Background
Ulcerative colitis (UC) and Crohn’s disease (CD) are subtypes of inflammatory bowel disease (IBD) [
1]. Symptoms of UC and CD might include abdominal pain, diarrhea and/or fever [
2], and UC and CD are caused by environmental interactions, genetic factors, and lifestyle factors [
3]. Therapeutic approaches to treating colitis include administration of anti-inflammatory drugs and immunomodulators, surgery, and therapies focused on controlling the immune cell-response cytokine pathway; however, treatments are limited because of several severe side effects such as allergies and lymphoma [
4]. For this reason, new therapeutic options are urgently needed to provide more effective and safe treatment, and several researchers have focused on the use of complementary and alternative medicines, such as natural products or traditional herbs. However, reliable data regarding the efficacy and safety of such treatments are lacking [
5].
Our novel formula KIOM-MA-128 (K-M-128) was generated by fermenting KIOM-MA using probiotics (
Lactobacillus rhamnosus). KIOM-MA is composed of
Glycyrrhizae Radix (the roots and rhizomes of
Glycyrrhiza uralensis),
Polygoni Cuspidati Rhizoma (the root of
Polygonum cuspidatum Sieb. et Zucc.),
Sophorae Flavescentis Radix (the root of
Sophora flavescens Ait.),
Cnidii Rhizoma (the rhizome of
Cnidium officinale Makino),
Arctii Fructus (the dried fruit of
Arctium lappa L.),
Ginseng Radix Alba (the root of
Panax ginseng C. A. Meyer),
Scrophulariae Radix (the root of
Scrophularia ningpoensis Hemsl.),
Zizyphi Semen (the seeds of
Zizyphus spinosa Hu),
Angelica Gigantis Radix (the root of
Angelica gigas Nakai), and
Saposhnikovia Radix (the root of
Saposhnikovia divaricata Schischkin), which have all long been used in natural pharmaceutical treatments in Asia. Previous studies have shown that the non-fermented formula, KIOM-MA, has anti-inflammatory and anti-allergic effects [
6,
7]. Furthermore, we have found that K-M-128, the bioconversion product, has anti-atopic dermatitis effects as well as anti-cancer effects [
7,
8]. However, the intracellular mechanism underlying the anti-allergic effects remains unclear, although we have elucidated that K-M-128 inhibits an antigen/IgE-induced response through the reduction of cPLA
2, COX-2 and other signaling molecules [
9]. In recent research, K-M-128 prevented against IL-6 induced intestinal barrier disruption via the regulation of tight junction proteins in a colon cancer cell line [
10]. Therefore, we investigated whether K-M-128 can regulate acute colitis in vivo. To accomplish this aim, we investigated the effects of K-M-128 in a DSS-induced mouse colitis model and evaluated whether they underlie the protective effects of this formula against colitis, inflammation, and damage to tight junctions in colonic crypts.
Methods
Preparation of KIOM-MA-128
The preparation of K-M-128 has been previously described [
6,
11]. Briefly, the KIOM-MA formula (
Glycyrrhizae Radix,
Polygoni Cuspidati Rhizoma,
Sophorae Flavescentis Radix,
Cnidii Rhizoma,
Arctii Fructus,
Ginseng Radix Alba,
Scrophulariae Radix,
Zizyphi Semen,
Angelica Gigantis Radix, and
Saposhnikovia Radix) was purchased from the Korea Medical Herbs Association (Yeongcheon, Korea), and the identification was confirmed by Porf. KiHwan Bae (The College of Pharmacy, Chungnam National University, Daejeon, Korea) [
7,
11]. A total of 1.84 kg of this formula (the ratios of each constituent of KIOM-MA are shown in Table
1) was placed in 18.4 L of water and then extracted by boiling for 3 h at 115 °C. The fermentation process was conducted such that the autoclaved KIOM-MA extract was added to the 1% broth media including
Lactobacillus rhamnosus (1X10
8 CFU/ml) at 37 °C for 48 h under micro-aerobic conditions followed by filtration through a 60 μm nylon filter (Millipore, Bedford, MA, USA), and the yield was 20.44%. After fermentation, the supernatant of K-M-128 was filtered and then freeze dried. The collected K-M-128 powder was kept at −20 °C until use. The freeze-dried powder was dissolved in saline, and its solution was prepared before every oral administration.
