Abstract:

Objective To discuss the effect of azathioprine（AZA） on ferroptosis in spermatocytes of the mice induced by reduced glutathione（GSH） peroxidase 4 inhibitor RSL3，and to clarify the potential mechanism. Methods The spermatogonia GC-2 cells of the mice were randomly divided into control group （no treatment）， RSL3 group （treated with 10 nmol·L^-1 RSL3 for 24 h）， RSL3+ferroptosis inhibitor （Ferrostatin-1， Fer-1） group （treated with 10 nmol·L^-1 RSL3 for 24 h+2 μmol·L^-1 Fer-1 for 12 h）， RSL3+low dose of AZA group （treated with 10 nmol·L^-1 RSL3 for 24 h +5 μmol·L^-1 AZA for 12 h）， RSL3+medium dose of AZA group （treated with 10 nmol·L^-1 RSL3 for 24 h+10 μmol·L?1 AZA for 12 h）， and RSL3+high dose of AZA group （treated with 10 nmol·L^-1 RSL3 for 24 h+20 μmol·L^-1 AZA for 12 h）. The MTT method was used to detect the activities of the GC-2 cells in various groups after treated with different concentrations of AZA and RSL3； the GSH and GSSG levels in the GC-2 cells were detected by GSH and oxidized glutathione（GSSG） detection kits； the malondialdehyde（MDA） levels in the GC-2 cells in various groups were detected by MDA detection kit； Western blotting method was used to detect the expression levels of long-chain acyl-CoA synthetase 4 （ACSL4）， heme oxygenase-1（HO-1）， and glutathione peroxidase 4（GPX4） proteins in the cells in various groups； immunofluorescence staining was used to detect the expressions of ACSL4 protein in the cells in various groups. Results Compared with control group， the differences in activities of the GC-2 cells in 5， 10， and 20 μmol·L?1 AZA groups had no significant differences（P>0.05）， while the activities of the GC-2 cells in 30 and 40 μmol·L?1 AZA groups were significantly decreased（P<0.01）； therefore， the AZA concentration was selected to be within 20 μmol·L?1. Compared with control group， the differences of the activities of the GC-2 cells in 1， 5， and 10 nmol·L?1 RSL3 groups had no significant differences（P>0.05）， while the activities of the GC-2 cells in 50， 100， 500， and 1 000 nmol·L?1 RSL3 groups were significantly decreased（P<0.05 or P<0.01）； therefore， the RSL3 concentration was set to be within 10 nmol·L?1. The GSH and MDA detection kits results showed that compared with control group， the levels of GSSG and MDA in the GC-2 cells in RSL3 group were significantly increased（P<0.05）， while the GSH levels were significantly decreased（P<0.05）； compared with RSL3 group， the levels of GSSG and MDA in the GC-2 cells in RSL3+Fer-1 group and RSL3+AZA group were significantly decreased （P<0.01）， while the GSH levels were significantly increased（P<0.01）. The Western blotting results showed that compared with control group， the expression levels of ACSL4 and HO-1 proteins in the GC-2 cells in RSL3 group were significantly increased（P<0.05）， while the expression level of GPX4 protein was significantly decreased（P<0.01）； compared with RSL3 group， the expression levels of GPX4 protein in the GC-2 cells in RSL3+Fer-1 group and RSL3+AZA group were significantly increased（P<0.05）， while the expression levels of ACSL4 and HO-1 proteins were significantly decreased（P<0.01）. The immunofluorescence staining results showed that compared with control group， the expression amount of ACSL4 protein in the GC-2 cells in RSL3 group was significantly increased， and compared with RSL3 group， the expression amounts of ACSL4 protein in the cells in RSL3+Fer-1 group and RSL3+AZA group were significantly decreased. Conclusion AZA can alleviate the ferroptosis-induced by RSL3 in spermatocytes of the mice.

Key words: Azathioprine, Ferroptosis, Spermatocyte cells, Glutathione peroxidase 4 inhibitor, Acyl CoA synthetase long-chain family member 4