The Mechanism Of Long-term Bone Marrow Suppression Induced By Ionizing Radition And The Radioprotective Effects Of MnTE On Bone Marrow Exposure To Ionizing Radition

The mechanism of Long-term Bone Marrow Suppression Induced by Ionizing Radiation and The Radioprotectiveeffects of MnTE on bone marrow Exposure to Ionizing RaditionObjective Ionizing radiation (IR) causes not only acute tissue damage but also late effects including long-term bone marrow (BM) injury. The induction of long-term BM injury is primarily attributable to the induction of hematopoietic stem cells (HSCs) senescence. However, the molecular mechanisms by which IR induces HSCs senescence have not been clearly defined, nor has an effective treatment been developed to ameliorate the injury. Thus, we investigated these mechanisms and the radioprotection of Mn (Ⅲ) meso-tetrakis (N-ethylpyridinium-2-yl) porphyrin (MnTE) on TBI induced BM injury in this study.Methods 4-8 weeks old 57BL/6-Ly-5.2 were classified into three groups: control group, radiation group and radiation plus drug group, radiation was 6.5 Gy total body irradiation (TBI) and drugs includes diphenylene iodonium and MnTE. Bone marrow mononulcer cells(BM-MNCs) isolated form these mice were used to perform competitive repopulation assay in vivo and CFU-GEMM individual colonies were developed by culturing BM-MNCs in MethoCult GF M3434 methylcellulose medium after 11 days were used to performcytogenetic analyses. and hematopoietic progenitor cells (HPCs) sorted out by flow cytometry were used to examine the following tests:the level of ROS by 2', 7'-dichlorofluorescin diacetate (DCFDA), the DNA damage and DNA double strand breaks by using 8-hydroxydeoxyguanine and H2AX staining, respectively; Colony forming function were analyzed by in vitro colony-forming cell assay, cobblestone area-forming cell assay and single cobblestone area-forming cell assay. Apoptosis was detected by Annexin V/7-AAD; The expression of NOXs, p16 and p53, were assayed by RT-PCR or immunostaining.Results The results showed that exposure of mice to a sublethal. dose of total body irradiation (TBI) induced a persistent increase in reactive oxygen species (ROS) production in HSCs selectively. The induction of chronic oxidative stress in HSCs was associated with sustained increases in oxidative DNA damage, DNA double-strand breaks (DSBs), inhibition of HSC clonogenic function, and induction of HSC senescence but not apoptosis. The induction of chronic oxidative stress in HSCs by TBI is probably attributable to the up-regulation of NADPH oxidase 4 (NOX4), because irradiated HSCs expressed an increased level of NOX4, and inhibition of NOX activity with diphenylene iodonium but not apocynin significantly reduced TBI-induced increases in ROS production, oxidative DNA damage, and DNA DSBs in HSCs and dramatically improved HSC clonogenic function.Then, we examined if Mn (Ⅲ) meso-tetrakis (N-ethylpyridinium-2-yl) porphyrin (MnTE), a superoxide dismutase mimetic and potent antioxidant, can mitigate TBI-induced long-term BM injury in a mouse model. Our results showed that post-TBI treatment with MnTE significantly inhibited the increases in ROS production and DNA damage in HSCs and reduction in HSC frequency and clonogenic function induced by TBI. In fact, the clonogenic function of HSCs from irradiated mice after MnTE treatment was comparable to that of HSCs from normal controls on a per HSC basis, suggesting that MnTE treatment inhibited the induction of HSC senescence by TBI. In addition, MnTE treatment can mitigate TBI induced defects in the abilities of HSCs to maitain quiescence, proliferate and survive in vitro. This suggestion is supported by the findings that MnTE treatment also reduced the expression of p16Ink4a (p16) mRNA in HSCs induced by TBI and improved the long-term and multi-lineage engraftment of irradiated HSCs after transplantation.Conclusions TBI selectively induces a persistent increase in reactive oxygen species (ROS) production in HSCs only. The induction of chronic oxidative stress in HSCs by TBI is probably attributable to the up-regulation of NADPH oxidase 4 (NOX4)。Chronic oxidative stress in HSCs leads to the loss of HSCs self-renewal and the induction of HSC senescence via up-regulation of p16, which leads to long-term bone marrow suppression. TBI-induced long-term BM suppression can be mitigated by antioxidant treanment. We found MnTE can mitigate IR-induced HSC senescence inhibiting the ROS-p16 pathway and be used to a therapeutic agent to mitigate TBI-induced long-term BM suppression by TBI.