| In recent years,the utilization of nuclear and related technology is increasingly used in multiple fields,such as medicine,energy,industry and military.Meanwhile,the potential risk of acute radiation-induced injuries is remarkably elevated.Once nuclear attack or massive casualty scenario occurs,a large number of civilians and first responding emergency crews need radiation protection.Therefore,there is a substantial unmet need for identifying and developing a broad-spectrum systemic radioprotective agents that are safe,effective,easily administered and broad time-window of prophylactic protection for the purpose of national security and medical application.Acute radiation syndrome?ARS?is a collection of health effects due to exposure to more than 1Gy of irradiation over a short period of time.The ARS is categorized into three sub-syndromes including hematopoietic,gastrointestinal and neural acute radiation syndrome,i.e.H-ARS,GI-ARS and neural-ARS depending on the irradiation dose and the most prominent signs and symptoms.Hematopoietic tissue within bone marrow?BM?is one of the most radiosensitive tissues.A critical loss of hematopoietic progenitor and stem cell?HPSC?populations induced by radiation results in H-ARS,which is characterized by marrow suppression with multi-lineage blood cytopenias,leading to life-threatening haemorrhage and sepsis.However,the approved radiation countermeasures for H-ARS are scarce and the prophylaxis agents for H-ARS have yet to be approved.Higher radiation dose triggers GI-ARS,during which massive crypt cells die and intestinal epithelia barrier breakdowns,which further causes dehydration,electrolyte disturbances sepsis and death within two weeks.To date,no radioprotectant has been approved specially for GI-ARS and there is no cure for GI-ARS.The other human organs such as skin,oral mucosa,lung,kidney et al are pretty sensitive to radiation besides BM and gastrointestine,manifesting as acute,early-arising injuries or delayed effects of acute radiation exposure?DEARE?.Radiation-induced multiple tissue and organ injuries?RI-MTOI?may deteriorates to radiation-induced multiple organ dysfunction syndrome?RI-MODS?,which is the difficulties in current treatment of ARS patients.Unfortunately,few reports on countermeasures of RI-MTOI can be found.Ironizing radiation?IR?or radical generated by the radiolysis of water can disrupt the structure of macromolecules such as proteins,lipids and DNA,which results in metabolic and functional alteration and further in cell injury or death.Free radical mediated injuries are considered as main radiation toxicity and DNA is considered as one of the most critical macromolecular targets for IR-induced cell injury and death.Amifostine?WR2721?is a thiol compound that scavenges free radicals and has well-documented,radioprotective attributes.Different animal models have proved WR2721 be potent countermeasure for nearly all normal tissues exposed to irradiation.Because of debilitating side/toxicity effects,the narrow time frame?15–30 min?for radioprotection,unfavorable routes of oral administration,WR2721 has been approved for radiotherapy/chemotherapy-induced normal cell injuries but not approved for nuclear/radiological emergency.Dimethyl sulfoxide?DMSO?is an organic compound containing sulfinyl group that acts as a free radical scanvenger.Numerous literatures have proven DMSO be an effective function of anti-inflammation,analgesia,anti-anxiety,diuresis,antibacterial activity.FDA approved DMSO for the treatment of interstitial cystitis in 1978.In 1962Ashwood-Smith reported that intraperitoneal injection of DMSO improved mice survival exposed to lethal dose irradiation.We recently reported that DMSO protected against radiation-induced mucositis.DMSO is absorbed rapidly and is rarely metabolized in vivo when orally administered and its low systemic toxicity by oral application has been demonstrated for several species,including primates,which led us to further experimentation with DMSO as an oral radioprotector.In the present study,we thoroughly evaluated oral DMSO's radioprotection against ARS including hematopoietic and gastrointestinal injuries.Besides,the characteristics of RI-MTOI in long-term survival mice exposed to high dose irradiation as well as potential radioprotection of