Quercetin ameliorates oxidative stress-induced apoptosis of granulosa cells in dairy cow follicular cysts by activating autophagy via the SIRT1/ROS/AMPK signaling pathway

Abstract

Background: Follicular cysts contribute significantly to reproductive loss in high-yield dairy cows. This results from the death of follicular granulosa cells (GCs) caused by oxidative stress. Quercetin is known to have significant antioxidant and anti-apoptotic effects. However, the effect of quercetin on follicular cysts has yet been elucidated. Therefore, this study aimed to explore the anti-oxidant and anti-apoptosis effects and potential molecular mechanisms of quercetin in H₂O₂-induced primary cow GCs and 3-nitropropionic acid (3-NPA)-induced mouse model of oxidative stress and thus treat ovarian cysts in dairy cows.

Results: In this study, compared with estrus cows, cows with follicular cysts showed heightened levels of oxidative stress and increased follicular cell apoptosis, while autophagy levels were reduced. A model of oxidative stress was induced in vitro by H₂O₂ and showed significant increases in apoptosis together with reduced autophagy. These effects were significantly ameliorated by quercetin. Effects similar to those of quercetin were observed after treatment of cells with the reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC). Further investigations using chloroquine (autophagy inhibitor), rapamycin (autophagy activator), selisistat (SIRT1 inhibitor), and compound C (AMPK inhibitor) showed that chloroquine counteracted the effects of quercetin on oxidative stress-induced apoptosis, while rapamycin had the same effect as quercetin. In addition, the SIRT1/AMPK pathway inhibitors antagonized quercetin-mediated mitigation of the effects of oxidative stress on increased apoptosis and reduced autophagy. Consistent with the results in vitro, in mouse ovarian oxidative stress model induced by 3-NPA, quercetin activated autophagy through the SIRT1/AMPK signaling pathway, while alleviating oxidative stress damage and inhibiting apoptosis in mouse ovaries.

Conclusions: These findings indicate that quercetin can inhibit apoptosis in GCs and restore ovarian function by activating autophagy through the SIRT1/ROS/AMPK signaling pathway, suggesting a new direction for the treatment of ovarian follicular cysts in high-yield dairy cows.

Keywords: Apoptosis; Autophagy; Follicular cyst; Oxidative stress; Quercetin.

Fig. 1

Levels of serum hormones in follicular cysts of dairy cows. A Ultrasonographic images of follicles from estrus cows and those with follicular cysts; scale bar = 1 cm. B The diameters of estrus and cystic follicles. C FSH, D LH, E E₂, F P4, G E₂/P4, and H IGF levels in the serum of estrus and follicular-cyst dairy cows (n = 7). C, estrus dairy cows; FC, follicular cyst dairy cows. Data are shown as mean ± SD; compared with the estrus group, ^**P < 0.01, ^***P < 0.001, ns, not significant

Fig. 2

Increased levels of apoptosis and oxidative stress in GCs from dairy cows with follicular cysts. A HE staining showing histological changes in estrus and cystic follicles; Original magnification = 200 and 400 × ; scale bars = 100 and 50 μm. B The ROS, MDA, and T-AOC levels in the sera of estrus and follicular-cyst dairy cows. C The ROS, MDA, and T-AOC levels in the fluid from estrus and cystic follicles. D and E Relative expression of apoptosis-related proteins (Bcl-2, Bax, and Cl-Cas3) in GCs of estrus and cystic follicles, shown by Western blotting (n = 3). C, estrus dairy cows; FC, follicular-cyst dairy cows. The loading control was β-actin, and data are presented as mean ± SD. ^*P < 0.05, ^***P < 0.001

Fig. 3

Quercetin inhibits oxidative stress-induced apoptosis in GC through antioxidant effects. A Immunofluorescence staining of FSHR and CYP19A1 were used to identify GCs in vitro, and PBS was used to replace the antibody in the NC group; scale bar = 100 μm. NC, negative control. B GC viability after treatment with H₂O₂ (50, 100, 200, 400 µmol/L) for 3 h (n = 6). C and D Relative protein expression of apoptosis-associated proteins (Bcl-2, Bax, and Cl-Cas3) in GCs treated with H₂O₂ (50, 100, 200 µmol/L), shown by Western blotting. E GC viability after treatment with quercetin (5, 10, 20, 40, 80 µmol/L) for 3 h (n = 6). F and G Relative protein expression of Bcl-2, Bax, and Cl-Cas3 in GCs treated with H₂O₂ (200 µmol/L) and/or quercetin (5, 10 and 20 µmol/L), shown by Western blotting. H Total ROS levels in GCs treated with H₂O₂ (200 µmol/L) and/or quercetin (20 µmol/L)/NAC (5 mmol/L), measured by DCFH-DA; scale bar = 100 μm. I and J Relative protein expression of Bcl-2, Bax, and Cl-Cas3 in GCs treated with H₂O₂ (200 µmol/L) and/or quercetin (20 µmol/L)/NAC (5 mmol/L), shown by Western blotting. Que, quercetin; NAC, N-acetylcysteine. The loading control was β-actin, and data are presented as mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001

