Anticolitic Effect of the Rhizome Mixture of Anemarrhena asphodeloides and Coptidis chinensis (AC-mix) in Mice

During a screening program to search the anticolitic herbal medicines, 80% ethanol extract of the rhizome of Anemarrhena asphodeloides (AA) was found to potently inhibit the expression of proinflammatory cytokines TNF-α and IL-1β, as well as the activation of NF-κB in LPS-stimulated colonic macrophages, followed by that of the rhizome of C. chinensis (CC). AA also potently inhibited TNBS-induced colitic markers, shortening of the colon and increase of macroscopic score, myeloperoxidase activity, TNF-α, IL-1β, and IL-6, in mice. The synergistic effect of CC against the anticolitic effect of AA was investigated. CC synergistically inhibited the anticolitic effect of AA. AC-mix (AA+CC, 1:1) potently inhibited them. AC-mix also inhibited the activation of NF-κB, as well as the expression of TNF-α, IL-1β, IL-6, iNOS and COX-2. The effects of AC-mix against oxazolone-induced colitis were investigated in mice. AC-mix also potently inhibited oxazolone-induced inflammatory markers, colon shortening, macroscopic score, myeloperoxidase activity, NF-κB activation and proinflammatory cytokines. Overall, the anti-colitic effect of AC-mix was superior to that of mesalazine. Based on these findings, AC-mix may improve colitis by inhibiting NF-κB activation.


INTRODUCTION
Infl ammatory bowel disease (IBS) refers to two chronic relapsing diseases, ulcerative colitis and Crohn's disease, that cause infl ammation of the intestines (Shanahan, 2002;Binder, 2004). Their pathogenic mechanism is assumed to be an immune response against gut constituents, such as intestinal bacterial toxins, exposed in intestinal environment (Rafi i et al., 1999;Atreya et al., 2000). Among these toxins, bacterial endotoxins, such as lipopolysaccharide (LPS) and fl agellin, that can cause colonic infl ammation stimulate the mucosal immune system (Radema et al., 1991;Rafi i et al., 1999). This induces infl ammatory mediators such as pro-infl ammatory cytokines, activating the infl ammatory reaction via distinct signaling pathways through Toll-like receptors (TLRs) and/ or cytokine receptors (Jung et al., 1995;Cario and Podolsky, 2000). Therefore, to cure infl ammatory diseases, natural products regulating infl ammatory mediators have been attempted (Paradkar et al., 2004;. The rhizome of Anemarrhena asphodeloides (family Lili-aceae) is an antipyretic, antiphlogistic, sedative, and diuretic agent in traditional Chinese medicine. Its main constituents are saponins. Of these saponins, timosaponins improve learning and memory in rats (Hu et al., 2005;Li et al., 2007) and inhibit platelet aggregation factor-induced platelet aggregation (Dong and Han, 1991). Xanthones, such as mangiferin, inhibit lung infl ammation and pruritus in mice Rivera et al., 2011), inhibited LPS-induced iNOS and COX-2 expression in RAW264.7 cells (Shin et al., 2008) and protect scopolamine-induced brain impairment in mice . However, the anti-colitic effect of AA remains to be thoroughly studied. The rhizome of Coptidis japonica (family Ranunculaceae), whose main constituent is an isoquinolone alkaloid berberine, has been used to treat patients who have gastroenteritis and diarrhea in tradition Chinese medicine (Bae et al., 1998;Zhou and Mineshita, 2000). Although its closely related plants are C. chinensis, C. teeta, and C. sinensis, their constituents are different to that of C. japonica. Nevertheless, they contain berberine. Berberine was proved to have biological activities

Isolation and culture of intestinal macrophages
For the isolation of macrophages in intestine, the Payer's patches were isolated from the mouse intestines and were digested in DMEM containing 1 mg/ml dispase, 0.25 mg/ml collagenase A, and 25 U/ml DNAse (Roche Diagnostic, Indianapolis, IN, USA) at 37 o C for 20 min with shaking and passed through a 70 μm cell strainer (BD Biosciences, San Diego, CA, USA). The cells were harvested by centrifugation and washed with PBS containing 4% FBS. The cells were resuspended in cold IMag buffer (0.05% bovine serum albumin and 2 mM EDTA in PBS) and viable cells were counted with trypan blue. The colonic macrophages were isolated from colonic cells by using a biotin-labeled anti-mouse F4/80 antibody and streptoviridin magnetic beads (Invitrogen).
To examine the anti-infl ammatory effect of test agents, intestinal macrophages were incubated in the absence or presence of test agents with 50 ng/ml LPS. The cytoxicities of these agents in the cell viability were measured using the crystal violet method.

