The Immune Regulation of Melanin From Gallus gallus domesticus Brisson Against Cyclophosphamide-Induced Immunosuppression

Abstract

Black-bone silky fowl (Gallus gallus domesticus Brisson), medicinal food homology, utilizes to enhance human immunity. However, it remains unclear whether Black-bone silky fowl melanin (BSFM), one of its bioactive components, could affect immune function. The purpose of this study is to examine the immunoregulatory effect and the underlying mechanism of BSFM in the cyclophosphamide-induced immunosuppressive mice model. The findings revealed that BSFM could significantly increase white blood cells (WBC) in peripheral blood; upregulate the expression of IL-4, TNF-α, and M-CSF in the plasma; and reduce tissue damage. Mechanistically, proteomics has revealed that BSFM therapy substantially affected the quantity of 29 proteins (Mtatp6, Cst3, Pglyrp1, Igkc, and other targets), which mostly participate in the phosphatidylcholine catabolic process, positive regulation of type IIa hypersensitivity, lipid catabolic process, and neutrophil chemotaxis. Metabolomics indicated that BSFM reduced the levels of Octanoylglucuronide, Gly-Gly, and N-alpha-acetyl-ornithine and modulated arginine biosynthesis. Furthermore, BSFM treatment modified the composition of gut microbiota and increased the relative abundance of Prevotella, S24-7, Olsenella, Lactococcus, hgcl-clade, Parasutterella, and Acetobacter. A significant correlation modified the composition of gut microbiota among inflammation-associated parameters, gut microbiota, and various metabolites (DMs) through Pearson correlation analysis. These findings suggest that BSFM holds promise in enhancing the human immune system and may serve as a complementary therapy in conventional chemotherapy.

Keywords: black‐bone silky fowl; gut microbiota; immune response; immunosuppressed mice; melanin.

FIGURE 1

Characterization of BSFM. (a) The mass spectrum of PTCA and PDCA formed at potassium permanganate oxidation of BSFM. (b) TEM map of BSFM. (c) Kinds and content of metals in BSFM.

FIGURE 2

BSFM improved symptoms of Cy‐induced immunosuppression in mice. (a) Establishment of the immunosuppressive mice model and drug treatment. (b) The thymus index. (c) The number of WBC. (d) The number of RBC. (e) The content of HGB. (f) The percentage of HCT. Data were presented as the mean ± standard deviation (n = 10). NC, normal control; MC, Cy model; LM, low dose of BSFM (100 mg/kg); MM, medium dose of BSFM (200 mg/kg); HM, high dose of BSFM (400 mg/kg); PC, rhG‐CSF positive control.

FIGURE 3

BSFM treatment adjusts cytokines balance and promotes splenic lymphocyte proliferation. (a) The levels of IL‐4. (b) The levels of IL‐10. (c) The levels of M‐CSF. (d) The levels of TNF‐α. (e) H&E‐stained images of spleen tissues (scale bars: 100 μm, 20 μm); yellow arrow in figure was marked as the location of spleen cell. (f) Representative immunohistochemistry profiles of CD3⁺ T in spleen with a scale bar of 100 μm and 50 μm. (g) Quantitation of the area of white pulp per field using Image J software. (h) Quantitation of positive area per field using Image J software. Data were presented as mean ± standard deviations (n = 6–10). NC, normal control; MC, Cy model; LM, low‐dose of BSFM (100 mg/kg); MM, medium‐dose of BSFM (200 mg/kg); HM, high‐dose of BSFM (400 mg/kg); PC, rhG‐CSF positive control. ****p < 0.001.

FIGURE 4

BSFM regulated the development of myeloid tissue of Cy‐induced immunosuppressed mice. (a) Heatmap of the common DEPs. (b) The expression of Igkc and Cst3 in myeloid tissue using Western blot. n = 3 in each group. (c) The relative levels of Igkc. (d) The relative levels of Cst3. NC, normal control; MC, Cy model; HM, high‐dose of BSFM (400 mg/kg).

FIGURE 5

BSFM regulated plasma metabolism (n = 6). (a) Principal component analysis (PCA) results of NC, MC, and HM groups. (b) Venn diagram showing the overlap of the metabolites identified in the plasma among the three groups. (c–e) Differential metabolites characterized in the plasma and their change trends after BSFM treatment. NC, normal control; MC, Cy model; HM, high dose of BSFM (400 mg/kg).

FIGURE 6

Effects of oral administration of BSFM on gut microbiota composition. (a) Genus level. (b–h) Seven species of bacteria at the genus level. NC, normal control; MC, Cy model; HM, high dose of BSFM (400 mg/kg).

FIGURE 7

Statistical analyses using the Spearman correlation coefficient were performed to examine the relationship. (a) BIs and DMs. (b) DMs and genus. The intensity of color represents the degree of association. *p < 0.05, **p < 0.01, ***p < 0.001.