Ixeris dentata NAKAI Reduces Clinical Score and HIF-1 Expression in Experimental Colitis in Mice

Ixeris dentata (ID) is an herbal medicine used in Asian countries to treat indigestion, pneumonia, hepatitis, contusions, and tumors; however, its effect on intestinal inflammation is unknown. Thus, we investigated the effect of ID in the dextran sulfate sodium (DSS) model of colitis in female BALB/c mice; animals were evaluated after seven days of DSS treatment. DSS-treated mice showed considerable clinical signs, including weight loss, reduced colon length, colonic epithelial injury, infiltration of inflammatory cells in the colon tissue, and upregulation of inflammatory mediators. However, administration of ID attenuated body weight loss, colon shortening, and the increase in disease activity index score. ID also significantly decreased the colonic mucosal injury and the number of infiltrating mast cells. Moreover, ID inhibited the expressions of cyclooxygenase-2 and hypoxia-inducible factor-1α in colon tissue. Taken together, the results provide experimental evidence that ID might be a useful therapy for patients with ulcerative colitis.


Introduction
Ulcerative colitis (UC) is a chronic inflammatory disorder of the colon and rectum with intervals of acute exacerbation. Its etiology remains unknown, although results from recent studies suggest that proinflammatory cytokines initiate the inflammatory response [1]. Patients with UC have been reported to have increased levels of interleukin-(IL-) 6 in the intestinal mucosa [2,3] and of tumor-necrosis-factor-(TNF-) in blood, colonic tissue, and stool [4]. Two cyclooxygenase (COX) isoenzymes have been recognized: COX-1, a constitutive enzyme, which generates prostaglandins (PGs) that protect the stomach and kidney against damage, and COX-2, an inducible enzyme induced by inflammatory stimuli, such as cytokines, and capable of generating PGs that contribute to the pain and swelling of inflammation [5,6]. The expression of COX-2 is also elevated in the inflamed mucosa of patients with UC [7]. Moreover, metabolism is altered in inflamed mucosal tissues, secondary to decreased mucosal perfusion caused by infiltration of inflammatory cells. The resultant 2 Evidence-Based Complementary and Alternative Medicine hypoxia [8,9] activates hypoxia-inducible-factor-(HIF-) 1, a transcription factor that links inflammatory pathways [10,11]. Although corticosteroids are effective in bringing about clinical remission, severe adverse effects can sometimes lead to their discontinuation; thus, alternative treatments are needed. Ixeris dentata NAKAI (ID) is a traditional herbal medicine used in Korea to treat indigestion, pneumonia, hepatitis, and tumors [12]. It has also been reported to protect against kainic-acid-induced oxidative stress in the mouse brain by regulating glutathione concentration [13] and to inhibit the anaphylactic response induced by compound 48/80 or IgE [14]. Moreover, lactic acid fermentation of ID increased its potency against IgE-induced allergic diseases [15]. However, it is unknown whether ID can reduce intestinal inflammation.
The dextran sulfate sodium (DSS) model of UC has been well characterized morphologically and biochemically. DSS induces an acute colitis characterized by bloody stools, ulcerations, and infiltration of inflammatory cells [16]. Histologically, DSS produces submucosal erosions, crypt abscesses, and epithelioglandular hyperplasia. It is generally believed that DSS is directly toxic to gut epithelial cells of the basal crypts and affects the integrity of the mucosal barrier [17]. Hence, the DSS-induced colitis model is particularly useful for studying the contribution of inflammatory mechanisms in colitis. To provide experimental evidence that ID might be a useful therapy for patients with UC, we examined the effects of ID on DSS-induced colitis. The specific aims were to assess the effect of ID on clinical signs of colitis, including weight loss, colon shortening, diarrhea, and occult/gross bleeding, and to investigate the effect of ID on proinflammatory mediators in the colon of DSS-treated mice.

Animals and Reagents.
Female BALB/c mice (6 weeks old) were obtained from Da-Mool Science (Taejeon, Republic of Korea). Mice were acclimatized in a specific pathogen-free environment under controlled conditions (22 ± 2 ∘ C under a 12 h light/dark cycle) for at least one week. All animal studies were carried out in accordance with the regulations issued by the Institutional Review Board of Wonkwang University (confirmation number: WKU11-10). DSS (mol wt: 36,000-50,000) was purchased from MP Biomedicals (Solon, OH, USA). Purified anti-mouse IL-6, recombinant mouse IL-6, and biotinylated anti-mouse IL-6 antibodies were obtained from BD-Pharmingen (San Diego, CA). Specific antibodies against COX-2, HIF-1 , and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemical reagents were purchased from Sigma (St. Louis, MO).

