Protective Effection And Mechanism Of Tea Polyphenols On Tri-Ortho-cresyl Phosphate-induced Mouse Ovarian Granulosa Cells Injury

Objectives:Tri-ortho-cresyl phosphate(TOCP)is widely used in industry and daily life,TOCP can cause damage to female reproductive system,but its mechanism is not clear.This study intends to investigate the potential mechanism of TOCP-induced damage of ovary and ovarian granulosa cells in mice and determine the role and the mechanism of TP in TOCP-induced damage of mouse ovary and ovarian granulosa cells.Methods:Western blot was utilized to investigate the effect of TOCP and TP pretreatment on autophagy in mouse ovarian tissue and ovarian granulosa cells.The reactive oxygen species test kits were used to detect whether TOCP induces oxidative stress of mouse ovarian tissue and ovarian granulosa cells,and whether TP inhibited TOCP-induced oxidative stress of mouse ovarian tissue and ovarian granulosa cells.MTT assay was utilized to observe the effect of TOCP and TP pretreatment on viability of mouse granulosa cells.Transmission electron microscopy(TEM)were used to detect the impact of TOCP and TP pretreatment on autophagy of mouse granulosa cells.Results:Female Kunming mice were intragastrically(i.g.)administered with 0,100,200 or 400 mg/kg/day TOCP for 14 days.Compared with the control group,TOCP could increase the ratio of autophagy protein LC3-II/LC3-I and the protein levels of Beclin1 and Atg5 in mouse ovarian tissue,which indicated that TOCP can induce autophagy of mouse ovarian tissue.Meanwhile,TOCP could dramatically increase the MDA level,whereas the content of GSH and the activities of antioxidant enzymes SOD and GSH-PX were markedly decreased in the TOCP-treated mice.These results indicated that TOCP could induce oxidative stress in mouse ovarian tissue.However,TP pretreatment could inhibit the induction of oxidative stress and autophagy by TOCP in mouse ovarian tissue,These results suggest that TP could inhibit mouse ovarian tissue autophagy via inhibiting oxidative stress.Primary mouse ovarian granulosa cells were treated with 0,0.125,0.25 or 0.5 mM TOCP for 48 h.MTT assay showed that TOCP significantly inhibited the viability of mouse ovarian granulosa cells in a dose-dependent manner(P<0.05).Meanwhile,TOCP significantly increased the ratio of LC3-II/LC3-I and the expression of Beclin1 and Atg5 in the cells.These results indicated TOCP could induce autophagy of mouse ovarian granulosa cells.TOCP could dramatically increase the MDA level,whereas the content of GSH and the activities of SOD and GSH-PX were markedly decreased in the TOCP-treated cells,implying that TOCP could induce oxidative stress in mouse ovarian granulosa cells.However,TP pretreatment could rescue the inhibition of viability by TOCP(P<0.05).TP could inhibit the induction of oxidative stress.Western blot showed that TP could also decrease the ratio of LC3-II/LC3-I and the expression of Beclin1 and Atg5 in the TOCP-treated cells.Compared with the control group,a large number of autophagic vacuoles were found in the TOCP-treated mouse ovarian granulosa cells,while TP could significantly decrease the number of autophagic vacuole in the cells.These results suggest that TP could inhibit autophagy of mouse ovarian granulosa cells via inhibiting oxidative stress.Conclusion:These results showed TOCP could induce autophagy and cause damage of ovarian tissue and granulosa cells of mice via oxidative stress,TP might play a protective role in the ovarian tissue and granulosa cells by inhibiting TOCP-induced autophagy via oxidative stress.