2.3.2. Macroscopic Scores

Seven days after colitis induction, rats were sacrificed, and laparotomy was performed and the appearance of colon was then examined. Distal colon was rapidly removed, opened longitudinally and gently washed with saline. For each specimen wet weight (mg)weight/length (cm) ratio was calculated as indicator of colonic edema. For macroscopic damage, each animal was scored by Appleyard and Wallace classification system score [23], The sum of scores for the ulceration (0 = No mucosal damage, 1 = Localized hyperemia but no ulcers, 2 = Ulcers without hyperemia/bowel wall thickening, 3 = Ulcers with hyperemia/bowel wall thickening at 1 site, 4 =Two or more sites of ulceration or inflammation, 5 Area of damage (necrosis) extended >1 cm along length of colon, 6–10 Area of damage extended >2 cm along length of colon; increasing the score was by 1 for each additional cm involved) for the adhesions (0 = No adhesions, 1 = Minor adhesions (no di fficult separation of the colon from the other tissue), 2 = Major adhesions) and for the evaluation of thickness (Wall thickness(x) of bowel calculated in mm) were calculated.

#### 2.3.3. Analysis of Microscopic Inflammatory Damage

The colon tissues of rats were fixed in 4% formaldehyde (Sigma–Aldrich, Inc., St. Louis, MO, USA), embedded in para ffin for histological studies and sectioned (5 μm thick). The slices were stained with haematoxylin and eosin (Bio-Optica Milano SpA, Milano, Italy). Histological sections were examined in a blinded fashion, and photos of sections were taken by light microscope (Olympus BX50, Olympus Optical Co., Tokio, Japan). The microscopic damage following score was calculated the Hunter et al. [24] classification method: by adding the histological finding (0 = normal, 1 = minimal, 2 = mild, 3 = severe), the degree of inflammatory infiltration (0 = normal, 1 = minimal, 2 = mild, 3 = severe), the layers infiltrated (0 = normal, 1 = minimal, 2 = mild, 3 = severe), the mucosal damage (0 = normal,1 = minimal, 2 = mild, 3 = severe), and the edema in the mucosa (0 = absent, 1 = present).

#### *2.4. Assay of MPO Activity*

The activity of myeloperoxidase (MPO) is an important marker for inflammatory damage. The MPO activity was estimated spectrophotometrically using hydrogen peroxide and o-dianisidine as substrate for MPO enzyme, as previously described [20,21] by following a method of Moreels et al. [25] The absorbance value was read at 460 nm (Beckman-Coulter Inc, Brea, CA, USA). MPO activity was expressed as units per gram tissue (U gram tissue−1), taken that 1 unit of enzyme reduces 1 μmole hydrogen peroxide (H2O2) per minute.

#### *2.5. ELISA Assay for Pro-Inflammatory Cytokines*

Commercial ELISA Kits (Cloud-Clone Corp, Wuhan, Hubei, China) was used for the evaluation of colonic amounts of interleukin-1β and interleukin-6 according to the manufacturer's instructions, as previously described [20,21].

#### *2.6. NF*κ*-B, iNOS and COX-2 mRNA Analysis by Real-Time PCR*

Total RNA was extracted from colon tissues (10 mg) using RNAeasy Mini kit (Qiagen, Valencia, CA, USA). 2 ng of total RNA were reverse transcribed into cDNA using RT FirstStrand kit (Qiagen, Valencia, CA, USA). The amplification of synthesized cDNAs was performed using SYBR Premix Ex Taq II (TaKaRa, Bio Inc., Foster City, CA, USA) and StepOne Real-Time instrument (Applied Biosystems, Foster City, CA, USA). Gene expression of inducible NOS (iNOS), COX-2 and beta actin, a housekeeping gene that is not subject to regulation, was performed in triplicate, using specific primers and amplification conditions. The oligonucleotide primer sequences were reported in Table 1.


#### **Table 1.** Primers used for qRT-PCR.

The program for PCR was 1 cycle of 95 ◦C (10 min), followed by 45 cycles of amplification. Consisting of denaturation at 95 ◦C (15 s), annealing at 60 ◦C (30 s), and extension for at 72 ◦C (30 s). For terminal elongation period, the samples were incubated at 72 ◦C (additional 10 min) at the end of the final cycle. The expression level was calculated from the PCR cycle number (CT) where the increased fluorescence curve passes across a threshold value. The relative gene expression of the target genes was calculated using 2−ΔΔCt approximation method algorithm.

#### *2.7. Analysis of Reactive Oxygen Species (ROS) Generation*

The conversion of non-fluorescent DCFH-DA to 2, 7 dichlorofluorescein (DCF) was evaluated, as previously described [20,21], to monitor the amount of hydrogen peroxidase in the sample. Sample was analyzed by fluorimeter (Microplate reader WallacVictor 2-1420 Multilabel Counter; PerkinElmer, Inc., Waltham, MA, USA) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

#### *2.8. Nitric Oxide (NO) Concentration Assay*

Nitric oxide concentration was measured using the Griess reaction [30]. Briefly after homogenization of rat colon tissue (10 mg) with 1 mL PBS, pH 7.2 and centrifugation (14,000 rpm, for 30 min, 4 ◦C) the supernatant was incubated with equal volume of Griess reagen<sup>t</sup> (room temperature, 15 min in the dark) and the absorbance was measured at 550 nm with a spectrophotometric Microplate reader (WallacVictor 2 Multilabel Counter, Perkin Elmer, Apeldoorn, The Netherlands) NO concentration was evaluated by using a standard curve.

#### *2.9. Data Analysis and Statistical Tests*

Results are shown as the mean ± SEM: '*n*' indicates the number of animals. Statistical analysis was performed using GraphPad Prism 6.0 software, and sets were assessed by one-way ANOVA followed by Tukey's multiple comparison test. *p* value < 0.05 means statistically significant. To compute the sample size G\*Power version 3.1.2 [31], was used, given power (1 − β) = 0.8.
