Bioactive Lignan Honokiol Alleviates Ovarian Oxidative Stress in Aging Laying Chickens by Regulating SIRT3/AMPK Pathway

Abstract

Aging is not only a key internal cause of age-related diseases in humans but also poses a threat to the productivity of farm animals with longer breeding cycles, such as laying chickens. Various measures were taken to prolong the laying period by reducing oxidative stress to improve poultry ovarian functions. Within the mitochondria, SIRT3, a member of the Sirtuin family, plays an important role in post-translational modifications and the regulation of protein activities involved in energy metabolism and oxidative response. This study aimed to investigate the alleviating effect of a bioactive lignan Honokiol (HKL) on oxidative stress in aging chicken ovaries in order to retard decline in egg production. The results showed that HKL treatment restored the abnormal balance between cell proliferation and apoptosis, and it enhanced the antioxidant capacity of the H₂O₂-induced small white follicles (SWFs) by activating the SIRT3/AMPK pathway. Moreover, HKL significantly increased total egg production, the number of yellow follicles, and the mRNA expression of yolk synthesis and deposition-related genes, serum estrogen, and antioxidant levels. These findings suggest that HKL holds promise in enhancing the egg productivity of aging laying chickens by promoting yolk deposition and reducing ovarian oxidative stress.

Keywords: AMPK; Honokiol; Sirtuins; chicken; ovarian aging; oxidative stress.

Figure 1

Effect of HKL on cell proliferation and apoptosis in the H₂O₂-induced SWFs. (A) (A1) H&E staining. (A2) IF staining. BrdU-positive cells (red) for evaluating cell proliferation. (A3) TUNEL assay. TUNEL-positive cells (green) for evaluating cell apoptosis. Nuclei were stained with DAPI (blue). GL (red arrowheads): granulosa layer. Scale bar: 100 μm or 50 μm. (B) The mRNA expression of proliferation-related genes ((B1–B3) PCNA, CDK2, and CCND1) and apoptosis-related genes ((B4–B6) BCL-2, Bax, and Caspase-3) in SWFs. Data are expressed as mean ± SEM (n = 4). Different lowercase letters indicate significant differences (p < 0.05). The expression results are relative to the control group level.

Figure 2

Effect of HKL on antioxidant capacity and SIRT3/AMPK pathway in the H₂O₂-induced SWFs. (A) Determination of antioxidant capacity and energy metabolism ((A1–A6) MDA, GSH, CAT, T-SOD, T-AOC, and ATP) in the H₂O₂-induced SWFs (n = 4). (B) Relative expression of SIRTs mRNA after H₂O₂ (B1) or HKL (B2) treatment in SWFs (n = 3). (C) Relative expression of SIRT3 mRNA (C1), SIRT3, and pAMPK proteins (C2–C4) after different treatments in SWFs (n = 3). Data are expressed as mean ± SEM. Different lowercase letters indicate significant differences (p < 0.05); * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. The expression results are relative to the control group level.

Figure 3

The suppression of 3-TYP on the promotion of cell proliferation and inhibition of cell apoptosis by HKL. (A) (A1) H&E staining and (A2) IF staining. BrdU-positive cells (red) for evaluating cell proliferation. (A3) TUNEL assay. TUNEL-positive cells (green) for evaluating cell apoptosis. Nuclei were stained with DAPI (blue). GL (red arrowheads): granulosa layer. Scale bar: 100 μm or 50 μm. (B) Effect of different treatments on the expression of proliferation-related genes ((B1–B3) PCNA, CDK2, and CCND1) and apoptosis-related genes ((B4–B6) BCL-2, Bax, and Caspase-3) in SWFs (n = 4). Different lowercase letters indicate significant differences (p < 0.05). The expression results are relative to the H₂O₂ group level.

Figure 4

The suppression of 3-TYP upon the promotion of antioxidant capacity and activation of the SIRT3/AMPK pathway by HKL. (A) Determination of antioxidant capacity and energy metabolism ((A1–A6) MDA, GSH, CAT, T-SOD, T-AOC, and ATP) in the H₂O₂-induced SWFs after 3-TYP treatment (n = 4). (B) Relative expression of SIRTs mRNA (B1) (n = 4); relative expression of SIRT3 and pAMPK proteins (B2–B4) in SWFs (n = 3). Data are expressed as mean ± SEM. Different lowercase letters indicate significant differences (p < 0.05).

