Autophagy In Hematopoietic System Is Radioprotective

Objective:The role of autophagy in the protection of hematpoietic system against radiationinjury remains unexplored. This study is designed to clarify whether ionizingradiation-induced DNA damage repair in hematopoetic system is dependent onautophagy, and to explore the underlying mechanism by which autophagy protectshematopoietic system in response to irradiation.Methods:First, the level of autophagy of bone marrow cells was pre-activated by the use ofdrugs in vitro followed by irradiation, and then cell proliferation, apoptosis and DNAdamage of the irradiated cells were detected. Next, autophagy defective mice (withdepletion of atg7gene) were used as a loss of autophagy function animal model andwild-type mice were administrated with rapamycin to active autophagy, followed bymeasuring peripheral hemogram, the ratio of hematopoietic stem/progenitor cells andcolony-forming ability after irradiation.Results:It was observed that the proliferation inhibition, apoptosis and DNA damage ofhematopoietic cells by irradiation were reversed by ex vivo autophagy activation.Activation of autophagy by rapamycin reduced the apoptotic death of bone marrow cells,but inhibition of autophagy with bafilomycin A1increased the apoptosis of bonemarrow cells exposed to radiation. Ex vivo activation of autophagy by rapamycindecreased the radiation-induced DNA damage of bone marrow cells, and inhibition ofautophagy with bafilomycin A1caused the opposite effect, as seen by examination ofthe DSB DNA damage marker γH2AX by flow cytometry and confocal microscopy. Inviv，activation of autophagy by administration of rapamycin protected mouse peripheralblood cells, primarily white blood cells and lymphocytes, red blood cells, and plateletsin the short-term postradiation period. The protective effect remained significant inwhite blood cells and lymphocytes, even at postirradiation day100. In vivo activation ofautophagy by rapamycin reduced irradiation-caused DNAdamage in bone marrow cells, including differentiated hematopoietic cells, late hematopoietic progenitor cells (Lin-),and hematopoietic stem and progenitor cells (LSKs), as shown by a reduced γH2AXlevel. Radiation-exposed cells treated with rapamycin retained their colony-formingability. Activation of autophagy partially rescued hematopoietic stem and progenitorcells, as seen by a higher number of LSK cells in the autophagy-enhanced group onpostirradiation day3.To confirm the above observations, radiation damage in the hematopoietic systemof a conditional autophagy defect mouse model (atg7f/f; Mx1-Cre) were examined. Thismodel lacks atg7and has defective atg7-dependent autophagy, as evidenced by loss ofLC3processing. Loss of autophagy caused greater damage in the short-termpostirradiation period in white blood cells, red blood cells, and lymphocytes. Plateletsnumber showed a transient increase at6h. Atg7-/-Mice were pretreated with vehicle orrapamycin for five times before γ-irradiation. The counts of WBC, LYM, RBC and PLTall have no statistical differences between the two groups. And it has also no statisticaldifferences in the DNA damage lever in the two groups. In contrast to the reduced DNAdamage seen after autophagy activation, loss of autophagy in HSPCs (LSKs) greatlyincreased in vivo DNA damage. Unlike what was seen after rapamycin activation ofautophagy, loss of autophagy worsened the irradiation-impairment of thecolony-forming ability of the cells. Loss of autophagy also reduced the percentage ofHSPCs in total bone marrow mononuclear cells following irradiation. Irradiationdamaged the vascular sinusoids in the spleen, but activation of autophagy by rapamycinmaintained the sinusoids; in contrast, loss of autophagy in autophagy defective miceworsened the spleen sinusoid impairment caused by irradiationTo understand the radioprotective mechanism of autophagy in the hematopoieticsystem, ROS levels and DNA damage repair activity in the context of activation or lossof autophagy were examined. Activation of autophagy by rapamycin improved ROSclearance in total bone marrow cells and HSPCs from the mice exposed to radiation.Finally, hierarchical cluster analysis of DNA repair pathway essential geneexpression in LSK of different groups were conducted. In three groups, Relativemessenger RNAs of BER, NER, MR, HR, NHEJ and cell cycle pathway expression inLSK were measured by real-time-Q-PCR.19genes were involved in NER, and8geneswere highly expressed in Rap group than which in Ctrl group.21genes were involvedin BER, and12genes were highly expressed in Rap group than those in Ctrl group.12 genes were involved in MR, and6genes were highly expressed in Rap group than thosein Ctrl group.Our results showed that treatment of mice with rapamycin resulted in significanttranscriptional suppression of the genes involving in the homologous recombination(HR) and nonhomologous end joining (NHEJ) with exception to Pold3, Rad52, Rpa1,Lig4and Poll. But the relative transcription levels of almost all genes in DNA damagerepair system in the Atg7-deleted mice were higher than the other two groups.However, opposite to the tracriptional results from the PCR array, it showeddown-regulation of the major HR proteins in the BM cells of autophagy defectivemouse group, including BRCA1, P95/NBS1, MRE11and Rad51, and alsodown-regulation of the major NHEJ proteins, Lig4, k80and XRCC4in western blot. Incontrast, most of these DSB damage repair proteins were upregulated when autophagywas activated by rapamycin, confirming the comprehensive role for autophagy inradioprotection of the hematopoietic system.Conclusion:Autophagy reduces the DSBs, protects LSK pool, enhances bone marrow cellproliferation, reduces apoptosis; Autophagy reduces ROS generated by radiationexposure; Autophagy confers regulatory proteins in HR and NHEJ pathways;Autophagy protects spleen after IR.