Spermatogenesis Dysfunction Induced By PM_2.5 From Automobile Exhaust Via The ROS-mediated MAPK Signaling Pathway

Objective:To explore the underlying mechanisms of spermatogenic dysfunction induced by PM_2.5.5 exposure,and then cause cellular oxidative stress damage,regulate the MAPK pathway to disrupt the integrity of the blood-testis barrier,elucidate the causes and molecular mechanisms of male reproductive function damage and infertility caused by PM_2.5 exposure.Methods:Collect fine particulate matter(PM_2.5)derived from automobile exhaust,establish a SD rat model of PM_2.5.5 exposure and Sertoli cell primary culture system.The first part?vivo study?:Clean-grade 6-week old SD male rats?180-200 g?were randomly divided into the control group?0.9%saline perfusion via tracheal intubation?;the PM_2.5 exposure group(PM_2.5suspension was perfused with 20 mg/kg.bw via tracheal intubation)and the intervention group(PM_2.5 suspension was perfused with 20mg/kg.bw via tracheal intubation,and vitamin C 100mg/kg.bw and vitamin E 50mg/kg.bw via administration by gavage.Each group was treated continuously for 4weeks,once a day,stopped for 1 day a week.After PM_2.5.5 exposure,the mating test was carried out to clarify the decline of reproductive function,the testicular and epididymis tissues were taken for morphological and sperm quality detection.The expression and localization of BTB?blood-testis barrier?-related proteins?including ZO-1,Occludin,?-catenin,and Cx43?were detected by Western blot?WB?and laser confocal microscopy,the expression changes of peroxide and apoptosis-related factors and MAPK signaling pathway activity were measured as well.The second part?vitro study?:Clean-grade 20-day-old SD pups were used for primary culture of the Sertoli cells,observing cellular morphology and cells identification were performed before the experiments,CCK8 was used to select the optimum concentration and time for PM_2.5.5 exposure.Sertoli cells were randomize Divided into the control group?without any treatment?;the PM_2.5.5 exposed group(culture medium with 50ug/ml PM_2.5;the intervention group(culture medium with 50ug/ml PM_2.5,and vitamin C100 ug/ml and vitamin E 50 ug/ml were added to the medium as well);the MAPK inhibitor group(culture medium with 50ug/ml PM_2.5,and the especial inhibitors of p38,JNK,ERK were added to the medium respectively)for 24 hours.The changes of oxidative stress and apoptosis index in Sertoli cells were detected.The BTB-related proteins?including ZO-1,Occludin,N-cadherin,?-catenin?and cytoskeleton of Sertoli cells were detected by WB and laser confocal microscopy.The changes of expression of MAPK signaling pathway and downstream signaling factors in Sertoli cells were observed after inhibitors adding.Results:Part ?:The abnormal morphology of testicular tubules,decreased sperm count,increased sperm deformity rate,and obvious male reproductive dysfunction were observed after PM_2.5.5 exposure.Significant decrease in the expression of BTB-related proteins?such as ZO-1,Occludin,?-catenin,and Cx43?were detected,resulting disrupting the integrity of the blood-testis barrier and affecting the normal production of sperm.After PM_2.5.5 exposure,the ROS-MAPK-Nrf2 signaling pathway is activated,and cellular apoptosis is increased,resulting in reproductive dysfunction,however,in the vitamins intervention group,the number and quality of sperm were significantly improved,the expression of BTB-related proteins were increased,the oxidative stress response was significantly reduced,the MAPK signaling pathway was inhibited,and apoptosis was significantly reduced.Part ?:Obvious oxidative stress and increased cellular apoptosis were triggered after PM_2.5 exposure in Sertoli cells,the expression of BTB-related proteins?such as ZO-1,Occludin,N-cadherin,?-catenin?were significantly decreased with the cytoskeletal protein F-actin was abnormally disordered after exposure.PM_2.5 exposure significantly activated MAPK signaling pathway.The inhibitors of P38,JNK and ERK blocked MAPK pathway and increased Nrf-2 expression in different degrees.Vitamins intervention alleviated the oxidative stress of Sertoli cells induced by PM_2.5.5 exposure,increased the expression of BTB-related proteins,and partially restored the order of cytoskeletal protein.Conclusion:PM_2.5 exposure originated from automobile exhaust causes male reproductive dysfunction obviously,the toxic effects were associated with causing oxidative stress in Sertoli cells,activating ROS-MAPK-Nrf-2 pathway,leading increased cellular apoptosis,thus destroying integrity of blood-testis barrier,which is critical for the normal production of sperm.Combination of vitamin C and vitamin E alleviates the oxidative stress response of Sertoli cells induced by PM_2.5 exposure,inhibits the MAPK pathway,restores the antioxidant capacity of Sertoli cells,remodels the blood-testis barrier,and restores male spermatogenesis.