Twenty-four Wistar albino were included in this study. Rats were divided into three groups. Group 1 (n = 8) was sham group. Group 2 is the group of chemically induced by intrarectal administration of trinitrobenzene sulfonic acid (TNBS) resulting in colitis. Group 3 (n = 8) is the group that was treated 7 days before and 7 days after with H. erinaceum resulting in colitis. The activity of colitis was evaluated macroscopically and microscopically in rats. In other words, nitric oxide (NO) levels, malondialdehyde (MDA), interleukin 6 (IL-6), nuclear factor-kappa B (NF-κB) and, tumor necrosis factor-α (TNF-α) in addition to the myeloperoxidasem (MPO) activities was determined.
The rate of TNBS-induced colitis caused to increase the level of MDA activities meaningfully in the colitis group than the control group. The results indicated that MDA (p = 0.001), NO (p = 0.001), IL-6 (p = 0.001), MPO (p = 0.878), TNF-α (p = 0.001), and NF-κB levels of treatment group decreased in the blood and colon tissues because of the H. erinaceum treatment when compared to the colitis group. H. erinaceum treatment was related to the declining of MDA, NF-κB, NO, IL-6, and TNF-α levels.
Inflammatory bowel disease (IBD) is a chronic inflammatory disease of unknown cause, and effective treatment options have not been discovered [1]. Recent data increasingly indicate that signaling molecules and pathways are responsible for the onset and progression of the disease. There is evidence that the increased regulation of inflammatory mediators, such as cytokines, chemokines, and adhesion molecules, plays an essential role in both human and experimental colitis models [2,3]. Nuclear factor-kappa B (NF-κB) is responsible for the gene expression regulation of inflammatory mediators such as tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), and nitric oxide synthase (iNOS) [4,5]. NF-κB is a key molecule involved in intestinal inflammation [6]. Studies that investigate the effect of anti-inflammatory drugs on the insula in IBD are difficult. For this reason, studies use animal models to investigate approaches to reduce inflammation and to activate various pathways.
Hericium erinaceus (HE), known as lion’s mane, is a medicinal mushroom and has been historical used in traditional Chinese medicine. HE mycelium and fruit bodies show great promise as substances that improve health such as brain and nerve health. The animal and in vitro experiments that investigated substances extracted from HE showed good results. In particular, the mycelium extract has been used clinically as an anti-inflammatory agent and in cytotoxic and cardiovascular antineoplastic research as an antibacterial and neuroprotective agent [7,8]. HE represents an important opportunity to treat Alzheimer’s and Parkinson’s diseases because HE is used as an anti-inflammatory medicine and promotes nerve growth factor gene expression.
The aim of this study was to evaluate the protective effect of HE treatment in a TNBS-induced colitis rat model.
METHODS
Twenty-four Wistar albino rats (225 to 275 g) were included in this study. Rats were kept in wire-mesh bottomed cages under a 12-hour light/12-hour dark cycle at a constant room temperature (22ºC ± 2ºC). A standard chow diet and water were provided. The study was approved by the Istanbul University ethics committee (number 2013-88).
Chemical reagent
2,4,6-Trinitrobenzene sulfonic acid (TNBS) was purchased from Sigma-Aldrich. The chemical formula of TNBS is C6H3N3O9S. Its other names are picrylsulfonic acid and trinitrobenzene. It has extreme oxidative properties. HE plays an antioxidative role because of its active substances and reducing ability. Its ethanol and hot water extracts have very high antioxidant activity in vitro. HE was purchased from Health for Life Trading Co., Ltd. HE (250 and 500 mg/kg/day body weight) was administered orally to rats.
Induction of colitis
Rats were slightly anesthetized with ether after a 24-hour fast. A 5-French polyurethane cannula was placed into the anus, and the tip was inserted to 8 cm proximal to the anal verge [9]. TNBS (10 mg/0.25 mL), which was dissolved in 50% ethanol, was inoculated into the colon through the cannula to induce colitis. Then, the rats were headstand to observe complications. The rats with complications were excluded from this study, and a new experimental subject was included in this study.
