| Research background:In the field of radioprotection and treatment,the treatment of severe ionizing radiation injury has always been the key problem of radiation medicine research.The main scientific challenges in this field are as follows:First,it is the lack of new radioprotection targets with clear mechanism.Second,there is a lack of effective and low toxic radioprotective agents for the treatment of radiation injury.At present,the best radioprotective drug approved by FDA in the world is WR-2721,which has been equipped by the U.S.army.However,due to its obvious side effects,the use of WR-2721 was limited to a certain extent.In Chinese army,there are two main types of radioprotective drugs as follows:Estrogen derivatives(500 injections,523 tablets)and Rubia cordifolia extract(408 tablets).However,these drugs not only have a long history,but also have poor efficacy.At present,most of the radioprotective drugs in the research stage have some shortcomings,such as unclear effect,unclear mechanism,and high toxicity and so on.Therefore,it is of great clinical significance to study the mechanism of radiation injury and find the high effective and low toxicity treatment measures for radiation injury.At the same time,at the national strategic level,improving the prevention and control level of high-dose radiation injury in China is in line with the needs of national security,and has very important national defense value.In 2008,it is reported by the Science magazine for the first time that Doctor Burdelya LG et al used CBLB502,which is a modified agonist of TLR5 signal pathway,to activate TLR5 signaling molecules,and then activate NF-κB,which played a very strong radioprotective effect in animal models of radiation injury.This groundbreaking research result opens up a new research direction for the prevention and control of radiation damage,and makes TLRs receptor family attract great attention in the field of radiation prevention and control,which has become a new hot spot in the field of radiation prevention and control.After reporting the study of TLR5,other members of TLRs family(such as TLR2,TLR4 and TLR9)have been gradually discovered and studied the radioprotective effects.At present,the research of TLR2 and TLR4 pathway in the protection of ionizing radiation injury is more in-depth.It has been confirmed that TLR2 and TLR4 are key new targets for the protection and treatment of ionizing radiation injury,and lots of new research results have been gradually reported.However,there are still two significant problems that restrict the application of targeting TLR2/4 researches.Firstly,it is reported that the TLR2/4targeted ligands cannot have both high efficiency and low toxicity at the same time.For example,the TLR4 ligand LPS has high protective effects but also high toxicity.Various modifications were carried out to reduce the toxicity;the result shows that the radioprotective effects will reduce accompanied by the reduction of toxicity,while the TLR2 ligands pam2 and pam3 have small side effects but weak protective potency.Secondly,these target ligands have good preventive effect before irradiation,but poor therapeutic effect after irradiation,which cannot meet the needs of severe radiation injury treatment.Therefore,although the report on TLR2/4 pathways bring good prospects for radiation protection research,the technical problem of curing severe ionizing radiation injury has not yet been completely solved.In view of the above two problems,in order to search for TLR2 or/and TLR4targeted ligands with significant radiation damage prevention and low toxicity,a new TLR2/NOD2 double targeted ligand CL429,which has clear protection and treatment effect on ionizing radiation damage,was screened from TLR2/4 related ligands through previous literature search,theoretical analysis and a large number of pre experiments.On the basis of previous research,we carried out a preliminary study on the role and mechanism of CL429 in the prevention and treatment of ionizing radiation injury.Firstly,the low toxicity of CL429 was confirmed from the whole animal level and cell level,and the optimal dosage of CL429 in mice and cells was found out respectively.Secondly,by the mouse model of acute radiation sickness,the preventive and therapeutic effects of CL429 were compared with WR2721 and LPS,as well as