1. Introduction
Inflammatory bowel disease (IBD) is characterized by diarrhea, bloody stools, abdominal pain, and weight loss. It is an umbrella term that can be used to refer to Crohn’s disease and ulcerative colitis (UC) [
1]. Albeit more commonly found in Western countries, emerging evidence suggests that IBD is increasingly prevalent in South America, Eastern Europe, Asia, and Africa [
2,
3]. IBD was originally considered to manifest in genetically vulnerable individuals, and there are currently 163 loci known to contribute to the onset of IBD. Examples of these genes are
NOD2 (which is related to innate sensing of bacteria) [
2,
4],
IL23R (which regulates inflammatory response to microbes) [
2],
CARD9 (which integrates signals from innate immune receptors and activates different pathways of cytokines in response) [
5], and
ATG16L1 (which controls autophagy) [
2]. Apart from genetic factors, environmental factors such as smoking, hygiene, dietary intake, and antibiotics usage are also considered to affect the onset of IBD [
4].
Various animal models have been introduced to mimic the symptoms of IBD, including chemically induced animal models using dextran sodium sulfate (DSS) [
6,
7,
8,
9] or 2,4,6-trinitrobenzene sulfonic acid [
10], bacteria-induced models such as Salmonella-induced colitis [
11], and genetically modified animals such as Il-10 [
12] or T cell receptor α [
13] knock-out mice. Among these models, DSS-induced colitis was noted to produce colitis symptoms that resemble those of UC in humans [
11]. The DSS-induced model is also notable for being easy to reproduce and manipulate [
14]. DSS-induced colitis in an animal model that is particularly prone to inflammation, such as C57BL/6 mice, was also noted to progress into chronic colitis instead of showing signs of mucosal healing after DSS was removed [
15,
16]. This animal model was suggested to be a good candidate to study the progression of acute epithelial inflammation into the subepithelial fibrosis in UC [
17].
Rice bran fermented with
Aspergillus kawachii and a mixture of lactic acid bacteria (fermented rice bran (FRB)), in particular, has been proven to be able to prevent the detrimental intestinal inflammation caused by DSS administration [
7]. Islam et al. [
7] initiated an FRB-supplemented diet in mice, 4 days before inducing intestinal inflammation by introducing DSS in their drinking water. FRB supplementation was found to elevate the amounts of short-chain fatty acids and tryptamine, a microbial metabolite from tryptophan in the intestine, which in turn might regulate the intestinal barrier tight junction and intestinal homeostasis. This effect was suggested to be induced partially due to the bioactive compounds of rice bran, such as polysaccharides [
18], glycoproteins [
19], γ-oryzanol, phytosterols, and vitamin E [
20]. Fermentation of rice bran (RB) has been suggested to make its bioactive compounds more accessible and easier to metabolize [
7,
21,
22]. A previous study has shown that fermentation enhances the amount of the total phenolic content in RB [
22]. Fermentation also increases the amount of tryptophan [
7], which is suggested to be able to ameliorate intestinal inflammation [
8]. Therefore, it is likely that FRB supplementation can be used not only as a preventive measure but also as a therapeutic agent against an ongoing intestinal inflammation.
While a previous study already investigated the beneficial effect of FRB supplementation against the onset of DSS-induced inflammation, it was also hypothesized that this effect was partially achieved via FRB prebiotic function [
7]. FRB was suggested to be able to promotes the symbiotic environment in mice intestine, thereby preventing the development and progression of DSS-induced colitis [
7]. However, changes in the composition of intestinal microbiota population are commonly established in patients with UC. Furthermore, the presence of intestinal microbiota and their metabolites were shown to alter intestinal defense mechanism against mucosal inflammation [
23]. Another study also reported impaired DNA integrity due to oxidation which persisted even after the intestinal inflammation has subsided [
24]. Hence, the usage of FRB supplementation as a therapeutic treatment rather than a preventive measure may not work as intended. This is an important distinction to make, especially in the case of IBD, since treatments in IBD are generally aimed to prevent a flare-up of an ongoing inflammation and to hinder its progression into an irreversible state such as stricture and altered colonic motility and permeability [
25].
Chronic inflammation in the intestine often results in intestinal fibrosis, as is often the case in patients with IBD. Fibrosis is characterized by excessive deposition of the extracellular matrix components, such as collagen, basement membrane proteins, and fibronectin, in response to chronic inflammation [
26]. While fibrosis may be considered a part of the normal wound healing process, uncontrolled and persistent fibrosis in injured tissues can cause anatomical alterations and loss of function [
27]. Intestinal fibrosis was found to cause strictures due to scar formation and tissue distortion [
26,
27,
28,
29]. IBD-associated fibrosis often leads to the loss of intestinal motility and permeability, causing loose stool or diarrhea even in the absence of inflammation [
25]. Intestinal fibrosis can also develop into intestinal blockage [
29], which requires treatment such as dilation and insertion of stents [
30] or even surgical intervention [
29]. FRB in diet has been shown to prevent the onset of intestinal inflammation [
7], and, thus, it might be able to reduce the risk of intestinal fibrosis. In the current study, we observed whether dietary supplementation with FRB would be able to enhance the recovery from DSS-induced intestinal inflammation in mice. We also evaluated FRB function in preventing the onset of intestinal fibrosis due to DSS-induced chronic inflammation in mice.
