Inflammatory bowel disease (IBD), a chronic and relapsing inflammation of the gastrointestinal tract, encompasses two types of intestinal inflammation: ulcerative colitis (UC) and Crohn’s disease [1
]. UC is a complex and debilitating disorder that is characterized by the presence of discontinuous lesions in the rectal and colonic mucosa [2
]. While the incidence rate of UC is high in Western countries, its occurrence in East Asian countries, such as Korea and Japan, has also increased in recent years on account of an increasing intake of a Westernized diet, characterized by high protein and fat content, as well as excessive sugar intake, with lower fiber consumption [3
Although the etiology and pathogenesis of UC remain obscure, increasing evidence suggests that the dysregulation of mucosal immunological function is responsible for the initiation and propagation of this disease. UC is predominantly triggered by the epithelial invasion of intestinal microbiota due to the loss of epithelial layer integrity [5
]. The resulting overproduction of pro-inflammatory cytokines, e.g., tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-6, induces colonic tissue damage and ulceration of the colon [6
]. The efficacy of conventional treatments varies and the commonly used drugs have long-term side effects [7
]. Thus, an effective preventive agent should be investigated, that would reduce inflammation for extended periods of time. Prebiotics are safe, indigestible food constituents, benefitting the host by selectively enhancing the growth of commensal microbiota in the gastrointestinal tract, that have been used for the treatment of IBD [8
Rice bran (RB) is a rich source of bioactive components, e.g., dietary fiber, vitamins, free amino acids, and antioxidants, which have a potential to promote gastrointestinal health [9
]. RB is presently available in most regions of the world as a by-product of rice polishing. The usage of RB for human consumption is limited, however, and RB is discarded or used as animal feed [10
]. Such RB treatments as fermentation, enzymatic processing, or fractionalization have been employed to increase its quality or render it edible for humans, in the form of a dietary supplement. Processed RB or fermented rice bran (FRB) are foodstuffs that are more enriched in such ingredients as protein, fiber, and phenolic compounds, than the usual raw bran [11
]. Therefore, it is extremely important to investigate the basic physiochemical and functional properties of FRB and to identify the mechanisms of action for its application as a nutraceutical.
Several studies have investigated the anti-colitis effects of processed RB in animal models, focusing on rice bran oil [12
], brown rice fermented by Aspergillus oryzae
], enzyme-treated rice fiber [8
], and RB fermented by Saccharomyces cerevisiae
and Lactobacillus plantarum
]. Processed RB is enriched for different ingredients to varying degrees because of different fermentation processes and preparation methods. Among these enriched ingredients, an essential amino acid, tryptophan, is considered an effective candidate agent against UC [7
]. In addition, in the gut, tryptophan metabolites of the microbiota, e.g., indole-3-aldehyde, kynurenine, indole-3-acetic acid, and tryptamine, can act as ligands for aryl hydrocarbon receptor (Ahr), a transcription factor that regulates IL-22
gene expression, controls autoimmunity processes, and promotes rapid recovery from colitis [15
FRB may also stimulate the microbial production of short-chain fatty acids (SCFAs), especially acetic acid (AA), propionic acid (PA), butyric acid (BA), and lactic acid (LA), which are strongly associated with the colonic health in human [16
]. SCFAs are produced by the microbiota in the course of breaking down complex carbohydrates, such as fiber, and are a primary source of energy for the enteric epithelium [16
]. The intestinal microbiota has a profound impact on the host immune system; dysbiosis of bacterial populations and suppression of SCFA production has been implicated in UC [17
]. Increased SCFA levels promote colonic epithelial cell proliferation, stimulate mucin production, and epithelial cell integrity. In particular, BA has been shown to induce colonic regulatory T cells and limit the innate immune cell-driven inflammation; it also has the potential to eliminate mutated epithelial cells through the induction of apoptosis [17
]. These activities contribute to the maintenance of colonic homeostasis by regulating the intestinal barrier integrity [18