DMSO to RI-MTOI were observed.Adult stem cells?ASC?play a core role in BM or intestine regeneration.Therefore,alleviating radiation-induced ASC injury,cutting down ASC death and enhancing ASC regeneration are the key strategies for preventing H-ARS and GI-ARS.P53 or PUMA gene knockout mice have less apoptotic HSC and are more resistant to radiation,which indicates p53-PUMA pathway a critical role in mediating HSC apoptosis.In addition,radiation exposure can lead to long-term BM damage owing to defects in HSC self-renewal capacity and HSC senescence.Whether DMSO could relieve short-term or long-term BM suppression through inhibiting HSC apoptosis,preserving HSC ability after radiation exposure remains to be elucidated.Radiation-induced normal hematopoietic tissue injuries are more common in radiotherapy.A radioprotective agent,which cannot distinguish HSC and leukemia stem cell?LSC?,would impair the benefits of radiotherapy,it deserves intensive study to investigate the radioprotective activity of DMSO to LSC.Intestinal stem cells?ISC?locate in the base of crypts.Lgr5 is the cell marker for ISC population,recent study implicates Lgr5 cells are indispensable for intestinal regeneration after irradiation.High-energy ray induces several different types of damage to DNA,among them,double strand breaks?DSBs?are the most detrimental DNA damage to a cell.It is widely believed that DMSO can decrease DSBs through radical scavenging.Recent research reported that low concentration DMSO cannot decrease radiation-induced DSBs but can promote DNA damage repair in vitro.We recently reported DMSO facilitate mouse lingual epithelium stem cell DNA DSBs repair.Whether DMSO has similar effect to ISC remains to be clarified.The role of crypt cell apoptosis in GI-ARS is controversial.On the one hand,deletion of PUMA blocks radiation-induced epithelia cell apoptosis and enhances crypt regeneration;on the other hand,deletion of p53 could inhibit radiation-induced apoptosis but not mitotic death,aggravating GI-ARS instead.Whether DMSO can protect ISC by blocking radiation-induced apoptosis are poorly understood.To investigate the effects and underlying mechanisms of DMSO on radioprotection to H-ARS and GI-ARS.Firstly,we established GI-ARS or H-ARS mouse model by whole-body irradiation?WBI?and confirmed the radioprotective effects of oral DMSO medication.Next,we took mouse intestinal tract or BM subjected to gamma ray irradiation as main research subjects,evaluated quantity and function of HSC using cytometry,colony-forming unit assay in vitro and competitive repopulation assay?CRA?in vivo;observed cell apoptosis using flow cytometric caspase3/7 and p53-/-,PUMA-/-mouse,to explore the mechanism of DMSO's protection to H-ARS.Then we observed cell proliferation using Brd U immunohistochemistry staining?IHC?;observed cell apoptosis using Tunel IHC,cleaved caspase 3 IHC;evaluated DNA damage and repair using?H2AX and 53BP1 IHC;directly observed ISC using Lgr5-EGFP-IRES-cre/ERT2?Bmi1-cre ERT2?R26R-EYFP mouse and vibratome slicing,to to explore the mechanism of DMSO's protection to GI-ARS.Besides,we assessed effects of DMSO on mouse multi-tissue and organ DEARE through observation to long-term survival mice exposed to high dose irradiation,to evaluate the effect of DMSO to irradiated leukemia cells,we developed Mll-AF9 cell-induced AML mouse model.The main results and conclusions were summarized as follows:1,DMSO promote BM cells regeneration and ameliorates pancytopenia induced by irradiation.Oral DMSO meditation prior to irradiation conferred 100%survival of mice receiving lethal dose?9.0Gy?and super-lethal dose?9.5Gy?irradiation.The DMF calculated was 1.24 with 95%confidence interval 1.22–1.26.DMSO protected against a lethal dose from 15min to 4h before irradiation.Oral DMSO not only protected radiation-induced acute HPSC injury,but also ameliorate long-term BM surppression following irradiation in mice.In addition,during a 2-month DMSO oral gavage to mice at dose of highest radiorptective efficiency?10g/kg?we found no obvious abnormity in growth rate,peripheral blood,multi-organ physiological function,which indicates a single dose of oral DMSO is pretty safe.2,Flow cytometric analysis of Caspase 3/7 in BM cells showed that DMSO did not inhibit LKS cells apoptosis and the HPSC