| BackgroundWith the increasing use of nuclear energy,the protection against exposure to ionizing radiation has gradually gained attention.It is found that ionizing radiation can cause DNA damage in living cells,which in turn leads to damage to cellular genetic material and even cell death.The radiation sensitivity of various tissues and organs of the human body is quite different.The testis is very sensitive to ionizing radiation.Large doses of exposure to ionizing radiation can directly lead to spermatogonial cells,spermatogonial stem cell death,and intact tubule endothelial injury and etc..Therefore,looking for a new method of radiation protection for the male reproductive system is an urgent problem to be solved.Toll-like receptors(TLRs)are kinds of pattern recognition receptors involved in various pathophysiological processes such as antigen recognition,tumor immunity,and antiviral.In the process of finding effective radiation protection methods,it has been identified radiation protection effects can be achieved by activating certain TLRs.In particular,the radiation protection effect of TLR4 is most prominent,which indicates that TLR4 may play a key role in the radiation protection among TLRs families.However,the traditional TLR4 agonist LPS is a component isolated from the bacterial cell wall and has strong immunogenicity.Its alpplication in radiation peotection is seriously restricted due to its obvious side effects.In a subsequent study,an attenuated TLR4 agonist,monophosphoryl lipid A(MPLA),was successfully prepared by chemical synthesis using LPS as a prototype.MPLA is currently used as an immunoadjuvant for tumor immunity.In preliminary work,our team found that MPLA can produce obvious radiation protection effect on mouse bone marrow,intestine and spleen by activating TLR4 with small toxicity,suggesting its great application prospect in clinical radiation protection.The pre-experimental results of this subject showed that MPLA also produced radiation protection effect on mouse testis.However,the radiation sensitivity and distribution of TLRs in different tissues and cells varies greatly,therefore,using the single theory to explain the role of TLRs in radiation protection can not meet the increasingly refined research,and is not conducive to the transformation of research results into applications.In this study,we preliminarily elucidated the radiation protection effect and mechanism of MPLA in male reproductive system based on the full consideration of biological integrity and intercellular differences.ContentsFirstly,Exploring the radiation protective effect of MPLA on mice testis and mice spermatogonia.We mainly explore the radiation protection effect of MPLA at cell aspect and animal aspect.At the cellular aspect,we selected mouse spermatogonia as the research object,and mainly explored the effect of MPLA on its cell proliferation ability.At the animal aspect,we detected effects of MPLA on tissue structural damage,cell cycle,endocrine function,and spermatogenesis after testicular irradiation in mice.Secondly,Exploring mechanism of MPLA on radiation protection of mouse testis and spermatogonia.To investigate the molecular mechanism of MPLA's protective effect on testicular radiation in mice,we examined the apoptosis of mice testis cells after MPLA treatment,as well as the activation level of DNA damage repair pathways and apoptotic pathway in mouse testis and spermatogonia.And by using knockout mice,the pathway dependence of MPLA action is clarified.In previous study,we found that MPLA produced radiation protection effect on mouse testis,but no radiation protection effect was found on spermatogonia.We hypothesized that due to the different expression and activation of TLR4 in various tissues,there may be some target organ and cells with significant activation of TLR4,and may indirectly protect against testicular and spermatogonia.In order to find the above target organs and target cells,we examined and confirmed the expression and activation level of TLR4 in various tissues,and looked for the target organs with the most obvious activation of TLR4 after MPLA treatment.After targeting the target organ,we further looked for target cells with significant TLR4 activation.After locking the target organ,we further looking for target cells with significant TLR4 activation.Next,we established the co-culture system of target cells and mice spermatogonia to clarify the effect of target cells on the radiation sensitivity of mice spermatogonia,we further explored subcellular effectors of target influencing spermatogonia radiation sensitivity,and finally determine that macrophage-derived exosomes play a radiation protection role on spermatogonia.We also examined the DNA damage repair pathway and apoptosis pathway activation level in spermatogonia after co-culture with macrophage or macrophage-derived exosome treatment.In order to further explore the specific components in exosomes that play a role in radiation protection,we examined the protein expression changes of macrophage-derived exosomes after MPLA treatment by protein array method,and finally determined the protein function by blocking the protein effect.And by blocking the protein effect,the specific composition of the protein that play roles in radiation protection is finally determined.MethodsFirstly,exploring the radiation protective effect of MPLA on mouse testis and mouse spermatogonia.At the cell aspect,the cell proliferation ability was detected by colony formation assay;at the animal aspect,testicular H&E staining was used to explore the tissue damage after testicular irradiation;mice sperm production ability was examined by unilateral epididymis sperm counting;cell cycle arrest after was detected by flow cytometry;the effects of radiation on mouse testicular endocrine function were determined by ELISA kit detection of mouse testis and serum testosterone.Secondly,looking for target organs and target cells of TLR4 activation after MPLA administration,and exploring its relationship with testicular radiation damage protection.In this section,we used western blot to determine TLR4 expression level in different organs and cells,and we used TLR4 immunofluorescence staining to detect the distribution of TLR4 in testis.p-P65 immunofluorescence staining was used to find the TLR4 activation target organs,and in target organs,F4/80 and p-P65 immunofluorescence co-staining was used to determine the target cell.After inentify the target cell,we established co-culture system consist of target cell and mice spermatogonia.We detect spermatogonia proliferation ability in co-culture system by colony formation assay.We