Immunomodulatory Activities of a Fungal Protein Extracted from Hericium erinaceus through Regulating the Gut Microbiota

Abstract

A single-band protein (HEP3) was isolated from Hericium erinaceus using a chemical separation combined with pharmacodynamic evaluation methods. This protein exhibited immunomodulatory activity in lipopolysaccharide-activated RAW 264.7 macrophages by decreasing the overproduction of tumor necrosis factor-α, interleukin (IL)-1β, and IL-6, and downregulating the expression of inducible nitric oxide synthase and nuclear factor-κB p65. Further researches revealed that HEP3 could improve the immune system via regulating the composition and metabolism of gut microbiota to activate the proliferation and differentiation of T cells, stimulate the intestinal antigen-presenting cells in high-dose cyclophosphamide-induced immunotoxicity in mice, and play a prebiotic role in the case of excessive antibiotics in inflammatory bowel disease model mice. Aided experiments also showed that HEP3 could be used as an antitumor immune inhibitor in tumor-burdened mice. The results of the present study suggested that fungal protein from H. erinaceus could be used as a drug or functional food ingredient for immunotherapy because of its immunomodulatory activities.

Keywords: Hericium erinaceus; anti-inflammation; functional food ingredient; fungal immunomodulatory protein; gut microbiota; immunotherapy.

Figures

Figure 1

Effect of crude protein extracts from Hericium erinaceus on trinitrobenzenesulfonic acid solution (TNBS)-induced inflammatory bowel disease (IBD) rats. (A) The technical route of this study; (B) the fresh fruiting bodies of H. erinaceus and the protein electrophoresis; (C) the hematoxylin and eosin-staining and immunohistochemistry results; (D) the Disease Activity Index scores (calculated according to the weight loss, stool consistency, and blood in feces) and observation of colons of the TNBS-induced IBD rats. Control is the normal group without any treatments, Model is the TNBS-induced IBD rats, HEP is the crude protein extract-treated group after TNBS enema, and 5-aminosalicylic acid (5-ASA) is the positive control group treated with 100 mg/(kg ⋅ day) of 5-ASA after TNBS enema. Values were expressed as means ± SDs. #P < 0.05 vs the control group, *P < 0.05, **P < 0.01 vs the model group, indicating significant differences compared with the model group.

Figure 2

Effects of HEP on trinitrobenzenesulfonic acid solution (TNBS)-induced rats. Normal group; model group, induced by TNBS enema; HEP group, the crude protein extract-treated group after TNBS enema; and positive control group, treated with 100 mg/(kg ⋅ day) of 5-aminosalicylic acid after TNBS enema. After treatment for 14 days, cytokines interleukin (1L)-1α (A), 1L-2 (B), 1L-8 (C), 1L-10 (D), 1L-11 (E), IL-12 (F), tumor necrosis factor (TNF)-γ (H), TNF-α (G), vascular endothelial growth factor (VEGF) (I), MIP-α (J), macrophage colony-stimulating factor (M-CSF) (K), and myeloperoxidase (MPO) (L) were produced. The assays were carried out according to the procedures recommended in the enzyme-linked immunosorbent assay kit manual. Values were means ± SDs of three independent experiments. #P < 0.05 vs the normal group, *P < 0.05, **P < 0.01 vs the TNBS-treated group.

Figure 3

Effects of HEP3 on proinflammatory cytokine productions in lipopolysaccharide (LPS)-activated RAW 264.7 cells, and effects on the d-galactose-induced HIEpiC senescent cells. Cell viability was measured by quantitative colorimetric methylthiazolyl tetrazolium (MTT) after incubation with HEP3 for 48 h [(A), a]; cells were preincubated with 0.05–0.20 mg/mL HEP3 for 4 h, and then treated with 1 μg/mL LPS for 24 h. Using an ELISA kit, tumor necrosis factor-α [(A), b], interleukin (IL)-1β [(A), c], IL-6 [(A), d], nitric oxide [(A), e], and inducible nitric oxide synthase [(A), f] in the supernatant were detected. [(B), a–e] The cells were treated with 40 mg/mL of d-galactose for 72 h combined with different concentrations of HEP3, and the number of senescent cells (blue-stained cells) was detected using β-galactosidase staining. [(B), f] The cells were treated with different concentrations of HEP for 24 h, and the cytotoxicity was detected by an MTT assay. [(B), g–i] The activities of malondialdehyde, total superoxide dismutase, and glutathione peroxidase of the cells. Values were means ± SDs of three independent experiments. #P < 0.05 vs the normal group, *P < 0.05, **P < 0.01 vs the model group, indicating significant differences compared with the model group.

