Objective:To discuss the inhibitory effect of diosmetin(DIO)on the ferroptosis induced by the glutathione peroxidase(GSH-Px)inhibitor(1S,3R)-RSL3(RSL3)in spermatocytes GC-2 of the mice,and to clarify the mechanism.Methods:The GC-2 cells were divided into control group,RSL3 group,RSL3+0.8 nmol·L-1 DIO group,RSL3+4.0 nmol·L-1 DIO group,RSL3+20.0 nmol·L-1 DIO group,and RSL3+ferroptosis inhibitor Ferrostain-1(Fer-1)group(200 nmol·L-1 Fer-1).The cells were treated with 0,1,5,10,50,100,500,and 1 000 nmol·L-1 RSL3 solutions,and 0,0.5,0.1,1.0,5.0,10.0,and 50.0 μmol·L-1 DIO solutions,respectively.Additionally,the GC-2 cells were divided into blank group,model group,and treatment group.The GC-2 cells in treatment group were further divided into 0.8,4.0,and 20.0 nmol·L-1 DIO groups,as well as RSL3+0.8 nmol·L-1 DIO group,RSL3+4.0 nmol·L-1 DIO group,and RSL3+20.0 nmol·L-1 DIO group.MTT method was used to detect the survival rates of the GC-2 cells in various groups.The GC-2 cells were treated with 100 nmol·L-1 RSL3 for 0,6,12,24,36,and 48 h;Western blotting method was used to detect the expression levels of ferroptosis-related proteins in the GC-2 cells in various groups;kits were used to detect the activities of superoxide dismutase(SOD),levels of malondialdehyde(MDA),and ratios of glutathione(GSH)to glutathione disulfide(GSSG)in the GC-2 cells in various groups;immunofluorescence method was used to detect the fluorescence intensities of acyl-CoA synthetase long-chain family member 4(ACSL4)protein in the GC-2 cells in various groups.Results:The MTT method results showed that compared with 0 nmol·L-1 RSL3 group,the survival rates of the GC-2 cells in 50,100,500,and 1 000 nmol·L-1 RSL3 groups were significantly decreased(P<0.01);compared with 0 μmol·L-1 DIO group,the survival rates of the GC-2 cells in 0.5,1.0,5.0,10.0,and 50.0 μmol·L-1 DIO groups were significantly decreased(P<0.01),and 100 nmol·L-1 RSL3 with DIO concentration<0.1 μmol·L-1 were selected for the subsequent experiments.Compared with blank group,the survival rates of the GC-2 cells in model group was significantly decreased(P<0.01);compared with model group,the survival rates of the GC-2 cells in RSL3+20.0 nmol·L-1 DIO group was significantly increased(P<0.01).The Western blotting results showed that compared with 0 h,the expression level of GPX4 protein in the GC-2 cells was significantly decreased after treated with RSL3 for 6 h(P<0.01),and the expression level of HO-1 protein was significantly increased after treated with RSL3 for 12 h(P<0.05);after treated with RSL3 for 12 h,the expression levels of GPX4 and FTH1 proteins were significantly decreased(P<0.05 or P<0.01);after treated with RSL3 for 24 h,the expression levels of GPX4 and HO-1 proteins were significantly decreased(P<0.05 or P<0.01);after treated with RSL3 for 36 and 48 h,the expression levels of HO-1 protein were significantly decreased(P<0.01).Therefore,100 nmol·L-1 RSL3 and for 12 h were selected as the experimental condition for the subsequent experiments.Compared with control group,the MDA level in the GC-2 cells in RSL3 group was significantly increased(P<0.01),and the SOD activity and GSH/GSSG ratio were significantly decreased(P<0.05).Compared with RSL3 group,the SOD activities in the cells in RSL3+0.8 nmol·L-1 DIO group,RSL3+4.0 nmol·L-1 DIO group,RSL3+20.0 nmol·L-1 DIO group,and RSL3+Fer-1 group were significantly increased(P<0.05 or P<0.01).The MDA levels in the cells in RSL3+20.0 nmol·L-1 DIO group and RSL3+Fer-1 group were significantly decreased(P<0.05 or P<0.01),and the GSH/GSSG ratio in the cells in RSL3+4.0 nmol·L-1 DIO group,RSL3+20.0 nmol·L-1 DIO group,and RSL3+Fer-1 group were significantly increased(P<0.05 or P<0.01).The immunofluorescence observation results showed that compared with control group,the fluorescence intensity of ACSL4 protein in the GC-2 cells in RSL3 group was significantly increased;compared with RSL3 group,the fluorescence intensities of ACSL4 protein in the cells in RSL3+0.8 nmol·L-1 DIO group,RSL3+4.0 nmol·L-1 DIO group,RSL3+20.0 nmol·L-1 DIO group,and RSL3+Fer-1 group were significantly decreased.The Western blotting results showed that compared with control group,the expression level of HO-1 protein in the cells in RSL3 group was increased(P<0.05),and the expression levels of GPX4 and FTH1 proteins were significantly decreased(P<0.05 or P<0.01);compared with RSL3 group,the expression levels of HO-1 protein in the cells in RSL3+0.8 nmol·L-1 DIO group,RSL3+4.0 nmol·L-1 DIO group,RSL3+20.0 nmol·L-1 DIO group,and RSL3+Fer-1 group were significantly decreased(P<0.05 or P<0.01),and the expression levels of GPX4 and FTH1 proteins were significantly increased(P<0.05 or P<0.01).Conclusion:DIO can alleviate the RSL3-induced ferroptosis in the GC-2 spermatocytes of the mice,and its mechanism may be related to the inhibition of HO-1 protein expression and the upregulation of expressions of GPX4 and FTH1 proteins.