Table 1
The weight ratios of each constituent in KIOM-MA-128
1 |
Glycyrrhizae Radix
| 1 |
2 |
Polygoni Cuspidati Rhizoma
| 1 |
3 |
Sophorae Flavescntis Radix
| 2 |
4 |
Cnidii Rhizoma
| 1 |
5 |
Arctii Fructus
| 2 |
6 |
Ginseng Radix Alba
| 2 |
7 |
Scrophulariae Radix
| 2 |
8 |
Zizyphi Semen
| 1 |
9 |
Angelica Gigantis Radix
| 1 |
10 |
Saposhnikovia Radix
| 1 |
Chemicals and reagents
Dextran sodium sulfate was purchased from MP Biomedicals (Santa Ana, CA, USA), and hematoxylin and eosin solutions were obtained from Sigma Aldrich (St Louis, MO, USA). The ELISA kits for the detection of mouse tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were purchased from eBioscience (San Diego, CA, USA), and RIPA lysis buffer was obtained from Millipore (Darmstadt, Germany). Phosphatase and protease inhibitor cocktails were purchased from Roche (Basel, Swiss). A BCA protein quantification kit, fluorescence-tagged antibody and anti-Zonula occludens-1 (ZO-1) antibody were obtained from Thermo Fisher Scientific (Waltham, MA, USA), and anti-F4/80 antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
Animals
Male ICR mice (6 weeks) were purchased from Samtako Inc. (Osan, Korea), divided into 4 groups including 6 mice per group under specific pathogen-free conditions (21–24 °C and 40–60% relative humidity) with a 12 h light/dark cycle, and provided with standard rodent food (Orientbio Inc., Sungnam, Korea) and water. All procedures for the animal study were approved by the Korea Institute of Oriental Medicine Institutional Animal Care and Use Committee (KIOM-IACUC) and were conducted in accordance with US guidelines (NIH publication #83–23, revised in 1985).
Induction of DSS-induced colitis
After acclimation, the mice were orally administered (oral gavage) saline with K-M-128 (125 or 250 mg/kg). The treatment of DSS was provided with or without 5% DSS-containing water ad libitum for 6 days; then, the mice were provided DSS-free water for an additional 2 days. The mice were sacrificed with CO2 gas at the end of the experiment; the colon lengths were measured, and proteins were extracted from the colonic tissues.
Measurements of body weight, colon length, and disease activity index (DAI)
During the experimental schedule, the mouse body weights and the disease activity index (DAI) were measured daily before the oral administration of K-M-128. The colonic lengths were measured according to photographs after the animals were sacrificed. The DAI was determined as the sum of the diarrheal and bloody stool scores [
12]. The DAI scoring system is described in Table
2.
Table 2
Disease activity index (DAI) scoring system. The score was determined based on the characteristics of two stool types, and the sum of the scores of two parameters was defined as the DAI score
0 | Normal stool | Normal colored stool |
1 | Mildly soft stool | Brown stool |
2 | Very soft stool | Reddish stool |
3 | Watery stool | Bloody stool |
Large-intestine endoscopy and histological analysis
On days 5 and 8 of the experiment, we investigated the colons of mice anesthetized with isoflurane by endoscopy. Endoscopies in all mice were performed using a mini-endoscope (670 mm length and 2.8 mm diameter, OLYMPUS, Shinjuku-ku, Tokyo, Japan) with a visible light source, and high-resolution images were obtained. After the endoscopy procedure on day 8, whole blood was collected from the abdominal vein of mice, and the animals were then killed for tissue collection. The isolated colons were fixed with 4% paraformaldehyde solution, embedded in paraffin block, and sectioned using a microtome. Histological sections were stained with a hematoxylin and eosin solution or incubated with antibodies to detect F4/80, a macrophage marker, and ZO-1, a tight junction protein.
Enzyme-linked immunosorbent assay for IL-6 and TNF-α
Collected whole blood was left at 4 °C overnight and then centrifuged (3000×g at 4 °C) for 15 min. The separated serum was stored at −80 °C until use. The levels of IL-6 and TNF-α in the serum were determined by ELISA kits according to the manufacturer’s instructions.
Immunoblotting analysis
Mouse intestinal protein was extracted with RIPA lysis buffer with phosphatase and protease inhibitor cocktails. After protein quantification with a BCA kit, the levels of ZO-1 in the protein lysates were analyzed by immunoblotting analysis using anti-ZO-1 antibody (220 kDa). Briefly, proteins of equal amount (20 μg/20 μl) were separated by 8% and 12% SDS-PAGE gels and then transferred to PVDF membranes. The membranes were incubated with a 1:200 dilution of ZO-1 antibody at 4 °C overnight. The following day, the membranes were incubated with secondary antibodies and detected using the ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA).