Three groups with an equal number of animals were formed. Group 1 was the sham group (n = 8). After catheterization, the anesthetized rats were substituted with 0.9 mL of 0.8% NaCl through the rectum. Group 2 was the colitis group (n = 8). To induce colitis, after catheterization through rectum, the mixture (35 mg/0.2 mL TNBS and 0.4 mL 50% ethanol) was administered to the anesthetized rats. Group 3 was the treatment group (n = 8). After catheterization and colitis induction, 35 mg/0.2 mL and 0.4 mL 50% of ethanol were administered to the anesthetized rats rectally administered TNBS. Additionally, the rats received HE (250 and 500 mg/kg/day body weight) orally 7 days before colitis induction and 7 days after colitis induction. The colon weight and colon length of each rat were recorded.
Surgical procedure
The surgical procedure under sterile conditions was applied to the experimental group on the 7th day of treatment. Intramuscular administration of 70 mg/kg ketamine hydrochloride and 7 mg/kg xylazine was used to anesthetize all Wistar albino rats. General anesthesia and spontaneous respiration were used. Povidone iodine was used to clean the abdominal region. The part of the colon with induced colitis was transected distal to the rectum as low as possible, and a nearly 9 cm long colonic segment was collected. The segment was opened longitudinally, and 0.8% saline solution was used to rinse the feces. For the analysis of biochemical parameters, 4 mL blood was collected. For biochemical and histopathological research, tissue samples were collected and transferred to a 0.8% NaCl solution or 9% formaldehyde solution. Blood samples were centrifuged and stored at –80ºC for biochemical research. Last, rats were sacrificed using a lethal dose of thiopental sodium
Microscopic score
The colon was divided into two parts for pathologic and biochemical studies after the macroscopic evaluation of the mucosa. The histopathological study was performed at Istanbul University. Colon tissue was separated into 5 mm sections and fixed in 9% formaldehyde [10]. After embedding in paraffin and staining with hematoxylin-eosin, 5 mm sections were prepared and evaluated with a light microscope. Inflammatory infiltration, thrombosis, apoptosis, necrosis, and vascular congestion were analyzed. All tests and microscopic alterations in the mucosa were evaluated from 0 to 3 by a histopathologist blinded to the groups.
Biochemical evaluation of the tissue and blood
For the biochemical assays, blood and terminal ileum were obtained. Blood samples were collected using a sterile needle by intracardiac puncture. Next, blood samples were obtained, centrifuged and separated into serum samples. Nitric oxide (NO), NF-κB, TNF-a, malondialdehyde (MDA), myeloperoxidasem (MPO), and IL-6 were evaluated. Tissues were rinsed with serum, weighed and homogenized for biochemical investigation. Distal colon samples were collected for biochemical evaluation. To detect inflammation, IL-6, MPO and TNF-a were evaluated. To determine the rate of apoptosis, NO and MDA, which can indicate the extent of oxidative damage, were investigated.
MPO measurement
Tissues were homogenized, incubated in 0.5% hexadecyl-trimethylammonium bromide (pH 5.5) and 0.026% ortho-dianisidine dihydrochloride, and 0.018% H2O2 was added. The reaction time was half an hour. The reaction was validated with sodium azide. All tests were performed twice and checked.
MDA measurement
MDA and thiobarbituric acid were examined by using a spectrophotometric procedure. Then, 0.2 mL of serum, 25 µL of butylated hydroxytoluene (pH 7.4) and 0.8 mL of tamponated phosphate were mixed. The prepared solution was mixed with 0.5 mL of 30% trichloroacetic hydro barbituric acid, put on ice for 2 hours and then centrifuged at 25°C for 15 minutes at 2,000 ×g. After centrifugation, ethylene diamine, tetra acetic thiobarbituric acid (0.070 mM) and 0.9% thiobarbituric acid (0.20 mL of 0.1 mol/L) was added to each mL. The supernatant was reserved in boiled water and left to cool at room temperature for 15 minutes. Spectrometric measurement was performed for the last prepared supernatant at 532 nm wavelength. The outcomes were recorded as nmol/mL.
