Effects Of 1α,25（OH）₂D₃ On PFOS-Induced Male Reproductive Toxicity

Background and Objective:The impact of environmental pollution on the ecological environment and human health have aroused considerable attention.Perfluorooctanesulfonic acid（PFOS）is a persistent organic pollutant that has been proven to cause various toxic effects.In this study,a mouse model of PFOS exposure in vivo and a mouse Sertoli cell model of PFOS exposure in vitro were established,and the 1α,25-dihydroxy vitamin D3[1α,25（OH）₂D₃]was used as the intervention factor.The purpose of this study was to explore whether PFOS induced oxidative stress in male reproductive system,and examine whether 1α,25（OH）₂D₃ exhibited protective effects through Kelch-like ECH-associated protein 1（Keap1）-Nuclear factor E2-related factor 2（Nrf2）-Antioxidant responsive element（ARE）signal pathway.This research provides a new target for preventing and treating PFOSinduced male reproductive toxicity and provides a novel strategy for natural antioxidant intervention.Methods:This study includes three sections.（1）The effects of 1α,25（OH）₂D₃ on PFOS-induced reproductive toxicity in male ICR mice.ICR male mice were randomly divided into three groups:control group（CON group,N=6）,PFOS exposure group（PFOS group,N=5）,and PFOS combined with 1α,25（OH）₂D₃ treatment group（PD group,N=5）,The PFOS and PD groups were treated with 10 mg/kg PFOS by gavage,and the CON group was treated with the same volume of 0.5%Tween20 by gavage.Mice in PD group were injected subcutaneously with 1 μg/kg 1α,25（OH）₂D₃ every other day,and the other groups were given the corresponding volume of 1×PBS as vehicle control.After 4 days of pretreatment with 1α,25（OH）₂D₃,PFOS was combined with 1α,25（OH）₂D₃ for 30 days.The mice were sacrificed at the end of the experiment,and the serum,testis,and epididymis tissues of the mice were collected for biochemical and pathological examination.The organ coefficients of testis and epididymis were calculated,the relative sperm concentration and sperm viability of mice were detected by automatic cell counter,the sperm morphology was observed by optical microscope after eosin staining,and the rate of sperm abnormality was calculated.The serum testosterone content was detected by ELISA.Hematoxylin and eosin（HE）staining was used to observe the morphology of testicular tissue.The mRNA levels of acrosome reaction-related genes（ACR,EQTN,PRSS21,CSNK2A2,SPACA1.ZPBP）in sperm and testosterone synthesis-related genes（STAR,CYP11A1）in testicular tissue were measured by Real-time PCR.The content of Malondialdehyde（MDA）was determined by TBA colorimetry,and the Total antioxidant capacity（T-AOC）was determined by ABTS colorimetry.WST-1 colorimetry was used to measure the activity of Superoxide dismutase（SOD）,and DTNB colorimetry was used to measure the contents of Glutathione（GSH）and Glutathione oxidized（GSSG）.The protein expressions of Vitamin D Receptor（VDR）,Keap1,Nrf2 and their downstream molecules in the testis were determined by Western blot.（2）The effects of 1α,25（OH）₂D₃ on PFOS induced testicular Sertoli cell injury.Testicular Sertoli cells（TM4 cells）were cultured in vitro and pretreated with 1α,25（OH）₂D₃ at 10 nM and 100 nM for 6 hours,and then combined with PFOS at 100 μM and 200 μM for 24 hours.The cell viability was detected by CCK-8 assay.The content of MDA was determined by TBA colorimetry,T-AOC by ABTS colorimetry,SOD activity by WST-1 colorimetry,and GSH and GSSG contents by DTNB colorimetry.DCFH-DA fluorescent probe was used to measure the production of Reactive oxygen species（ROS）in TM4 cells.VDR gene was silenced by RNA interference in TM4 cells.The mRNA and protein expression levels of VDR,Keap1,Nrf2 and their downstream molecules in TM4 cells were determined by Realtime PCR and Western blot.（3）The regulatory mechanism of PFOS on Keap1.TM4 cells were treated with small molecule inhibitors Cycloheximide（CHX）or MG-132,and the mRNA and protein expression levels of Keap1 in TM4 cells were detected by Real-time PCR and Western blot in vitro cell model.The possible binding sites of PFOS and Keapl proteins were screened by computer simulation software Molecular Operating Environment（MOE）.Results:（1）The effects of 1α,25（OH）₂D₃ on PFOS induced reproductive toxicity in male ICR mice.①Organ coefficients:compared with CON group,the organ coefficients of the testis and epididymis in PFOS group was significantly decreased.②Sex hormone levels:PFOS exposure resulted in a decreased testosterone level,and 1α,25（OH）₂D₃ intervention resulted in an increased testosterone level,but the results were not statistically significant.