Study On The Radiation Protection Effect Of FMLP And Its Mechanism

Background:In recent years,with the development of nuclear technology,ionizing radiation has become one of the pollution factors in the process of social development,and human exposure to large doses of ionizing radiation will lead to a variety of adverse biological effects,so it is very important to develop radiation protection drugs against ionizing radiation damage.Recent studies have found that bacterial structural components(such as lipopolysaccharide,flagellin,etc.)can inhibit cell apoptosis caused by radiation and enhance the body's resistance to radiation.In our laboratory's previous studies,it was found that stimulation of TLR4 ligand lipopolysaccharide(LPS)in wild-type mice increased the tolerance to radiation.LPS is one of the main components of the cell wall of gram-negative bacteria,which can activate the body's immune response.Although LPS has a good antiradiation effect,LPS can cause toxic reactions in infected organisms.Because of its toxic and side effects,we screened a large number of LPS analogues through preliminary experiments.It was found that the leukocyte chemotactic peptide n-formyl methionyl-leocylphenylalanine could induce an inflammatory response similar to that of LPS stimulation.It has been reported in the literature that FMLP is critical to the cellular functions of neutrophils and monocytes,including adhesion,chemotaxis and release of superoxide anions.This suggests that FMLP may have a potential radiation protection effect by affecting cell function and regulating immune function of the body.Based on the above research background,we carried out a study on the radiation protection effect of FMLP and its preliminary mechanism.Purposes:1.To evaluate the toxic effect of FMLP,and to study the protective effect of FMLP on in vitro cells,radiation-sensitive tissues and mice.2.Carry out research on the mechanism of FMLP radiation protection,and deeply explore the molecular mechanism of FMLP radiation resistance.Methods:1.Study on toxicity and radiation protection effect of FMLP(1)Study on toxicity of FMLP Cell level: after FMLP at different concentrations was applied to human intestinal epithelial cells(HIEC),bone marrow primary cells(BMC)of C57BL/6 mice and Spleen cells of C57BL/6 mice,cck-8 method was used to detect cell viability.(2)Radiation protection effect of FMLP on cells in vitro.Cell viability tests showed that FMLP increased the viability of HIEC,BMC and Spleen cells;In the comparison of the effects of various protective agents,FMLP has the best protective effect,with the survival rate reaching more than 80%.HIEC,BMC and Spleen cell were selected for flow cytometry apoptosis test.The results showed that FMLP improved the survival rate of all three types of cells to a certain extent,and the survival rate of HIEC cells increased by 20%-30%.(3)Protective effect of FMLP on radiation-sensitive tissues.FMLP can significantly increase the number of peripheral blood white blood cells and bone marrow nucleated cells in irradiated mice,reduce the apoptosis rate of BMC in vivo,and improve the spleen organ index in irradiated mice.FMLP can also protect intestinal tissues and reduce the apoptosis of intestinal cells.(4)Radiation protection effect of FMLP on whole animals.The survival rate and survival time of C57BL/6 mice irradiated at different doses were recorded to evaluate the radiation protection effect of FMLP on the whole animal.2.Preliminary study on the molecular mechanism of FMLP radiation protection.(1)The radiation protection mechanism of FMLP was preliminarily studied in multiple knockout mice.Primary splenic cells and bone marrow pathological sections of wild-type(WT),TLR2 knockout(KO),TLR4 KO and My D88 KO mice were used to investigate whether the radiation protection effect of FMLP depended on TLRs and its downstream signaling pathways.(2)FPR1 and FPR2 knockout stable cell lines were constructed to study the radiation protection mechanism of FMLP.(3)Quantitative PCR was used to study the effect of FMLP on m RNA levels of primary radiation-protective cytokines IL-6,IL-11,CCL-2,IL-12 and GM-CSF in spleen of exposed mice.(4)The expression levels of radiation-protective cytokines IL-6,IL-11,CCL-2,IL-12 and GM-CSF in mice were detected by ELISA.(5)RNA sequence technology was used to screen potential radiation-protective differential genes,and then quantitative PCR was used to further detect the expression of related genes.Results:1.Results of studies on toxicity and radiation protection effect of FMLP(1)Toxicity of FMLP.CCK-8 showed no significant toxicity of FMLP to HIEC,BMC and Spleen cell at concentrations below 16 M,and showed a slight effect of promoting cell viability.0.5-1 M was selected as the optimal dose.In the comparison of the toxicity of FMLP with WR-2721 and LPS,the results of the survival experiment showed that the acute toxicity of FMLP was between LPS and WR-2721.(2)Radiation protection effect of FMLP on in vitro cells.Cell viability tests showed that FMLP increased the viability of HIEC,BMC and Spleen cells;In the comparison of the effects of various protective agents,FMLP has the best protective effect,with the survival rate reaching more than 80%.HIEC,BMC and Spleen cell were selected for flow cytometry apoptosis test.The results showed that FMLP improved the survival rate of all three types of cells to a certain extent,and the survival rate of HIEC cells increased by 20%-30%.(3)Protective effect of FMLP on radiation-sensitive tissues.FMLP significantly increased the number of white blood cells and bone marrow nucleated cells in peripheral blood of irradiated mice,reduced the apoptosis rate of BMC in vivo in mice,Increase spleen organ index of mice after exposure,FMLP also protects bone marrow and intestinal tissues and reduces intestinal cell apoptosis.(4)Radiation protection effect of FMLP on whole animals.The survival of mice proved that the survival rate of the irradiation group was longer than that of the irradiation group alone under the condition of total body irradiation at different doses.The radiation protection effect of FMLP was higher than that of WR-2721.2.Preliminary molecular mechanism of FMLP radiation protection(1)The primary study on FMLP in knockout mice does not depend on TLRs for radiation protection.(2)A stable cell line was constructed to study the radiation protection mechanism of FMLP,and it was found that the radiation protection effect of FMLP mainly depended on FPR1.(3)Quantitative PCR results showed that FMLP can promote m RNA transcription levels of cytokines such as IL-6,IL-11,CCL-2,IL-12 and GM-CSF in spleen primary cells of exposed mice.(4)ELISA showed that FMLP could up-regulate the expression of IL-6,IL-11,CCL-2,IL-12,GM-CSF and other radiation-protective cytokines in mice.(5)A total of 593 differentially expressed genes were analyzed by RNA sequence(sequencing)in mouse spleen cells,including 343 up-regulated genes and 250 downregulated genes.Then,significant difference Gene analysis,GO(Gene Ontology)analysis and KEGG biopathway enrichment analysis were performed by heat map,volcano map,We found that Ccnb1,Ccnb2,Cdk1,Mcm2,Mcm7 and other regulatory factors closely related to cell cycle progression were up-regulated,Combining with the reference literature,then we used quantitative PCR to verify the changes of Aurka,Aurkb,Ccnb1,Ccnb2,Cdk1,Mcm2,Mcm7 and other factors,indicating that these genes were up-regulated.Conclusion:1.The toxicity of FMLP is between LPS and WR-2721.FMLP plays a certain role in radiation protection at three levels: in vitro cells,radiation-sensitive tissues and in vivo mice.2.FMLP's radiation protection does not depend on the TLRs signaling pathway,Mainly through the FPR1 signaling pathway.Promotes transcription of hematopoietic cytokines such as IL-6,IL-11,CCL-2,IL-12 and GM-CSF.FMLP affect the immune response in mice after exposure and play a role in radiation protection.At the same time,it was found that FMLP could also play a radiation protection role by up-regulating Aurka,Aurkb,Ccnb1,Ccnb2,Cdk1,Mcm2,Mcm7 and other genes related to cell cycle progression in mice.