Therapeutic effects of lentinan on inflammatory bowel disease and colitis‐associated cancer

Abstract In this study, we investigated the therapeutic potential of lentinan in mouse models of inflammatory bowel disease (IBD) and colitis‐associated cancer (CAC). Lentinan decreased the disease activity index and macroscopic and microscopic colon tissue damage in dextran sulphate sodium (DSS)‐induced or TNBS‐induced models of colitis. High‐dose lentinan was more effective than salicylazosulfapyridine in the mouse models of colitis. Lentinan decreased the number of tumours, inflammatory cell infiltration, atypical hyperplasia and nuclear atypia in azoxymethane/DSS‐induced CAC model. It also decreased the expression of pro‐inflammatory cytokines, such as IL‐13 and CD30L, in IBD and CAC model mice possibly by inhibiting Toll‐like receptor 4 (TLR4)/NF‐κB signalling and the expression of colon cancer markers, such as carcinoembryonic antigen, cytokeratin 8, CK18 and p53, in CAC model mice. In addition, lentinan restored the intestinal bacterial microbiotal community structure in IBD model mice. Thus, it shows therapeutic potential in IBD and CAC model mice possibly by inhibiting TLR4/NF‐κB signalling‐mediated inflammatory responses and disruption of the intestinal microbiotal structure.


| INTRODUCTION
Inflammatory bowel disease (IBD) is a disease that affects millions of people and includes ulcerative colitis (UC) and Crohn's disease (CD).
IBD is a critical risk factor for colitis-associated cancer (CAC), which was first recognized by Crohn and Rosenberg in 1925 and accounts for one-sixth of all deaths in UC patients. [1][2][3] However, currently available drugs to treat IBD and prevent CAC are not satisfactory.
Hence, new and improved drugs are necessary.
IBD is influenced by genetic and environmental factors, infections and immune system disorders. 4 TLRs are the key mediators of innate host defenses in the intestine that maintain mucosal immune homoeostasis. Toll-like receptor 4 (TLR4) signalling induces the inflammatory response through the activation of nuclear factor kappa-lightchain-enhancer of activated B cells (NF-κB). 5 Recent evidence shows that intestinal microbiota also plays a critical role in the pathogenesis of IBD. 6 For centuries, Chinese culture has treasured mushrooms as a health food, an "elixir of life." 7 Mushrooms have been consumed worldwide to maintain general human health because of the general understanding that they have antioxidant, anticancer, antidiabetic, antiallergic, immunomodulating, cardioprotective, anticholesterolaemic, antiviral, antibacterial, antiparasitic, antifungal, detoxification and hepatoprotective effects; they also protect against inflammatory processes and tumour development. [8][9][10][11] The consumption of dietary mushrooms also decreases breast cancer risk in postmenopausal women. 12 A number of bioactive compounds have been identified in mushroom species.
The main constituents are polysaccharides. 13 A hot-water mycelial extract, called lentinan, has been widely used as a preventive and therapeutic agent for cancer patients in Japan and China. [14][15][16] It is a polysaccharide with β-1,3-linked glucose backbone and β-1,6-linked glucose branches. 17 Lentinan shows beneficial effects in DSS-induced acute colitis mouse model because of its intestinal anti-inflammatory activity. 18 However, the therapeutic potential of lentinan in chronic UC, CD and CAC remains unknown. We have suggested that lentinan can exhibit therapeutic effects both on UC and CD and prevent CAC. Therefore, we investigated the therapeutic efficacy of lentinan in chemically induced acute and chronic colitis and CAC mouse models.

