Long-term supplementation of genistein improves immune homeostasis in the aged gut and extends the laying cycle of aged laying hens

Abstract

Aging is associated with alterations in gut function, including intestinal inflammation, leaky gut, and impaired epithelial regeneration. Rejuvenating the aged gut is imperative to extend the laying cycle of aged laying hens. Genistein is known to have beneficial effects on age-related diseases, but its precise role in homeostasis of the aged gut of laying hens remains to be elucidated. In this study, 160 45-wk-old Hyline Brown laying hens were continuously fed a basal diet or a diet supplemented with 40 mg/kg genistein until they reached 100 wk of age. The results revealed that long-term genistein supplementation led to an improvement in the egg production rate and feed conversion ratio, as well as an increase in egg quality. Moreover, the expression levels of senescence markers, such as β-galactosidase, P16, and P21, were decreased in the gut of genistein-treated aged laying hens. Furthermore, genistein ameliorated gut dysfunctions, such as intestinal inflammation, leaky gut, and impaired epithelial regeneration. T_reg cell-derived IL-10 plays a crucial role in the genistein-induced regulation of age-related intestinal inflammation. This study demonstrates that long-term consumption of genistein improves homeostasis in the aged gut and extends the laying cycle of aged laying hens. Moreover, the link between genistein and T_reg cells provides a rationale for dietary intervention against age-associated gut dysfunction.

Keywords: Genistein; IL-10; aged laying hen; gut dysfunction; regulatory T cell.

Figure 1

Genistein improves the egg-laying performance of aged laying hens. Graph showing the variation trends in (A) egg production rate, (B) egg mass, (C) feed intake, (D) feed conversion ratio in different groups during the 50 to 60, 60 to 70, 70 to 80, 80 to 90, and 90 to 100-wk stages; n = 8. (E–H) Graph showing egg production rate, egg mass, feed intake, and feed conversion ratio in different groups during the 50 to 100-wk stages; n = 8. Data are mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001 (2-tailed t test).

Figure 2

Genistein enhances the egg quality of aged laying hens. Graph showing the variation trends in (A) eggshell thickness, (B) eggshell strength, (C) Haugh units, (D) albumen height, and (E) yolk color of laying hens in different groups; n = 8. Data are mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001 (2-tailed t test).

Figure 3

Genistein ameliorates intestinal cellular senescence of aged laying hens. (A) Immunohistochemical of β-Galactosidase in the ileum of laying hens. Scale bars, 200 μm. (B) Graph showing MFI of β-Galactosidase; n = 6. (C, D) qRT-PCR analysis of the markers of cellular senescence (p16 and p21) of ileum of laying hens. Data are mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001 (2-tailed t test).

Figure 4

Genistein improves the intestinal epithelial barrier function of aged laying hens. (A) H&E-stained section of the ileum of laying hens. Scale bars, 200 μm. Graph showing villus height and crypt depth of ileum; n = 6. (B) Graph showing histological scores of the ileum; n = 6. (C) PAS-stained section of the ileum of laying hens. Scale bars, 50 μm. (D) Graph showing the number of goblet cells per 200 μm of the ileum; n = 6. (D) qRT-PCR analysis of the markers of enterocytes (SI), goblet cells (Muc2), Paneth cells (Lyz), and ISCs (Lgr5 and Olfm4) of ileum of laying hens. (E) FITC-dextran concentration from the serum of laying hens; n = 8. (F) qRT-PCR analysis of the tight junctions (Z0-1, Occludin, and Claudin-1) of ileum of laying hens; n = 8. Data are mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001 (2-tailed t test).

Figure 5

The effects of genistein on intestinal lamina plasma cells-mediated humoral immunity of aged laying hens. (A) Flow cytometry analysis of Bu-1⁺, Bu-1⁺IgY⁺, Bu-1⁺IgA⁺, and Bu-1⁺IgM⁺ frequency in ileal LPLs of laying hens. (B–E) Graph showing the percentage of Bu-1⁺, Bu-1⁺IgY⁺, Bu-1⁺IgA⁺, and Bu-1⁺IgM⁺ cells in ileal LPLs of laying hens; n = 6. Data are mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001 (2-tailed t test).

Figure 6

Genistein ameliorates intestinal inflammaging of aged laying hens. (A) Heatmap of mRNA expression of 18 inflammatory cytokines from the ileum of laying hens. The red color denotes a positive correlation, while the blue color denotes a negative correlation. The intensity of the color is proportional to the expression level of genes. Data are mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001 (2-tailed t test). (B–D) ELISA analysis showing expression of TNF-a, IL-6, and IL-10 in the ileum of laying hens; n = 6. Data are mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001 (2-tailed t test).

Figure 7

Genistein promotes the expression of T_reg cell-derived IL-10 in the aged gut of laying hens. (A) Flow cytometry analysis of IL-10⁺, IL-10⁺CD11c⁺ (dendritic cell), IL-10⁺CD19⁺ (B cell), IL-10⁺CD4⁺ (T cell), and IL-10⁺CD11b⁺ (macrophage) frequency in ileal LPLs of laying hens. (B) Graph showing the percentage of IL-10⁺, IL-10⁺CD11c⁺, IL-10⁺CD19⁺, IL-10⁺CD4⁺, and IL-10⁺CD11b⁺ cells in ileal LPLs of laying hens; n = 6. (C) Flow cytometry analysis of IL-10⁺CD4⁺CD25⁺ (T_reg cell), IL-10⁺CD4⁺RORγt⁺ (Th17 cell), and IL-10⁺CD4⁺GATA-3⁺ (Th2 cell) frequency in ileal LPLs of laying hens at 100 wk old. (D) Graph showing the percentage of IL-10⁺CD4⁺CD25⁺, IL-10⁺CD4⁺RORγt⁺, and IL-10⁺CD4⁺GATA-3⁺ cells in ileal LPLs of laying hens at 100 wk old; n = 6. Data are mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001 (2-tailed t test).

Figure 8

The graphical abstract. Genistein ameliorates gut dysfunction such as intestinal inflammation, leaky gut, and impaired epithelial regeneration. Moreover, T_reg cell-derived IL-10 plays a crucial role in the genistein-induced regulation of age-related intestinal inflammation. Long-term genistein supplementation improved production performance and egg quality in aged laying hens.