The Protective Effect And Mechanism Of ETAT-PPM1B In Radiation Induced Hematopoietic Stem Progenitor Cell Damage

Objective:Ionizing radiation(IR)is becoming more widespread in the productive life of humans.The risk of damage from IR is greatly elevated.IR causes pathological changes in almost all organs and systems,causing damage to various systems of the body.Among them,hematopoietic tissue is highly sensitive to ionizing radiation and one of the earliest appearing and most severe injuries in the body after being subjected to radiation.Ionizing radiation triggers the body’s bone marrow(BM)hematopoietic dysfunction,peripheral blood leukocytes with lymphopenia.This is mainly due to the damage of bone marrow hematopoietic stem and progenitor cells(HSPCs),which cannot maintain the normal hematopoiesis of the body.Presently,there is a lack of effective means to protect the hematopoietic system after radiation injury.The prevention and alleviation of hematopoietic system damage caused by ionizing radiation has become an important topic that needs to be addressed urgently.In this study,we aimed to(1)establish a mouse model of radiation injury to the hematopoietic system by IR,(2)develop a mouse model of radiation injury protection to the hematopoietic system by using eTAT protein phosphatase 1B(PPM1B)intervention,(3)explore whether eTAT-PPM1B can attenuate the hematopoietic system damage caused by ionizing radiation,(4)investigate the underlying mechanism.The present study provides a new approach and strategy for preventing and protecting the radiation injury to the hematopoietic system caused by IR.Method:In vivo experiment:1.SPF grade 8-week-old C57BL/6 mice were randomly divided into 2 groups:an unirradiated control group and an irradiated group.Radiation-induced hematopoietic injury model was established by subjecting mice to 4 Gy of whole-body X-ray radiation,the control group was not subjected to radiation.After14 days,the right femur and tibia of the mouse were taken for flow cytometry analysis,and the left femur and tibia were fixed and sectioned,followed by Hematoxylin and eosin(HE)staining.2.Labeling eTAT/eTAT PPM1B protein with Cy5-NHS eater,SPF grade8-week-old C57BL/6 mice were randomly divided into two groups:control(eTAT group)and treatment(eTAT-PPM1B group).The eTAT and eTAT-PPM1B groups of mice received pre-labeled empty plasmid eTAT and eTAT-PPM1B proteins via tail vein injection,respectively.Three hours after the end of injection,bone marrow was harvested from the bilateral femurs and tibias of the mice and analyzed by flow cytometry.3.Unirradiated SPF grade 8-week-old C57BL/6 mice were randomly divided into two groups:the control group(eTAT group)and the administration group(eTAT-PPM1B group).The eTAT group and eTAT-PPM1B group received two injections of eTAT or eTAT-PPM1B protein(5mg/kg)in the tail vein,with an interval of four hours between the two injections.After the injection,the mice in both groups continued to receive routine feeding.After 14 days,the right femur and tibia of the mouse were taken for flow cytometry analysis,and the left femur and tibia were fixed and sliced for subsequent HE staining.4.SPF grade 8-week-old C57BL/6 mice after radiation were randomly divided into two groups:the control group(4Gy-eTAT group)and the treatment group(4Gy-eTAT-PPM1B group).The mice received 4Gy X-ray radiation and received two tail vein injections of eTAT/eTAT-PPM1B(5mg/kg)at 2 and 6 hours.After the injection,the two groups of mice continued to receive routine feeding for 14 days,and the subsequent analysis was the same.In vitro experiment:1.The distribution of eTAT-PPM1B protein in bone marrow cells cultured in vitro:The eTAT/eTAT-PPM1B protein was labeled with Cy5-NHS eater and incubated overnight at 4℃.Add the incubated protein to the pre laid BM,and take photos under a fluorescence microscope after 3 and 24 hours to observe the fluorescence staining.Take cells for flow cytometry analysis.2.Effects of radiation on numbers of c-kit~+cells:to observe the dose relationship among the effects of radiation on c-kit~+cells,cells were irradiated with 1 Gy,2 Gy,3Gy,and 4 Gy of X-rays.Numbers of cells in each group were determined at 24 hours after radiation.3.The effect of different concentrations of eTAT-PPM1B on the number of c-Kit~+cells after radiation:eTAT-PPM1B with concentrations of 0,25,50,and 100nmol/m L were added to the culture medium containing c-Kit~+cells,respectively.After 3 hours,the cells were subjected to 2Gy radiation,and after 24 hours,cell counts were performed.4.Changes in the expression levels of programmed cell death and