| Reagents
Lentinan was obtained from Kaifeng Pharmaceutical Co. (Henan, China). The structure of lentinan is shown in Figure S1  and bred under specific pathogen-free conditions. The experiments were performed according to a protocol approved by the Animal Ethics Committee of the Tianjin International Joint Academy of Biotechnology and Medicine. The mice were allowed to acclimatize for 7 days before being used in any experiments. Different DSS concentrations and administration cycles induce acute or chronic colitis model in mice. [19][20][21] Rectal administration of TNBS induces pathology similar to human CD. 22 Mice were divided into the following six groups (n = 10/group) for the DSS-induced mouse model: normal control (Normal); model control (Model); SASP (Sulfasalazine); and low-, medium-and high-dose lentinan (5, 10 and 20 mg/kg, respectively) groups. For the TNBS-induced colitis model, 19,20 an additional solvent control group (Ethanol) was added in addition to the above groups. DSS-induced acute colitis was induced in C57BL/6 mice by oral administration of 5% DSS in drinking water for 8 days. DSSinduced chronic colitis was induced in C57BL/6 mice by administering 1% DSS orally for 21 days according to previously published protocols. Fresh DSS solution was prepared every day. Normal control mice were given drinking water only. TNBS-induced colitis model was induced in BALB/c mice by administering an enema of 120 mg/ kg TNBS in 50% ethanol. 22,23 The normal control and solvent control groups were administered with enemas of normal saline and 50% ethanol respectively. A schematic overview of the animal model established in this study and the lentinan administration methods used are shown in Figure 1A. C57BL/6 mice were used for DSS-induced colitis because cryptitis and crypt abscesses can be found in half of the DSS-treated C57BL/6JOlaHsd mice, while in BALB/cAnNHsd mice, cryptitis was rarely observed. Reepithelization of the distal part of the colon can be found as early as day 5 of DSS treatment in C57BL/6JOlaHsd but not in BALB/cAnNHsd mice. 24 Moreover, BALB/c mice were used for induction of colitis by TNBS because BALB/c strains are susceptible to TNBS induction, while C57BL6 are resistant. 20,25

| Colitis model and lentinan treatment
The long-term retention test of lentinan showed that lentinan was stable under the conditions of temperature (23-27°C) and relative humidity (50%-70%) for 12 months. The study of tissue distribution of lentinan in mice after lentinan intraperitoneal injection showed that lentinan was mainly distributed in the spleen and liver and less distributed in the heart, lungs and kidneys (the above data were pro- widely used as an anti-colitis agent in human IBD therapy and serves as a positive control in studies of experimental colitis models. 28,29 It is indicated in the treatment of mild-to-moderate UC and as adjunctive treatment of severe UC. 30 The C57BL/6 mice of the model and normal control groups were pretreated with 20 mg/kg lentinan and ddH 2 O daily for 7 days. Then, the model group mice were administered with 5% DSS in drinking water for 7 days, whereas the normal control mice were administered with normal drinking water only. The mice were monitored for body weight, stool consistency and bloody stool every day until they were killed. The colitis assessments were performed as described in the Data S1.

| Western blot detection
Detailed information is provided in the Data S1.

| Immunofluorescence analyses
Detailed information is provided in the Data S1.

| Immunohistochemical analyses
Detailed information is provided in the Data S1.

| Dual-luciferase reporter assays
Detailed information is provided in the Data S1.

| 16S rRNA sequencing of intestinal microbiota
The detailed protocol is shown in the Data S1.

| Statistical analysis
Data were analysed using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA) and expressed as the mean ± SD. Differences between groups were analysed by the Mann-Whitney U test and one-way analysis of variance. P < 0.05 was considered statistically significant.  Figure 1B). The disease activity indexes (DAIs) of both lentinan-treated and SASP groups were lower than that of the model group ( Figure 1C). Moreover, the medium-and high-dose lentinantreated groups had lower DAIs than the SASP group ( Figure 1C).
Furthermore, lentinan and SASP treatments prevented colon length shortening. The colon length was longer in the high-dose lentinan group than in other groups, indicating that high-dose lentinan was more effective in maintaining colon length than SASP in the DSSinduced acute colitis model (8.57 ± 0.40 cm vs 7.91 ± 0.43 cm, P = 0.002; Figure 1D). These results demonstrate that lentinan attenuates colitis more effectively than SASP.

| Lentinan alleviates colonic damage in the IBD mouse model
We observed the disappearance of mucosal folds, visible erosion, ulcers, mucosal hyperaemia and oedema in the colon tissues of DSS/ TNBS-induced IBD model mice (Figure 2A Figure 2B). This result suggested that lentinan decreased colitis severity more effectively than SASP.

| Lentinan pre-treatment prevents DSS-induced mouse models of colitis
We also investigated if lentinan prevented DSS-induced mouse models of colitis by treating a group of mice with lentinan (20 mg/kg) before DSS induction. The lentinan pre-treated mice showed lower DAIs  Figure 3D). In addition, the histological scores of lentinan pretreated mice were lower than those of the model group mice (1.75 ± 0.96 vs 5.25 ± 0.96, P = 0.002; Figure 3E). Lentinan pre-treated mice also showed more intact mucous epithelium and lower neutrophil infiltration than untreated mice ( Figure 3E).