Fig. 4

Quercetin inhibits oxidative stress-induced apoptosis in dairy cow GCs through autophagy activation. A and B Relative protein expression of autophagy-associated proteins (BECN1 and LC3B2) in GCs of estrus and cystic follicles, shown by Western blotting (n = 3). C, estrus dairy cows; FC, follicular-cyst dairy cows. C and D Relative protein expression of BECN1 and LC3B2 in GCs treated with H₂O₂ (50, 100, 200 µmol/L), shown by Western blotting. E and F Relative protein expression of BECN1 and LC3B2 in GCs treated with H₂O₂ (200 µmol/L) and/or quercetin (20 µmol/L), shown by Western blotting. G and H Relative protein expression of BECN1 and LC3B2 in GCs treated with H₂O₂ (200 µmol/L) and/or quercetin (20 µmol/L)/NAC (5 mmol/L), shown by Western blotting. I GCs treated with H₂O₂ (200 µmol/L) and/or quercetin (20 µmol/L)/NAC (5 mmol/L). After the transfection of GCs with adenovirus plus autolysosome quantitation via mCherry-GFP-LC3, autophagy levels were visually observed; scale bar = 50 μm. J and K Relative protein expression of BECN1 and LC3B2 were determined in GCs treated with H₂O₂ (200 µmol/L) and quercetin (20 µmol/L)/RAPA (100 nmol/L) and CQ (50 µmol/L) by Western blotting. Que, quercetin; NAC, N-acetylcysteine; RAPA, rapamycin; CQ, chloroquine. The loading control was β-actin, and data are presented as mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ns, not significant

Fig. 5

Quercetin inhibits GC apoptosis and activates autophagy by regulating the phosphorylation of AMPK. A and B Relative protein expression of P-AMPK and AMPK in GCs treated with H₂O₂ (200 µmol/L) and/or quercetin (20 µmol/L)/NAC (5 mmol/L), shown by Western blotting. C and D Relative protein expression of BECN1, LC3B2, Bcl-2, Bax, and Cl-Cas3 in GCs treated with H₂O₂ (200 µmol/L), quercetin (20 µmol/L), and/or CC (50 µmol/L), shown by Western blotting. Que, quercetin; NAC, N-acetylcysteine; CC, compound C. The loading control was β-actin, and data are presented as mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001

Fig. 6

Quercetin inhibits ROS accumulation in GCs of dairy cows through SIRT1 and regulates autophagy through AMPK. A Total ROS levels in GCs treated with H₂O₂ (200 µmol/L), quercetin (20 µmol/L), and/or EX527 (10 µmol/L), measured by DCFH-DA; scale bar = 100 μm. B and C Relative protein expression of SIRT1, P-AMPK, and AMPK in GCs treated with H₂O₂ (200 µmol/L), quercetin (20 µmol/L), and/or EX527 (10 µmol/L), shown by Western blotting. D and E Relative protein expression of BECN1, LC3B2, Bcl-2, Bax, and Cl-Cas3 in GCs treated with H₂O₂ (200 µmol/L), quercetin (20 µmol/L), and/or EX527 (10 µmol/L), shown by Western blotting. Que, quercetin; EX527, selisistat. The loading control was β-actin, and data are presented as mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001

Fig. 7

Quercetin reduces oxidative stress-induced apoptosis in mouse GCs by autophagy activation through the SIRT/ROS/AMPK signaling pathway. A Weights of 3-NPA- and/or quercetin-treated mice. B Ovaries of 3-NPA- and/or quercetin-treated mice. C Ovary weights of 3-NPA- and/or quercetin-treated mice. D Histological changes in the ovaries of 3-NPA- and quercetin-treated mice; original magnification = 40 × . E ROS, F MDA, and G T-AOC levels in the ovaries of 3-NPA- and quercetin-treated mice. H TEM images of autophagic structures in the GCs of 3-NPA- and quercetin-treated mice; scale bar = 500 nm. I Immunofluorescence staining of Cl-Cas3 and LC3B2 in the ovaries of 3-NPA- and quercetin-treated mice; original magnification = 40 ×. J and K Relative protein expression of Bcl-2, Bax, Cl-Cas3, SIRT1, P-AMPK, AMPK, BECN1, and LC3B2 in the ovaries of 3-NPA- and quercetin-treated mice, shown by Western blotting. The loading control was β-actin, and data are presented as mean ± SD. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001