Figure 4

Effect of HEP3 on the cyclophosphamide-induced immunotoxicity mice. Body weight changes (A), thymus index (B), and spleen index (C), neutral red engulfment (D), splenocyte proliferation (E), platelet (F), and white blood cell (G), the tissue structure of the spleen (H), the CD3+(I), CD4+/CD8 (J), CD4+(L), CD8+(M), CD28+/CD8 (K), and naive T cells (N). CT is the control group treated with just vehicle, CTX is the cyclophosphamide-induced group (intraperitoneal injection of 80 mg/kg) group, HEP3-D is the group treated with 100 mg/kg HEP3 and intraperitoneal injection of 80 mg/kg cyclophosphamide, and HEP3-G is the group treated with 200 mg/kg HEP3 and intraperitoneal injection of 80 mg/kg cyclophosphamide. Values were means ± SDs of six independent experiments. #P < 0.05 vs the control group, *P < 0.05, **P < 0.01 vs the CTX, indicating significant differences.

Figure 5

Influence of cyclophosphamide on the cecal microbiota of mice. (A) The Venn diagram; (B) the PCA analysis of operational taxonomic units; (C) heat map of 16S rRNA gene sequencing analysis of cecal content at the genus level. Z denotes the control (normal) group just treated with vehicle, and M is the cyclophosphamide-induced (intraperitoneal injection of 80 mg/kg) group.

Figure 6

Effects of HEP3 on the microbiota of cecal contents in rats with cyclophosphamide-induced immunotoxicity. (A) The rarefaction curve; (B) the principal component analysis of operational taxonomic units; (C) the classification and abundance of cecal contents at the phylum level; (D) the Venn diagram of OTUs; (E) the sample species classification tree. Z denotes the control group just treated with vehicle, M is the cyclophosphamide-induced (intraperitoneal injection of 80 mg/kg) group, D is the 100 mg/kg HEP3-treated group, and G is the 200 mg/kg HEP3-treated group. Values were means ± SDs of six independent experiments. #P < 0.05 vs the control group, *P < 0.05, **P < 0.01 vs the cyclophosphamide-induced group, indicating significant differences.

Figure 6

Effects of HEP3 on the microbiota of cecal contents in rats with cyclophosphamide-induced immunotoxicity. (A) The rarefaction curve; (B) the principal component analysis of operational taxonomic units; (C) the classification and abundance of cecal contents at the phylum level; (D) the Venn diagram of OTUs; (E) the sample species classification tree. Z denotes the control group just treated with vehicle, M is the cyclophosphamide-induced (intraperitoneal injection of 80 mg/kg) group, D is the 100 mg/kg HEP3-treated group, and G is the 200 mg/kg HEP3-treated group. Values were means ± SDs of six independent experiments. #P < 0.05 vs the control group, *P < 0.05, **P < 0.01 vs the cyclophosphamide-induced group, indicating significant differences.

Figure 7

Effect of HEP3 on the microbiota classification and abundance detected by 16S rRNA gene sequencing analysis of cecal contents from the mice with cyclophosphamide-induced immunotoxicity at the genus level (A,B). Effect of HEP3 on KEGG pathways of gut microbiota in mice with cyclophosphamide-induced immunotoxicity (C,D). Z denotes the control group just treated with vehicle, M is the cyclophosphamide-induced (intraperitoneal injection of 80 mg/kg) group, D is the 100 mg/kg HEP3-treated group, and G is the 200 mg/kg HEP3-treated group.

