Effects Of Fluoride On IL-17A And The Signaling Pathway In Mice Testis

[Objective]Over the decades,the study of fluoride showed that the damage of fluoride on male reproductive system was manifold.The research was to explore the effect of fluoride on the expression on IL-17A and the signaling pathway related genes and proteins,and to further explore the reproductive toxicity mechanism of fluoride to the male reproductive system and provide theoretical basis for the design of scientific target drug.[Method]103 adult BALB/c mice were divided randomly into 5 groups which were control group,low fluoride group,medium fluoride group,high fluoride group and EAO group,three mices were used for testis homogenate,each group of 25.Control group and EAO group mice were drank distilled water,and mice in fluoride groups were provided distilled water containing 25,50 and 100mg/L NaF,The EAO group model by using subcutaneous inject testicular homogenate to make model.During the whole experiment,each group guaranteed its free intake and free drinking water.After 150 days exposure,mice were killed and testes,epididymis,semen,femur and other major organs,a part of the testes with Bouins reagent were fixed,the other part of the testes,decapsulated testes gained to collected the testicular interstitial fluid and testicular interstitial cell to detect the related indicators,fluorine ion selective electrode method were determinated the fluorine content,HE staining to observe testicular tissue morphological structure changes in EAO group,by enzyme-linked immunosorbent assay(ELISA),Immunohistochemical technology(IHC)and Realtime polymerase chain reaction(PCR)to detect IL-17A and the signaling pathways regulated genes and proteins expression.[Result]1.Compared with the control group,the fluorine content of femur was significantly increased in each fluoride treated groups,and it extremely difference in low fluoride group,medium fluoride group and the high-fluoride group(P<0.01).2.Testis HE staining results of EAO group showed that,the spermatogenic epithelium arranged in an orderly and tight of testis in control group,and the presence of inflammatory cells is almost invisible in testicular interstitial,There were no cells in the functional cavity of the seminiferous tubule and filled with sperm;Compared with control group,the spermatogenic epithelium arranged was disorganized and loose,and formed a lot of vacuoles of testis in the EAO group,there are lots of inflammatory cells in the testicular interstitial and some of the germ cells fall into the cavity of the seminiferous tubule.3.Compared with control group,the major organ coefficients were no significant difference in each fluoride group,but in EAO group,the testis organ coefficients significantly decreased(P<0.01),the epididymis organ coefficients significantly increased(P<0.05),and the spleen organ coefficient significantly increased(P<0.01),heart and kidney organ coefficients were no significant difference;Sperm viability,motility and density were declining in each group,sperm viability and motility were significant difference in fluoride treated group(P<0.05),EAO group were significantly decreased(P<0.01),and the sperm density was significant difference in the high fluoride group and in the EAO group(P<0.05,P<0.01).4.Compared with the control group,the ELISA results showed that the level of IL-17A in the testicular interstitial tissue fluid of the mice were significantly increased in both the high-fluoried group and the EAO group(P<0.05).There was a linear correlation between the fluoride content of the mice and the content of IL-17A in the testicular interstitial tissue fluid and the linear correlation coefficient R2=0.5090.5.Compared with the control group,the protein expression areas of IL-17RA and IL-17RC mainly in testicular interstitial and seminiferous epithelium,the result of analysis optical density value,the protein expression levels of IL-17RA and IL-17RC were significant increased in the middle and high fluoride group and EAO group(P<0.05,P<0.01).6.The expression of related genes in the IL-17 signaling pathway through qRT-PCR,compared with the control group,the mRNA expression of IL-17A,IL-17RA and IL-17RC was increased and especially in high fluoride group were significantly increased(P<0.05,P<0.01),and were highly increased in EAO group(P<0.01).Actl mediated MAPKS pathway,the gene level of Actl was significantly increased in in high fluoride group and EAO group(P<0.05,P<0.01),the gene level of TRAF6 was no difference.In ERK pathways,the mRNA expression of ERK2 and ERK5 were significantly decreased in medium fluoride group and high fluoride group(P<0.05,P<0.01),while in the EAO group was no difference;In JNK pathways,the mRNA expression of JNK1 and JNK3 were significantly increased in high fluoride group(P<0.05),while the mRNA expression of JNK2 was significantly increased in EAO group(P<0.01);In P38 pathways,the mRNA expression of P38MAPK was only significantly increased in EAO group(P<0.01),and was not significant in each fluoride group.Actl mediated NFKB pathway,the gene expression of TAK1 showed a downward trend,but not significant.Actl mediated C/EBP pathway,the gene expression of C/EBP was significantly increased in EAO group(P<0.01),and all not obvious change in each fluoride group.[conclusion]The results show that the inflammatory cytokine IL-17A content significantly increased in mice testicular interstitial fluid,and further detect the related genes expression on IL-17 signaling pathways,IL-17 signal mediated Actl-MAPKs signal related genes significantly changes,this results showed that at the genetic level,IL-17 mediated Actl-MAPKs pathway may be induced the immune response of the testes after the fluoride treated.