inflammation related genes in c-Kit~+cells after radiation:Mouse c-Kit~+cells were divided into a control group(0Gy)and a radiation group(4Gy).The radiation group received 4Gy X-ray radiation,while the control group did not receive radiation.24 hours later,real-time fluorescence quantitative PCR(q RT-PCR)was used to detect changes in the expression of programmed death and inflammation related genes.5.The effect of eTAT-PPM1B on the number and related gene expression of c-Kit~+cells after radiation:c-Kit~+cells were divided into eTAT group and eTAT-PPM1B group.Two groups of cells were treated with eTAT or eTAT-PPM1B(50nmol/m L),and then subjected to 2Gy radiation after 3 hours.Cell counts were performed 24 hours after administration,and changes in the expression of programmed death and inflammation related genes were detected by q RT-PCR.Results:1.The results of HE staining of the bone marrow at 14 days after 4Gy radiation showed that compared with the non-radiation group,the arrangement of cells in the bone marrow cavity of the radiation group was relatively loose,and the number of bone marrow cells decreased(P<0.01).The proportion and quantity of various HSPCs in the radiation group BM showed varying degrees of changes,with a decrease in the number of Lin~-cells(P<0.01),a decrease in the proportion and quantity of HPCs cells(P<0.001),a decrease in the number of HSCs cells(P<0.05),a decrease in the proportion and quantity of MPP1 cells(P<0.01),and an increase in the proportion and quantity of MPP2 cells(P<0.05).The proportion of apoptosis in Lin~-cells,HPCs,LSK cells,HSCs,MPP1,and MPP3.4(P<0.05)increased to varying degrees.2.The eTAT-PPM1B can be injected into the bone marrow through the caudal vein.Obvious fluorescence was also detected after incubating eTAT and eTAT-PPM1B in vitro,indicating that eTAT and eTAT-PPM1B can be well delivered to the target location both in vivo and in vitro.3.Compared with the non-radiation group,the inflammatory factors IL-18(P<0.001)and IL-1βin the radiation group,the expression of RIPK3(P<0.001),MLKL(P<0.0001),and Caspase-3(P<0.0001)genes related to apoptosis and programmed cell death increased.4.The eTAT protein and eTAT-PPM1B protein had no effect on the density and number of cells in the bone marrow cavity of non-irradiated mice;The eTAT protein and eTAT-PPM1B protein had no effect on the cell ratio and number of bone marrow HSPCs in non-irradiated mice,did not increase the apoptosis rate of HSPCs after administration.5.The eTAT-PPM1B can reduce bone marrow damage in 4Gy irradiated mice:compared with the eTAT group,eTAT-PPM1B treatment resulted in an increase in cell density and quantity in the irradiated bone marrow cavity(P<0.05).The proportion of Lin~-cells,HSCs,and MPP1 was higher in the eTAT-PPM1B group after injection than in the eTAT group(P<0.05);There was no statistically significant difference in other HSPCs between the two groups,but there was an upward trend in the proportion of LSK and MPP3.4 cells after eTAT-PPM1B injection;There was no difference in the proportion of apoptosis between the eTAT group and the eTAT-PPM1B group in irradiated HSPCs mice.6.In vitro experiments showed that compared with the radiation group,the number of cells significantly decreased 24 hours after 2Gy X-ray irradiation(P<0.05);Compared with the 0nmol/m L control group,there was no significant difference in the number of cells after administration.7.The number of c-Kit~+cells after irradiation was significantly lower than that of non-irradiated cells,but there was no significant change in non-irradiated cells after administration;The number of cells treated with eTAT-PPM1B after radiation was higher than that in the eTAT group(P<0.05),indicating that eTAT-PPM1B has a radiation protective effect on c-Kit~+cells under in vitro administration.8.The expression of programmed cell death gene RIPK3,apoptosis-related gene Puma and inflammatory cytokine IL-1βwere down-regulated by q RT-PCR after irradiation(P<0.05).There was no significant difference in MLKL,Bcl2 and TNF-αamong administration groups(P>0.05).This suggests that the protective mechanism of eTAT-PPM1B against HSPCs in bone marrow may be related to reducing inflammatory response,inhibiting apoptosis and programmed cell death.Conclusions:1.Radiation can reduce the number and proportion of various hematopoietic stem/progenitor cells in bone marrow activate the necrotic apoptotic pathway and inhibit bone marrow.2.The eTAT-PPM1B protein can effectively transfer PPM1B protein into bone marrow hematopoietic stem cells to protect radiation-damaged hematopoietic stem progenitor cells.