Statistical Analysis
All statistical analyses were performed with SPSS version 18, and graphs were drawn with GraphPad Prism version 5. Experimental values are given as the means ± SEM. The significant difference was determined by one-way ANOVA test. p values less than 0.05 were regarded as statistically significant.
Discussion
UC is a colonic inflammation caused by several factors, and chemical-induced animal models are useful to discover the mechanism and to establish novel clinical approaches in colitis treatment. Patients with UC account for half of all patients with IBD in the United States, and the symptoms of UC can be improved through various treatments. However, these treatments have numerous side effects, and drug tolerance is observed in some patients [
15,
16]. Therefore, a new therapeutic method remains to be developed and is currently needed.
Administering DSS in the drinking water to mice or other animals for several days has direct toxic effects on epithelial cells in the intestinal crypts and on the integrity of the barrier, recapitulating the major symptoms of acute and chronic colitis. Therefore, the DSS-induced colitis model is useful for researching the contributions of immune mechanisms in colitis. Previous studies have shown that DSS administration for 7 or 10 days induces acute colitis, a loss of body weight, shortening of the colon, inflammatory gene expression, and phenotypes of histological staining [
17,
18]. Therefore, we induced acute colitis by DSS administration in the drinking water for 6 days in ICR mice. Then, we confirmed the typical symptoms of UC, such as loss of body weight, intestinal shortening, and crypt damage, and found that these factors were reduced by treatment with K-M-128. Therefore, our formula suppresses the onset of UC.
Then, we identified inflammatory mechanisms in the colitis model. Macrophage infiltration and cytokine secretion have major roles in intestinal inflammation; macrophages and cytokines control multiple aspects of the inflammatory response. Macrophages have a crucial role in innate and adaptive immune responses. F4/80, which recognizes a murine macrophage surface protein, has been used to detect macrophage infiltration in the immune response. In our data, macrophages recognized by F4/80 were widely distributed in colon tissues, and the levels of inflammatory cytokines such as TNF-α, IL-6 and IL-1β were elevated in the sera of DSS-treated mice. In support of this finding, recruited macrophages are known to be associated with the secretion of TNF-α, IL-6 and IL-1β [
19]. Therefore, TNF-α, IL-6 and IL-1β are very important cytokines in IBD because their levels are increased in the mucosa and serum in IBD, including UC and CD. For this reason, many studies have examined the regulation of cytokines as potential targets in therapeutic approaches [
16,
20]. In the present study, we confirmed that DSS treatment induced immune responses such as macrophage infiltration and cytokine secretion, whereas K-M-128 repressed the pro-inflammatory cascade. Our data indicated that the mechanism of K-M-128 was related to the reduction of the macrophage infiltration and cytokine secretion from the mucosa into the serum.
We anticipated that these inflammatory responses would be associated with the barrier function of intestinal crypts. The intestinal epithelial barrier has important protective effects against external insults such as antigens, toxins, or infections [
21]. Several studies have suggested that the down-regulation of occludin, one of the tight junction proteins, is associated with IBD such as UC and CD [
22,
23]. In addition, tight junction protein-1, also known as ZO-1, is associated with intracellular tight junctions. Thus, when DSS induces acute colitis, ZO-1 expression is also reduced, and intestinal permeability is subsequently increased [
24,
25]. Therefore, the regulation of ZO-1 expression might be important in the treatment and prevention of colitis diseases. In this study, we found that the integrity of epithelial crypts relies on paracellular tight junctions through the expression of ZO-1 protein. If our formula can restore damaged epithelial mucosa, it might be associated with tight junction proteins. As shown in our results, K-M-128 prevented the down-regulation of tight junctions and the damage of intestinal crypts. ZO-1 expression in the K-M-128 (250 mg/kg) group was elevated compared with the control group. These results might indicate that K-M-128 had a better effect on intestinal barrier function. Moreover, our formula restored the barrier permeability dysfunction caused by IL-6 [
10].
Conclusion
We conclude that our novel formula K-M-128 has anti-colitis effects through the regulation of paracellular tight junctions. Therefore, K-M-128 suppresses the inflammatory response by inhibiting the penetration of pro-inflammatory factors into the intestinal mucosa. K-M-128 might be further developed as an effective preventive approach to treat intestinal inflammation.
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