Flammulina velutipes stem regulates oxidative damage and synthesis of yolk precursors in aging laying hens by regulating the liver-blood-ovary axis

Abstract

Egg production levels in late laying hens are negatively correlated with increasing age. Decreased liver and ovarian function in aging laying hens is accompanied by decreased antioxidant capacity, reproductive hormone levels, and follicular development, resulting in decreased synthesis of yolk precursors. The golden needle mushroom (Flammulina velutipes) has been reported to exhibit anti-inflammatory, antioxidant, and hypolipidemic properties. We aimed to reveal the therapeutic effects of F. velutipes stem (FVS) on liver-blood-ovary axis and investigate the underlying mechanisms. A total of 360 sixty-seven-wk-old laying hens were randomized into 4 treatment groups: 1) basal maize-soybean meal diet (CON); 2) basal maize + 20 g/kg FVS (2% FVS); 3) basal maize + 40 g/kg FVS (4% FVS); and 4) basal maize + 60 g/kg FVS (6% FVS). FVS groups demonstrated significantly increased egg production and ovarian development compared with the CON group. The addition of FVS increased the levels of antioxidant enzymes (GSH-Px, T-SOD, and T-AOC) in the liver, serum, and ovaries and decreased malondialdehyde levels by regulating the expression of proteins related to the Keap1-Nrf2/ARE signaling pathway. Additionally, FVS significantly decreased ovarian apoptosis by regulating Bax, Bcl-2, and caspase3 mRNA and protein expression levels. FVS significantly increased the expression levels of estradiol, progesterone, luteinizing hormone, and follicle stimulating hormone and their respective receptors. With increased levels of estradiol transported to the liver through the bloodstream, targeted binding to estrogen receptor (ER)-α and ER-β led to significant increases in ApoVLDL II, ApoB, and VTG II mRNA expression associated with yolk precursor synthesis. FVS decreased the levels of triglyceride and total cholesterol and significantly increased the expression of lipid metabolism, and transport-related mRNAs (FAS, PPAR-a/γ, and MTTP) in the liver. Therefore, the dietary supplementation of FVS can maintain the productive performance of aging laying hens by alleviating the degree of oxidative stress and regulating the transport of functional substances along the liver-blood-ovary axis, thereby improving the synthesis of yolk precursors.

Keywords: Flammulina velutipes stem; liver–blood–ovary signal axis; oxidative stress; reproductive hormone; yolk precursor synthesis.

Figure 1

FVS affected the Nrf2-Keap1/ARE pathway in the liver and ovary. (A) The mRNA abundance of Nrf2, Keap1, and downstream genes in the liver and ovary. (B) Effects of FVS on the protein levels of Nrf2, Keap1 and HO-1 in the liver. (C) Effects of FVS on the protein levels of Nrf2, Keap1 and HO-1 in the ovary. Abbreviations: Nrf2, nuclear factor E2-related factor 2; Keap1, kelch-like ECH-associated protein 1; sMaf, small Maf protein; HO-1, heme oxygenase 1; NQO1, NAD(P)H quinone oxidoreductase 1; GPX1, glutathione peroxidase 1; SOD1, superoxide dismutase 1; CON, laying hens fed with a basic diet; 2%FVS, laying hens fed with a basic diet supplemented with 20 g/kg FVS; 4%FVS, laying hens fed with a basic diet supplemented with 40 g/kg FVS; 6%FVS, laying hens fed with a basic diet supplemented with 60 g/kg FVS. The results are expressed as the mean ± SEM (n = 4); *P < 0.05 and **P < 0.01.

Figure 2

Influence of FVS on the follicular parameters and histology of the ovary in hens. (A) Hematoxylin-eosin (HE) staining of the hen's ovary tissue. (B) Statistical analysis of the number of primordial follicles and pre-ovulatory follicles index in ovary. Scale bar = 200 and 500 μm. Abbreviations: AF, atresia follicle; CON, laying hens fed with a basic diet; GF, growing follicles; GC, granulosa cells; M, medulla; PF, primary follicles; 2%FVS, laying hens fed with a basic diet supplemented with 20 g/kg FVS; 4%FVS, laying hens fed with a basic diet supplemented with 40 g/kg FVS; 6%FVS, laying hens fed with a basic diet supplemented with 60 g/kg FVS. The results are expressed as the mean ± SEM (n = 6); *P < 0.05 and **P < 0.01.