the preventive and therapeutic effects of CL429 combined with TLR2 ligand pam3,NOD2 ligand MDP and pam3+MDP,respectively.The radioprotective effects of CL429 were further confirmed from the aspects of bone marrow hematopoietic tissue and small intestine tissue.Under the premise of clear radioprotective effects,it is revealed that CL429 plays a radioprotective role in the whole animal and cell level from the perspective of TLR2 and NOD2 signaling pathway,which may mainly rely on tlr2-myd88-nf-κB signaling pathway and partly rely on NOD2signaling pathway,and CL429 may not play a radioprotective role through transcriptional activation of TLR2 and NOD2.Finally,RNA sequencing was used to identify the new key effector molecules and their related signaling pathways in the protection of CL429 against radiation injury,and the mechanism of radiation protection of CL429 was further explored by heatmap,reads distribution map,Go and KEGG enrichment pathway analysis.The main innovations of this project are as follows:firstly,a new TLR2/NOD2double targeted ligand Cl429 has been successfully screened through literature research,theoretical analysis and pre experiment,which not only has the preventive and therapeutic effect of radiation damage,but also has the high efficiency and low toxicity.Secondly,the effect and principle of prevention and treatment of ionizing radiation damage on Cl429 has been comprehensively explored from the level of cells,tissues and the whole animal.Finally,it is proved that the protection of Cl429 against radiation injury mainly depends on TLR2 and partly on NOD2.Furthermore,the signal pathway(tlr2-myd88-nf-κb)and the key role component(S100A9)related to the prevention and treatment of ionizing radiation injury of Cl429 are successfully screened by RNA sequencing,which provides a new research idea and intervention approach for the prevention and treatment of radiation injury.Research objectives:1.A new TLR2/NOD2 double targeting ligand CL429 with high efficient and low toxic was screened through largely literature consult,theoretical analysis and preliminary experiments,and its toxic dose was found out from the overall animal level and in vitro cell level,which laid the foundation for the research of CL429 as a potential new radioprotective agent.2.The best dose and mode of administration for radiation protection of CL429 was verified at the whole animal level,the radiation protection effect of CL429 is clarified from the whole animal level and radiation sensitive tissues and organs,which provides experimental basis for CL429 to become a possible radioprotective agent with high efficiency and low toxicity.3.The scientific basis of CL429 regulating TLR2/NOD2 signaling pathway was elucidated to prevent and treat ionizing radiation injury,a new target molecule of CL429was found to protect against radiation injury through the second generation sequencing.And the mechanism of CL429 was clarified to prevent and treat radiation injury from multiple perspectives,so as to explore a new research path for the key issue of ionizing radiation injury protection.Research contents:1.The screening of a novel dual targeting ligand CL429 and its acute toxicity to cells and mice.(1)The screening of novel double targeting ligand CL429:13 kinds of targeted ligands related to TLRs family members were selected through previous literature research and theoretical analysis,and the cell viability was detected by CCK-8 method after 8 Gy irradiation of HIEC cells.(2)Acute toxicity test of CL429 on cells:Gradient concentrations of CL429(0μg/ml,50μg/ml,100μg/ml,200μg/ml,400μg/ml,800μg/ml,1600μg/ml,3200μg/ml)were added to the logarithmic growth phase of HIEC cells.After 24 hours of culture,the OD value was detected by CCK-8 method to evaluate the cell viability.(3)Acute toxicity test of CL429 on mice:C57BL/6 mice were randomly assigned into two groups(n=6):CL429 group and LPS group.Both groups were given intraperitoneal injection with the dosage of 25mg/kg body weight(500μg/mouse),and then the survival rate of mice within 48 hours after administration was observed continuously.2.Radioprotective effects of CL429 on animals(1)Finding out he best radioprotective dose of CL429 on mice:Six week old male SPF C57BL/6 mice were divided into 8 groups(control group,1μg/mouse+1μg/mouse,5μg/mouse+5μg/mouse,10μg/mouse+10μg/mouse,20μg/mouse+20μg/mouse,50μg/mouse+50μg/mouse,100μg/mouse+100μg/mouse,200μg/mouse+200μg/mouse).The mice were injected intraperitoneally for 24 hours and 2 hours before irradiation(7.5Gy)respectively.The survival of mice was observed continuously for 30days.