organ after 7 days of irradiation.(2)Ionizing radiation-induced injury model in mice:C57BL/6 mice were divided into 2 groups,and both groups were given 4Gy X-ray whole-body irradiation and injected with siPUMA plasmid and EGFR control plasmid,respectively,administered at a concentration of 5μg/g,and monitored for 7 days by tail vein injection 6h before,6h after,and 24h after irradiation.(3)Ionizing radiation-induced injury model in mice:C57BL/6 mice were divided into unirradiated group(NO-IR group)and ionizing radiation group(IR group).The IR group was given X-ray whole-body radiation,on the basis of which the IR group mice were divided into 2 groups with siPUMA plasmid and EGFR control plasmid injection,respectively.The administration concentration of the two plasmids was 5μg/g.The plasmids were administered by tail vein injection 6h before,6h after and 24h after irradiation.The corresponding indexes were taken from each organ after 7 days of irradiation.2.Detection of the ratio and number of hematopoietic cells(1)Detection of hematopoietic stem progenitor cell ratio and number:The percentages and numbers of Lin~-cells,LK cells(Lin~-Sca~-1-c-kit~+),LSK cells(Lin~-Sca-1~+c-kit~+),and Lin~-Sca1~+cells in the bone marrow of each group were analyzed by flow cytometry.(2)Percentage of T cells in thymus:The percentages of CD4~+CD8~+T cells,CD4~-CD8~-T cells(DN1～DN4),and early thymic T progenitor cells(ETP,c-kit~+CD44~+CD25~-)in the thymus of each group of mice were analyzed by flow cytometry.(3)Percentage and numbers of B cells in spleen:The percentages and numbers of mature B cells(B220~+),marginal B cells(MZB,B220~+CD21/35~+CD23~-)and follicular B cells(FOB,CD21/35~+CD23~+)in the spleen of each group of mice were analyzed by flow cytometry.(4)Percentages of each blood cell in peripheral blood:The percentages of CD11b~+cells,B220~+cells,CD3~+cells and Gr-1~+cells in peripheral blood of each group of mice were analyzed by flow cytometry.3.Morphological detection:The density of bone marrow cells within the bone marrow of each group was compared through HE staining.4.Apoptosis-related gene expression level detection:the expression of apoptosis-related genes such as Puma,Bax,Bak was detected by q PCR.Results:1.In vitro cell transfection assays demonstrated that plasmid DNA entering cells can produce exosomes containing siPUMA.In vivo mouse plasma exosome assays demonstrated that plasmid siPUMA in mice can produce exosomes containing siPUMA.2.Under physiological conditions,the differences in the proportions of each hematopoietic cell in bone marrow,spleen,thymus,lymph nodes and peripheral blood were comparable between EGFR plasmid-injected mice and siPUMA plasmid-injected mice(P>0.05).3.The distribution of cells in the bone marrow of siPUMA mice delivered in vivo after irradiation was denser(P<0.05).The numbers of Lin~-cells,the proportion and numbers of LK cells and the proportion of LSK cells in the mice of the siPUMA plasmid injection group were significantly higher than the values of the corresponding parameters in the mice of the EGFR group(P<0.05 or P<0.01).4.The proportion of splenic Mature B cells and MZB cells was more in mice injected with siPUMA plasmid after irradiation than in mice injected with EGFR plasmid(P<0.01).There was no significant difference in the proportion of FOB cells between the two groups(P>0.05).5.There was no significant difference in the proportion of T cells in each stage of the thymus.The proportion of B220~+cells in lymph nodes and the proportion of various types of blood cells in peripheral blood were comparable between the two groups of mice after irradiation(P>0.05).6.The q PCR results showed that the expression of Puma,Bax,and Bak genes was reduced in mature B cells in the spleen of mice injected with siPUMA plasmid after irradiation compared with mice injected with EGFR plasmid after irradiation.Conclusion:1.Puma siRNA plasmids injected into the tail vein of mice are able to produce exosomes containing siPUMA.2.In vivo delivery of siPUMA accelerated body weight recovery in mice after irradiation.3.In vivo delivery of siPUMA promoted the recovery of hematopoietic stem progenitor cells in the bone marrow and B cells in the spleen of mice after radiation,and the protective effect of mature B cells in the spleen was mediated by inhibiting Puma expression and attenuating radiation-induced apoptotic damage.