Research On Toxicity And Apoptotic Mechanism Of Lead Acetate In Mice Leydig Cells In Vitro

As environmental lead pollution and occupational lead exposure are becoming more serious, reproductive toxicity of lead in humans and animals is given much attention, but the mechanism of reproductive toxicity of lead is not very clear. Leydig cells as the stent of spermatogenic cells can provide the necessary nutrients, synthesize and secrete androgen binding protein which provide high concentrations of fine androgen environment for spermatogenic cells and is a very important body of male germ cells which have a certain hagocytic function. Therefore, this study, choosing mouse Leydig cells as the research object, discussed the injury and mechanisms of lead to male germ cells. For providing experimental and theoretical basis by further research and development of new lead antidote, this study will provide a new starting point for the prevention and treatment of livestock caused by a heavy metal reproductive dysfunction.Objective:The aim of the study was to explore the mechanism of apoptotic death receptor pathway through researching the effects of the alkali type lead acetate (C4H8O6Pb2) on the apoptosis of mice leydig cells in vitro.Method:Culture mice leydig cells in vitro. After subcultured by using pancreatic enzyme digestion, the cell was divided into five groups when the cell density is80%. Add(0,2,10,50μmol/L) of lead acetate respectively to infect mice leydig cells. After the intervention of24hours,MTT method was used to detect cell proliferation.SOD activity, MDA content and GSH-Px activity were measured by NBT chromogenic assay, TBA assay and indirect method respectively. ABTS assay was used to detect total antioxidant capacity. Hoechest33258and Annexin-FITC/PI staining assay were used to detect apoposis. Western blotting analysis of Caspase-8, FAS, FAS1expression, and caspase-8blocker inhibitory effect of lead acetate leads to apoptosis.Result:leydig cells grew well at36℃,5%CO2and the logarithmic growth phase of leydig cells was between12h and36h Lead acetate (C4H8O6Pb2) inhibits proliferation of mice leydig cells.2μM、10μM、50μM lead acetate caused oxidative damage through significantly decreased antioxidant enzymes SOD, GSH-Px activity and total antioxidant capacity(T-AOC) and increased MDA content. Studies have shown that lead acetate can induce cell death, the highest rate of cell apoptosis is up to33.87%and the cell apoptosis of TM3cells death receptor pathway is via FAS/FAS-L pathway, mainly in:a significant increase of FAS,FAS-L,Caspase-8protein content compared with control group,and Caspase-8inhibitors (C21H34N4O10) significantly inhibited lead acetate(C4H8O6Pb2) induced apoptosis in mice leydig cells. Conclusion:Lead acetate (C4H8O6Pb2) inhibited proliferation of mice leydig cells,caused cell oxidative damage and induced apoptosis via the FAS-mediated death receptor pathway.