| Lentinan inhibits TLR4-induced inflammation in vitro
Mucosal immune disorders lead to IBD as a result of TLR4 activation results in NF-κB activation and cytokine expression in macrophages. 32 We investigated if lentinan inhibited lipopolysaccharide (LPS)-stimulated TLR4 signalling and reduced IBD progression.
As shown in Figure 5A

LPS-induced TLR4 activation increases calcium influx followed by
CaMKII activation. 33 We observed that lentinan treatment decreased calcium influx in a concentration-dependent manner ( Figure 5B). This finding suggested that lentinan decreased TLR4 signalling. Furthermore, Western blot, immunofluorescence and immunohistochemistry experiments showed that lentinan reduced the translocation of NF-κB-p65 to the nucleus ( Figure 5C-E). Dual-luciferase reporter gene assay also demonstrated that lentinan reduced NF-κB activity ( Figure 5F). We also tested the efficacy of lentinan treatment in another colon epithelial carcinoma cell line, Caco-2, and found that lentinan inhibited TLR4 pathway and nuclear translocation of NF-κB ( Figure S2).

| Lentinan decreases the levels of IL-13 and CD30L in LPS-induced inflammation in vitro
The inflammatory cytokine antibody assay showed that lentinan also decreased the expression of the downstream inflammatory cytokines regulated by NF-κB. As shown in Figure 6A, LPS induced 40 different types of inflammatory cytokines in RAW 264.7 macrophages, but lentinan treatment reduced the levels of these LPS-induced inflammatory cytokines. Most significantly, 1 mg/mL of lentinan decreased the expression of IL-13 and CD30L, which are critical inflammatory factors ( Figure 6A,B). Clustering analysis demonstrated that the inflammatory cytokines in lentinan-treated RAW 264.7 cells stimulated by LPS were more similar to control cells without LPS stimulation ( Figure 6B).

| Lentinan reduces the expression of IL-13 and CD30L and colon cancer markers in CAC mouse models
Immunohistochemical analysis of colorectal tissues from the CAC mouse models showed that lentinan decreased the expression of IL-13 and CD30L as well as the colon cancer markers such as CEA, CK8, CK18 and p53 ( Figure 6C,D).

| Lentinan restores the intestinal floral structure
IBD is facilitated by immune hyper-responsiveness towards certain intestinal microbiota, which is critical in stimulating mucosal F I G U R E 6 Lentinan inhibits the expression of inflammatory cytokines and colon cancer markers. A, Cytokine antibody chip assay analysis of the concentrations of cytokines from RAW264.7 cells treated with 1 mg/mL LPS in the presence or absence of 1 μg/mL lentinan, detected by cytokine antibody chip assay. B, Grouping analysis of the cytokine antibody chip assay results. C, Representative images of IHC staining of the cancer markers CEA, CK8, CK18 and p53 (all in brown) and the cytokines IL-13 and CD30L (both in red) in colon tissues from CAC model mice treated with or without lentinan. The nuclear staining is coloured blue. D, Quantitative analysis of cancer markers and inflammatory cytokines based on the IHC staining in 5C. Data are represented as mean ± SD. *denotes P < 0.05; **denotes P < 0.01 immune disorders. 34 Figure 7B). This finding is consistent with a previous study that reported a decrease of Firmicutes and an increase of Proteobacteria in IBD patients. 37,38 Cluster analysis showed that the gut microbiota at the genus level in the lentinantreated mice was more similar to normal mice without colitis induction ( Figure 7C). Specifically, gut microbiota such as Eubacterium and Parabacteroides were reduced to nearly normal levels after lentinan treatment ( Figure 7D). The diagram in Figure 7E suggested that lentinan rebalances the intestinal microbiota in mice or protects the good bacteria and intestinal mucosa from harmful bacteria or other agents.

