Effects Of Spermidine On Autophagy And Antioxidant Function Of Ovary And Granulosa Cells In Mice

Spermidine(SPD),as one of the natural polyamines,has the ability to combine with negatively charged biological macromolecules such as nucleic acids,proteins and biofilms to prevent oxidative stress and eliminate reactive oxygen species.So far,the research on antioxidant function of spermidine is mainly concentrated in neural tissue,but the mechanism of spermidine’s involvement in regulating ovarian antioxidant function and the further influence on its reproductive and production performance is relatively unclear..Exogenous supplementation of spermidine can activate autophagy to resist oxidative stress and delay aging,and many anti-aging properties of spermidine have causal relationship with its role in ensuring protein stability by stimulating protective autophagy of cells.In conclusion,we speculate that spermidine can enhance the antioxidant function of the body by affecting the autophagy level of tissues and cells.Therefore,in this study,the ovarian tissue and granulosa cells of ICR mice were treated with spermidine,and the effects of spermidine on autophagy,anti-oxidative stress of ovaries and granulosa cells were studied.The protective effects of spermidine on mice ovaries and granulosa cells were investigated from in vivo and in vitro.The research on the mechanism of chemical stress lays a foundation for the mechanism of spermidine in anti-oxidative stress of ovary and granulosa cells by inducing autophagy.The main results are as follows:(1)After feeding female ICR mice with spermidine for 3 months,there was no significant difference in daily gain and ovarian index between the two groups(p > 0.05).The number of atresia follicles and corpus luteum in spermidine treated mice were significantly lower than those in control group(p < 0.05).(2)Spermidine can participate in the regulation of ovarian polyamine homeostasis in female ICR mice by mediating the expression of key genes related to polyamine metabolism;spermine content in ovaries of spermidine treated mice was significantly higher than that of control group(p < 0.05),while putrescine and spermidine content had no significant change(p > 0.05);the relative expression of APAO gene in ovaries of spermidine treated mice was significantly decreased(p < 0.05);the relative expression of SPDS,SPMS and SMO genes was significantly lower than that of control group(p < 0.01);There was no significant difference in the relative expression of SSAT gene between the two groups(p > 0.05).(3)Spermidine can significantly increase the antioxidant capacity of mouse ovarian and follicular granulosa cells and reduce ovarian lipid peroxidation;the total antioxidant capacity of ovaries in spermidine-treated mice was significantly increased(p < 0.05),the activities of SOD and CAT were significantly higher than those in control group(p < 0.05);the level of MDA was significantly lower than that in control group(p < 0.05);the activity of GSH-px had no significant change(p > 0.05).The total antioxidant capacity and antioxidant enzymes activity of granulosa cells treated with Spermidine at different concentrations did not change significantly after 24 hours(p > 0.05);after 48 hours,the activities of T-AOC,CAT and SOD in spermidine treatment group increased significantly(p < 0.05);after 72 hours,the activities of T-AOC,CAT and SOD in granulosa cells treated with Spermidine at 0.1 and 1.0 μmol/L continued to increase,and were significantly higher than those in control group.(p < 0.01);CAT activity increased significantly in 10 μmol/L treatment group(p < 0.05),but had no significant effect on total antioxidant capacity and SOD activity of granulosa cells(p > 0.05).(4)Spermidine induces autophagy in ovarian and follicular granulosa cells of ICR mice;the expression of Beclin 1 and LC3 II/I protein in the ovaries of spermidine treated mice increased significantly(p < 0.05),while the expression of p62 protein decreased significantly(p < 0.05)compared with the control group,After 48 hours of spermidine treatment,the expression of Beclin 1 and LC3 II/I protein in spermidine treatment group was significantly higher than that in control group(p < 0.05),and the expression of p62 protein decreased significantly;the expression of Beclin 1 and LC3 II/I protein in spermidine treatment group was significantly higher than that in control group(p < 0.05).the p62 protein in 0.1 μmol/L spermidine treatment group decreased significantly(p <0.05).After 72 hours of treatment,0.1,1.0 and 10.0 μmol/L spermidine increased the expression of Beclin 1 and LC3 II/I protein(p < 0.05)and decreased the expression of p62protein(p < 0.05).(5)Prolonged exposure of spermidine at low concentation can significantly increase the activity of follicular granule cells and promote granulosa cell proliferation;after 24 hours treatment with different spermidines,the activity of granulosa cells did not change significantly(p > 0.05).1 μmol/L spermidine could significantly promote the proliferation of granulosa cells(p < 0.05).After 48 hours of treatment,spermidine at lower concentration(0.1 μmol/L)for a long time could also significantly increase cell proliferation and cell viability(p < 0.05).After 72 hours of treatment,0.1 μmol/L spermidine promoted granulosa cell proliferation and significantly increased cell viability(p < 0.05),and 10.0 μmol/L spermidine significantly decreased granulosa cell viability(p <0.05).The results showed that spermidine could regulate the polyamine metabolism in ovaries of female mice by mediating the expression of key genes related to polyamine metabolism,induce autophagy of ovarian and follicular granulosa cells in mice,improve cell antioxidant capacity,reduce follicular atresia and promote the proliferation of follicular granulosa cells.