Preparation of TNBS-induced colitic mice
TNBS-induced colitis was induced by the administration of 2.5% (w/v) TNBS solution (100 μl, in 50% ethanol) into the colon of lightly anesthetized mice via a thin round-tip needle equipped with a 1-ml syringe . The normal group was treated with vehicle alone. The needle was inserted so that the tip was 3.5-4 cm proximal to the anal verge. To distribute the agent within the entire colon and cecum, mice were held in a vertical position for 30 s after the injection. Using this procedure, >95% of the mice retained the TNBS enema. If the mice quickly excreted the TNBS-ethanol solution, it was excluded from the remainder of the study. Test agents (dissolved in 2% tween 80 solution, 10 or 20 mg/kg) or vehicle alone was orally administered once a day for 3 days after TNBS treatment. The mice were sacrifi ced the next day after the fi nal administration of test agents. The colon was quickly removed, opened longitudinally, and gently cleared of stool by PBS. Macroscopic assessment of the disease grade was scored according to a previously reported scoring system (0, no ulceration and no infl ammation; 1, ulceration without hyperemia; 2, no ulceration and local hyperemia; 3, ulceration and infl ammation at one site only; 4, two or more sites of ulceration and infl ammation; 5, ulceration extending more than 2 cm), and the colon tissue was then used for ELISA and immunoblotting .

Preparation of oxazolone-induced colitis in mice
Oxazolone-induced colitis was studied in mice as previously described (Wirtz et al., 2007). To presensitize the mice, a 1.5 cm×1.5 cm fi eld was shaved on the back between the shoulders, and 200 μl of a 3% (w/v) solution of oxazolone in acetone/olive oil (4:1) was applied. Eight days after presensitization, mice were rechallenged intrarectally under ethyl ether general anesthesia with 100 μl of 1% oxazolone in 50% ethanol to induce colitis or 50% ethanol alone (vehicle control). Test agents (AA, 10 and 20 mg/kg; AC, 10 and 20 mg/ kg; mesalazine, 10 mg/kg dissolved in 2% tween 80) were orally administered once a day for 15 days after oxazolone administration. The mice were sacrifi ced on next day after the fi nal administration of test agents. The colon was quickly removed, opened longitudinally, and gently cleared of stool by PBS. Macroscopic assessment of the disease grade was http://dx.doi.org/10.4062/biomolther.2013.048 scored according to a previously reported scoring system and the colon tissue was then used for ELISA and immunoblotting .

Histological exam
The colons were fi xed in 10%-buffered formalin solution, embedded in paraffi n using standard methods, cut into 8-μm sections, stained with hematoxylin-eosin, and then assessed under light microscopy .

Colon tissue preparation
The colon tissues were excised, perfused with ice-cold perfusion solution (0.15 M KCl, 2 mM EDTA, pH 7.4), and homogenized in 50 mM Tris-HCl buffer (pH 7.4) . The homogenates were centrifuged at 10,000×g at 4 o C for 30 min and the supernatants used for further studies.

Assay of myeloperoxidase activity
An aliquot (50 μl) of the colon supernatant was added to a reaction mixture of 1.6 mM tetramethyl benzidine and 0.1 mM H 2 O 2 and incubated at 37 o C; the absorbance was obtained at 650 nm over time . Myeloperoxidase activity was defi ned as the quantity of enzyme degrading 1 μmol/ml of peroxide at 37 o C and expressed in unit/mg protein. The protein content was assayed by the method of Bradford (1976).

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For the immunoblot analyses of p-IRAK1, p-IKKβ, p-p65 and β-actin, the colon tissue homogenates or the colleted cells were resuspended in 1 ml of RIPA lysis buffer containing 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail . After centrifugation, the supernatant was used for the immunoblot assay. The proteins from collected cells were subjected to electrophoresis on 8-10% sodium dodecyl sulfate-polyacrylamide gel and then transferred to nitrocellulose membrane. Levels of p-IRAK1, p-IKKβ, p-p65 and β-actin were assayed as previously described. Immunodetection was performed using an enhanced chemiluminescence detection kit.