Figure 3

FVS alleviated ovarian apoptosis via the Bax/Bcl-2 and caspase3 pathway. (A) Effects of FVS on the relative expression of apoptosis-related genes in the ovary tissues. (B and C) Effects of FVS on the protein levels of Bax, Bcl-2 and caspase3 in the ovary. Abbreviations: CON, laying hens fed with a basic diet; 2%FVS, laying hens fed with a basic diet supplemented with 20 g/kg FVS; 4%FVS, laying hens fed with a basic diet supplemented with 40 g/kg FVS; 6%FVS, laying hens fed with a basic diet supplemented with 60 g/kg FVS. The results are expressed as the mean ± SEM (n = 4); *P < 0.05 and **P < 0.01.

Figure 4

Effects of FVS on the levels of serum reproductive hormone and their receptors. (A) The levels of serum E₂, FSH, LH and P₄. (B) The levels of mRNA expression of hormone receptors in the ovary of hens. Abbreviations: CON, laying hens fed with a basic diet; E₂, estradiol; ER-β, estrogen receptor-β; ER-α, estrogen receptor-α; FSH, follicle-stimulating hormone; FSHR, follicle-stimulating hormone receptor; LH, luteinizing hormone; LHR, luteinizing hormone receptor; P₄, Progesterone; 2%FVS, laying hens fed with a basic diet supplemented with 20 g/kg FVS; 4%FVS, laying hens fed with a basic diet supplemented with 40 g/kg FVS; 6%FVS, laying hens fed with a basic diet supplemented with 60 g/kg FVS. The results are expressed as the mean ± SEM (n = 6); *P < 0.05 and **P < 0.01.

Figure 5

Effect of FVS on liver histomorphology and heptic function damage. (A) Hematoxylin-eosin (HE) staining of the hen's liver tissue. (B) Effect of FVS on hepatic AST and ALT levels. Scale bar = 500 μm. Abbreviations: AST, aspartate transaminase; ALT, alanine transaminase; CON, laying hens fed with a basic diet; CV, central vein; IC, inflammatory cells; LD, lipid droplet; VD, vacuolar degeneration; 2%FVS, laying hens fed with a basic diet supplemented with 20 g/kg FVS; 4%FVS, laying hens fed with a basic diet supplemented with 40 g/kg FVS; 6%FVS, laying hens fed with a basic diet supplemented with 60 g/kg FVS. The results are expressed as the mean ± SEM (n = 4); *P < 0.05 and **P < 0.01.

Figure 6

Effect of FVS on the synthesis capacity of yolk precursors and their receptors. (A) Effect of FVS on yolk precursors, TG, T-CHO and VLDLR levels. (B) Effect of FVS on mRNA expression levels of genes related to yolk precursor synthesis and lipid metabolism. Abbreviations: ApoB, apolipoprotein B; ApoVLDLⅡ, apolipoprotein VLDL Ⅱ; TG, triglycerides; T-CHO, total cholesterol; VLDLy, very low-density lipoprotein Y; VTG, vitellogenin; VLDLR, very low-density lipoprotein receptor; VTGⅡ, vitellogenin Ⅱ; CON, laying hens fed with a basic diet; 2%FVS, laying hens fed with a basic diet supplemented with 20 g/kg FVS; 4%FVS, laying hens fed with a basic diet supplemented with 40 g/kg FVS; 6%FVS, laying hens fed with a basic diet supplemented with 60 g/kg FVS. The results are expressed as the mean ± SEM (n = 6); *P < 0.05 and **P < 0.01.

Figure 7

Schematic diagram outlining the regulation of reproductive function in aging laying hens by Flammulina velutipes mushroom stem through the liver-blood-ovary axis.