Preparation of ID.
ID extract was prepared by decocting with distilled water for three hours (100 g/L). The residue was filtered, lyophilized, and maintained at room temperature. The yield of dried extract from the starting materials was about 7.8%. Dried extract was diluted in saline and filtered through 0.22 m syringe filter.

Disease Activity Index (DAI).
Intestinal disease activity was assessed based on the weight loss, the presence of diarrhea accompanied by blood and mucus, and colonic shortening [18]. DAIs were calculated by scoring weight loss, diarrhea, and rectal bleeding, based on the scoring system ( Table 1) described by Murthy et al. [19]. Weight loss was defined as the difference between initial and final weights and diarrhea as the absence of fecal pellet formation and the presence of continuous fluid fecal material in the colon. Rectal bleeding was assessed based on the presence of diarrhea containing visible blood and on the presence of gross rectal bleeding. DAI values were calculated using the following formula: DAI = {(weight loss score) + (diarrhea score) + (rectal bleeding score)}/4. The DAI was determined by three investigators blinded to the protocol. The clinical parameters used in the present study were chosen to represent the subjective clinical symptoms observed in human UC.

Cytokine
Assays. Levels of IL-6 and TNF-in the serum and tissue were measured using an enzyme-linked immunosorbent assay (ELISA), as previously described [20]. Briefly, 96-well plates were coated with 100 L of anti-mouse monoclonal antibodies (1.0 mg/mL at pH 7.4 in phosphatebuffered saline [PBS]) and incubated overnight at 4 ∘ C. After additional washes, 50 L of sample or IL-6 and TNFstandard was added and incubated at room temperature for two hours. Plates were then washed, and 0.2 g/mL of biotinylated anti-mouse antibody was added and incubated at room temperature for two hours. After washing the plates, avidin-peroxidase was added, and plates were incubated for 30 min at 37 ∘ C. The plates were then washed again and ABTS substrate was added. Color development was measured at 405 nm using an automated microplate ELISA reader. Standard curves were prepared using serial dilutions of recombinant antibodies. Protein concentrations Evidence-Based Complementary and Alternative Medicine 3 Normal colonic mucosa 1 Loss of one-third of the crypts 2 Loss of two-third of the crypts 3 Lamina propria covered with a single layer of epithelial cells with mild inflammatory cell infiltration 4 Erosions and marked inflammatory cell infiltration were measured using bicinchoninic acid (BCA) protein assay reagent (Sigma).

Western Blot Analysis.
Distal colons were homogenized in lysis buffer (iNtRON Biotech, Republic of Korea) and centrifuged at 13,000 rpm for five min. The supernatants were transferred to fresh tubes, and protein concentrations were determined using BCA protein assay reagent (Sigma). Lysates (50 g of protein) were separated by 10% SDS-PAGE and transferred to membranes (Amersham Pharmacia Biotech, Piscataway, NJ), which were blocked with 5% skim milk in PBS-Tween-20 (PBST) for 1 h at room temperature. Membranes were incubated overnight with primary antibodies against COX-2 and HIF-1 and washed 3 times with PBST. Blots were incubated with secondary antibodies for one hour at room temperature; antibody-specific proteins were visualized using an enhanced chemiluminescence detection system (Amersham Corp. Newark, NJ, USA). Protein densities were quantified by densitometry.

Histological
Processing. All trimmed rectums were fixed in 10% neutral buffered formalin. After paraffin embedding, 4 m sections were prepared. Representative sections were stained with hematoxylin and eosin (H&E) for examination under light microscopy or with toluidine blue to detect mast cells.

Microscopic
Scoring. The histological damage on the prepared, cross trimmed H&E stained samples were evaluated by two pathologist observers who were blinded to the experimental groups according to the modified criteria ( Table 2) from Hamamoto et al. [21]. Briefly, the mucosa damages were scored 0-4 based on the loss of crypt (mucosa) and infiltration of inflammatory cells (max. score = 4).

The Effects of ID on Clinical Signs in DSS-Induced Colitis.
DSS caused a decrease in body weight (Figure 1(a)) and colon length (Figures 1(b) and 1(c)) at day 7 by 19.5% and 47.8%, respectively, compared to the control group. Both ID and SFZ alleviated the DSS effects on body weight loss and colon shortening (Figures 1(a)-1(c)). ID and SFZ also attenuated the DSS-mediated increase in DAI scores (Figure 1(d)).

