Female Sprague-Dawley rats (180-200g) were purchased from Experimental Animals Co. LTD of Chongqing Tengxin Bier (GradeⅡ, SYXK-(YU), 2009-0002). Rats were housed under constant environmental conditions with temperature (25 ± 1℃) and humidity (50 % ± 10 %) for a week before the experiment. They were kept under a 12 h light/12 h dark cycle (08:00–20:00, light) and free access to food and water. All rats were fasted for 48 h before experiments with free access to water. Coprophagy was prevented by keeping the animals in cages with gratings as the floors. The distribution of animals in the groups and the treatment allotted to each group with ten animals (seventeen groups) were randomized.
17 groups were treated as follows: Group 1: the rats in normal control were fed with 0.8 ml normal saline (0.9% NaCl) by gavage, rats in Group 2 to Group 17 were fed with 0.8 ml anhydrous alcohol by gavage. Group 2 served as model control, Groups 3 to Groups 17 were administered 8-BBR-Cn (suspended in distilled water) at dose of 25, 50 and 100 mg/kg body weight respectively. Groups 3 to Groups 17 were (25, 50 and 100 mg/kg) BBR group, (25, 50 and 100 mg/kg) 8-BBR-C4 group, (25, 50 and 100 mg/kg) 8-BBR-C8 group, (25, 50 and 100 mg/kg) 8-BBR-C12 group and (25, 50 and 100 mg/kg) 8-BBR-C16 group respectively.
The study was designed in accordance with ARRIVE guidelines. All experiments approved by the Ethical Committee of Animal Care of the Third Military Medical University under the bioethical allowance number SYXK-(Yu) 2007-0005 and complied with the Guide for Care and Use of Laboratory Animals.
Reagents and chemicals
PGE2 assay kit was obtained from Shanghai Baiwo technology co. LTD (Shanghai, China). NOS assay kit was obtained from Nanjing Jiancheng bioengineering institute (Jiangsu, China), other reagents and chemicals were of analytical reagent grade. Berberine (purity > 98%) was obtained from Xi'an Realin Biotechnology Co., Ltd. (Xi'an, China). 8-BBR-Cn (n=4, 8, 12 and 16, purity > 98%) were synthesized and provided by our laboratory 15.
Ethanol-induced gastric lesions
The rat model with gastric ulcer was established according to literatures 18, 19. The rats were fed with 8-BBR-Cn (n=0, 4, 8, 12 and 16) by gavage and two hours later the rats were fed with 0.8 ml anhydrous ethanol. One hour after the administration of ethanol, the animals were sacrificed and examined for lesions in the stomachs.
Stomach was incised along the greater curvature and examined for ulcers in the glandular region. Usually, circular lesions consisted of long, dark red bands running vertically down the corpus of the stomach were observed but, sometimes, linear lesions were also seen. The lesions were scored on a 0-10 scale according to the estimated percent of corpus mucosa covered with lesions. Stomachs of rats were subjected to visual macroscopic examination (Fig. 1) and ulcer score was calculated. The gastric tissues were stored at -80 ℃ before biochemical analysis.
Determination of ulcer index (UI) and percentage inhibition
UI and percentage inhibition in ethanol-induced rats were calculated. The stomach was cut open along the greater curvature and the inner surface was examined by dissecting microscope. The UI was calculated according to the scoring method of Tan et al20. Percentage ulcerated surface (US), was calculated as: US (mm2) = (total area covered by ulcers ÷ total corpus mucosal surface)×100. The following score was used in order to calculate ulcer indices: 0, no ulcer; 1, US≤0.5; 2, 0.5≤2.5; 3, 2.5≤5; 4, 5≤10; 5, 10≤15; 6, 15≤20; 7, 20≤25; 8,25≤30; 9, 30≤35; 10, US>35. The UI for each animal was then calculated as the mean ulcer score. Percentage inhibition was calculated according to the formula method of Hariprasath et al21. The percentage of inhibition was calculated by the following formula: [ (UI control − UI treated)÷UI control]×100.
The mucosa was scrapped and rapidly rinsed with ice-cold saline. The tissue was weighed and homogenized in 10 volumes of phosphate buffer (0.1 M, pH-7.4). The homogenate was centrifuged (3000×g, 15 min, 4 ℃) and the supernatant was transferred into a new test tube. The supernatant was processed for PGE2 estimation using the enzyme linked immunosorbent assay kit, following the manufacturer's instructions. Results were expressed as pg PGE2/ml.
Determination of NOS activity in mucosal tissue
The stomach was removed and gently rinsed with 0.9 % NaCl. Mucosa was scrapped and rapidly rinsed with ice-cold saline. The tissue was weighed and homogenized in 10 volumes of phosphate buffer (0.1 M, pH-7.4). The homogenate was centrifuged (3000×g, 15 min, 4 ℃) and the supernatant was used for NOS activity determination by NOS assay kit, following the manufacturer's instructions. The activites of iNOS and TNOS were measured directly and the cNOS activity was caculated by subtracting iNOS activity from TNOS activity. The OD value was determined by a spectrophotometer (U-3010, Hitachi, Japan) at 530 nm. Results were expressed as U NOS/mg prot (A unit of NOS was defined as 1 nmol NO produced by reacting 1 min in 1 mg of tissue protein at 37℃).
Protein concentration of the supernatants in tissue sample was determined by spectrophotometry using a commercial assay kit by Coomassie blue method.
Data from this study were analyzed by the version 17.0 of the SPSS programme and reported as mean ± SEM of three separate experiments for each sample. Statistical comparisons were performed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple range test.