Protein Hydrolyzates from Changbai Mountain Walnut (Juglans mandshurica Maxim.) Boost Mouse Immune System and Exhibit Immunoregulatory Activities

Abstract

The Changbai Mountain walnut (Juglans mandshurica Maxim.) is a rich source of essential amino acids. Walnut dregs are byproducts of edible oil production and primarily used as fodder and fertilizers. We systematically examined the effect of three types of walnut protein hydrolyzates-albumin, glutelin, and globin-on the immune system of mice and aimed to provide the theoretical basis for developing and utilizing J. mandshurica Maxim. protein resources. In comparison with the normal control mice, those treated with different doses of walnut proteins showed improved immune indices, including organ index, spleen lymphocyte proliferation, macrophage activity, number of CD4⁺ and CD8⁺ T cells, immunoglobulin A (IgA) and secretory IgA content, and mRNA and protein expression levels of cytokine factors. Our results indicated that these walnut proteins may have positive effects on the immune system and perform their immunomodulatory functions by inducing splenic enlargement. These findings support the use of walnut proteins as nutritional sources to boost the immune system.

Figure 1

Electrophoretic banding protein generated by SDS-PAGE of walnut proteins. 1, 2, 3, and 4 swim lanes correspond to standard proteins, albumin, glutelin, and globin.

Figure 2

Effects of walnut proteins on pinocytosis of peritoneal macrophages. Peritoneal macrophages were prepared from mice treated with walnut proteins at different doses. A distilled water-treated group was used as the negative control and thymopeptide was used to treat the positive control group. The phagocytosis index was measured using the accumulation of neutral red in cells via a colorimetric assay, which represented the phagocytic activity of the peritoneal macrophages of each protein-treated group. Values are expressed as mean ± standard deviation (SD). Asterisks represent statistically significant differences; ^∗P < 0.05 and ^∗∗P < 0.01 compared with the control group.

Figure 3

Proliferative analysis of splenocytes from mice treated with the walnut proteins at different doses. Approximately 5 × 10⁵ splenocytes were isolated from the protein-treated mice. A distilled water-treated group was used as the negative control, and thymopeptide was used as the positive control. The stimulation index represents the proliferative capacity of splenocytes from each group after treatment with the walnut proteins. Values are expressed as mean ± SD. Asterisks represent statistically significant differences; ^∗P < 0.05 and ^∗∗P < 0.01 compared with the control group.

Figure 4

Effects of the oral administration of walnut proteins on T-lymphocyte subpopulations. Splenocytes were separated from protein-treated mice after oral administration at varying doses. Approximately 1 × 10⁷ splenocytes were used to analyze the percentages of CD3⁺/CD4⁺ (a) and CD3⁺/CD8⁺ (b) cells by flow cytometry. The control group was orally given distilled water and the positive group was given thymopeptide during the experimental period. (c) normal control group; (d) positive group; (e) Glo + L group; (f) Glo + M group; (g) Glo + H group; (h) Alb + L group; (i) Alb + M group; (j) Alb + H group; (k) Glo + L group; (l) Glu + M group; (m) Glu + H group. Values are expressed as mean ± SD. Asterisks represent statistically significant differences; ^∗P < 0.05; ^∗∗P < 0.001 compared with the control group.

Figure 5

Effects of walnut proteins on immunoglobulin A (IgA) and secretory IgA (sIgA) levels in the mouse intestine. The distilled water-treated group was used as the negative control and thymopeptide was used as the positive control. The levels of IgA and sIgA in the intestine were determined by ELISA after the oral administration of the walnut proteins at varying doses. Values are expressed as mean ± SD. Asterisks represent statistically significant differences; ^∗P < 0.05; ^∗∗P < 0.01 compared with the control group.

Figure 6

Effects of the oral administration of walnut proteins on the expression of IFN-γ (a) and IL-6 (b) and effects of the oral administration of albumin on mRNA expression of IFN-γ (c) and IL-6 (d). The control group was orally given distilled water and the positive group was given thymopeptide during the experimental period. The levels of IL-6 and IFN-γ were determined by ELISA after the oral administration of the walnut proteins at varying doses. Values are expressed as mean ± SD. Asterisks represent statistically significant differences; ^∗P < 0.05; ^∗∗P < 0.01 compared with the control group.

Figure 7

The flow chart of the mechanism of action of hydrolyzate proteins of walnut.