The Radioprotection And Mechanism Of Novel TLR Ligand Zymosan-A

Background:In recent years,nuclear energy and ionizing radiation have been widely used in civilian,medical and military fields.Radiation-induced damage has become one of the key scientific and technological issues,which urgently needed to be solved.It is urgent to study and find new targets of radiation protection and to develop new and effective radioprotectant.In the year of 2008,Burdelya et al firstly reported that the agonist of toll like receptor5?TLR5?showed strong radioprotective effects in mice.TLRs bring a new development direction to radioprotection.TLR2,TLR4,TLR5,and TLR9 have critical roles in radio-resistance.Previous studies proved that TLR2/TLR4/TLR9 displayed significant radioprotective effects on mice.Zymosan-A,which is extracted from the cell well of the yeast Saccharomyces cerevisiae,has been demonstrated as a potent ligand of TLR2 and TLR4.Zymosan-A is glucan linked by?-1,3 glycosidic bonds.Pre-experimental results show that it has obvious radiation protection effects.In this study,we explored the radioprotective effects of Zymosan-A and its potenial mechanism.The research project is divided into three parts,including the toxicity test of Zymosan-A,the radioprotective effects of Zymosan-A and the potenial radioprotective mechanism of Zymosan-A.Purposes:1.To systematically study the radioprotective effects of Zymosan-A on cells and whole animals.2.To explore the mechanism of radiation protection of Zymosan-A and to try to screen new potential targets for radiation protection.Methods:1.Chemical and reagents.Zymosan-A,purchased from Sigma,was resolved in normal saline?NS?.2.Cells culture and treatment.Human intestinal epithelial cells?HIEC?,human umbilical vein endothelial cells?HUVEC?,and human B lymphocyte cells?AHH-1?were obtained from ATCC.HIEC and AHH-1were cultured in RPMI 1640 with 10%FBS at 37?in a 5%CO2 humidified chamber.HUVEC was cultured in DMEM with 10%FBS at 37?in a 5%CO2 humidified chamber.Cells were treated with Zymosan-A?40?g/ml?12h and2h before irradiation.3.The toxicity test of Zymosan-A.Mice was treated with Zymosan-A?0 mg/mice,5.0 mg/mice,10.0 mg/mice,20.0 mg/mice?by peritoneal injection,and then the survival of mice was recorded.After treated with Zymosan-A at various concentration for 24 hours,HIEC cells and HUVEC cells were subjected to the assay of CCK-8.4.The model of acute radiation injury.⁶⁰Co?the radiation center,the faculty of Naval Medicine,Second Military Medical University,China?was used to irradiate mice and cells.5.The radioprotective effects of Zymosan-A in vivo.Mice were treated with Zymosan-A?50 mg/Kg,dissolved in NS?or NS by peritoneal injection at 24 h and 2 h before TBI.Then the survival was recorded.6.The radioprotective effects of Zymosan-A in vitro.Cell were treated with Zymosan-A or NS at 12 h and 2 h before irradiation.Then cell viability was analyzed by CCK-8.The cell apoptosis was analyzed by Apoptosis Detection Kit?Invitrogen,Carlsbad,California,USA?.7.The radioprotective effects of Zymosan-A on radiation-sensitive tissues.The femurs of mice were removed and fixed in 4%paraformaldehyde at 1,5,10,15,30 day post irradiation,and then the femurs were stained with Hematoxylin and Eosin?HE?.The number of white blood cells?WBC?was counted by blood cell analyzer?Mindray?.The relative number of bone marrow cells?BMCs?was counted by flow cytometry?Beckman Cytoflex?.The spleen coefficient means the ratio of the spleen weight?g?to body weight?g??spleen weight/body weight?.8.The level of radioprotective cytokines.The levels of IL-6,IL-11,IL-12 and TNF-?were analyzed by Enzyme-linked immunosorbent assay?ELISA?.All steps were operated according to the instruction.9.The radioprotective effects of Zymosan-A on hematopoietic stem cell.Bone marrow cells?BMCs?were isolated freshly 24 hours after TBI.The proportion of HSCs and HPCs with Lin/c-Kit/Sca-1 phenotype was determined by flow cytometry?Beckman Cytoflex?.10.The analysis of?-H2AX foci.Immunofluorescence analysis was used to detect?