异菌脲对小鼠精母细胞GC-2铁死亡的影响OA

Objective:To discuss the effect of iprodione(Ipr)on ferroptosis in mouse spermatocyte GC-2 cells,and to clarify its possible mechanism.Methods:The MTT method was used to detect the viability of GC-2 cells after treatment with 0,0.001,0.010,0.100,1.000,10.000,and 100.000 μmol·L-1 Ipr for 24 h.Additional GC-2 cells were taken and divided into blank control group,1.0 μmol·L-1 Ipr group,2.5 μmol·L-1 Ipr group,and 5.0 μmol·L-1 Ipr group(treated with Ipr solutions at final concentrations of 0,1.0,2.5,and 5.0 μmol·L-1,respectively,for 24 h).Fluorescence microscope was used to observe the intracellular reactive oxygen species(ROS)generation and mitochondrial membrane potential changes;Kit methods were used to detect the superoxide dismutase(SOD)activity,malondialdehyde(MDA)level,and the reduced glutathione(GSH)/oxidized glutathione(GSSG)ratio in the GC-2 cells in various groups;Western blotting method was used to detect the expression levels of ferroptosis-related proteins in the GC-2 cells in various groups after Ipr exposure;immunofluorescence method was used to detect the fluorescence intensity of heme oxygenase-1(HO-1)protein in the GC-2 cells in various groups.Results:The MTT assay results showed that compared with 0 μmol·L-1 Ipr group,the viabilities of the GC-2 cells in 1.000,10.000,and 100.000 μmol·L-1 Ipr groups were significantly decreased(P＜0.01);thus,1.0,2.5,and 5.0 μmol·L-1 Ipr were selected for subsequent experiments on GC-2 cells.The fluorescence analysis results showed that compared with blank control group,the ROS generation in the GC-2 cells in 2.5 and 5.0 μmol·L-1 Ipr groups was increased and the mitochondrial membrane potentials were decreased;there were no significant changes in the MDA level,SOD activity,and GSH/GSSG ratio in the GC-2 cells in 1.0 μmol·L-1 Ipr group(P＞0.05);compared with blank control group,the MDA levels in the GC-2 cells in 2.5 and 5.0 μmol·L-1 Ipr groups were significantly increased(P＜0.05 or P＜0.01),and the SOD activities and GSH/GSSG ratios were significantly decreased(P＜0.01).The Western blotting results showed that compared with blank control group,after 24 h of treatment,the expression levels of glutathione peroxidase 4(GPX4)and ferritin heavy chain 1(FTH1)proteins in the GC-2 cells in 1.0 μmol·L-1 Ipr group showed no significant changes(P＞0.05);compared with blank control group,after 24 h of treatment,the expression levels of GPX4 and FTH1 proteins in the GC-2 cells in 2.5 and 5.0 μmol·L-1 Ipr groups were significantly decreased(P＜0.05 or P＜0.01);compared with blank control group,after 24 h of treatment,the expression levels of HO-1 protein in the GC-2 cells in 1.0,2.5,and 5.0 μmol·L-1 Ipr groups were significantly increased(P＜0.01).The immunofluorescence results showed that compared with blank control group,the fluorescence intensities of HO-1 protein in the GC-2 cells in 1.0,2.5,and 5.0 μmol·L-1 Ipr groups were significantly enhanced.Conclusion:Ipr induces ferroptosis in mouse spermatocyte GC-2 cells,and its mechanism may be related to the up-regulation of HO-1 protein expression and the down-regulation of GPX4 and FTH1 protein expressions.