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.
Mushrooms are rapidly becoming recognized as a promising source of novel proteins. Fungal immunomodulatory proteins (FIPs) are small-molecule proteins extracted from the fruiting body of some higher basidiomycetes (mushrooms). FIPs have similar structure and immune function as lectins and immunoglobulins, which were first extracted from Ganoderma lucidum in 1989. Different kinds of FIPs were extracted from G. lucidum, G. tsugae Murrill, Flammulina velutipes, and Volvariella volvacea continuously (1–4). FIPs have exhibited many beneficial functions in previous studies, including antitumor (5), antiallergy (6, 7), and the ability to stimulate immune cells to produce cytokines (8, 9). Several proteins as lectins (10), lignocellulolytic enzymes (11–14), protease inhibitors (15, 16), and hydrophobins (17–19) have shown unique features and could offer solutions to several medical and biotechnological problems (such as microbial drug resistance, low crop yields, and demands for renewable energy). These stunning properties along with the absence of toxicity render these biopolymers ideal compounds for developing novel functional foods or nutraceuticals with the increase in consumers’ consciousness and demand for healthy food. Large-scale production and industrial application of some fungal proteins prove their biotechnological potential and establish higher fungi as a valuable, although relatively unexplored, source of unique proteins.
Hericium erinaceus, belonging to the division Basidiomycota and class Agaricomycetes, is both an edible and medicinal mushroom. It is popular across the continents for its delicacy and is used as a replacement for pork or lamb in Chinese vegetarian cuisine. It is rich in active constituents such as diterpenoid compounds, steroids, polysaccharides, proteins, and other functional ingredients, which are used as good natural plant resources (18). Previous studies have shown the effectiveness of H. erinaceus in improving cognitive impairment (20), stimulating nerve growth factors (21) and nerve cells (22), improving hypoglycemia (23), and protecting against gastrointestinal cancers (24, 25). They are also processed into different kinds of products (beverage, cookies, oral liquid, and so on) sold in supermarkets and drugstores. Until now, little has been studied about the proteins from H. erinaceus (26). A previous study revealed, using Coomassie Brilliant Blue G-250 method, that the content of total proteins in H. erinaceus was up to 20 mg/100 g, indicating that the proteins in H. erinaceus might be good active ingredients and hence should not be ignored. Therefore, the aim of this study was to evaluate the immunomodulatory activities of FIPs extracted from the fruiting bodies of H. erinaceus using cells and animal experiments and to reveal the underlying mechanism. This study might lay a foundation for the application of the nutritional and medicinal value of H. erinaceus.
Materials and Methods
Plant Material and Protein Extraction
The fresh fruiting bodies of H. erinaceus were collected from the Research Laboratory of Edible Mushrooms of Guangdong Institute of Microbiology, China, in June 2015, and identified by Prof. Xie Yizhen of the Guangdong Institute of Microbiology.
Fresh fruiting bodies (5,000 g) of H. erinaceus were pureed in a blender (Philips, HR2095/30, ROYAL PHILIPS, Amsterdam of Holland), and extracts were prepared by the methods shown in the Presentation S1 in Supplemental Material. The solutions were combined, filtrated after acidification to pH 4.3 with dilute acetic acid, and then mixed with (NH4)2SO4 to 80% saturation. The resulting solution was kept in a refrigerator at 4°C overnight and then centrifuged at 5,000 rpm for 20 min at 4°C. The supernatant was removed. The precipitation was dissolved in 5 mL of pH 8.0 Tris–HCl buffer and lyophilized in a vacuum freeze dryer (Alphai-4LD plus, Marin Christ, Osterode, Germany) for crude protein extraction. The next purification was done using the membrane separation technology combined with the activity evaluation experiment in rats with trinitrobenzenesulfonic acid solution (TNBS)-induced inflammatory bowel disease (IBD).
Animals
This study used 5- to 6-week male Sprague-Dawley rats (weighing 180–220 g), 4- to 5-week male BALB/c mice (weighing 16–20 g), and Kunming male mice (weighing 18–22 g); all purchased from the Animal Center of the Guangdong Medical Laboratory Animal Center, Guangzhou, China. The animals were kept in the specific-pathogen-free Animal Laboratory of Guangdong Institute of Microbiology, in a temperature (23 ± 1°C) and humidity (55 ± 10%) controlled room under a 12-h light/dark cycle (lights off at 1700 p.m.). The animals were given free access to food and water that were sterilized. The experimental protocols were approved by the Animal Ethics Committee of Guangdong Institute of Microbiology, and all experimental procedures conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize the number of animals used.
