Effect of fluoride at different concentrations on proliferation and apoptosis of mouse Leydig cells in vitro

Objective To explore the effect of fluoride at different concentrations on proliferation and apoptosis of mouse Leydig cells in vitro.Methods Mouse Leydig cells were collected by trypsinization to form monoplast suspension,centrifuged to float again,and then cultured in vitro.The cell numbers were counted,the cell suspension density adjusted,and the cells were inoculated into 96-shadow mask according to 1×106 per milliliter.All cells were cultured routinely.When the cell fusion ratio reached 80% and the cells did not start to differentiate,the cells were divided into 4 groups,cultured in DMEM-F12 medium containing 0,5,10,20 mg/ L fluoride,respectively.After each group was treated for 0,24,48,72,96,and 120 h,MTT test was used to detect the cell proliferation in each group.Flow cytometry(FCM) with annexin V-PI double staining was applied to detect the apoptosis in Leydig cells of each group after treatment for 48 h.Results 1) After the cells were treated for 24 h and 48 h,fluoride(20 mg/L) significantly inhibited proliferation of the Leydig cells,but 5 mg/L and10 mg/L had a less effect on the Leydig cells.After the cells were treated for 72 h,fluoride(10 mg/L) significantly inhibited proliferation of the Leydig cells.After the cells were treated for 96 h and 120 h,fluoride(5 mg/L,10 mg/L,20 mg/L) significantly inhibited Leydig cell proliferation.2) When the cells were treated by fluoride for 48 h,apoptosis was induced in the Leydig cells of all groups.The Leydig cells showed a highest late apoptosis rate at 10 mg/L fluoride,while the highest early apoptosis rate in Leydig cells was after 20 mg/L fluoride treatment.Conclusion Fluoride can effiectively inhibit proliferation and promote apoptosis of Leydig cells in mice.