The Mechanism Of Exosome-mediated IR Abscopal Effect In Leading To Pyroptosis Of Sertoli Cells

ObjectiveMale infertility accounts for a large proportion of infertility.Its common causes include chemotherapy,radiation,iatrogenic injury and so on.The United Nations Scientific Committee on the effects of atomic radiation（UNSCEAR）proposed that the main source of ionizing radiation（IR）exposed to humans is medical radiation.With the increase of medical-related radiation,the quality of male semen has decreased significantly in recent decades.Aiming at reducing the impact of medical radiation sources on the reproductive system,local precision radiotherapy such as three-dimensional conformal radiation therapy（3DCRT）has been advocated in recent years to limit the irradiation field and the radiation beam intensity of the therapeutic machine,so as to reduce the radiation to important organs outside the target area.It is worth noting that conformal radiotherapy and precision radiotherapy may also have a certain impact on tissues and organs near the irradiation site or distant tissues and organs.This effect is called radiation-induced abscopal effect.Therefore,for medical workers and patients exposed to ionizing radiation for a long time,it is particularly important to explore the underlying mechanism of male infertility caused by distant effects of ionizing radiation at the molecular and cellular level and find new prevention and treatment methods.Previous studies have shown that whole lung fractionated radiation can cause testicular Sertoli cell damage in male mice through abscopal effect,and local ionizing radiation can cause sperm DNA damage and epigenetic changes through abscopal effect as well.Moreover,some scholars have proposed that local radiotherapy/ionizing radiation can affect the steady-state structure of cell membrane,promote the production of extracellular vesicles（EVs）,including exosomes,and transport them to other parts of the body outside the radiation exposure area through the circulatory system,so as to affect the functions of other tissues and organs.Spermatogenesis in testicular spermatogenic epithelium is protected by the ultrastructure of specific cells composed of testicular Sertoli cells.The Sertoli cell spermatogenic epithelial barrier,also known as blood-testis barrier（BTB）,is mainly composed of tight junctions（TJs）,gap junctions（GPs）,basal ectoplasmic specialization（ES）and desmosomes.As one of the closest blood tissue barriers in mammals,BTB can prevent harmful endogenous or exogenous substances from entering the seminiferous tubules and provide a stable seminiferous microenvironment for spermatogenesis.This study aims at exploring the role of exosomes in mediating the abscopal effect of local ionizing irradiation,and further study its cellular and molecular biological mechanisms in affecting BTB and Sertoli cells.This study would be accomplished by establishing a scientific and reasonable local ionizing radiation animal model and the co-incubation in vitro model of Sertoli cells and plasma exosomes of local ionizing radiation mice.It might provide a new potential target and theoretical basis for the prevention and treatment of male infertility caused by abscopal effect of ionizing radiation.Methods1.The whole lung irradiation male mice model was established by Co⁶⁰-γradiation source.The models were established by 2Gry,4Gry,6Gry and 8Gry irradiation doses for 3consecutive days.2.Take the testicular tissue of the above mouse model after 24 hours and 48 hours of irradiation in different dose groups and observe the changes of testicular tissue under different dose time conditions by H&E staining.3.Extract the plasma exosomes of the above mouse models,identify the characteristics of the exosomes,and incubate them with TM4 cell line.Explore the dose-time condition of TM4 cell injury by using ROS detection and cytotoxicity detection experiments.4.Extract the plasma exosomes of the mouse model irradiated by the above 2Gry dose for three consecutive days,detect the protein mass spectrometry and apply bioinformatics analysis,and then obtain the proteomic data results.Collect the testicular tissue of mouse model,stain the testicular tissue with immunofluorescence（IF）and immunohistochemistry（IHC）to detect BTB related proteins（Occludin,F-actin,ZO-1 andα-Tubulin）expression.5.Immunofluorescence staining was performed on the testicular tissue of the above mouse model to detect the expression of germ cell markers（GCNA 1）,Sertoli cell markers（GATA4 and ERM）,and macrophage markers（F40/80）,so as to explore the specific types of cell injury.Prepare the above mouse model testicular tissue sperm cell suspension,detect the changes of spermatogenic cycle by PI staining flow cytometry,and detect the changes of testosterone and inhibin B levels in mouse serum by ELISA.6.The testicular tissue of mice with local ionizing radiation was detected by Me DIP promotor methylation arrays and m RNA transcriptome arrays,and the methylation and transcriptome data were jointly analyzed.The key genes of testicular injury caused by distant effect of ionizing radiation were screened by RT-PCR and WB experiment,and the specific cellular localization of key genes was detected by immunofluorescence co localization experiment.7.The ultrastructural changes of TM4 cells co-incubated with plasma exosomes of local ionizing radiation mice