-H2AX foci.The images of cell smears were obtained using an Olympus BX60 fluorescent microscope?Olympus America Inc.,Center Valley,PA,USA?equipped with a Retiga 2000R digital camera?Q Imaging Inc.,Surrey,BC,Canada?.11.RNA sequencing and functional enrichment analysis.Total RNA was isolated from BMCs using Trizol?Invitrogen,USA?24 hours after radiation.Sequencing was performed at Guangzhou Ribo Bio Co.Ltd.with the Illumina HiSeq 2500.All the subsequent analyses were performed using the clean data.All the differentially expressed genes were used for heat map analysis,Gene Ontology Analysis,and KEGG ontology enrichment analyses.Results:The toxicity test of Zymosan-A.The most effective dose of Zymosan-A is lower than the dose of LD50.Zymosan-A exhibited a significant radioprotective effect in vivo and in vitro.The survival rates of Zymosan-A group were increased after TBI,and the dose reduction factor?DRF?was 1.25?Zymosan-A vs.NS?.Zymosan-A promoted cell viability and inhibited cell apoptosis caused by radiation.Zymosan-A protected the hematopoietic system against radiation-induced damageThe radiation damage on the structure of the bone marrow was alleviated in the Zymosan-A group.The number of nucleated cells was greater in the Zymosan-A group than in the NS group.Moreover,the bone marrow recovered much faster in the Zymosan-A than in the NS group.We also found that treatment with Zymosan-A increased the number of PWBCs,BMCs,and spleen coefficients.Zymosan-A induced radioprotective effects via TLR2.Zymosan-A protected TLR4 KO mice from radiation-induced death but had no radioprotective effects to the TLR2 KO mice.These findings consistently indicated that Zymosan-A induced radioprotective effects via TLR2 but not via TLR4.Zymosan-A up-regulated IL-6,IL-11,IL-12,and TNF-?in vivo.By detecting the serum cytokines'levels in the mice pretreated with Zymosan-A,we found that the serum levels of IL-6,IL-11,IL-12 and TNF-?were upregulated in vivo 24 h post-radiation.Zymosan-A inhibited BMCs apoptosis caused by radiation.we detected the apoptosis of BMCs 24 hours after radiation and found that the BMCs apoptosis rate increased after radiation,while the apoptosis rate was decreased significantly in BMCs from mice which treated with Zymosan-A.Zymosan-A up-regulated the levels of GM-CSF,G-CSF,IL-12,and IL-6 in BMCs.By using flow cytometry,we found that Zymosan-A up-regulated the levels of GM-CSF,G-CSF,IL-12,and IL-6 in BMCs.Zymosan-A protected cells from radiation induced DNA damage.Zymosan-A reduced the number of?-H2AX foci per cell at 0,0.5,and 2 hours post-irradiation,which means that Zymosan-A protected cells from radiation-induced DNA damage.Identification of DEGs between IR+NS and IR+Zymosan-A groupsBy using the RNA sequencing technology,total of 131 differentially expressed genes were identified?[log2 Fold Change]>0.8 and q value<0.05?,including 30 up-regulated genes and 101 down-regulated genes in the IR+Zymosan-A groups,compared to IR+NS groups.DEGs Gene Ontology Analysis between IR+NS and IR+Zymosan-A groupsGene Ontology Analysis was used to investigated changes in the patterns of genes between IR+NS and IR+Zymosan-A groups.The DEGs were classified into three functional groups:biological process group,cellular component group,and molecular function group.The inflammatory response,nucleotide-binding oligomerization domain containing 1pathway,and nucleotide-binding oligomerization domain containing 2 pathway were significantly enriched in biological process group.The MHC class 1 protein complex,Golgi cisterna,and endoplasmic reticulum exit site were significantly enriched in cellular component group.Within the molecular function group,the TAP-binding,beta-2-microglobulin binding,and peptide antigen binding were significantly enriched.Signaling pathway enrichment analysis of DEGs between IR+NS and IR+Zymosan-A groupsThe DEGs were mapped to 18 pathways in the KEGG database.Moreover,the DEGs were classified into 5 classifications including cellular processes,environmental information,human diseases,metabolism,and organismal systems.Within the environmental information group,TNF signaling pathway and NF-kappa B signaling pathway were significantly enriched.Conclusions:1.The effective dose of Zymosan-A was much lower than the lethal dose of Zymosan-A in vivo and in vitro.2.Zymosan-A exhibited great protective effects against ionizing radiation in vivo and in vitro.3.Zymosan-A protected bone marrow cells from radiation-induced apoptosis,up-regulated IL-6,IL-12,G-CSF,and GM-CSF in BMCs.Moreover,Zymosan-A might exhibit radioprotective effect through regulating immune system process and inflammatory response,in which TLR2 showed a key role.In addition,Zymosan-A also protects cells from ionizing radiation-induced DNA damage.These results provide new insights into the radioprotective effects of Zymosan-A.