Nrf2 Signaling Pathway Mediates the Protective Effects of Daphnetin Against D-Galactose Induced-Premature Ovarian Failure

Abstract

Oxidative damage can lead to severe ovarian dysfunctions and even premature ovarian failure. Nrf2, a significant transcription factor that regulates the oxidative stress response of cells, declines with age. Daphnetin, as a kind of natural Chinese herbal medicine, can activate Nrf2 and further promote the antioxidant defense of cells. However, whether Daphnetin treatment can protect ovary from premature ovarian failure and the specific mechanism involved are not understood. This study aimed to investigate the protective function of Daphnetin against the ovarian aging induced by D-galactose in wild-type and Nrf2^-/- mice. Female C57BL/6 mice with Wild-type and Nrf2^-/- were divided into five groups separately and the premature ovarian failure model were established by D-galactose and then Daphnetin and VE were given for treatment. After 42 days, ovaries tissue and serum were collected for biochemical determination, H&E staining, Immunohistochemical staining and western blot analysis. In the WT-POF group, ovarian function was broke, and the expression of the ovarian senescence-associated protein P16 and the level of oxidative stress were significantly increased, while the expression of the anti-senescence protein klotho was significantly decreased. In addition, the expression of Nrf2 and the antioxidases GCLC, HO-1 and NQO1 were decreased, but TXNIP and NLRP3 were significantly increased. Furthermore, the characteristics of premature ovarian failure were more significant in Nrf2 knockout mice than in wild-type mice, especially the expression of NLRP3 and TXNIP. Moreover, daphnetin, an Nrf2 activator, rescued d-gal-induced POF in a dose-dependent manner, while the protective effect was weakened or even lost in Nrf2 knockout mice. Our results suggested that daphnetin is likely to be a candidate drug for premature ovarian failure treatment and it is mostly possible referred to the molecular mechanism of increasing Nrf2 expression and inhibiting NLRP3 activation in the ovarian aging process.

Keywords: NLRP3; Nrf2; Txnip; daphentin; premature ovarian failure.

FIGURE 1

Establish a mouse model of POF. (A) The body weight changes of mice in control and D-gal group (low, middle and high dose) were recorded during the process of inducing the POF model. (B,C) The serum FSH and E2 levels were tested in four groups. (D,E) The levels of GSH and SOD in the ovarian tissues were tested. (F,G) The proportion of primordial follicle. All data are shown as means ± SEMs, n = 5. Statistical significance: *p < 0.05, **p < 0.01.

FIGURE 2

Nrf2 knockout significantly damaged the ovarian function and antioxidant capacity of POF mice. (A) Immunoblotting analysis of Nrf2 protein. (B) Using vaginal cytology evaluating the oestrous cycle and days spent in pro-oestrus (P), oestrus (E), metoestrus (M), and dioestrus (D) over the course of 12 consecutive days. (C,D) The serum FSH and E2 levels were tested. (E,F) The levels of GSH and SOD in ovarian tissues. (G,H) NLRP3 protein expression was analyzed after immunohistochemistry. (I,J) The level of P16 and klotho were analyzed. The data in B-H are shown as means ± SEMs, n = 5. The data in A, I-J are shown as means ± SEMs, n = 3. Statistical significance: **p < 0.01, ^## p < 0.01.

FIGURE 3

Protective effects of Daph on the ovarian function and follicular development. (A) The structure of Daph monomer and immunoblotting analysis of Nrf2 protein expression after Daph was administrated. (B) Construct mice model according to the method as shown. (C,D) Serum FSH and E2 were tested in the control, D-gal, Daph and VE groups. (E,F) Follicles in five groups were observed after H&E staining and the numbers of follicles at different stages of maturation were summarized. The data in C-F are shown as means ± SEMs, n = 5. The data in A are shown as means ± SEMs, n = 3. Statistical significance: *p < 0.05, **p < 0.01, ^# p < 0.05, ^## p < 0.01.

FIGURE 4

Effects of Daph on p16 and klotho expression. (A,C) The IHC staining showed cellular locations of p16 and klotho proteins in different groups. (B,D) The p16 and klotho expressions were quantitatively analyzed. (E,F) Expressions of p16 and klotho were verified by western blot. The data in (A–D) are shown as means ± SEMs, n = 5. The data in (E–F) are shown as means ± SEMs, n = 3. Statistical significance: *p < 0.05, **p < 0.01, ^## p < 0.01.

FIGURE 5

Effect of Daph on oxidative stress-related pathways in the ovary. (A,B) The total SOD enzyme activity and the GSH level were measured in ovarian tissues. (C) The levels of Nrf2, GCLC, NQO1 and HO-1 protein expression in ovarian tissues were observed via Western blotting. (D) The protein expression levels were quantitatively analyzed. (E,F) TXNIP and NLRP3 protein expression in the five groups using IHC staining. All data are shown as means ± SEMs, n = 5, besides (C,D) which data are shown as means ± SEMs, n = 3. Statistical significance: *p < 0.05, **p < 0.01, ^# p < 0.05, ^## p < 0.01.

FIGURE 6

Knockout of Nrf2 abolished the protective capability of Daph for ovarian function and follicular development. (A,B) The serum FSH and E2 levels were tested in the five groups in Nrf2^−/− mice. (C,D) GSH and SOD expression in ovarian tissues was tested. (E) Follicles were observed after H&E staining. (F) Summarizing the numbers of follicles at different developmental stages. All data are shown as means ± SEMs, n = 5. Statistical significance: **p < 0.01.

FIGURE 7

Knockout of Nrf2 abolished the regulatory capability of Daph for ovarian senescence-associated protein expression. (A–D) The IHC staining showed the p16 and klotho expressions in the five groups in Nrf2^−/− mice. (E,F) Expressions of p16 and klotho were measured through western blot. The data in (A–D) are shown as means ± SEMs, n = 5. The data in (E,F) are shown as means ± SEMs, n = 3. Statistical significance: **p < 0.01.

FIGURE 8

Knockout of Nrf2 abolished the adjustive power of Daph on oxidative stress-related pathways. (A,B) The Nrf2, NQO1 and HO-1 protein expression levels in ovarian tissues were tested by Western blotting. (C,D) TXNIP and NLPR3 protein expression level analysis via immunohistochemistry. The data in (A,B) are shown as means ± SEMs, n = 3. The data in (C,D) are shown as means ± SEMs, n = 5. Statistical significance: **p < 0.01.

FIGURE 9

Scheme summarizing the protective effects of Nrf2 activation on D-gal-induced POF via inhibiting TXNIP/NLRP3 inflammasome activation. Under D-gal condition, Nrf2 downregulation increases the accumulation of ROS, and then increase TXNIP/NLRP3, p16 and FSH and decrease klotho, SOD, E2 and GSH, leading to POF. Furthermore, Daph inhibits the accumulation of ROS and the activation of TXNIP/NLRP3 inflammsome via activating Nrf2, has a protective effect on D-gal-induced premature ovarian failure.