The present study was approved by the Institutional Ethics Committee for Animal Experiments (2014/A-41). All experiments were performed at the Inonu University Experimental Animals Production Center (IUEAPC; Malatya, Turkey). The study protocol was executed in accordance with the United States Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, publication no. 5377-3, 1996), on the principles of animal research. A total of 32 Wistar albino female rats (3–4 months old; weight, 200±20 g) were used. Rats were purchased from the IUEAPC and randomly divided into groups (n=8/group). Each rat was kept in a separate cage. All rats were fed a standard rat pellet and tap water diet, with 12-h fasting employed prior to the experiment. Rats were maintained in an environment with a 12:12 light-dark cycle at 21±2°C room temperature and 60±5% humidity.

AA was used to induce acute colitis in rats. Rats were administered 4% AA at 24-h intervals for three days with a 6G nelaton catheter, under mild anesthesia [ketamine hydrochloride (50 mg/kg) and xylazine (5 mg/kg) mixture]. A catheter was inserted 6 cm into the anus, and 1 ml AA was administered followed by 1 ml air, after which rats were maintained in the Trendelenburg position for 15 min. Dxp was administered as a single dose of 500 mg/kg for three days, under mild anesthesia (as described above). An insulin injector (Beybi Plastik Fabrikasi Sanayi A.Ş, Istanbul, Turkey) was used to deliver Dxp into the peritoneum from the right lower quadrant of the abdomen. Dosages of AA and Dxp were adjusted according to previous dose-response studies (4,11).

A total of 32 rats were randomly divided into four equal groups (n=8); i) group I, the control group, 1 ml saline solution (0.9%) was administered intrarectally; ii) group II, rats received 4% AA (1 ml/day into the colon intrarectally) as a single dose for three consecutive days; iii) group III, rats received 4% AA (1 ml/day into the colon intrarectally) as a single dose for three consecutive days, and from day four, a single dose of Dxp (500 mg/kg; Bepanthene ampule^®; Bayer AG, Leverkusen, Germany) was administered intraperitoneally (IP) for three days; and iv) group IV, 500 mg/kg Dxp was administered IP as a single dose for three consecutive days.

On day 7, laparotomy was performed under high-dose anesthesia (70 mg/kg ketamine and 8 mg/kg xylazine; i.m.), and tissue specimens were subsequently collected from the area 10-cm distal from the colon. Oxidative stress markers, including malondialdehyde (MDA), total oxidant status (TOS), oxidative stress index (OSI), and anti-oxidant system markers, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione (GSH) and total anti-oxidant capacity (TAC) levels, were analyzed. Histopathological examination was performed under light microscopy.

For light microscopic analysis, samples harvested from the distal colon were fixed in 10% formalin for 48 h, dehydrated in an ascending alcohol series, and subsequently embedded in paraffin. Using a microtome, paraffin blocks were prepared for sectioning at 5-µm thickness. Sections were stained with hematoxylin and eosin to assess the general morphology and Periodic acid-Schiff was employed for goblet cell secretion assessment.

Assessment of colonic injury was performed using the following criteria: Mucosal epithelium (ulceration); lamina propria (polymorphonuclear cell infiltrate); crypts loss, submucosa (hemorrhage, edema, infiltration of inflammatory cells); and caspase-3 immuno-reactivity (according to the extent of cell staining in all layers of the colon). Colonic mucosal damage was evaluated on a 0–4 scale by two histologists, who were blinded to the experimental group. Grading criteria were as follows: Grade 0, normal appearance; grade 1, slight injury (0–25% involvement); grade 2, moderate injury (25–50% involvement); grade 3, severe injury (50–75% involvement); and grade 4, extensive full thickness injury (>75%) (12). The microscopic grade of each tissue was calculated as the sum of the scores given to each criteria; therefore, the maximum score that could be obtained was 28. Surface goblet cells and mitosis figures in crypts were counted using a Leica Q Win image analysis system (Leica Microsystems Ltd., Milton Keynes, UK) in 20 areas under ×40 objective.