]. The intestinal barrier integrity is crucial for maintaining the beneficial relationship between the host and the intestinal microbes; as it not only constitutes a physical barrier, preventing the entry of invading microorganisms, but also provide a defense mechanism by sensing pathogenic microorganisms or their toxins [19
Intercellular tight junction (TJ) complexes consist of multiple proteins, including occludin (OCLN) and claudin (CLDN), which mainly determine the intestinal barrier integrity [20
]. TJs are located at the apical ends of the lateral membrane of the epithelial cells; the loss of TJ barrier integrity is associated with the initiation and development of UC [20
]. Another characteristic of human UC is a pronounced depletion of mucin-producing goblet cells and the mucus layer, correlating with an increased microbiota-induced colonic inflammation and disease pathology [22
Dextran sodium sulfate (DSS) is commonly used in rodent models to chemically induce the intestinal inflammation [15
]. DSS administration leads to weight loss, bloody diarrhea, and immune cell infiltration, and to increased production of inflammatory mediators, similarly to human colitis [6
The purpose of the present study was to investigate the effects of dietary FRB supplementation on UC pathology in a murine model of DSS-induced UC. We used a unique FRB, in which RB was fermented by both Aspergillus kawachii and Lactobacillus sp., notably enriched with free tryptophan and tryptamine. We hypothesized that FRB supplementation would mitigate the symptoms of colitis and its clinical severity by enhancing the indole derivatives and colonic SCFAs. To evaluate the protective effect of FRB, we also determined the serum pro-inflammatory cytokines and colonocyte pro-inflammatory gene expression, which include genes responsible for tight junction’s barrier integrity, mucin production, and inflammatory cell infiltration in the inflamed sites. Our results indicate a protective effect of dietary FRB in DSS-induced UC in the mouse model, linked to modulation of colonic SCFAs and pro-inflammatory gene expression.
Dietary components have been recently revealed to exert remarkable beneficial effects on host physiology, and are currently targeted, or have high potential to be targeted, as treatments for human disease [28
]. UC is a gastrointestinal disease with rapidly increasing world prevalence. It is important to identify the dietary compounds and its mechanisms to ameliorate the progression of inflammation during UC. In the current study, we demonstrated that FRB completely protects the animals against DSS-induced UC, by improving body weight and stool consistency, as well as decreasing intestinal bleeding, compared to the control and RB diets. Histological analysis revealed lower crypt damage and inflammatory cell infiltration in FRB group animals than in RB and control group animals.
Pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, amplify the inflammatory cascade of inflammatory mediators, destructive enzymes, and free radicals that cause tissue damage [29
]. We observed elevated levels of TNF-α and IL-6 in the sera of RB and control group animals but not in FRB group animals. Furthermore, dietary FRB significantly inhibited the transcripts of these cytokines in the colonic tissue, similarly to another study [12
Chemokines act as chemoattractants for neutrophils during acute inflammation and prolonged inflammation [30
]. In the current study, we detected a significantly higher colonic expression of Ccl2
, and Cxcr3
genes in the control and RB groups (p
< 0.05, RB vs. control) compared to the FRB group, which supports the protective effect of FRB in colitis.
MPO is an important bactericidal marker in UC; elevated MPO levels are responsible for greater neutrophil influx [15
]. In the current study, MPO activity was significantly lower in the FRB group (p
< 0.05 vs. control) than in the RB group (p
> 0.05 vs. control). This indicated that the DSS-induced neutrophil infiltration in the colonic mucosa was suppressed by FRB supplementation.
The severity of chronic gut inflammation may result from a sustained overproduction of reactive oxygen metabolites and acute lipid peroxidation [31
]. In the current study, we also observed a significant elevation of TBARS levels in the control and RB groups, which indicate severe tissue damage in the RB group (p
> 0.05 vs. control), but not in the FRB group (p
< 0.05 vs. control).