were observed by transmission electron microscope.The expression of inflammasome composition NLRP3 and the formation of ASC speck were verified by Immunocytochemistry（ICC）.8.Extract the protein of TM4 cells co-incubated with plasma exosomes of local ionizing radiation mice and the protein of testicular tissue of model mice.And detect the pyroptosis pathway NLRP3/Gsdma2/Caspase-1/IL-1βby WB experiment.9.Construct stable transfected TM4 cell line with Gsdma2 knockdown by lv-Gsdma2-RNAi and verify the effect of Gsdma2 on caspase-1 and IL-1βby WB and PCR experiments.And the extracellular level of IL-1βwas detected by ELISA.10.Using cytokine protein detection arrays to detect the changes of 200 types of cytokines in the testicular tissue of the above mouse model.Then bioinformatics analysis was conducted to find the key regulatory pathway in the upstream of pyroptosis process.NF-κB inhibitor and WB experiments was used to verify it.Results1.We successfully established the whole lung IR mice model with 2Gry fractionated IR in 3 consecutive days.The results of H&E staining showed that there were obvious swelling and loosening of seminiferous tubules,detachment of basal cells from basement membrane and block of spermatogenesis in testicular tissue.The toxicity of exosomes extracted from the plasma of that mice model to TM4 cells reached the highest level after48 hours co-incubation.2.Proteomics of mouse plasma exosomes under local ionizing radiation,combined with BTB related proteins in mouse testis（occludin,F-actin,ZO-1 andα-Tubulin）immunofluorescence（IF）and immunohistochemistry（IHC）staining suggest that local ionizing radiation may lead to the destruction of the ultra-fine connection of BTB in mice through the abscopal effect mediated by exosomes.3.Under the above conditions,local ionizing radiation caused significant damage to mouse testicular tissue,significantly down regulated the expression of GCNA1,GATA4and ERM,and significantly up-regulated F40/80,suggesting a significant decrease in germ cells and Sertoli cells and an increase in interstitial macrophage infiltration.4.Under the above conditions,local ionizing radiation led to a significant decrease in the proportion of testicular sperm cells entering S phase in male mice,and there was no significant change in the levels of serum testosterone and inhibin B,suggesting that the spermatogenic cycle was blocked and the process was sex hormone-independent.5.Methylation microarray and m RNA transcriptome microarray were used to detect the testicular tissue of mice exposed to local ionizing radiation.The results showed that the abscopal of local ionizing radiation could significantly change the methylation level of gene promoter and the level of gene transcriptome in mouse testicular tissue.6.Nine candidate genes such as Gsdma2 and F730043M19Rik were found by combined analysis of methylation and transcriptome data.RT-PCR and WB experiments showed that the expression of Gsdma2 changed most significantly in the testis of established mice model.7.In the immunofluorescence co-localization experiment,Gsdma2 and GATA4showed significant co-expression,suggesting that Gsdma2 may be the key gene of testicular Sertoli cell damage caused by local ionizing radiation.8.Through transmission electron microscope experiment,we observed"pore-forming phenomenon"in TM4 cells co-incubated with plasma exosomes of local ionizing radiation mice.Immunocytochemistry（ICC）experiment confirmed the assembly of NLRP3/ASC inflammasome,suggesting the possible existence of pyroptosis happening in Sertoli cells.9.WB experiment revealed the pyroptosis pathway NLRP3/Gsdma2/Caspase-1/IL-1βin TM4 cells co-incubated with plasma exosomes of local ionizing radiation mouse.This phenomenon was also verified by WB experiment in testicular tissue of local ionizing radiation mouse model.10.Further studies showed that Gsdma2 cleaved Caspase-1 and IL-1βin the process of Sertoli cell pyroptosis mediated by local ionizing radiation exosomes,with the release of cleaved-IL-1βto extracellular space.11.The results of cytokine detecting arrays and bioinformatics analysis suggest that NF-κB signaling pathway was significantly up-regulated in the testis of local ionizing radiation mice.Combined with the results of WB experiment,NF-κB functions as a key regulator in the upstream of TM4 pyroptosis process.ConclusionLow dose fractionated local（whole lung）ionizing radiation can lead to the disruption of BTB ultrastructure and Sertoli cell death through exosome mediated abscopal effect,and then cause sex hormone independent spermatogenic cycle arrest.The abscopal effect of local ionizing radiation can significantly change the methylation and transcription level of a bunch of gene base promoters in mouse testis.Among them,the methylation level of Gsdma2 gene promoter is reduced,with high expression in Sertoli cells.Local ionizing radiation induces pyroptosis of Sertoli cells through exosome mediated abscopal effect,in which Gsdma2 plays a key role.NLRP3/ASC inflammasome complex activates gsdma2protein after assembly,and then Gsdma2 cleaves caspase-1 and IL-1β,with inflammatory factor cleaved-IL-1βreleased to extracellular space.Transcription factor NF-κB plays a key regulatory role in the pyroptosis process.Gsdma2 may become a potential gene target for the prevention and treatment of male infertility caused by ionizing radiation.