NO measurement
The serum NO level was analyzed with Griess reagent. For the first phase, nitrate reductase was used to degrade the nitrate in the serum. For the second phase, nitrogen purple was improved by Griess reagent. Zinc sulfate was added to this mixture and centrifuged at 10,000 ×g for 4 minutes. The measurement was performed using azo dye by chromatographic spectrometer. The outcomes were recorded as nmol/mL.
TNF-α and IL-6 measurements
An enzyme-linked immunosorbent assay was used to measure TNF-a and IL-6. The lowest values that could be determined were 0.12 pg/mL and 0.03 pg/mL for TNF-a and IL-6, respectively.
NF-κB measurement
The colon of rats was embedded in 10% buffered formalin. Paraffin blocks were cut into 5 µm slices. The paraffin-embedded specimens were deparaffinized and postfixed in 100% acetone for 5 minutes to block endogenous peroxidase activity. Antigen retrieval was performed, and the sections were incubated with primary rabbit polyclonal anti-NF-κB antibody (anti-rabbit P50Ab-2) for 30 minutes at room temperature. Then, the slides were treated with biotinylated secondary antibody and streptavidin-conjugated horseradish peroxidase. Hematoxylin was used to stain for visualization [11].
Statistical evaluation
The results were evaluated as the mean and standard deviation. All data are expressed as the mean ± SD. Kruskal-Wallis and analysis of variance were used for statistical analysis, and p < 0.05 was accepted as significant (SPSS version 25.0 for Windows, IBM Co., Armonk, NY, USA).
Macroscopic colitis score
The macroscopic score was markedly increased in all rats administered TNBS compared with the rats in the sham group (p < 0.001). Macroscopic injury of the colon on the 7th day after TNBS administration indicated hyperemia, ulceration, colonic wall thickness and severe adhesions between the colon and other organs. HE treatment markedly reduced the macroscopic score in TNBS-treated animals (p < 0.001).
To investigate colitis formation, the length of the colon and the weight of the colon in the sham, HE treatment and nontreatment groups were compared. In the HE-treated group, the length of the colon was similar to that of the sham group, and the weight of the colon was similar to that of the sham group. Colon weight was increased, and colon length was decreased in the untreated group
Microscopic colitis score
The microscopic score was markedly higher in all rats administered TNBS than in the rats in the sham group (p < 0.001) (Fig. 2). After treatment with HE and TNBS, the pathologic score decreased markedly (p < 0.001). Fig. 3 shows that HE administration resulted in less mucosal injury, healed mucosal structure, and epithelial integrity. According to immunohistochemistry analysis, NF-κB expression was higher in the rats from group 2 higher than in rats from group 3.
Biochemical results
The colonic MDA levels are shown in Fig. 4. The oxidative damage parameters (NO and MDA levels) were compared among the groups. The level of the sham group was markedly different from that of the treatment group and colitis group. The blood and tissue levels of NO and MDA were markedly decreased in the HE-treated group compared with the colitis group (blood NO, p = 0.001; tissue NO, p = 0.001; blood MDA, p = 0.001; tissue MDA, p = 0.001). The comparison of oxidative damage is shown
IL-6 and TNF-α levels in blood and tissue were analyzed as inflammatory parameters. The levels in the treatment group and colitis group results were markedly different from the levels in group 1 (p = 0.001). Compared with the colitis group, group 3 showed substantially decreased levels. Another parameter of inflammation in tissue is MPO level. The MPO levels of the treatment and colitis groups were not significantly different (p = 0.878). The inflammatory damage results are shown in Table 2.
The level of NF-κB expression in the untreated group was high (p < 0.01). The NF-κB expression level was lower in the HE treatment group than in the TNBS-induced colitis model