Although the fermentation process may enrich the protein and dietary fiber content in FRB [11
], it is not known which specific FRB component contributes to the reduction of colitis. Here, during fermentation, FRB becomes enriched almost 10 times in tryptamine, which may act as an Ahr ligand [15
]. Ahr has been highlighted as an immunological regulator in DSS-induced inflammation by promoting the differentiation of Th17, or regulatory T cells, and IL-22 production by Th22 T cells, which in turn regulate the epithelial barrier function and intestinal homeostasis [32
]. The serum tryptophan levels were higher in the FRB group (p
= 0.0101 vs. control), but not in the RB group (p
> 0.05 vs. control). Low tryptophan levels are responsible for severe IBD complications and, thus, tryptophan containing diet supplementation comprises a novel therapeutic strategy for IBD treatment [7
Although high levels of dietary fiber and prebiotics increase the intestinal SCFA concentration [33
], few studies investigated the stimulation of SCFA production during UC by FRB. In the current study, fecal and colonic SCFA levels were significantly higher in the FRB group than in control and RB groups, as shown in Table 2
. The differences in SCFA levels, especially BA and LA levels, clearly reflect the UC-ameliorating potency of FRB compared to RB. Generally, the absorption of SCFAs by the colonic cell is rapid, and the colon absorbs more than 95% of the SCFAs produced [34
]. Cell-surface G-protein coupled receptors (GPRs), such as GPR41 and GPR43, are activated by SCFAs and exhibit anti-inflammatory effects to minimize inflammation. SCFAs, in particular BA, are known to induce apoptosis of T cells by inhibiting histone deacetylase, thus eliminating the source of inflammation in the colon [35
]. The amount and relative abundance of SCFA in the colon may be considered as biomarkers of a healthy status [37
]. Prebiotic substrates that selectively promote the growth of beneficial microbiota also induce changes in SCFA production in patients with irritable bowel syndrome [38
]. Thus, FRB supplementation may alter the gut microbiota.
AA, PA, and BA enhance the mRNA levels of intestinal TJs genes, the major determinants of intestinal barrier integrity, and play an important role in intestinal defense, as well as in the maintenance of intestinal homeostasis [18
]. Thus, the increased SCFAs levels by FRB supplementation may contribute to promote symbiotic intestinal environment, and prevent the development and progression of UC.
TJ proteins, especially OCLN, and CLDN1 and 4, are important for epithelial barrier integrity and seem to play a pivotal role in intestinal homeostasis [39
]. Our data suggest that FRB supplementation significantly enhances the expression of genes encoding TJ proteins in the inflamed colonic tissue. Protection of the epithelial TJ barrier integrity by FRB involves the suppression of a chronic and robust activation of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6. We also observed that the expression of a gene encoding another pro-inflammatory cytokine, IL-17
, which is responsible for augmenting autoantibody production in UC [40
], was lower in the FRB group than in the RB and control groups. It is tempting to speculate that the modulation of epithelial-immune interactions by dietary FRB supplementation might present a novel preventive strategy for UC.
The mucus layer protects the gastrointestinal mucosa from mechanical, chemical, and microbial challenge [41
]. Mucin 2 (encoded by Muc2
) is the most prominent mucin secreted by the goblet cells [41
]. High Muc2
mRNA expression was observed only in the FRB group. Thus, in the control and RB groups, reduced Muc2
expression in the intestine leads to abnormal tissue morphology, marked by an increased thickness of the gut mucosa, increased inflammatory cell infiltration, and robust pathogenesis of UC.
RB fermentation by A. kawachii
increases the flavonoid content in FRB [42
]; flavonoids are not only responsible for the smell and flavor, but also regulate innate immunity, inhibit the production of pro-inflammatory cytokines and, thus, reduce the severity of experimental colitis [43
]. An overview of the mechanisms of action of FRB underlying the protection against DSS-induced colitis is shown in Figure 6
In the present study, the effect of whole RB on UC is unclear. RB is rich in bioactive components, such as ferulic acid [12
]. Ferulic acid has both antioxidant and anti-inflammatory properties and its anti-colitis effect in a DSS model has been reported [12
]. Here, we have analyzed TBARS levels and it was found to be elevated in the RB group compared to that in the FRB group (Figure 2
F). Thus, a dual fermentation process may augment the antioxidant properties in FRB.
In the future, more evidence on different FRB fractions and active components should be collected to clarify their mechanism of action. Metabolomics analysis might also reveal multiple potential protective mechanisms of FRB, including immunomodulation to prevent UC. We anticipate that FRB will emerge as an excellent source of nutraceuticals, as well as a functional food.