Abstract:

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.

Key words: Ferroptosis, Mouse spermatocyte GC-2, Iprodione, Glutathione peroxidase 4, Ferritin heavy chain 1, Heme oxygenase-1