The Role Of ROS/NLRP3 Inflammasome In Testicular Injury Induced By Molybdenum And Cadmium In Sheep

To investigate the effects of testis toxicity and ROS/NLRP3 inflammasome activation under Mo and Cd co-exposure in sheep.（NH₄）₆Mo₇O₂₄·4H₂O and Cd Cl₂ are selected as the experimental Mo and Cd source respectively.Two-month-old male sheep and primary Sertoli cells are used as models.24 male sheep are divided into control group,Mo group（45mg/kg·BW Mo）,Cd group（1 mg/kg·BW Cd）and Mo+Cd group（45 mg/kg·BW Mo+1mg/kg·BW Cd）.All groups are administered for 50 days.The sheep primary Sertoli cell culture model is established by two-step enzymatic digestion and treated with Mo（500μM）and Cd（4μM）for 24 h in vitro.After Mo and/or Cd exposure,collection and analysis of basic testicular data,H&E staining,TEM analysis investigate the morphological changes,IF analysis is used to observe the expression of junction related proteins and NLRP3,antioxidant kit is used to detect oxidative stress indicators,RNA-Seq and western blotting detect junction related and NLRP3 inflammasome pathway-related gene and protein levels.The levels of IL-18 and IL-1βin the supernatant are detected by ELISA kit and the intracellular ROS level is observed by fluorescence microscope in vitro,and ROS scavenge and NLRP3 inflammasome inhibitor are added to investigate the molecular mechanism of BTB dynamics and inflammatory response in vitro.Results show that Mo and/or Cd exposure evoke structural destruction in sheep testis,inhibit the m RNA and protein expression of CX-43,OCLN,ZO-1（P<0.05）,decrease the activities of T-SOD and CAT（P<0.05）,increase the content of MDA（P<0.05）and the ROS level（P<0.05）and activate NLRP3 inflammasome（P<0.05）and eventually increase the release of inflammatory factors lead to inflammatory injury.In Mo and Cd-induced BTB impairment models of SCs,treatment with the ROS scavenger BHA or NLRP3 inflammasome inhibitors MCC950normalize BTB integrity,inhibit ROS production and inflammatory reaction（P<0.05）.Collectively,the combination of Mo and Cd promotes ROS production and inflammatory reaction,damages the integrity of the BTB,leads to testicular injury and male reproductive toxicity via activating ROS/NLRP3 inflammasome pathway.