Cell Culture
The RAW 264.7 macrophages, HIEpiC, and CC531 cell lines were obtained from the Shanghai Aolu Biological Technology Co., Ltd. (China). They were maintained in Dulbecco’s modified Eagle medium or RPMI-1640 supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere of 95% air and 5% CO2 and seeded into a 75-cm2 culture dish. On reaching 80% confluence, the cells underwent digestive transfer culture after fusion growth at a density of 5 × 104 cells/mL.
Anti-inflammatory Evaluation of IBD Model Rats
After 7 days of adaptation period, the animals were randomly divided into four groups [100 mg/(kg ⋅ day): proteins extracted from H. erinaceus (HEP), model, normal, and 5–aminosalicylic acid groups], with six rats in each group, and housed three per cage. The rats were fed a standard diet, and water was available freely. After 24 h of fasting, the rats were anesthetized by intraperitoneally injecting 2% sodium pentobarbital (0.2 mL/100 g). The rats were intubated (using latex tubing of 2 mm diameter, lubricated with edible oil before use) from the anus, gently inserting the tubing into the lumen about 8.0 cm. Then, 150 mg/kg of TNBS (dissolved in 50% ethanol; Sigma-Aldrich, MO, USA) solution was injected through the latex tubing, and the rats were hung upside down for 30 s to enable the mixture to fully seep into the lumen without leakage. The rats in the HEP group were treated by intragastric administration after 1 day of TNBS induction.
After 14 days of treatment, the rats were anesthetized by intraperitoneally injecting 2% sodium pentobarbital (0.25 mL/100 g). The blood plasma was collected by the abdominal aortic method, and the serum by centrifugation (1,500 rpm, 10 min). Then, the serum was used to monitor the production of the cytokines interleukin (1L)-1α, 1L-2, 1L-8, 1L-10, 1L-11, and IL-12; tumor necrosis factor (TNF)-γ and TNF-α; vascular endothelial growth factor (VEGF); human macrophage inflammatory protein-1α (MIP-α); and macrophage colony-stimulating factor (M-CSF) and myeloperoxidase (MPO). The colons obtained from the rats were fixed in 4% paraformaldehyde at pH 7.4 for further pathological observation.
Immunomodulatory Activity on RAW 264.7 Macrophages
After incubating RAW 264.7 macrophages with HEP3 (0–200 μg/mL) for 4 h, followed by an additional 24 h of treatment with lipopolysaccharide (LPS; 1 μg/mL), the supernatant was used to monitor the production of the cytokines 1L-1β, 1L-6, TNF-α, and nitric oxide (NO), and the intracellular levels of inducible nitric oxide synthase (iNOS) and nuclear factor-κB (NF-κB) p65. The assays were carried out according to the procedures recommended in the enzyme-linked immunosorbent assay (ELISA) kit manual, which was purchased from USCN Life Science Inc. (Wuhan, China).
Effect on the Cyclophosphamide Immunosuppressant Mice Model
The animals were randomly divided into four groups (n = 10): normal, model, and HEP3-treated with 200 and 100 mg/(kg ⋅ day) groups. The immunosuppressant mice were induced by intraperitoneally injecting cyclophosphamide [cyclophosphamide-induced group (CTX), 80 mg/kg] once a day, for 3 days, while the mice in the normal group were intraperitoneally injected with saline as a control. All mice had free access to tap water and food (ad libitum). On day 14, the mice were sacrificed, and the serum, spleen, and cecal contents were isolated for further analysis.
Prebiotic Effect of HEP3 on TNBS-Induced Mice
All animals were randomly divided into nine groups (n = 9): control, model, model and high-dose antibiotics, HEP3 [100 mg/(kg ⋅ day)], Bifidobacterium, HEP3 and high-dose antibiotics, HEP3 and Bifidobacterium, Bifidobacterium and high-dose antibiotics, and HEP3 and Bifidobacterium and high-dose antibiotics. All the antibiotics were given for 4 days. Then, IBD was induced with TNBS, followed by 7 days of drug treatment and induction with TNBS again, and finally followed by another 4 days of drug treatment. The model mice were prepared using TNBS (150 mg/kg) enema according to the procedure described in Section.
After treatment, the mice were anesthetized by intraperitoneally injecting 2% sodium pentobarbital (0.25 mL/100 g). The blood plasma was collected by the abdominal aortic method, and the serum by centrifugation (1,500 rpm, 10 min). Then, the serum was used to monitor the production of cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), TNF-γ, 1L-10, IL-12, 1L-17α, 1L-4, TNF-α, and VEGF. The colons and spleens obtained from the rats were fixed in 4% paraformaldehyde at pH 7.4 for further pathological observation, and the cecum contents were collected for 16s rRNA analysis.
