EIQ was kindly supplied by Prof. Whang (Herb Medicine Laboratory, Chung-Ang University, Seoul, Korea) and was dissolved in distilled water for animal treatments. 2,4,6-Trinitrobenzene sulfonic acid (TNBS), sodium carboxymethylcellulose (CMC-Na), 5-aminosalicylic acid (5-AS), EDTA, EGTA, urethane, hexadecyl trimethyl ammonium bromide (HETAB), hydrogen peroxidase, dimethylformamide were purchased from Sigma (St.Louis, MO, USA). EIE was kindly provided by Dong-A Pharmaceutical Co. Ltd (Yong-In, Korea) and was dissolved in CMC-Na for animal treatments. TNF-α and NO assay kits were purchased from Cayman Chemical (Ann Arbor, MI, USA).

Male Sprague-Dawley rats weighting between 180 and 200 g were supplied by Samtako Bio (Osan, Korea). The animals were group-housed in cages with wire-net floors in a room controlled for temperature (24~25℃) and humidity (70~75%) and were fed a normal laboratory diet (Samtako Bio). Rats were fasted for 24 h prior to the induction of colitis, but were allowed free access to tap water throughout. For experiments, animals were randomized into groups of n=6. The experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Institute of Chung-Ang Pharmaceutical Science (Chung-Ang University, Seoul, Republic of Korea).

Colitis was induced according to the procedure described by Morris et al. [22]. Following a 24 h fast, rats were anesthetized with and intra peritoneal injection of urethane (0.5 g/ml) and a medical-grade polyurethane cannula (external diameter, 2 mm) was inserted into the anus and advanced to 8 cm proximal to the anal verge. TNBS was dissolved in ethanol (50% v/v) at 120 mg/ml and 0.25 ml was instilled into the colon through the cannula to induce acute colitis. Following instillation, the animals were maintained in a head-down position for 2~3 min to prevent leakage of the instillation. A normal control group received 0.9% saline in place of TNBS. EIE was suspended in 0.5% CMC-Na and EIQ were orally administered at the doses indicated in the Results section in a volume of 2 ml/kg body weight. Animals were dosed 48, 24, and 1 h prior to the TNBS instillation and then again 24 h later. Animals were sacrificed 48 h after induction of colitis. As a positive control, one group of TNBS-treated animals received 25 mg/kg 5-aminosalicylic acid (5-AS) in a volume of 2 ml/kg. Rats in a normal control group received 0.9% saline by intra colonic route in place of TNBS solution in a comparable volume. All rats were monitored daily for behavior, body weight, and stool consistency.

The severity of colitis was evaluated by an independent observer who was blinded to the treatments. Animals were sacrificed 48 h after TNBS instillation. The distal 10-cm portion of the colon was removed and cut longitudinally, rinsed in physiological saline to remove fecal residue, and weighed. Macroscopic inflammation scores were assigned based on morphological features of the colon (score 0~10), the presence of adhesions (score 0~2), and stool consistency (score 0~1) according to the criteria of Bobin-Dubigeon et al. [9]. Pieces of inflamed colon were collected and frozen at -80℃ until measurements of biochemical parameters.

The MPO assay was modified from previously described method [23]. An 1 ml aliquot of leukocyte suspension from extracted from colon was centrifuged at 600 g at 4℃ for 7 min. The pellet was suspended in 1 ml of 80 mM sodium phosphate buffer, pH 5.4, containing 0.5% (wt/v) hexadecyl trimethyl ammonium bromide (0.5% HETAB), sonicated and centrifuged at 12,000 g at 4℃ for 15 min. Duplicate 30 µl samples of the resulting supernatant were aliquoted into wells of a 96-well micro titer plate. The reaction was stopped by adding 30 µl of 1.46 M sodium acetate, pH 3.0 to each well. MPO activity was calculated by measuring the absorbance of samples at 620 nm and comparing to a MPO standard.

The concentration of nitrite, an indicator of NO production, was measured in the supernatants of colonic tissue homogenized (TMZ-20DN homogenizer, Taemin, Korea) with PBS, pH 7.2, containing 1% (wt/v) bovine serum albumin (BSA). Equal volumes of homegenate and Griess reagent (1% sulphanilic in 5% phosphoric acid and 0.1% N-[1-naphtyhl]-ethylendiamine dihydrochloride) were mixed and the absorbance was read at 450 nm. NO levels were assayed with a quantitative nitric oxide assay kit (Cayman Chemical, Ann Arbor, MI, USA).

The total GSH content of colonic tissue was determined using a modification of the method of Beutler et al. [24]. A mitochondrial fraction was prepared. Samples were added to metaphosphoric acid and allowed to stand for 5 min to precipitate proteins. Phosphate buffer and 5'5-dithiobis-2-nitro-benzoic acid were added for color development. GSH was determined by measuring absorbance at 415 nm and absolute concentrations were calculated using a GSH standard (Sigma).