(2)Comparison of radioprotective effect of CL429,WR2721 and LPS:Six week old male SPF C57BL/6 mice received intraperitoneal injection of CL429(2.5 mg/kg),WR2721(150 mg/kg),PBS control group and LPS(2.5 mg/kg)at two dose points(7.5Gy and 9 Gy)24 hours and 2 hours before irradiation,respectively,or once immediately after irradiation.The survival of mice was observed for 30 days after irradiation.(3)Comparison of radioprotective effect between CL429 and Pam3,MDP and Pam3+MDP:The C57BL/6 mice were given CL429,Pam3(50 ng/kg),MDP(5 mg/kg),Pam3+MDP and PBS control group by celiac injection at 9Gy dose respectively 24 hours and 2 hours before irradiation or once immediately after irradiation.The survival of mice was observed continuously for 30 days after irradiation.3.Protective effect of CL429 on radiation injury of bone marrow hematopoietic system(1)Pathological analysis of bone marrow HE staining:Male C57BL/6 mice in CL429 group and PBS control group received 7.5Gy total body irradiation after twice administration.The femoral heads were taken for pathological sections on the 1st,5th and10th day after irradiation and analyzed by HE staining.(2)Detection of apoptosis of mouse bone marrow cells:Male C57BL/6 mice got total body irradiation of 7.5Gy and 9Gy respectively after twice administration.Bone marrow cells were harvested 24 hours after irradiation.The apoptosis rate of nucleated cells in bone marrow tissue was detected by flow cytometry FITC annexin V/PI double staining.(3)Effect of CL429 on the proportion of hematopoietic stem cells and progenitor cells in mouse bone marrow:Bone marrow cells of male C57BL/6 mice were collected 24hours after 7.5Gy total body irradiation.The proportion of hematopoietic stem cells(lin-c-kit+Sca-1+,LKS+)and hematopoietic progenitor cells(lin-c-kit+Sca-1-,LKS-)in bone marrow nucleated cells was detected by immunofluorescence flow cytometry.(4)Determination of colony forming unit in the spleen of mice after hematopoietic stem cells transplantation:The donor mice received 4Gy total body irradiation after administration.The recipient mice received 7.5Gy total body irradiation,and 24 hours later the BMCs(1×10~5 cells/mouse)were transplanted into the recipient mice.The splenic nodules of mice were observed 9 days after bone marrow transplantation.4.Protective effect of CL429 on radiation injury of intestinal tissue(1)Pathological analysis of small intestine with HE staining:The mice in CL429group and PBS control group received 9Gy total body irradiation after twice administration.Small intestine tissues were taken for HE staining pathological analysis on the 1st,3rd,5th day after irradiation.(2)Immunohistochemical detection of Ki67 cell proliferation in small intestinal crypt:Male C57BL/6 mice in CL429 group and PBS control group received 9Gy total body irradiation after twice administration.Small intestine tissues were taken for immunohistochemically Ki67 staining at 3.5 and 5 days after irradiation to detect the apoptosis of small intestine crypt cells.(3)The situ apoptosis of crypt cells was detected by TUNEL staining:Male C57BL/6 mice in CL429 group and PBS control group received 9 Gy total body irradiation after twice administration.At 24 hours after irradiation,small intestinal tissues were taken for immunohistochemistry TUNEL staining to detect in situ apoptosis of small intestinal crypt cells.(4)Culture of intestinal organs in mice:The small intestinal crypt of male C57BL/6mice was taken for organ culture and stimulated with CL429.After 12 hours,the organs were irradiated with 6.0 Gy.After irradiation,the organs were cultured for 7 days.The size and budding of the organs were analyzed statistically.5.Preliminary study on the mechanism of CL429 in preventing radiation injury(1)Effects of CL429 gene knockout mice after administration and irradiation on survival:Wild type mice administered PBS group(WT+PBS),wild type mice administered CL429 group(WT+CL 429),NOD2 knockout mice administered PBS group(NOD2 KO+PBS),NOD2 knockout mice administered CL 429 group(NOD2 KO+CL 429),TLR2 knockout mice administered PBS group(TLR2 KO+PBS),TLR2knockout mice administered CL429 group(TLR2 KO+PBS)KO+CL429),TLR2/NOD2 double knockout mice treated with PBS(TLR2/NOD2 DKO+PBS),TLR2/NOD2 double knockout mice treated with CL429(TLR2/NOD2 DKO+CL429).Cl429was delivered at a dose of 2.5mg/kg 24 hours and 2 hours before irradiation.After receiving 7.5Gy total body irradiation,the survival of mice was observed 30 days after irradiation.