Antiaging Protective Effect on the d-Galactose-Induced Senescent Cells
The HIEpiC cells were induced by 40 g/L d-galactose for 72 h and co-incubated with or without different concentrations of HEP (0–200 μg/mL). The methyl thiazolyl tetrazolium (MTT) assay was conducted to assess the cell viability. Senescence-associated β-galactosidase staining (operational procedure according to the kits’ instructions) was used to identify the senescent cells. The activities of malondialdehyde (MDA), total superoxide dismutase (T-SOD), and glutathione peroxidase (GSH-Px) were measured. The protein concentration of cells was determined using the Coomassie Brilliant Blue G250 assay. The enzyme activities, level of MDA, and protein content were all determined using the detection kits purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). The procedures were performed according to the manufacturer’s instruction. The levels were normalized to the protein concentration of each sample and expressed as a percentage of non-treated controls.
Antitumor Experiment
The CC531 cells were cultured in the RPMI-1640 medium (containing 10% calf serum), placed in an incubator at 37°C with 5% CO2 and saturated humidity. The culture medium was replaced every 2 days, and the adherent cells were digested using 0.05% trypsin when the cells reached 80% confluence after 7 days of adaptation period. The logarithmic-phase human prostate cancer cell line CC531 was prepared to a concentration of 1.0 × 107 cells/mL. Each mouse was injected subcutaneously with 0.2 mL of cell suspension.
Two weeks later, the minimum and maximum diameters of the tumor body were measured. Then, 24 moderately sized mice were chosen and divided into three groups, including HEP3 high-dose group [HH, 100 mg/(kg ⋅ day)], HEP3 low-dose group [HL, 50 mg/(kg ⋅ day)], and model group, with eight mice in each group, and another eight normal mice as the normal group. The volume of the dose was 0.2 mL per mice per day. The model and normal groups were given equivalent volume of phosphate-buffered saline (PBS). Three weeks later, the rats were anesthetized by intraperitoneally injecting 2% sodium pentobarbital (0.25 mL/100 g), decapitated, and dissected. The blood plasma was collected from the orbit, and the serum by centrifugation (1,500 rpm, 10 min). Then, the serum was used to monitor the production of tumor-associated cytokines TNF-α, interferon (IFN)-γ, M-CSF, transforming growth factor (TGF), and VEGF. All the assays were carried out according to the procedures recommended in the ELISA kit manual. The mice were sacrificed by cervical dislocation. The tumor tissue was stripped off, and the tumor inhibition rate (TIR) was calculated. The sample was stored in liquid nitrogen for further use.
Microbiome Analysis
Fresh fecal samples were collected before the fasting of the rats and stored at −80°C. Frozen microbial DNA isolated from mice cecal sample with the total mass ranging from 1.2 to 20.0 ng was stored at −20°C. The microbial 16S rRNA genes were amplified using the forward primer 5′-ACTCCTACGGGAGGCAGCA-3′ and the reverse primer 5′-GGACTACHVGGGTWTCTAAT-3′. Each amplified product was concentrated via solid-phase reversible immobilization and quantified by electrophoresis using an Agilent 2100 Bioanalyzer (Agilent, USA). After quantifying DNA concentration using NanoDrop spectrophotometer, each sample was diluted to a concentration of 1 × 109 mol/μL in the Tris–EDTA buffer and pooled. Then, 20 μL of the pooled mixture was used for sequencing with the Illumina MiSeq sequencing system according to the manufacturer’s instructions. The resulting reads were analyzed as described in a previous study (28).
Hematoxylin and Eosin (HE) Staining and Immunohistochemical Analysis
Tissues from the mice or rats were freshly excised and fixed in 10% triformol. Once the samples were fixed, dehydration, clarification, and inclusion were carried out. After the blocks were obtained, the sections were cut using a microtome (Microm HM325, Germany), with a thickness of 5 μm. Sections of hydrated and deparaffinized tissues were stained with HE followed by appropriate method for histological observation. From each colon description, 10 sections were analyzed by three independent observers (JM, EM, and RMC).
The paraffin-embedded slices of colon tissue (4 μm) were incubated overnight with anti-NF-κB p65, anti-Foxp3, anti-IL-10, and anti-TNF-α primary antibodies at 4°C; all the antibodies were purchased from Abcam (Cambridge, UK). The slices were then washed with PBS and incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. After washing with PBS again, the slices were developed using 3,3′-diaminobenzidine as a chromogen and counterstained with hematoxylin. Images were acquired using a Leica DM2500 system (Leica Microsystems, Germany).
Statistical Analysis
All data were expressed as means plus SDs of at least three independent experiments. The significant differences between treatments were assessed with one-way analysis of variance or Student’s t-test at P < 0.05 using the Statistical Package for the Social Sciences (SPSS; Abacus Concepts, CA, USA) and Prism 5 (GraphPad, CA, USA) software.