4. Discussion
The progression of DSS-induced inflammation and its development into intestinal fibrosis were observed in the current study. The presence of blood in the stool and diarrhea were observed on the fourth day after DSS administration was started (
Figure 1A). The mRNA levels of pro-inflammatory cytokines such as
Il-1β, Tnf-α, and
Il-6 (
Figure 3A–C) were found to increase after the administration of DSS was ceased, and their levels significantly decreased 5 weeks after DSS administration ended. Signs of epithelial loss were visible soon after DSS administration ended; however, its detrimental effect was at its peak 2 weeks after DSS administration was stopped (
Figure 2B). Five weeks after stopping the DSS administration, diarrhea was still found in the control group, and signs of crypt dysplasia were still visible, albeit intestinal epithelial regeneration had begun (
Figure 1A and
Figure 2E). This progression of DSS-induced colitis has been observed in other studies [
54,
55,
56]. DSS has been shown to form nanometer-sized vesicles with medium-chain-length fatty acids in the colon upon ingestion. These complexes fuse with the intestine cell membrane and deliver DSS into the cells, thereby disrupting the activity of the cells [
54]. DSS-induced inflammation starts to manifest several days after its ingestion and is marked by the spike of
Tnf-α in the colon [
55]. Tnf-α, followed by mucin depletion, leads to the erosion of intestinal epithelia, infiltration of inflammatory cells, and the upregulation of other pro-inflammatory cytokines such as
Il-1β,
Il-6, and
Il-17 at a later stage, along with crypt dysplasia [
15,
56]. Blood was visible in the stool several days after DSS-induced inflammation occurred, although it disappeared soon after DSS administration was stopped [
15]. On the other hand, diarrhea occurs at about the same time as the occurrence of fecal blood, and it persists weeks after the removal of DSS [
15,
48]. After stopping the administration of DSS, the intestinal epithelial layer started to recover, the amount of inflammatory cell infiltration decreased, and the intestinal crypts were regenerated. The levels of
Il-1β,
Il-6, and
Il-17 have been observed to remain high even after the removal of DSS [
15,
48,
56].
The mRNA level of
Il-10 (
Figure 4B) and
Il-22 (
Figure 5C) were also found to be at their peak after the DSS treatment was discontinued. The increase of
Il-10 and
Tgf-β, as anti-inflammatory cytokines, in the early days of DSS-induced inflammation has been described in a previous study, and their expression indeed decreased with the progression of colitis [
55].
Tgf-β is a pleiotropic cytokine, and its expression was reported to be increased in IBD tissues. It has been suggested that this cytokine is necessary to suppress inflammation in UC and promotes intestinal recovery [
57]. In the recovering intestine,
Tgf-β1 promotes the differentiation of fibroblasts into myofibroblasts by enhancing α-smooth muscle actin, modulating myofibroblast migration, and mediating myofibroblast extracellular matrix (ECM) production [
49,
58]. We found no statistical differences in the mRNA levels of
Tgf-β1 with diet treatments among all the groups (
Figure 8C); however, the combined effect of
Tgf-β and other cytokines and chemokines might have been a crucial factor in the deposition of ECM in the intestinal lining, which will affect the onset of fibrosis after DSS-induced inflammation.
Excessive deposition of ECM is considered to be one of the reasons for colon thickening and shortening after the onset of DSS-induced inflammation [
15,
48]. Following the administration of DSS, ECM deposition was apparent in the intestine (
Figure 6A). This symptom of disease progression has also been examined in other studies, in which ECM deposition was reported to be apparent in colon lamina propria, especially in the sites of severe crypt damage and inflammation after a single DSS cycle [
16]. Collagen deposition was also noted in submucosa and even in the focal region of the serosa layer during the recovery stage after a single administration of DSS treatment [
15,
16]. Since excessive collagen deposition may lead to the development of intestinal fibrosis, FRB seemingly reduced the risk of fibrosis due to DSS-induced colitis, by reducing the mRNA expression of
Col1a1 and
Col1a2 and increasing the level of
Mmp3 (
Figure 7A,B,D).
There are several ways through which
Tgf-β exerts its fibrogenic function. The canonical pathway of
Tgf-β signaling in fibrosis utilizes the activity of Smad2/3, and the non-canonical pathway involves the JAK1–STAT3 axis [
59,
60]. Dietary FRB supplementation decreased the level of Smad2/3 after 2 weeks. A longer period of supplementation decreased the levels of Smad2/3 in FRB group, yet increased its levels in RB group (
Figure 8A). FRB supplementation decreased the mRNA level of
Smad7 after 2 weeks and increased it back after 5 weeks of diet treatment, albeit this was not statistically significant (
Figure 8B). From these results, we can speculate that FRB supplementation is able to attenuate DSS-induced inflammation and the onset of fibrosis via the canonical pathway of
Tgf-β and Smad2/3 activation.