(2)Cell viability of knockdown cell line stimulated with CL429 after administration:HIEC cells in logarithmic growth phase were transfected with Lipofectamine 3000 to construct si-TLR 2,si-NOD 2 and si-TLR 2+si-NOD 2 cell lines respectively.Cl429was treated with 8Gy irradiation,and CCK-8 was used to detect cell viability 24 hours after irradiation.(3)The pathological analysis of bone marrow and gut of TLR2 KO mice treated with cl429 by HE staining:C57BL/6 and TLR2 KO mice were divided into four groups:WT+PBS,WT+cl429,TLR2 Ko+PBS,TLR2 Ko+cl429.3.5 days after irradiation,the bone marrow and small intestine of mice were taken for pathological analysis.(4)Detection of TLR2 and NOD2 m RNA expression in bone marrow cells by real-time PCR:Bone marrow nucleated cells were obtained from male C57BL/6 mice in both CL429 and PBS control group after twice administration.The m RNA expression levels of TLR2 and NOD2 were detected by real-time PCR.(5)Detection of protein expression related to TLR2-MYD88-NF-κB signaling pathway:The protein of HIEC cells was extracted at 0,2,4,8,12 and 24 hours after CL429 stimulation.The expression of TLR2,NOD2,My D88 and p-IKK were detected by WB.(6)Detection of radiation protective cytokines:Six week old male C57BL/6 mice received 7.5Gy total body irradiation after CL429 administration.After 24 hours,the peripheral blood serum of mice was collected.The expression levels of IL-6,IL-11,IL-12and TNFαin serum were detected by ELISA.(7)Identification of new key effector molecules and related signaling pathways in the protection of CL429 against radiation injury by RNA sequencing:Six week old male C57BL/6 mice received 7.5Gy total body irradiation after CL429 administration.Bone marrow cells and spleen were taken 24 hours after irradiation,and small intestine tissue was taken 72 hours after irradiation.After liquid nitrogen quick freezing,Shanghai Ouyi Biotechnology Co.Ltd.was entrusted to conduct RNA quality inspection and sequencing.The differentially expressed genes in CL429 group were analyzed,and the most differentially expressed target effector molecules were screened out.The differentially expressed genes were further analyzed by heat map,reads distribution map,Go and KEGG enrichment pathway analysis.(8)Q-PCR analysis of potential key molecules:C57BL/6 mice were randomly divided into two groups:control and cl429.Bone marrow,spleen and small intestine were taken 24 hours after irradiation to extract m RNA for Q-PCR analysis of potential key molecules S100A8,S100A9,Wnt5a,IGF2BP3,CCL2,CCL3,CCl4,ccnb1,ccnb2,CCND1,CCNE1,ccne2 and CDK1.(9)Cell viability analysis of primary mouse bone marrow and spleen cells after irradiation with recombinant protein:The primary cells of bone marrow and spleen from C57BL/6 mice were divided into six groups:control,cl429,S100A8,S100A9,Wnt5a and IGF2BP3(the concentration of recombinant protein was 50ng/ml).CCK-8 was used to detect the cell viability 24 hours after irradiation.(10)Effects of Cl429 on the survival of S100A9 KO mice and wild-type mice:Heterozygous S100A9 KO mice and wild-type C57BL/6 mice were divided into four groups(n=6):WT+PBS,WT+cl429,S100A9 Ko+PBS,S100A9 Ko+cl429.The mice received 9 Gy total body irradiation after intraperitoneal administration,and the survival of mice was observed 30 days after irradiation.6.SPSS 19 was used to analyze the research results,and mean±SD was used to express the results.T test was used to judge whether there was statistical difference between the two groups.Kaplan Meier survival curve was used to compare the survival between the two groups.The experimental data were plotted and analyzed by Graphpad prism software.Research results1.The screening of a novel dual targeting ligand CL429 and its acute toxicity to cells and mice.