| Objective: To explore the role of Nrf2/ARE-related pathways in the regulation of premature ovarian failure by constructing a mouse model of aging.Methods: 1.Observe the effects of different concentrations of D-gal on the ovaries of mice: 6-8 weeks old C57 BL female mice were randomly divided into three groups: a.D-gal group(low-dose): subcutaneous injection of D-gal 200mg/kg/d for 42 days;b.D-gal group(high-dose): subcutaneous injection of D-gal 600mg/kg/d for 42 days;c.control group: subcutaneous injection of saline in the same amount as the first two groups for 42 days.And observe mental state,food intake and body weight;serum follicle-stimulating hormone(FSH)and estradiol(E2)levels measurement;the proportion of primordial follicles and the Nrf2 expression of ovarian tissues in each group.2.Observe the ovarian status of mice in the control group and Nrf2-/-group: Next,we observe the serum FSH and E2 levels,superoxide dismutase(SOD)and glutathione(GSH)levels in ovarian tissues,the expression of Nrf2,senescence-associated protein P16 and anti-senescence protein klotho in ovarian tissues in the control group and Nrf2-/-group.3.Construction of wildtype and Nrf2-/-mouse aging model: 6-8 weeks old C57 BL and Nrf2-/-female mice were randomly divided into 3 groups: a.D-gal group: subcutaneous injection of D-galactose 600mg/kg/d for 42 days;b.Nrf2-/-group: subcutaneous injection of D-galactose 600mg/kg/d for 42 days;c.Control group: subcutaneous injection of saline in the same amount as the first two groups for 42 days.And measure the following indicators in each group of mice: the levels of serum FSH and E2;the levels of SOD and GSH,the expression of senescence-associated protein P16 and anti-senescence protein klotho measured by western blot and immunohistochemistry,the number of follicles at different developmental stages.4.The expression of Nrf2 and its downstream antioxidant enzymes: Western blotting was used to detect the expression changes of Nrf2 and its downstream antioxidant enzyme NQO1 and HO-1 in the ovarian tissues of mice.Results: 1.In the d-gal group(high dose),mice gradually showed reduced activity,reduced food intake,poor mental status and slow weight gain;this phenomenon was not observed in the control and d-gal group(low dose).In the d-gal group(low dose),the difference of serum FSH and E2 was not statistically significant compared with the control group(P>0.05);while FSH levels were significantly increased and E2 levels were significantly decreased in the d-gal group(high dose)compared with the control group,with statistically significant differences(P<0.01).In addition,the proportion of primordial follicles was significantly lower in the d-gal group(high dose)compared with the control group,and the difference was statistically significant(P<0.01).In addition,the expression of Nrf2 in the ovaries of the D-gal group(high dose)was significantly reduced,indicating that excessive ROS stimulation might contribute to the exhaustion of Nrf2.Hence,we selected 600 mg/kg/day of d-gal as the appropriate concentration to establish a mouse model of POF.2.Comparison between control group and Nrf2-/-group: FSH levels were significantly higher and E2 levels were significantly lower in the Nrf2-/-group compared with the control group,with statistically significant differences(P<0.05 or P<0.01);In addition,GSH and SOD levels were significantly lower in the Nrf2-/-group compared with the control group,indicating that ovarian antioxidant levels were damaged,the differences were statistically significant(P<0.01);Compared with the control group,the level of senescence-associated protein P16 was significantly increased and the expression of anti-senescence protein klotho was significantly decreased in the Nrf2-/-group,and the differences were statistically significant(P<0.01).The results above suggested that Nrf2 knockout significantly damaged the HPG axis and antioxidant capacity.3.D-gal POF group compared with the Nrf2-/-POF group: in the Nrf2-/-POF group,FSH levels were significantly increased(P<0.01)and E2 levels were significantly decreased(P<0.05)compared to the D-gal POF(high dose)group;GSH and SOD levels were significantly lower in the Nrf2-/-POF group compared with D-gal POF group,the differences were statistically significant(P<0.01);P16 level was significantly increased and Klotho expression was significantly decreased in the Nrf2-/-POF group compared with D-gal POF group,the differences were statistically significant(P<0.01);The follicles counting at different stages showed that the proportion of primordial follicles,primary follicles,and antral follicles was reduced,while the proportion of atretic follicles was significantly increased in the Nrf2-/-POF group compared with D-gal POF group;the differences were statistically significant(P<0.05 or P<0.01).These results suggest that Nrf2 knockdown contributed to a more significant state of POF induced by d-gal.4.Western Blot further examined the expression levels of Nrf2 and its downstream antioxidant enzymes(NQO1,HO-1).Compared with the d-gal POF group,the expression of Nrf2 and its downstream genes were significantly reduced in the Nrf2-/-POF group,and the differences were all statistically significant(P < 0.01).In conclusion,Nrf2 plays a critical role in the regulation of POF.Conclusion: 1.D-gal can cause premature ovarian failure in mice.2.The senescence situation of ovaries are even worse in Nrf2 knockout mice.3.Nrf2/ARE-related pathways may play a critical role in the regulation of premature ovarian failure in mice. |
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