Figure 5

The suppression of CC on promotion of cell proliferation and inhibition of cell apoptosis by HKL. (A) (A1) H&E staining. (A2) IF staining. BrdU-positive cells (red) for evaluating cell proliferation. (A3) TUNEL assay. TUNEL-positive cells (green) for evaluating cell apoptosis. Nuclei were stained with DAPI (blue). GL (red arrowheads): granulosa layer. Scale bar: 100 μm or 50 μm. (B) Effect of different treatments on the expression of proliferation-related genes ((B1–B3) PCNA, CDK2, and CCND1) and apoptosis-related genes ((B4–B6) BCL-2, Bax, and Caspase-3) in SWFs (n = 4). Data are expressed as mean ± SEM. Different lowercase letters indicate significant differences (p < 0.05). The expression results are relative to the H₂O₂ group level.

Figure 6

The suppression of CC on promotion of antioxidant capacity and activation of SIRT3/AMPK pathway by HKL. (A) Determination of antioxidant capacity and energy metabolism ((A1–A6) MDA, GSH, CAT, T-SOD, T-AOC, and ATP) in the H₂O₂-induced SWFs after CC treatment (n = 4). (B) Relative expression of SIRT3 mRNA (B1) (n = 4); relative expression of SIRT3 and pAMPK proteins (B2–B4) in SWFs (n = 3). Data are expressed as mean ± SEM. Different lowercase letters indicate significant differences (p < 0.05).

Figure 7

Effect of HKL on laying performance and ovarian development of D580 chickens. (A) Effect of feeding HKL on egg production and ovarian development: (A1,A2) total egg production, egg production rate; (A3,A4) weight of ovary and PHFs; and (A5) number of PHFs (n = 4). (B) Hierarchical development in D280, D580, and the HKL-fed D580 group: (B1) Follicles are arranged in order; (B2) the number of different follicles (n = 4). (C) The mRNA expression of yolk synthesis-related genes ((C1–C3) ApoB, ApoVLDLII, and VTGII) in D580 livers and yolk deposition-related genes ((C4–C6) VLDLR, LPL, and OCLN) in D580 SWFs (n = 4). Data are expressed as mean ± SEM. Different lowercase letters indicate significant differences (p < 0.05). ns: not significant. The expression results are relative to the control group level.

Figure 8

Effects of HKL on serum estrogen, liver function, and antioxidant levels in D580 chickens. (A) Effect of HKL on estrogen and liver indicators of D580 serum in vivo. E₂ (A1) and P₄ (A2) levels were determined by immunological methods in serum. ALB (A3) and GLB (A4) contents, and AST (A5) and GGT (A6) enzyme activities were determined by biochemical methods in serum (n = 4). (B) Determination of antioxidant capacity and energy metabolism ((B1–B6) MDA, GSH, CAT, T-SOD, T-AOC, and NAD⁺) in serum (n = 4). Data are expressed as mean ± SEM. Different lowercase letters indicate significant differences (p < 0.05).

Figure 9

Effect of HKL on the proliferation and apoptosis of SWFs and SIRT3/AMPK pathway in D580 chickens. (A) (A1) H&E staining. (A2) IF staining. PCNA-positive cells (red) for evaluating cell proliferation. (A3) TUNEL assay. TUNEL-positive cells (green) for evaluating cell apoptosis. Nuclei were stained with DAPI (blue). GL (red arrowheads): granulosa layer. Scale bar: 100 μm or 50 μm. (B) The mRNA expression of proliferation-related genes ((B1–B3) PCNA, CDK2, and CCND1) and apoptosis-related genes ((B4–B6) BCL-2, Bax, and Caspase-3) in SWFs (n = 4). (C) The expression levels of SIRT3 mRNA (C1) (n = 4), SIRT3 and pAMPK proteins (C2–C4) in SWFs (n = 3). Data are expressed as mean ± SEM. Different lowercase letters indicate significant differences (p < 0.05).