Statistical analysis
All data are expressed as the mean ± standard deviation (S.D.), with statistical signifi cance analyzed using one-way ANOVA followed by a Student-Newman-Keuls test.

RESULTS
During a screening program to search the anticolitic herbal medicines, 80% EtOH extract of AA was found to potently inhibit the expression of proinfl ammatory cytokines TNF-α and IL-1β in LPS-stimulated intestinal macrophages, followed by CC (Fig. 1). AA also inhibited the phosphorylation, of IRAK1,  IKKβ and IκBα and the activation of NF-κB.
The ability of AA to inhibit TNBS-induced colitis was tested in mice (Fig. 2). Intrarectal injection of TNBS signifi cantly caused colitis, manifested by shortening of the colon and increase of myeloperoxidase activity. AA also potently inhibited TNBS-induced shortening of colons and increase of myeloperoxidase activity. AA also inhibited TNBS-induced NF-κB activation, as well as expression of proinfl ammatory cytokines, TNF-α, IL-1β, and IL-6 (Fig. 3).
Next, we tested the synergistic effect of CC in the anticolitic effect of AA in mice (Fig. 4). Intrarectal injection of TNBS potently caused severe infl ammation, manifested by colon shortening and myeloperoxidase increasing. Treatment with AA or CC in TNBS-induced colitic mice signifi cantly inhibited the shortening of colons and the increase of myeloperoxidase activity. Treatment with AC-mix prepared by the addition of CC in AA inhibited the shortening of colons and the increase of myeloperoxidase activity more potently than those of AA or CC.
Next, we tested the anticolitic effect of AC in mice (Fig. 5). Intrarectal injection of TNBS caused severe infl ammation, manifested by shortened, thickened, and erythematous colons. TNBS also increased myeloperoxidase activity. Treatment with the combined mixture AC-mix in TNBS-induced colitic mice inhibited the shortening of colons and the increase of myeloperoxidase activity. AC-mix also inhibited NF-κB activation (Fig. 6). AC-mix at a dose of 20 mg/kg inhibited TNBSinduced TNF-α by 74.1%, IL-1β by 81.6%, and IL-6 expression by 82.8%. AC-mix also inhibited iNOS and COX-2 expression. However, it reversed IL-10 to 71.9% of the normal control group. The anti-colitic effect of AC-mix at a dose of 20 mg/kg was more potent than at a dose of 10 mg/kg. However, in our preliminary study treated with 10 or 50 mg/kg mesalazine to TNBS-induced colitic mice, we could not fi nd the signifi cant differences of anti-colitic effects, such as colon shortening and macroscopic score, between them (data not shown). Thus, the anti-colitic effect of AC-mix was superior to that of mesalazine.
Next, we investigated the effect of AC-mix against oxazolone-induced colitis in mice (Fig. 7). Treatment with oxazolone alone caused severe colonic infl ammation, including the  www.biomolther.org shortening of colon, loosing of bodyweight, and an increase of macroscopic score, myeloperoxidase activity, proinfl ammatory cytokine expression, and NF-κB activation. AC-mix potently ameliorated oxazolone-induced colon shortening and bodyweight loosing, as well as increased myeloperoxidase activity, proinfl ammatory cytokine expression and NF-κB activation. AC-mix also inhibited iNOS and COX-2 expression (Fig. 8). AC-mix at a dose of 20 mg/kg inhibited TNBS-induced TNF-α by 54.6%, IL-1β by 67.3%, and IL-6 expression by 80.0%. However, AC-mix reversed IL-10 to 89.7% of the normal control group. AC-mix also inhibited TNBS-induced IRAK1 and IKKβ phosphorylation, as well as NF-κB activation. Overall, the anti-colitic effect of AC-mix was superior to that of mesalazine.
In the present study, AA potently ameliorated TNBS-induced colitis in mice. Furthermore, AA inhibited infl ammation in LPSstimulated colonic macrophages by regulating the phosphorylation of IRAK1 and the activation of NF-κB. AC-mix, which was prepared by the addition of CC in AA, ameliorated TNBSor oxazolone-induced colitis via inhibiting the activation of NF-κB in the colons of mice. Thus, the anticolitic effect of AA was synergistically increased by the addition of CC. Oeverall, the anticolitic effect of AC-mix was superior to that of mesalazine, which is used commercially for colitis.
Based on these fi ndings, AC-mix may potently ameliorate colitis by synergistically inhibiting NF-κB signaling pathway.