(1)The screening of novel double targeting ligand CL429:In this subject,we found that the cell viability of CL429 group was slightly lower than that of LPS group,but significantly higher than that of other TLRs family members through previous literature research,theoretical analysis and CCK-8 test of HIEC cells after 8 Gy gamma irradiation.(2)Acute toxicity test of CL429 on cells:After adding CL429 with different gradient final concentration to the logarithmic growth phase HIEC cells,CCK-8 method was used to detect the cell viability after 24 hours of culture.It was found that the cell viability gradually increased with the increase of CL429 concentration when CL429 concentration was lower than 400μg/ml,and increased with the increase of CL429 concentration when CL429 concentration was 800μg/ml.Compared with the control group,the cell viability of the 1600μg/ml group decreased slightly but was still higher than that of the control group.While the cell viability of the 3200μg/ml group decreased significantly and was even lower than the control group,showing the significant toxicity of CL429.The optimal concentration of CL429 is 40μg/ml,so it can be considered that CL429 is safe or has no obvious toxic and side effects.(3)Acute toxicity test of CL429 on mice:By observing the survival of mice within 48hours after administration(25 mg/kg),it was found that all mice in CL429 group survived,while all mice in LPS group died within 36 hours.2.Radioprotective effects of CL429 on animals(1)Finding out he best radioprotective dose of CL429 on mice:After intraperitoneal injection of CL429 in gradient concentration twice and receiving 7.5Gy total body irradiation,the survival of mice was observed continuously for 30 days after irradiation.It was found that the optimal dose of CL429 for radiation protection test in mice was2.5mg/kg,which was 1/10 of the previous dose of acute toxicity test in mice.Therefore,it can be considered that the optimal dose of CL429 in mice was safe or unknown acute toxicity of the drug.(2)Comparison of radioprotective effect of CL429,WR2721 and LPS:The survival rate of mice in CL429 group and LPS group was 100%under the condition of 7.5Gy preirradiation prophylactic administration,which was significantly better than 80%in WR2721 group;with preventive administration of CL429 followed by 9Gy pre irradiation,the survival of mice in CL429 group was still100%,and still better than 90%in LPS group and 70%in WR2721 group;the survival of mice in CL429 group was 100%with 7.5Gy post irradiation treatment,which was better than 80%in LPS group and 60%in WR2721group;the survival of mice in CL429 group was 100%with 9Gy post irradiation treatment.The survival rate decreased to 50%,which was still better than 30%in LPS group and 0%in WR2721 group.(3)Comparison of radioprotective effect between CL429 and Pam3,MDP and Pam3+MDP:The survival rate of CL429 group was still 100%under the condition of preventive administration before 9Gy irradiation,and the order of preventive effect from high to low was CL429>Pam3+MDP>Pam3>MDP>PBS;after 9Gy irradiation,the survival rate of CL429 group was significantly reduced to 50%,and the order of therapeutic effect from high to low was CL429>Pam3>Pam3+MDP>PBS>MDP.3.Protective effect of CL429 on radiation injury of bone marrow hematopoietic system(1)Pathological analysis of bone marrow HE staining:Compared with PBS group,the damage of bone marrow cavity emptiness and blood sinus injury in CL429 group was significantly reduced,and the number of BMCs in CL429 group was much more than that in PBS group.(2)Detection of apoptosis of mouse bone marrow nucleated cells:The apoptosis rate of BMCs in each group increased significantly with the increasing of irradiation dose,while the apoptosis rate of BMCs in CL429 group was significantly lower than that in PBS group.(3)Effect of CL429 on the proportion of hematopoietic stem cells and progenitor cells in mouse bone marrow:Compared with PBS control group,the proportion of hematopoietic stem cells and hematopoietic progenitor cells in CL429 group was obviously increased.(4)Determination of colony forming unit in the spleen of mice after bone marrow transplantation:The number and volume of splenic nodules in CL429 group were significantly better than those in PBS control group.4.Protective effect of CL429 on radiation injury of intestinal tissue(1)Pathological analysis of small intestine with HE staining:Compared with CL429group,the number of small intestinal villi in PBS control group decreased,the morphology was abnormal,the mucosa became thinner,the crypt epithelial cells decreased,and the length of small intestinal villi was significantly shorter.