③Histology of the testis:PFOS exposure resulted in thinning of the seminiferous epithelium,reduction of the diameter of seminiferous tubules,reduction of the number of layers of seminiferous tubules,sparse and irregular arrangement,and even obvious vacuolization changes.After the intervention of 1α,25（OH）₂D₃,the seminiferous tubules restored their original tubular structure and arranged tightly,and vacuolization was significantly reduced.④Sperm quality:compared with CON group,the relative concentration and survival rate of sperm in PFOS group decreased significantly,the rate of sperm malformation increased,and the expression of acrosome reaction-related gene ACR decreased.Compared with PFOS group,the relative sperm concentration and survival rate in PD group were significantly increased,the rate of sperm malformation in PD group was decreased,and the gene expression of acrosome reaction-related genes EQTN,PRSS21,SPACA1 and ZPBP in the PD group was increased.⑤Oxidative stress related indicators:1α,25（OH）₂D₃ could significantly increase serum T-AOC,PFOS exposure significantly increased the proportion of GSSG in serum,while 1α,25（OH）₂D₃ intervention reduced the proportion of GSSG.⑥ Keap1-Nrf2-ARE signaling pathway related proteins:PFOS significantly increased the expression of Nrf2 and its downstream molecules Heme oxygenase-1（HO-1）,NADPH:quinone oxidoreductase 1（NQO1）,Superoxide dismutase 2（SOD2）,1α,25（OH）₂D₃ significantly reduced the expression of Nrf2 and its downstream molecules while activating VDR.These results suggest that 1α,25（OH）₂D₃ plays a redox balance role in preventing PFOS exposure induced reproductive system damage in ICR male mice by regulating the Nrf2-ARE signaling pathway.（2）The protective effects of 1α,25（OH）₂D₃ against PFOS induced Sertoli cell injury.①Cell viability:low concentration of PFOS（5 μM,10 μM,20 μM）treatment for 24 hours could significantly enhance the viability of TM4 cells,while high concentration of PFOS（100 μM,200 μM）could significantly inhibit the viability of TM4 cells.10 nM and 100 nM 1α,25（OH）₂D₃ intervention significantly improved the PFOS induced cell viability decline.②Oxidative stress related indicators:10 nM and 100 nM 1α,25（OH）₂D₃ intervention significantly improved the PFOS-induced increase in ROS production in TM4 cells.200 μM PFOS exposure significantly increased SOD activity and MDA content in TM4 cells,while 1α,25（OH）₂D₃ intervention significantly decreased SOD activity and MDA content.Exposure to 200 μM PFOS significantly increased GSSG content and decreased GSH content in TM4 cells,and 1α,25（OH）₂D₃ intervention improved the redox imbalance caused by PFOS.Compared with SiNC group,PFOS combined with 1α,25（OH）₂D₃ treatment significantly increased SOD activity and MDA content and decreased GSH content after SiVDR transfection.③Keap1-Nrf2-ARE signaling pathway related proteins:PFOS could significantly inhibit the protein expression of Keapl in TM4 cells and significantly increase the protein expressions of Nrf2 and its downstream.1α,25（OH）₂D₃ could significantly activate the protein expression of VDR and significantly improve the PFOS induced protein overexpression of Nrf2 and its downstream.Similar results were obtained by Real-time PCR.Compared with SiNC group,PFOS combined with 1α,25（OH）₂D₃ treatment significantly increased the protein expressions of Nrf2 and its downstream SOD2 in TM4 cells after the transfection of SiVDR.The results of Real-time PCR showed that PFOS combined with 1α,25（OH）₂D₃ treatment significantly increased the expression of Nrf2,NQO1,HO-1,and GPx4 after SiVDR transfection.These results suggested that 1α,25（OH）₂D₃ pretreatment could protect testicular Sertoli cells from the oxidative damage of PFOS,and significantly alleviate PFOS induced Nrf2-ARE signaling pathway activation in a VDR-dependent manner.（3）The regulatory mechanism of PFOS on Keap1.①Results of small molecule inhibitor treatment:PFOS accelerated the decrease of Keap1 protein expression after the treatment with protein synthesis inhibitor.Administration of MG-132 reversed the PFOS induced decrease in Keapl protein.②Computer software MOE molecular simulation docking results:The docking score（-5.7464 kcal/mol）and the number of docking sites were used to output the optimal conformation.There were two docking sites,Val608 and Thr560,and PFOS was located in the docking pocket of Keapl protein.These results suggested that PFOS may directly bind to the Va1608 and Thr560 sites of keapl and accelerate the degradation of Keap1 through the proteasome pathway.Conclusion:PFOS may accelerate the degradation of Keapl protein through the proteasome pathway,thereby over activating the Nrf2-ARE signaling pathway,resulting in oxidative stress and male reproductive toxicity.1α,25（OH）₂D₃ pretreatment could attenuate PFOS-induced male reproductive toxicity by improving the activation of Nrf2-ARE signaling pathway in a VDR-dependent manner.