survival from p53-and PUMA-deficient mice after irradiation was also protected by DMSO.Through Tunel and Cleaved Caspase 3 IHC assay,we found DMSO cannot inhibit apoptosis of crypt cell in different position and apoptosis of intestine lamina propria cell after 8-22Gy irradiation.In addition DMSO remarkably increased regenerated crypts in p53-/-and PUMA-/-mice exposed to whole-body irradiation.All these results indicate that DMSO protects HSC or ISC survival after irradiation independent of inhibiting apoptosis.3,DMSO promoted colony-forming ability and competitive repopulation ability of both in vivo and ex vivo DMSO-treated mouse BM cells after irradiation,all collectively suggests that DMSO directly protects radiation-induced damage to HSPCs.For Mll-AF9 leukemic cells irradiated ex vivo,DMSO can neither inhibit cell apoptosis nor enhance colony-forming ability.DMSO has no effect on leukemogenesis of DMSO-pretreated leukemic cells irradiated in vitro.Surprisingly,DMSO pretreatment prolonged AML mouse model survival time after radiotherapy,all these indicate that DMSO have no radioprotective activity to leukemia stem cells.Furthermore,DMSO treatment radioprotects irradiated human umbilical cord blood HPSCs.4,Oral DMSO meditation significantly increased regenerated crypts and prolonged survival time after high dose WBI.Pretreatment with DMSO resulted in 100%survival of mice that received BM transplantation?BMT?after 16-19Gy WBI,where the vehicle-treated mice died within 10 days.Amazingly,DMSO resulted in 40%survival of mice within 30-day observation after 22Gy WBI combined with BMT.Dose-survival plot shows that DMSO can push 7Gy the survival curve to the right,the DMF calculated was 1.48 with 95%confidence interval 1.46–1.50.DMSO treatment 12h prior irradiation remarkably increased regenerated crypts and DMSO treatment 6h prior irradiation conferred 70%survival after 16Gy WBI combined with BMT,which indicates that the maximum effective prophylactic administration time is more than 6h for DMSO.Compare to positive control drug WR2721,DMSO has the wider time frame and radiation dose frame for radioprotection.5,In the early time post irradiation,?H2AX and 53BP1 IHC assay showed that DMSO could promote crypt cell DNA damage repair,but unable to decrease radiation induced DSBs.Brd U IHC showed that DMSO had no effect on radiation-induced crypt cell cycle arrest 6-12h after 15Gy WBI but promoted more crypt cells reentering into S phase 24h after 15Gy WBI.Direct observation by vibratome slicing and laser confocal microscope found that DMSO could decrease Lgr5 ISC loss,promote Lgr5 ISC proliferation after irradiation.Lineage tracing in mice intestines further proved DMSO could promote Lgr5 ISC regenerating villus after irradiation.These results collectively indicate that DMSO decrease radiation-induced ISC death through facilitating DNA DSBs repair,which further enhance ISC regenerating intestine villus.6,Through long term observation to DMSO-treated survival mice that received 17-22Gy WBI combined with BMT,we found that DMSO prolonged mouse total survival time up to 200-500d,alleviated acute radiation-induced mouse lung,kidney,liver,heart delayed injuries,and we found no obvious abnormity in multi-organ physiological function,which indicates DMSO could alleviate RI-MTOI.In conclusion,in the present study,we firstly reported and thoroughly evaluated the radioprotection of oral administrated DMSO against GI-ARS and H-ARS,specially,to the best of our knowledge,the radioprotective effects of DMSO on GI-ARS outperform other radioprotective agents ever reported.Considering its low toxicity,high radioprotective efficacy,oral bioavailability,systemic radioprotection of multi-organ,extended time window of protection,high stability and low price,DMSO is an ideal candidate for human prophylactic use in nuclear/radiological emergency and medical radiation.Mechanistically,we found DMSO can preserve stem cell pool,enhance stem cell regeneration but not inhibit stem cell radiation-induced apoptosis,which may be the common mechanism underlying the radioprotective effects of DMSO.Our findings provide a new insight into the therapeutic method and the common mechanism of radiation-induced multi-organ injuries... |
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