(2)Immunohistochemical detection of Ki67 cell proliferation in small intestinal crypt:The positive rate of small intestinal crypt cells in CL429 group was significantly higher than that in PBS control group.(3)The situ apoptosis of crypt cells was detected by TUNEL staining:The number of TUNEL positive cells in intestinal crypt cells of PBS control group was significantly more than that of CL429 group after 9 Gy total body irradiation.(4)Culture of intestinal organs in mice:After CL429 stimulation and 6.0 Gy irradiation,it was observed that compared with PBS control group,the number of small intestinal crypt cells forming organ like cells and the number and volume of single organ like cells budding in CL429 treatment group were more.5.Preliminary study on the mechanism of CL429 in preventing radiation injury(1)Effects of CL429 gene knockout mice after administration and irradiation on survival:After receiving 7.5Gy total body irradiation twice before irradiation,the survival rate of mice in each group was 100%in WT+CL429 group>50%in NOD2 KO+CL429group>20%in TLR2 KO+CL429 group.(2)Cell viability of knockdown cell line stimulated with CL429 after administration:The results of Western blot showed that the expression of TLR2 and NOD2 protein was significantly down regulated after si RNA knockdown.The cells were further irradiated with 8Gy and the cell viability was detected by CCK-8(compared with PBS control group):si NC group>si-NOD2 group>si-TLR2 group>si-TLR2+si-NOD2 group.(3)The pathological analysis of bone marrow and gut of TLR2 KO mice treated with cl429 by HE staining:Compared with PBS irradiation group,Cl429 can effectively reduce the radiation damage of bone marrow and small intestine of wild-type and TLR2 KO mice.In Cl429 group,the radiation damage of bone marrow and small intestine of wild-type mice is less than that of TLR2 KO mice.(4)Detection of TLR2 and NOD2 m RNA expression in bone marrow cells by real-time PCR:Compared with PBS control group,CL429 could significantly up regulate TLR4,7,8,11 and 13 m RNA levels,but TLR2 and NOD2 m RNA levels were not significantly up-regulated,or even slightly down regulated.(5)Detection of protein expression related to TLR2-My D88-NF-κB signaling pathway:The results of Western blot showed that the expression of TLR2,NOD2 and My D88 protein were significantly up-regulated after CL429 stimulation,and then gradually decreased with time,while p-IKK was not up-regulated.(6)Detection of radiation protective cytokines:Compared with the blank control group,the results showed that the expression of IL-6 and TNFαin PBS group decreased to a certain extent,while IL-11 and IL-12 increased to a certain extent;the expression of IL-6,IL-11,IL-12 and TNFαin CL429 group increased significantly.(7)Identification of new key effector molecules and related signaling pathways in the protection of CL429 against radiation injury by RNA sequencing:Six week old male C57BL/6 mice received 7.5Gy total body irradiation after CL429 administration.Bone marrow cells and spleen were taken 24 hours after irradiation,and small intestine tissue was taken 72 hours after irradiation.After liquid nitrogen quick freezing,Shanghai Ouyi Biotechnology Co.Ltd.was entrusted to conduct RNA quality inspection and sequencing.Compared with the control group,the differentially expressed genes in CL429 group were analyzed,and the most differentially expressed target effector molecules were screened out.The differentially expressed genes were further analyzed by heat map,reads distribution map,Go and KEGG enrichment pathway analysis.(8)Q-PCR analysis of potential key molecules:Q-PCR was used to verify the potential key molecules S100A8,S100A9,Wnt5a,IGF2BP3,CCL2,CCL3,CCl4,ccnb1,ccnb2,CCND1,CCNE1,ccne2 and CDK1.Furthermore,four molecules S100A8,S100A9,Wnt5a and IGF2BP3 were screened out for its up-regulattion and stable expression.(9)Cell viability analysis of primary mouse bone marrow and spleen cells after irradiation with recombinant protein:The cell viability was detected after the primary bone marrow and spleen cells of mice were treated with recombinant protein and irradiated.The results showed that:in primary bone marrow cells,cell viability:S100A9>cl429>Wnt5a>S100A8>IGF2BP3>con;in primary spleen cells,cell viability:cl429>S100A9>IGF2BP3>con.