FRB might also affect the non-canonical pathway of
Tgf-β, as FRB supplementation sustained the mRNA levels of
Il-22 (
Figure 5C) and
Il-10 (
Figure 4B) and lowered those of
Tnf-α (
Figure 3B).
Il-22 promotes intestinal epithelial regeneration and wound healing, and therefore it also affects the development of intestinal fibrosis [
61,
62]. Furthermore,
Il-22 upregulates many antimicrobial proteins including Reg3γ and mucin via the expression of Stat3 [
8,
63].
Il-22 is known to have a synergistic effect with
Tgf-β, in increasing the proliferation of myofibroblasts and regulating the production of collagen [
61]. Additionally, Il-10 promotes the production of intestinal mucus by suppressing endoplasmic reticulum stress via attenuation of Stat1 and Stat3 [
64]. The STAT3-IL-10-IL-6 pathway has also been shown to regulate macrophage phenotype from pro-inflammatory to restorative [
65]. Similarly,
Tnf-α has been noted to promote collagen production via Tnfr2 and Stat3. The STAT3-IL-10-IL-6 pathway leads to the regulation of Timp-1 and Mmp2 activities, and therefore it affects collagen degradation. Additionally,
Tnf-α also interacts with Igf-1 to enhance myofibroblasts proliferation, which results in collagen accumulation [
66]. Therefore, it is understandable how FRB supplementation, which resulted in decreasing the level of
Tnf-α and maintaining the levels of
Il-10 and
Il-22, might have prevented collagen deposition and lowered the risk of fibrosis.
FRB supplementation also lowered the mRNA level of
Il-17 in the colon (
Figure 5B). The elevated level of
Il-17 and increased levels of other profibrogenic cytokines such as
Il-1β,
Tgf-β, and
Tnf-α were suggested to be involved in the pathogenesis of intestinal fibrosis [
50]. While
Tgf-β1 is shown to regulate Th17 development and differentiation,
Il-17 is also hypothesized to independently regulate the balance of matrix metalloproteinases and the tissue inhibitor of metalloproteinase (Mmp/Timp). This hypothesis was supported by a report which stated that anti-
Il-17 antibody treatment not only lowers the expression of
Il-1β,
Tgf-β, and
Tnf-α but also partially modulates the Mmp/Timp balance, indicating that other mechanisms must have been involved in the pathogenesis of intestinal fibrosis [
50].
Probiotic-derived soluble compounds which are either secreted by bacteria or released after bacterial lysis, often referred to as postbiotics, have been suggested as a novel strategy in maintaining the intestinal homeostasis. Previously, a protein secreted by
Lacticaseibacillus rhamnosus GG was reported to be able to protect mice from 3% DSS-induced colitis [
67]. GABA, protein p40, lactocepin 31, and other uncharacterized soluble molecules produced from
Levilactobacillus brevis BGZLS10-17 have also been suggested to modulate the immune response of Concanavalin A-stimulated mesenteric lymph node cells [
68]. Islam et al. [
7] stated that the level of tryptamine, a microbial metabolite of tryptophan, increased to about 10 times in fermented rice bran compared to non-fermented rice bran. Tryptamine, indole, and indole-acetic acid are secondary metabolites of gut microbiota, which can also function as ligands for the aryl hydrocarbon receptor (Ahr) [
69]. Ahr activity has been shown to alleviate colitis by regulating the levels of
Il-22 and
Il-17, and therefore Ahr has been suggested to maintain intestinal homeostasis by regulating the activities of intraepithelial lymphocyte, Treg, and Th17 cells [
8]. As dietary FRB supplementation was able to alter the mRNA expression of both
Il-22 and
Il-17 (
Figure 5B,C), it is possible that the tryptophan metabolites play a synergistic role with other bioactive compounds in FRB in ameliorating DSS-induced colitis and, subsequently, contribute to the prevention of the onset of fibrosis. Alauddin et al. [
22] also remarked that fermentation process altered the macronutrients composition of RB and FRB. Fermentation increased the amount of protein (RB, 14.4 g/100 g; FRB, 15.4 g/100 g), lipid (RB, 6.7 g/100 g; FRB, 10.0 g/100 g), and dietary fiber (RB, 4.4 g/100 g; FRB, 22.0 g/100 g). On the contrary, fermentation decreased the amount of carbohydrate (RB, 49.3 g/100 g, FRB, 13.1 g/100 g). These macronutrients alterations marked the activity of microorganisms that were used in the fermentation process. However, this estimation was performed by neglecting the biomass of microorganisms which is contained in FRB after the fermentation process. As the current study did not include a group in which mice were given supplementation of the mixture of
Aspergillus kawachii,
Lacticaseibacillus rhamnosus,
Levilactobacillus brevis, and
Enterococcus faecium, without the addition of rice bran, our study could not exclude the possibility that the presence of inactive microorganisms in FRB might have played a role in FRB function in assisting the intestinal recovery or preventing the onset of fibrosis post-DSS-induced colitis. This limitation of the current study should be addressed in future studies related to FRB.