For immunohistochemical analysis, sections of 4 µm thickness were placed on polylysine-coated slides. Following rehydration, samples were immersed in a citrate buffer (pH 7.6), heated in a microwave oven for 20 min, cooled for 20 min at room temperature, and subsequently washed with phosphate-buffered saline (PBS; three 2 min washes). Washed sections were immersed in 0.3% H₂O₂ for 7 min prior to washing again with PBS. Sections were incubated with a primary caspase-3 (CPP32) Ab-4 rabbit polyclonal antibody (cat no. RB-1197-R7; ready-to-use; Thermo Fisher Scientific, Inc.) for 30 min at room temperature, rinsed in PBS and subsequently incubated with biotinylated goat anti-polyvalent (cat. no. TP-125-BN; ready-to-use) antibody and streptavidin peroxidase (cat no. TS-125-HR) (both Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 10 min at room temperature. Staining was completed with chromogen substrate for 15 min and slides were counter-stained with Mayer's hematoxylin for 1 min, rinsed in tap water and dehydrated. Sections were examined by a histopathologist unaware of the experimental protocol using a Leica DFC 280 light microscope (Leica Microsystems, Ltd.). Caspase-3-positive cells in the cytoplasm were stained brown.

A total of 200 mg frozen colonic tissue was cut into sections using liquid nitrogen and homogenized in 10 volumes of ice-cold Tris-HCl buffer with respect to tissue weight (50 mmol/l; pH 7.4) using a homogenizer (Ultra Turrax IKAT18 basic homogenization; Werke, Staufen, Germany) for 3 min at 7,500 × g and 4°C. The supernatant solution was extracted with an equal volume of ethanol/chloroform mixture (3/5; v/v). Following centrifugation at 3,000 × g for 30 min at 4°C, the upper layer was used to analyze total tissue protein levels.

MDA content in the homogenates was determined spectrophotometrically by measuring the presence of thiobarbituric acid reactive substances (TBARS) (13). A total of 3 ml 1% phosphoric acid and 1 ml 0.6% thiobarbituric acid solution were added to 0.5 ml homogenate and pipetted into a tube. The mixture was heated in boiling water for 45 min and, following cooling, the colored part was supplemented with 4 ml n–butanol. Absorbance was measured using a spectrophotometer (UV-1601; Shimadzu Corporation, Kyoto, Japan) at 532 and 520 nm. Amount of lipid peroxide was calculated as TBARS of lipid peroxidation. Results were expressed in nmol/g tissue according to a prepared standard graph, prepared using the measurements of the standard solutions (1,1,3,3-tetramethoxypropane).

Protein content of the samples was determined by the method outlined by Lowry et al (14), using bovine serum albumin as a standard.

Total SOD activity was determined according to the method used by Sun et al (15), which is based upon the principle of the inhibition of nitroblue-tetrazolium (NBT) reduction by the xanthine-xanthine oxidase system as a superoxide generator. One unit of SOD was defined as the enzyme amount required to induce a 50% reduction in the NBT reduction rate. SOD activity was calculated as U/mg protein.

CAT activity was determined according to the method outlined by Aebi and Suter (16). The principle of the assay is based on the determination of the rate constant (k, s⁻¹) or H₂O₂ decomposition rate at 240 nm. Results are provided as k/g protein.

GPX activity was measured according to the method used by Paglia and Valentine (17). Briefly, in a tube containing NADPH, GSH, sodiumazide and glutathione reductase, an enzymatic reaction was initiated by the addition of H₂O₂, and the change in absorbance at 340 nm was recorded using a spectrophotometer. Activity was expressed in U/g protein.

Concentration of GSH in the homogenate was measured spectrophotometrically according to Ellman's method (17). Briefly, aliquots of tissue homogenate were mixed with distilled water and 50% trichloroacetic acid in glass tubes and centrifuged at 2,000 × g for 15 min at 4°C. Supernatants were mixed with 0.4 mol Tris buffer (pH 8.9) and 0.01 mol 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) was added. Following agitation of the reaction mixture, absorbance was measured at 412 nm within 5 min of the addition of DTNB against a blank sample that contained no homogenate. Absorbance values were extrapolated from a glutathione standard curve and provided as GSH (µM/g tissue).