(10)Effects of Cl429 on the survival of S100A9 KO mice and wild-type mice:Compared with s100A9ko+PBS group,the survival rate and survival time of s100A9ko mice treated with Cl429 were improved.Cl429 could partially rescue the radiation damage of s100A9ko mice,but the radiation protection effect of Cl429 on s100A9ko mice should be far lower than that of wild-type mice.Research conclusionFirstly,we successfully screened out a new TLR2/NOD2 dual targeting ligand from a number of TLRs and NODs family ligands,and confirmed that its toxicity level was significantly lower than that of LPS and WR2721 from the overall animal and cell level.The preventive and therapeutic effects of CL429 are better than LPS,WR2721,TLR2ligand Pam3,NOD ligand MDP and Pam3+MDP,which laid a solid foundation for CL429 to become a potential radioprotective agent with high efficiency and low toxicity.Then,the radioprotective effect of CL429 was confirmed from the tissue and organ level of bone marrow hematopoietic tissue and small intestine tissue.Cl429 can alleviate bone marrow cavity emptiness and blood sinus injury,inhibit the apoptosis of BMCs,promote the proliferation and differentiation of hematopoietic stem and progenitor cells,and has a positive role in promoting the recovery and reconstruction of bone marrow hematopoietic tissue after radiation injury.For the small intestine,CL429 can slow down the radiation-induced small intestinal villi breaking and shortening,morphological changes,mucosal thinning and the reduction of crypt epithelial cells,promote the proliferation and inhibit the apoptosis of small intestinal crypt cells.It is also confirmed that CL429 can effectively promote the formation of small intestinal crypt cells and their further development Proliferation and differentiation.Finally,we discuss the mechanism of CL429 in protecting against ionizing radiation injury.First of all,it was confirmed that CL429 may mainly rely on TLR2 and partly on NOD2 to prevent radiation injury at the whole animal level and in vitro cell level.Moreover,CL429 could not upregulate the m RNA levels of TLR2 and NOD2 after irradiation,which suggested that CL429 might not activate TLR2 and NOD2 through transcription.Furthermore,the expression of TLR2-Myd88-nf-κB signaling pathway related proteins were detect by Western blot.It was found that TLR2,NOD2 and My D88were significantly up-regulated at first,while p-IKK was not up-regulated.It suggests that CL429 may play a protective role against radiation injury by activating TLR2-Myd88-nf-κB signaling pathway.The results showed that cl429 could significantly up regulate IL-6,IL-11,IL-12 and TNFαby detection of the serum cytokines of mice after irradiation.In short,the protective effect of Cl429 against radiation injury mainly depends on TLR2,and partly depends on NOD2.Cl429 played a protective effect against radiation injury through tlr2-myd88-nf-κB signaling pathway and upregulation of radiopotective cytokines IL-6,IL-11,IL-12 and TNFα.Next,we screened out the related signal pathways TLR2 and NOD2 by RNA sequencing,which indicated that the protective effect of Cl429 against radiation injury was closely related to these two signaling pathways.At the same time,the potential key molecules were screened out.The key molecule S100A9 was successfully identified by Q-PCR of mouse bone marrow,spleen and small intestine,and the cell viability of primary mouse bone marrow and spleen cells after administration of recombinant protein.Finally,the survival rate of S100A9 knockout mice verified that S100A9 played an important role in the protective effect of cl429 against radiation injury.In conclusion,we demonstrated that the protective effect of Cl429 against radiation injury partly depends on NOD2,mainly depends on TLR2 through tlr2-myd88-nf-κB signaling pathway and upregulation of radiopotective cytokines IL-6,IL-11,IL-12 and TNFα.Meanwhile,we also successfully identified and confirmed that S100A9 played an important role in the protective effect of Cl429 against radiation injury,which provided a new research idea and intervention way for the prevention and treatment of radiation injury. |
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