Lipid peroxidation was determined according to the method of Buege and Aust, by measuring the formation of thiobarbituric acid reactive substances (TBARS) spectrophotometrically. Colonic tissues were collected, homogenized in PBS (pH 7.0). The samples were centrifuged at 12,000 g at 4℃ for 15 min then 0.3 ml supernatant was removed and mixed with 0.9 ml of 8% trichloroacetic acid (TCA). The sample was again centrifuged at 10,000 g at 4℃ for 5 min. An 1 ml aliquot of supernatant was added to 0.25 ml of 1% TBA, and the resulting solution was heated at 100℃ for 20 min. The tube was cooled, 2 ml of n-butanol was added, and the tube was vortexed for 90 sec. After centrifugation at 3,000 g at 4℃ for 5 min, 1 ml of the n-butanol phase was removed and used in the TBARS assay. Finally, the absorbance at 532 nm was measured and MDA content was calculated with MDA standards.

Protein concentrations were measured spectrophotometrically using the Bio-Rad assay kit (Bio-Rad Chemical Division, Richmond, CA). Absorption was monitored at 595 nm.

The data are expressed as the means±S.E.M. Statistical differences between means were analyzed by the Student's t-test, with p<0.05 considered significant.

Physical differences in rats were evident 48 h after intra colonic instillation of TNBS. Animals receiving only TNBS showed prostration, pilo erection and hypo motility, and lost significantly more body weight than control animals (Table 1).

The presence of adhesions to adjacent organs, as well as diarrhea, was frequently observed in TNBS-treated rats (Table 1). Macroscopic inspection of the cecum, colon, and rectum of TNBS-treated animals showed evidence of severe colonic mucosal damage, with edema, deep ulceration and hemorrhage (Fig. 2).

The macroscopic changes in the distal colon were also quantified (Fig. 3).

TNBS-treated rats also had significantly increased colon weight/length ratios (Fig. 4), an indicator of inflammation.

Treatment of animals with EIE (100 mg/kg) or EIQ (30 mg/kg) significantly reduced the loss in body weight, the presence of adhesions to adjacent organs, and diarrhea (Table 1). Administration of 5-AS also decreased the presence of adhesions compared with TNBS-treated animals. The ratio of colon weight/length was decreased significantly in rats treated with EIE (100 mg/kg) and EIQ (30 mg/kg) (Fig. 4).

The highest doses of EIE and EIQ also attenuated the extent and severity of colonic injury, as reflected in the lower macroscopic scores (Fig. 3).

In the initial inflammatory process in colitis, tissue damage caused by ROS or other factors induces infiltration of MPO-positive neutrophils. Thus, measurement of MPO activity can be used to quantitate neutrophil infiltration. MPO activity was significantly increased 48 h after instillation of TNBS compared with levels in control animals (Fig. 5).

Treatment of animals with EIE and EIQ significantly decreased MPO activity in a dose-dependent manner (EIE 50 and 100 mg/kg, EIQ 30 mg/kg). At doses of 50 and 100 mg/kg EIE, MPO activity was decreased by 51% and 63%, compared with the TNBS group. Similarly, treatment with 30 mg/kg EIQ reduced MPO activity to 52% of that observed in the control TNBS-treated group. Treatment with 5-AS reduced MPO activity to 62% of that observed in the TNBS group.

The level of NO level in the colons of animals treated with TNBS was significantly increased compared with the normal control group (Fig. 6), and this was reversed by treatment with 5-AS. Treatment with EIE and EIQ (100 mg/kg and 30 mg/kg, respectively) also significantly inhibited nitrite production (Fig. 6).

Colonic injury by acute TNBS administration was characterized by a significant increase of the pro-inflammatory Th1 cytokine TNF-α over the level seen in the normal control group. Treatment with EIE and EIQ caused a clearly dose-dependent reduction in TNF-α levels, although a statistically significant effect was observed only at the highest doses (100 mg/kg EIE, 30 mg/kg EIQ). EIE and EIQ reduced TNF-α level by 61% and 32% compared with the TNBS group (Fig. 7).

Instillation of TNBS causes colonic injury and increases MDA levels. To rebalance the intracellular redox state, GSH is oxidized with toxic lipids or H₂O₂ through the action of glutathione S-transferase (GST). Animals with TNBS-induced colitis had 54% reduction in GSH levels in the colon compared with the normal control animals (Fig. 8). However the GSH levels were restored by treatment of rats with EIE and EIQ. At doses of 50 and 100 mg/kg EIE, and 30 mg/kg EIQ, the GSH levels were restored by 35%, 84%, and 54% respectively, compared with the TNBS-treated group. Treatment of animals with 5-AS restored the GSH levels by 82%.

When colonic tissues are damaged by TNBS instillation, the increased reactivity of ROS with lipids leads to a significant increase in MDA levels. MDA levels were measured in the TNBS-treated rat colons as an indicator of lipid peroxidation. Compared with the untreated control animals, TNS instillation caused a significant increase in MDA, which was inhibited in a dose-dependent manner by treatment with EIE and EIQ (Fig. 9). Treatment of rats with 50 and 100 mg/kg EIE significantly decreased the MDA levels respectively. Similarly, animals treated with 10 and 30 mg/kg EIQ showed a reduction in MDA respectively. Treatment of animals with 5-AS decreased the MDA levels.