Autophagy Mitigates Radiation-induced Bone Marrow Hematopoietic DNA Damage Through Enhanced Stat3 Signaling

Objectives:1) To explore whether autophagy activation can promote DNA damage repair;2) To examine whether autophagic radioprotection of bone marrow hematopoietic cells depends on Stat3 signaling.Methods:1) Flow cytometry and confocal microscopy were used to detect the protein level of γ-H2 AX in the bone marrow mononuclear cells(BM-MNCs);2) Alkaline and neutral comet assay were used to examine the DNA strand breaks in BM-MNCs;3) Western blotting was conducted to detect the Stat3 and proteins relative to DNA repair in the cells pretreated with rapamycin;4) Quantitive-RT-PCR was used to detect the m RNA expression of Stat3 in Stat3 knockdown BM-MNCs;5) Image flow cytometry was conducted to examine the nuclear localization of p-Stat3(Y705);Results:1. Rapamycin(Rap) pretreatment reduced the radiation-induced DNA double-strand breaks(DSBs)Both in vivo and in vitro, the percentage of γ-H2AX-positive cells in rapamycin pretreated group was significantly less than those irradiated cells without rapamycin treatment. Single-cell alkaline comet assay showed that the tail moment of cells with rapamycin pretreatment before irradiation was shorter than those without rapamycin treatment(p<0.05), and neutral comet assay disclosed a similar results(p<0.05).2. Rapamycin attenuated radiation-induced DSB damage through autophagyWestern blotting showed that the autophagic level increased with the pretreatment of rapamycin in BM-MNCs. Baf A1, an inhibitor of autophagy, attenuated the radioprotective effect of rapamycin. Using an Atg7 defective mouse model, in which Atg7 was deleted in the hematopoietic cells, we observed that the radioprotection mediated by rapamycin was blocked when the autophagy was impaired.3. Autophagy can modulate Stat3 activity in bone marrow hematopoietic cellsRapamycin treatment enhanced the phosphorylation of Stat3, and this phosphorylation was crippled when autophagy was pharmacologically inhibited or genetically impaired with or without radiation exposure.4. Autophagy regulated Stat3 activity to promote the DSB repairWhen Stat3 signaling was blocked by inhibitors, Stattic or S3I-201, or by sh RNA targeting Stat3, autophagy was no longer able to mediate the radioprotection.Conclusions:Rapamycin pretreatment protects bone marrow hematopoietic cells from DNA double-strand break damage, and this protection is mediated by autophagy. Autophagic radioprotection of the bone marrow hematopoietic cells depends on Stat3 signaling.