TAC levels were determined using a novel automated colorimetric measurement method developed by Erel (18). In this method, a hydroxyl radical is produced by the Fenton reaction, which reacts with the colorless substrate, O–dianisidine, to produce a dianisyl radical that is bright yellow-brown in color. Following the addition of the sample, the oxidative reactions initiated by the hydroxyl radicals present in the reaction mix are suppressed by the antioxidant components of the sample. In turn, this prevents the color change and thereby provides an effective measurement of the total antioxidant capacity of the sample. This assay has been demonstrated to have excellent precision values (<3%). Results were presented as mmol Trolox equivalent/l.

TOS was determined using a novel automated measurement method, developed by Erel (18). Oxidants present in the sample oxidize the ferrous ion-O-dianisidine complex to a ferric ion. The oxidation reaction is enhanced by glycerol molecules, which are abundantly present in the reaction medium. The ferric ion forms a colored complex with xylenol orange in an acidic medium. Color intensity, which can be measured spectrophotometrically, is correlated to the total amount of oxidant molecules present in the sample. The assay was calibrated with H₂O₂ and the results were expressed in terms of µmol H₂O₂ equivalent/l.

The TOS to TAC ratio was accepted as the OSI, which is an indicator of the degree of the oxidative stress (20). The OSI value was calculated using the following formula: OSI (arbitrary unit)=TOS/TAC. The OSI value of the distal colon samples was also calculated as OSI (arbitrary unit).

Data were expressed as median (min-max) values or mean ± standard deviation depending upon the overall variable distribution. Normality was assessed using the Shapiro-Wilk test. Normally distributed data were analyzed by one-way analysis of variance, followed by Tukey's post-hoc tests. Non-normally distributed data were compared by Kruskal Wallis H test among the groups. When significant differences were determined, multiple comparisons were carried out using the Mann Whitney U test with Bonferroni correction. Statistical analyses were performed using SPSS version 22.0 for Windows (IBM SPSS, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

No animal mortality occurred during the experimental period. A decrease in the body weights was observed among the groups prior to and following the experiments (data not shown), however, this difference was not significant.

As demonstrated in Table I, a significant difference was detected in the MDA levels between group AA and the control group (P<0.05), and between the MDA levels in Group AA and Group AA + Dxp (P<0.05). A significant decrease was detected in SOD, CAT and GPX levels in Group AA, compared with the control group (P<0.05). When Group AA + Dxp and Group AA were compared, significant improvements were observed in SOD, CAT, GPX, and GSH parameters in Group AA + Dxp (P<0.05). Significant differences in TOS and OSI levels were also detected between Group AA and the control group (P<0.05). Furthermore, there were marked differences in TOS, TAC, and OSI levels, and a significant difference in TOS levels between Group AA and Group AA + Dxp (P<0.05).

Tissue sections of rats in the control and Dxp groups exhibited normal large bowel structures, and the colonic mucosa was observed as intact (Fig. 1A and B). Numerous goblet cells were identified on the surface epithelium and crypt in the control and Dxp groups (Fig. 2A and B). Notable histological damage occurred in the AA group (score, 12.5±2.3). The lamina epithelialis, lamina propria, muscularis mucosae, and the submucosa of the colon layers could not be distinguished from each other due to extensive inflammatory cell infiltration and ulceration (Fig. 3A). Diffuse inflammatory cells in the mucosa were predominantly composed of neutrophils. In addition to severe goblet cell depletion, an absence of crypts was observed (Fig. 3B).

The microscopic score of the AA + Dxp group was significantly reduced compared to the AA group (P<0.01); however, Dxp treatment did not completely alleviate lesions, it merely restricted them. Ulceration, inflammatory cell infiltration and loss of crypt were still present in the affected areas (Fig. 4A). In some areas, the mucosa remained intact in this group. In intact fields, goblet cells were detected on the surface and crypt epithelium (Fig. 4B). Additionally, the number of mitotic figures was significantly increased when compared with the AA group (P<0.0001).

Caspase-3 immunostaining was only detected in the epithelial cells on the luminal surface in the control and Dxp groups (Fig. 5A and B). Conversely, caspase-3-positive cells were observed only in epithelial crypt cells due to superficial epithelial ulceration in the AA group (Fig. 5C). The appearance of cells stained with caspase-3 in the AA + Dxp group was consistent with the control group (Fig. 5D). Results of histological grading and the number of goblet cells and mitotic figures in all groups are shown in Tables II and III.