Effects Of IL-17A Gene Knockout Or Self-recovery On Testicular Injury Induced By Fluoride In Mice

Testis is an important male reproductive organ.Plenty of studies have shown that fluoride(F)exposure can cause testicular damage,but the mechanism of the damage remains unclear.Therefore,in order to further explore the mechanism of F-induced testicular autoimmune reaction and the role of IL-17 A in it,this study established a 180 d sodium fluoride(Na F)exposure model of wild type(WT)C57BL/6J mice and IL-17 A knockout(IL-17A-/-)mice(C57BL/6J background),and used various techniques like hematoxylin-eosin(HE)staining,q RT-PCR,western blot,immunohistochemistry,ELISA and fluorescence staining.The mechanism of F-caused testicular damage and the alleviated mechanism of IL-17 A gene knockout on F-caused testicular damage were further studied by mRNA transcription sequencing technology.In addition,the 60 d and 120 d self-recovery model based on F exposure model of WT mice was used to explore whether the F-induced testicular injury could be recovered after a period of self-recovery(withdrawal period).Establishment of animal model: 72 WT mice were divided into control group(also known as C group)and F group,36 mice in each group.24 IL-17A-/-mice were divided into IL-17A-/-group(KC group)and IL-17A-/-+F group(KF group),12 mice in each group.Animals in control and IL-17A-/-groups drank distilled water.The mice in F and IL-17A-/-+F groups were received distilled water with 50 mg/L Na F.After 180 d(F exposure period),12 mice were selected randomly from each group were sacrificed for subsequent experiments.Evans blue was injected intraperitoneally to detect the blood-testis barrier permeability(n=3).We collected sperms from epididymal cauda and vas deferens for the detection of sperm quality(n=6),serum for the evaluation of autoantibodies(n=6)and femur to determine the fluorine concentration in bone(n=6).The testis was used for the evaluation of testicular histopathology(n=3)as well as expression of mRNA and protein(n=6).The remaining 24 mice in control group and F group were stopped fluoride exposure.And after self-recovery of 60 d and 120 d,12 mice in each group were randomly selected for the above treatment.In addition,the mice testis of 180 d F exposure period was used for mRNA transcriptome sequencing.(1)The results of 180 d F exposure model: the results indicated that F exposure for 180 d caused the increase of femur fluorine concentration,the decreased sperm quality including the reductions of sperm count and survival,the damaged testis histopathology which was characterized by the disordered arrangement of germ cells and the obvious cell shedding in the lumen of seminiferous tubules,the enhanced mRNA and protein expressions of inflammatory cytokines containing IFN-γ,IL-6,TNF-α,pro-IL-1β and IL-1β,the changes of autoantibody such as the appearance and increased content of anti-testicular autoantibodies in sera and the autoantibody deposition in spermatogenic epithelium and sperm of seminiferous tubule,the increased mRNA and protein expressions of IL-17 A and the activation of IL-17 A signaling cascade like the elevated mRNA and protein expressions of Act1,NF-κB,AP-1 and CEBPβ,and the increased protein expressions of IL-17 RC with a decrease of IκBα and TRAF6,the alterations of autoimmune related genes containing the decreased mRNA and protein expressions of AIRE,FOXP3 and CD28 with an increase of MHCⅡ,the reduced protein expression of CTLA4,the increased permeability of blood-testis barrier which was characterized by red fluorescence in the seminiferous tubule,and the decreased mRNA and protein expressions of blood-testis barrier-related genes and proteins containing the reduced mRNA and protein expressions of Occludin and ZO-1 with the decreased protein expressions of β-catenin and N-cadherin.Summarizing the results,it is found that among 57 primary indicators,F changed 43 indicators.After IL-17 A knockout,30 of 43 F-induced changes were alleviated.(2)The results of the self-recovery model of 60 d and 120 d: after 60 d of recovery,compared with the control group,17 indicators in F group were changed.After 120 d of recovery,compared with the control group,except the fluorine content of the femur and the protein expression of ZO-1,F-induced differences all returned to normal levels.(3)The results of mRNA transcriptome sequencing: the differentially expressed genes between F and C groups were found involved in cell adhesion molecules and tight junction pathways,and the genes including Cdh1,Cldn1 and Tjp3 in these two pathways were closely related to the blood-testis barrier.The differentially expressed genes between F vs C and KF vs F were enriched in arachidonic acid metabolic pathway together.Conclusion: F exposure induced testicular injury and decrease of sperm quality by autoimmune response.The mechanism of autoimmune response was involved with the activation of IL-17 A signaling pathway,the damage of tolerance to self‐antigens,the enhancement of antigen presentation and the damage of blood-testis barrier.IL-17 A gene knockout improved these F-induced changes to some extent.After withdrawal period,the F-caused testicular damages gradually restored to normal in a time-dependent manner.Arachidonic acid metabolic pathway may be an vital mechanism for IL-17 A gene knockout to alleviate testicular damage caused by F.