also used colony formation assay to detect spermatogonia proliferation ability after GW4869 treatment,macrophage supernatant treatment or macropgage derived exosome treatmentThirdly,exploring the radiation protection mechanism of MPLA on testis and spermatogonia.In cell aspect,we used western blot assay to detect molecular expression changes in DDR pathway and apoptosis pathway after co-culture treatment or exosome treatment.We used protein array method to detect differentially expressed proteins in exosomes after MPLA treatment.We blocked the effect of the target protein by applying protein neutralizing antibodies,and explored radiation sensitivity of spermatogonial cells after neutralizing antibody treatment by colony formation assay.In animal aspect,we used western blot assay to detect molecular expression changes in DDR pathway and apoptosis pathway.We used?H2AX immunofluorescence staining to detect the efficiency of DNA damage repair.By using TUNEL staining,we examined cell apoptosis level both in TLR4 deficient mice and TRIF deficient mice.We also used H&E staining to detect irradiation induced testis damage level in TLR4 deficient mice and TRIF deficient mice,to confirm pathway dependencies in which MPLA works.Finally,we used neutralizing antibody to neutralize identified proteins,and examined testis damage level after irradiation by H&E staining.ResultsFirstly,MPLA has a clear radiation protection effect on mouse testis,but has no obvious radiation protection effect on spermatogonial cells.We found that MPLA significantly reduced radiation-induced damage to testicular tissue structure,reduced radiation-induced diploid cell death in the testis,reduced radiation-induced decrease in sperm production,and decreased testicular secretion of testosterone.However,in in vitro experiments,we found that MPLA did not reduce the decline of spermatogonia proliferative capacity caused by irradiation.Secondly,MPLA has a radiation-protective effect on mouse testis by increasing the efficiency of DNA damage repair and reducing apoptosis.This effect is achieved through TLR4-TRIF pathway.We found that MPLA can effectively reduce the activation of ?H2AX in mice testis cells,increase the activation level of DNA-PKcs T2609,p-ATR,which is involved in DNA damage repair,reduce the activation level of proapoptotic molecule C-Caspase3,and reduce testis cell apoptosis at 16 h after irradiation.However,MPLA does not reduce the activation level of key molecules in the apoptotic pathway in spermatogonial after irradiation.We found that MPLA did not produce radiation protection effect in testis of TLR4 and TRIF deficient mice,indicating that the radiation protection of MPLA is achieved through the TLR4-TRIF pathway.Thirdly,spleen-derived macrophages are the target cells with most obvious TLR4 activation level after MPLA treatment.We found that TLR4 in the testis is mainly distributed in mesenchymal cells,while the expression of TLR4 in the key cell spermatogonia involved in spermatogenesis is extremely small.In addition,the expression level of TLR4 in the spleen is at a high level.By immunofluorescence staining of the TLR4 pathway activation marker p-P65 in various tissues,we found that cells in the spleen showed a high activation level of TLR4 pathway after MPLA administration.By immunofluorescence co-staining,we further found that the TLR4 pathway in spleen derived macrophages is highly activated.Fourthly,after MPLA stimulation,Macrophage-derived exosomes produced radiation protection effects on mouse testis and spermatogonia,and G-CSF and MIP-2 are key molecules for radiation protection in exosomes.We established a co-culture system of macrophages and spermatogonia,and found that the radiation sensitivity of spermatogonia was significantly reduced after treatment with MPLA,and the activation level of DNA damage repair pathway in spermatogonial cells was increased,and the activation level of apoptotic pathway was decreased.It suggests that a substance derived from macrophages after MPLA stimulation has a radiation protective effect on spermatogonia.Next,we stimulated macrophages with MPLA and cultured the spermatogonia cells with macrophage-derived supernatants.It was found that the supernatant also produced radiation protection effect on spermatogonia,and this effect disappeared after the treatment of vesicle inhibitor GW4869,which suggested that the exosomes in supernatant may be a key component of radiation protection.Next,we directly treated the spermatogonia with macrophage-derived exosomes after stimulation with MPLA,and found that exosomes can reduce the radiation sensitivity of spermatogonia,increase the activation level of spermatogonial DNA damage repair pathway,and reduce the activation of apoptosis pathway.In order to explore the specific components in exosomes which contribute to exosome mediated radiation protection,we detected the difference in protein expression of exosomes after MPLA treatment by protein array method,and found a variety of differentially expressed proteins including G-CSF and MIP-2.Finally,by using neutralizing antibodies,we found that after blocking the effects of G-CSF and MIP-2,the radiation protection effect of exosome was significantly weakened,indicating that G-CSF and MIP-2 may be the key moleculars in exosomes that participate in testes and spermatogonia radiation protection.ConclusionWe found that MPLA can produce radiation protection effect on mice testis by promoting DNA damage repair and alleviating apoptosis,and effectively alleviate the damage of tissue structure after testicular irradiation in mice.However,MPLA does not produce significant radiation protection effect on mice spermatogonia;we found that TLR4 expression level in spermatogonia is minimal,suggesting that MPLA may have a radioprotective effect on spermatogonia through TLR4 acting on other cells.We found that mouse macrophages exhibited high expression of TLR4,and TLR4 downstream pathway was highly activated after MPLA treatment.Mouse macrophage-derived exosomes after MPLA treatment produced obvious radiation protection effect on mouse spermatogonia.It also promotes spermatogonia DNA damage repair and reduces cell apoptosis after irradiation.We found that MPLA treatment resulted in significant changes in the expression levels of various protein components in macrophage-derived exosomes,and the expression of G-CSF and MIP-2 in mouse macrophage-derived exosomes increased significantly after MPLA stimulation,this two protein may be the key molecules that produce radiation protection effect on mice testis and spermatogonia. |
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