The Research Of Radioprotection And The Mechanism Of HKST On Radiation Injury

With the extensive application of ionizing radiation technology in national economy,medical and military fields,as well as the potential risks of nuclear power plant accidents,nuclear terrorist attacks and nuclear wars,how to prevent and control the body damage caused by ionizing radiation has become a worldwide problem.In addition,there are a large nμmber of high LET radiation in space radiation and nuclear powered ships,which also put forward higher requirements for radiation damage prevention.However,at present,there is no good prevention and treatment method for severe and acute radiation diseases caused by high dose irradiation or high LET radiation in the world.Therefore,the development of highly effective and low-toxic radiation protectors has become a key scientific and technological problem to guarantee nuclear safety and the safe use of nuclear energy,and become a major scientific problem to be solved urgently in radiological medicine.TLRs（toll-like receptor family）consist of highly conserved motifs and are specific for their ligands.Activation of these TLR related pathways might be exploited to study the ability of various TLR ligands in modification of radiation response.Recently,Studies on radiation biology have found that activation of TLR2,TLR3,TLR4,TLR5 and TLR9 have been shown to possess protective efficacy against lethal effects of ionizing radiation and are currently under different stages of development as radiation countermeasure agent for ARS which brings new hope for medical protection against ionizing radiation.Based on the specific structure characteristics of TLRs,specific biological functions and the distribution of TLRs in the body,the radiation protection effect of activating a TLR signal pathway alone is limited,and it is difficult to play a good role in protecting a variety of tissues and organs.Therefore,the search for a high efficiency,low toxicity of TLRs co-agonist,which can activate multiple TLR signaling pathways,correct the excessive activation of a single TLR caused by the body damage,is of extremely significance to exert the anti-radiation effect more efficiently and extensively,and to protect various tissues and organs as much as possible after irradiation.Heat Killed Salmonella typhimuriμm（HKST）is a highly effective co-agonist for TLR2,TLR4 and TLR5,and it effectively activates TLR2,TLR4,TLR5 and its downstream signaling pathway and inflammatory factor expression.Therefore,this project is mainly about the radioprotective effects of HKST,and the corresponding molecular mechanism.We do experiments about the hematopoietic immune system,the intestinal injury,and the lung injury afterγ-ray radiation in the animal level and cell level.And at the same time,we explore the anti-radiation effect of HKST on hematopoietic system and the gut after heavy ion irradiation.Heat Killed Salmonella typhimuriμm（HKST）is a highly effective co-agonist for TLR2,TLR4 and TLR5,and it effectively activates TLR2,TLR4,TLR5 and its downstream signaling pathway and inflammatory factor expression.Therefore,this study focused on HKST,the co-agonist of TLR2,TLR4 and TLR5.We pretreated HKST 12 hours before exposure,then clarify the protective effect of HKST on the hematopoietic immune system injury caused by gamma radiation in vivo and in vitro,analyze the protective effect of HKST on intestinal injury induced by gamma radiation.In order to further clarify the protective effect of HKST on gamma radiation induced lung injury,HKST was administered 12 hours before/after/both exposure.Finally,TLR4-/-mice,TLR2-/-mice and siTLR2,siTLR4 and siTLR5 cells were used to investigate the effect of TLR molecular knockout on HKST protective function,to clarify the molecular mechanism of HKST protective effect on gamma radiation.At the same time,we investigated the radiation protection of HKST on the hematopoietic system and intestinal tract after exposure to heavy ions.HKST has a protective effect against hematopoietic immune system radiation damage.Cell experiments showed that HKST significantly inhibited the apoptosis of HUVEC and L02 cells after gamma irradiation,improved cell survival rate,and repaired the DNA damage induced by gamma irradiation.At the animal level,HKST inhibited peripheral leukopenia and effectively improved survival after gamma irradiation.For bone marrow,the largest hematopoietic organ,HKST significantly alleviated gamma-irradiated bone marrow injury,significantly increased nucleated cell count,and increased the proportion of CD34+bone marrow cells in mice after irradiation.The radiation protection effect of HKST on the spleen was mainly manifested in the increase of the number of spleen white marrow after irradiation,the significant reduction of gamma-induced apoptosis of spleen cells,and the effective reversal of the imbalance of CD4+/CD8+in the spleen caused by gamma-rays.Serum cytokine ELISA showed that HKST significantly improved Th1/Th2 immune imbalance induced by gamma rays.In addition,¹²C⁶⁺irradiation in cells and mice significantly reduced the RAW264.7 cell apoptosis induced by ¹²C⁶⁺in HKST.At the animal level,HKST can alleviate bone marrow injury in mice exposed to 4Gy ¹²C⁶⁺whole-body radiation,reduce the number of apoptotic cells in femoral bone marrow,and reduce the expression of BAX and caspase-3 proteins in the apoptotic pathway.HKST provides protection against intestinal radiation damage.HKST can reduce intestinal injury after local exposure of 25Gy gamma rays in mice,and the protective effect is better than that of amphostin WR2721,the only radiation protection drug approved by FDA.Studies of 7Gy gamma-irradiated mice showed that HKST could effectively promote the proliferation of small intestinal crypt cells after gamma-irradiation and inhibit intestinal apoptosis induced by gamma-irradiation.In addition,HKST also had a protective effect on intestinal injury in mice exposed to 4Gy ¹²C⁶⁺whole-body radiation.Further,small intestinal organ culture showed that HKST could promote the growth of small intestinal organs and increase the germination rate,which confirmed the radiation protection effect of HKST in vitro.HKST has a protective effect against radioactive lung injury.After the local exposure of 15Gy gamma ray to the lungs of mice,it was found that HKST could reduce the pulmonary hyperemia and edema,reduce the pulmonary coefficient,and reduce the inflammatory response of the lung tissues of the mice.HKST could decrease collagen deposition,inhibite apoptosis,decrease levels of TGF-and its downstream signaling pathways,and ultimately inhibite gamma-induced epithelial mesenchymal transformation（EMT）.In terms of the mechanism,we treated RAW264.7 cells with HKST to observe NF-κB p65 nuclear transposition at different time points,and the results showed that HKST effectively activated NF-κB.Subsequently,antibody staining to NF-κB p65 was performed on mouse liver,spleen and testicular tissue sections.HKST was found to activate most liver and spleen cells more strongly than in testicular cells.Western blot results showed that HKST activated the TLR and MAPK signaling pathways.Finally,through TLR4-/-mice,TLR2-/-mice,and siTLR2,siTLR4,siTLR5 cells and other experiments,it was found that HKST mainly relies on TLR4 for radiation protection in vivo,partly depends on TLR2,and mainly relies on TLR4 and TLR5 for radiation protection in vitro.Altogether,HKST,as a potential radioprotective agent,has a good radioprotective effect on acute irradiation induced hematopoietic immune injury,intestinal injury and radioactive lung injury by activating TLR and MAPK signaling pathways.Therefore,the study on HKST,a common agonist of TLR2,TLR4 and TLR5,provides a theoretical basis for the research and development of highly effective and low-toxic protective agents,opens up new ideas for the prevention and treatment of severe radiation damage,and promotes the scientific and technological progress and discipline development in the field of nuclear energy and nuclear safety to a certain extent.In addition,for the first time,we focused on the prevention and treatment of heavy ion damage by HKST,which also provided theoretical support for the prevention and treatment of heavy ion damage.