The Effects of ID on Epithelial Injury and Mast Cells Infiltration in DSS-Induced Colitis.
Mucosal thickness is regarded as a parameter of mucosal integrity. DSS treatment caused epithelial injury, as evidenced by an approximate 60% in mucosal thickness (Figures 4(a) and 4(c)). Mucosal infiltration of inflammatory cells, including mast cells (Figure 4(a); arrows), was also detected in the DSS-treated group as compared with the control group (Figures 4(a) and 4(d)). ID and SFZ treatments attenuated these effects induced by DSS treatment (Figures 4(a), 4(c), and 4(d)). ID and SFZ treatments also reduced the DSS-mediated microscopic damage to the colonic tissue (Figure 4(b)).

Characterization of ID Constituents.
A chromatogram of ID along with the standard 3,4-dihydroxy cinnamic acid is shown in Figure 5; the regression ( 2 ) of calibration curve was 0.9998. Based on the calibration curve, the content of 3,4-dihydroxy cinnamic acid in ID was estimated to be 4 mg/g of water extract of ID.

Discussion
UC is a type of inflammatory bowel disease (IBD), the symptoms of which include abdominal pain, weight loss, and bloody diarrhea [22][23][24]. Most therapies for UC include glucocorticosteroids, sulfasalazine, and immunomodulators (such as azathioprine) [25,26]; however, these treatments can cause serious adverse effects. Although traditional herbal medicines have garnered much interest for their potential to treat inflammation, their pharmacological mechanisms of action have remained largely unresolved.
Here it is demonstrated that ID alleviates the clinical signsweight loss, colon shortening, diarrhea, and occult/gross bleeding-in the DSS-treated mouse model of UC. We also found that ID reduced epithelial injury, inflammatory cell infiltration into the colon tissue, and indices of microscopic injury. Moreover, ID prevented increases in the expression of COX-2 and HIF-1 and in the production of IL-6 and TNF-. Thus, these results suggest that ID effectively inhibits symptoms of colitis caused by DSS. Inflammatory cytokines such as IL-6, TNF-, and interferon-mediate the pathogenesis of murine colitis [27][28][29]. Cytokines and chemokines are secreted by immune cells like T lymphocytes and macrophages that infiltrate the inflamed region. Studies in patients with UC have shown that gene [30] and protein expression [2][3][4] of IL-6 and TNFare similarly increased in the rectal mucosa. Consistent with the notion that IL-6 and TNF-plays a causal role in the pathogenesis of UC, we found that ID suppressed the DSSinduced increase in IL-6 and TNF-in mouse serum.
During the inflammatory process, the COX-1mRNA and protein activity do not change, whereas COX-2 levels increase dramatically, leading to the production of proinflammatory PGs [31]. However, Okayama et al. [32] found that both COX-1 and COX-2 inhibitors exacerbate inflammation and ulceration in the colon. Nevertheless, selective inhibitors like lumiracoxib have been developed as nonsteroidal antiinflammatory drugs, many of which have been shown to be efficacious in a model of chemically induced colitis [33,34]. 5-aminosalicylates, another drug class used to treat IBD, exert anti-inflammatory effects by inhibiting COX-2 activation [35]. Our study showed that ID inhibited the DSS-induced increase in COX-2 activation. These results suggest that the anti-inflammatory effect of ID is attributable to the regulation of COX-2 in DSS-induced colitis.
Inflamed mucosal tissue of colitic mice is highly hypoxic, leading to the overexpression of HIF-1 [36]. Clinical studies have also verified the upregulation of HIF-1 in colonic tissue from patients with IBD [37,38]. And in HIF-1 overexpressing mice, Nuclear Factor-B activity and the expression of pro-inflammatory genes were sequentially elevated [39]. Moreover, HIF-1 was shown to directly bind to the COX-2 promoter, thereby regulating the expression of COX-2 protein in two colorectal carcinoma cell lines, HCT116 and HT29 [40,41]. It is therefore tempting to speculate that in the present study, the DSS-mediated increase in HIF-1 expression triggers the upregulation of COX-2. By virtue of its ability to inhibit the increased HIF-1 expression, ID therefore suppresses COX-2 activation and the ensuing inflammatory response.

Conclusions
We have shown that ID reduces the clinical signs and levels of inflammatory mediators in DSS-induced colitis in mice. This study provides experimental evidence to show that ID might be a useful therapy in the treatment of UC.

Authors' Contribution
Dae-Seung Kim and Jang-Ho Ko contributed equally to this work.