| DISCUSSION
IBD is a severe disease that results in persistent patient discomfort and poor quality of life. Most of the drugs that are commonly used to treat IBD are associated with adverse effects. 39,40 Therefore, there is an urgent need to find new drugs that effectively treat IBD without severe adverse effects.
Recently, edible ginger-derived nanoparticles were found to be effective in the prevention and treatment of IBD and CAC. 41,42 The immunomodulatory, anti-inflammatory and anti-carcinogenic effects of glucans, which are abundantly found in edible mushrooms, have been reported in numerous studies. These polysaccharides show high efficacy and low toxicity than other therapeutic agents in clinical use. 43,44 Lentinan is an anti-tumour polysaccharide from Lentinula edodes, which is highly effective against DSS-induced acute colitis. 20 In the present study, lentinan suppressed body weight loss and colon length shortening and improved histological scores in DSS-induced acute colitis, chronic colitis and TNBS-induced model mice. Highdose lentinan treatment provided better effects than the positive control drug SASP. Both the dose of lentinan and SASP used in this study were converted from the maximum clinical dose of the two drugs, and thus the treatment effects are comparable. These findings suggest that lentinan has immense therapeutic potential for IBD patients. Lentinan has been used clinically for many years and has very few and mild adverse effects. Lentinan prevented IBD in the present study. Moreover, lentinan inhibited tumour formation, infiltration of inflammatory cells and atypical hyperplasia in AOM/DSSinduced CAC mice model.
Studies have shown that environment, genetics and host immunity form a multi-dimensional, highly interactive regulatory triad controlling TLR function in the intestinal mucosa. Aberrant TLR signalling owing to environmental perturbations or genetic mutations or changes in host immunity cause acute and chronic intestinal inflammation that eventually lead to IBD and CAC if not effectively treated. 34,45,46 LPS is an endotoxin released by Gram-negative F I G U R E 7 Lentinan restores intestinal bacteria microbiotal community structure in acute colitis model mice. A, Bacteroidetes, Firmicutes and Proteobacteria were the dominant bacteria at the phylum level in the mouse faecal samples. Lentinan-treated mice were more similar to normal mice. B, Lentinan treatment recovered the Firmicutes/Proteobacteria ratio to nearly normal levels. C, Cluster analysis showed that the gut microbiota at the genus level in the lentinan-treated mice was more similar to normal mice without acute colitis. D, Gut microbiota such as Eubacterium and Parabacteroides reduced to nearly normal levels after lentinan treatment. E, Schematic showing the role of lentinan in protecting the intestinal bacteria microbiota and mucosa bacteria, which binds and activates TLR4 in macrophages. 47,48 This results in the activation of the MyD88 and TRIF intracellular signalling pathways, release of calcium ions and translocation of NF-κB, thereby inducing the expression of pro-inflammatory cytokines that can lead to IBD and CAC, if persistent. 49,50 The effects of lentinan on inflammation have been previously studied. Ahn found that lentinan attenuates AIM2 and non-canonical inflammation while increasing pro-inflammatory cytokines via TLR. 51 In the current study, we found that lentinan down-regulated LPSinduced TLR4-mediated NF-κB signalling pathway and the expression of various inflammatory cytokines, which are key factors in leucocyte extravasation, colitis and CAC. 52 For example, it reduced the expression of IL-13 and CD30L, which are two key factors in IBD and CAC. 53,54 We suppose that the inflammation-modulating properties of lentinan may vary depending on cell type and the inflammatory microenvironment. The glycan chains of lentinan may interfere with the binding of TLR4 to LPS and inhibit TLR4 signalling and inflammation.
Intestinal bacteria microbiota constitutes a complex microecosystem that plays a crucial role in preventing IBD and CAC.
Intestinal microbiotal imbalances trigger intestinal mucosal immune disorders. 55 The findings of this study show that lentinan promotes the recovery of the intestinal bacteria microbiotal community structures in IBD mice, which further attenuates the factors that induce IBD. We found that gut microbiota such as Eubacterium and Parabacteroides increased in IBD model mice, while lentinan reduced the relative abundance. There are conflicting reports on the role of Parabacteroides and Eubacterium in IBD. The abundance of Parabacteroides was increased in colitis-prone NHE3−/− mice but was decreased in colitis-prone IL22−/− mice. 56,57 Previous studies have found decreased and increased colonization of Eubacterium in UC and CD patients respectively. [58][59][60] This study may enrich the relevant knowledge of intestinal microbiota changes in IBD.

| CONCLUSIONS
Our study proved that lentinan is effective in treating IBD and CAC in mouse models. The inhibition of the TLR4 signalling pathway and recovery of intestinal bacteria microbiotal structures may be the potential pharmacological mechanism of lentinan in mouse models of IBD and CAC. Therefore, we demonstrate that lentinan has promising therapeutic potential in IBD and CAC treatment and prevention. 15PTGCCX00140).

CONFLI CT OF INTEREST
The authors declare that they have no competing interests.