Figure 7

Effect of HEP3 on the microbiota classification and abundance detected by 16S rRNA gene sequencing analysis of cecal contents from the mice with cyclophosphamide-induced immunotoxicity at the genus level (A,B). Effect of HEP3 on KEGG pathways of gut microbiota in mice with cyclophosphamide-induced immunotoxicity (C,D). Z denotes the control group just treated with vehicle, M is the cyclophosphamide-induced (intraperitoneal injection of 80 mg/kg) group, D is the 100 mg/kg HEP3-treated group, and G is the 200 mg/kg HEP3-treated group.

Figure 8

HEP3 extracted from Hericium erinaceus improved the pathological parameters of the trinitrobenzenesulfonic acid solution (TNBS)-induced mice. (A) The body weight changes; (B) the levels of lipopolysaccharide in serum; (C) the levels of cytokines GM-CSF, tumor necrosis factor (TNF)-γ, 1L-10, interleukin (IL)-12, 1L-17α, 1L-4, TNF-α, and vascular endothelial growth factor in serum; (D) the histopathological changes in colon; and (E) the histopathological changes in spleen. Control is the normal group; model is the TNBS-induced group; model and high-dose antibiotics; HEP3 [100 mg/(kg ⋅ day)], Bifidobacterium, HEP3 and high-dose antibiotics, HEP3 and Bifidobacterium, Bifidobacterium and high-dose antibiotics, HEP3 and Bifidobacterium and high-dose antibiotics.

Figure 9

Immunohistochemical staining of tumor necrosis factor-α (A), NF-κB p65 (B), interleukin-17 (C), and Foxp3 (D) in the colons of different experimental groups in inflammatory bowel disease mice after treatment with HEP3. Control is the normal group; model is the trinitrobenzenesulfonic acid solution-induced group; model and high-dose antibiotics; HEP3 [100 mg/(kg ⋅ day)], Bifidobacterium, HEP3 and high-dose antibiotics, HEP3 and Bifidobacterium, Bifidobacterium and high-dose antibiotics, HEP3 and Bifidobacterium and high-dose antibiotics.

Figure 10

HEP3 showed good prebiotic effects in trinitrobenzenesulfonic acid solution (TNBS)-induced mice. (A,B) The relative abundance at the phylum level; (C–F) the relative abundance at the family level. Control is the normal group; model is the TNBS-induced group; model and high-dose antibiotics; HEP3 [100 mg/(kg ⋅ day)], Bifidobacterium, HEP3 and high-dose antibiotics, HEP3 and Bifidobacterium, Bifidobacterium and high-dose antibiotics, HEP3 and Bifidobacterium and high-dose antibiotics. Values were means of six independent experiments.

Figure 11

Effects of HEP on the average tumor weight and the content of various cytokines in the CC531 cell tumor xenograft model mice. The tumor tissue of HL, HH, and model groups (A); the tumor inhibition rate (B); the contents of various cytokines (C) were detected using an enzyme-linked immunosorbent assay kit: tumor necrosis factor-α (a), PSA (b), interferon-γ (c), macrophage colony-stimulating factor (d), transforming growth factor (e), and vascular endothelial growth factor (f). Values were means ± SDs. #P < 0.05, ##P < 0.01 vs the normal group; *P < 0.05, **P < 0.01 vs the model group, indicating significant differences.

Figure 12

Dominant species interaction-associated network analysis results. Nodes represent the dominant species under the identity of different colors: the red line shows a positive correlation and the green line shows a negative correlation. More connections by nodes indicated more association with others. (A) Antibiotics rapidly declined the diversity and destroyed the stability of the whole ecological system; (B) HEP3 can main the diversity and stability of the whole ecological system in IBD model mice.

Figure 12

Dominant species interaction-associated network analysis results. Nodes represent the dominant species under the identity of different colors: the red line shows a positive correlation and the green line shows a negative correlation. More connections by nodes indicated more association with others. (A) Antibiotics rapidly declined the diversity and destroyed the stability of the whole ecological system; (B) HEP3 can main the diversity and stability of the whole ecological system in IBD model mice.