Animals. There were provided male Wistar rats (180-200 g) by the animal laboratory of the Medicinal Plants Research Center, Institute of Medicinal Plants (IMP), Karaj, Iran. Sixty rats were allocated in plastic cages for 3 days to adapt to a new condition in the animal house under ordinary circumstances (12:12 h light:dark cycle, humidity (55±7%), and room temperature (23±2°C)). Each group consisted of 6 animals. Their diet consisted of a standard diet and access to water ad libitum. This study approved by the ethical committee of Iran Islamic Azad University (ethics code: IR.IAU.PS.REC.1399.296)
Materials. Aminoguanidine (AG), a specific neuronal nitric oxide synthase (nNOS) inhibitor and L-NG-Nitro Arginine Methyl Ester (L-NAME), a non-specific NOS inhibitor(Sigma, St Louis, MO, USA). Licofelone (2-[6-(4-chlorophenyl)-2,2-dimethyl-7-phenyl-2,3-dihydro-1H-pyrrolizin-5-yl] acetic acid), a COX1/COX2 and 5-LOX inhibitor was a generous gift from Tofigh daru (Tehran, Iran). The ELISA kits for NF-κB and TLR-4 were purchased from ZellBio Company(Germany), and for SOD were purchased from Kiazist Company (Iran). All chemicals used in the study were of analytical grade.
Preparation of Licofelone. Licofelone was dissolved in slightly alkaline water, sonicated, and used for intraperitoneal (i.p.) injection into the rats (Eslami et al., 2016).
Study design: The study period for all groups was 3 days. Starting from the colitis induction day with acetic acid (1 mL, 4%, i.r.) into 9 groups (day 1), the remaining group was considered a sham. Drugs were administered once a day for two successive days. Rats are denied access to food for 24 hours before colitis induction day. Dosage of drugs Recommended based on previous studies (Eslami et al., 2016, Fakhraei et al., 2014a). In this study, 6 male rats in each group are used, 10 groups have been considered for the study, which includes (n=6):
- Sham group: Rats were treated with normal saline via Intraperitoneal injection.
- Control group: Rats received normal saline via Intraperitoneal injection.
- Dexamethasone group: Dexamethasone was administrated at 1 mg/kg, i.p., 24 hours after receiving acetic acid
- Licofelone-2.5 group: licofelone was administrated at 2.5 mg/kg i.p. 24 hours after receiving acetic acid
- Licofelone-5 group: licofelone was administrated at 5 mg/kg i.p. 24 hours after receiving acetic acid
- Licofelone-10 group: licofelone was administrated at 10 mg/kg i.p. 24 hours after receiving acetic acid
- AG-100: AG was administrated at 100 mg/kg i.p. 24 hours after receiving acetic acid
- L-NAME-10: L-NAME was administrated at 10 mg/kg i.p. 24 hours after receiving acetic acid
- Licofelone-10 + AG-100 group: AG was received 30 min before administering the maximum dose of licofelone
- Licofelone-10 + L-NAME-10 group: L-NAME was received 30 min before administering the maximum dose of licofelone
48 hours after induction of colitis, animals were euthanized by CO2. The abdomen was immediately opened, and the last 8 cm of the colon was removed and cleaned with normal saline. Colon segments were scored, as will be explained in more detail later. Segments were divided into two parts, one part was kept in 10 cc formalin 10% for microscopic evaluation. Another part was weighed and stored at -80 ° C until biomarker analysis.
Intraperitoneal injections. One of the most common methods in studies is i.p. injection, in which a drug is injected into the peritoneal cavity. It is an easy, convenient, and fast method with minimal stress for animals and higher absorption capacity than oral, IM, and SC routes. In addition, a large volume of solution (maximum 10 ml/kg) can be safely injected into rats in this way (Al Shoyaib et al., 2020).
Induction of colitis by Acetic acid. Acetic acid induces severe damage to epithelial tissue by creating local inflammation and destroying the integrity of the mucosa, so according to the pathogenesis and histological characteristics, acetic acid-induced colitis is an animal model that mimics inflammations seen in UC. Therefore, it is a valuable animal model for studying drugs with anti-inflammatory effects, such as Licofelone. Colitis was induced by intrarectal administration of acetic acid. Initially, rats were given 50 mg/kg pentobarbital sodium intraperitoneally to induce colitis, after which they were positioned on their right side and were given 1ml acetic acid 4% by injection through a polyurethane conduit for enteral feeding, after that, the rats were held in supine Trendelenburg position because of preventing anal leakage of acetic acid (Fakhraei et al., 2014a, Saadatzadeh et al., 2012).
Determination of ulcer index
Macroscopic evaluation. The severity of colonic damage caused by inflammation in the macroscopic examination was evaluated immediately after removing the colon using the following scoring system: 0 (normal appearance with no damage), 1 (localized hyperemia without ulceration), 2 (Linear ulcer without distinct inflammation), 3 (Linear ulcer with inflammation in one area), 4 (Two or more sites of ulceration and/or inflammation), 5 (Two or more significant sites of inflammation and ulceration or one major site of inflammation and ulceration extending >1 cm along the length of the colon) (Morris et al., 1989).
Microscopic evaluation. For microscopic evaluation, colon samples were collected and fixed in formalin 10%, embedded in paraffin, and prepared in 5 μm-thick paraffin sections. Sections were stained with hematoxylin and eosin, last, microscopic evaluation was performed by light microscopy (Stumhofer et al., 2006).
Biochemical assays
Sample preparation to evaluate inflammatory factors. Samples were weighed and homogenized in an ice bath in 10 volumes of 50 mM phosphate buffer with pH=7.4. Then 500 μl of the homogenized mixture was transferred to the microtubes for IL-1β and TNF-α tests, and the remaining was centrifuged at 3500g for 30 minutes, then supernatants were separated and transferred to the microtubes for myeloperoxidase (MPO) and NF-κB tests, and finally, all the solutions and the remaining precipitate from this initial centrifuge were frozen at -80 ° C until biomarker analysis.
Real-time reverse transcription-polymerase chain reaction (RT-PCR). In this method, the expression of the studied genes of all samples in the groups should be examined and compared with the positive, negative, and sham control groups. For this purpose, RNA was extracted using BioFACT's RNA extraction kit in a columnar method.
Then, using the nanodrop spectrophotometer technique, the quantitative evaluation of RNA was performed. The nanodrop device automatically measures the RNA concentration using optical absorption (OD) at 260/260 nm and calculates the desired sample concentration at the corresponding wavelength in nanograms per microliter. In addition, the purity of the resulting RNA is determined by the absorption ratio of 260 to 280, and a ratio greater than 1.6 is considered acceptable. First, 1 µl of RNA-containing solution is reduced to 100 µl with DEPC-treated water, and as mentioned earlier, its optical absorption by the spectrophotometer at 260 and 280 nm is read.
Then, using BioFACT's cDNA synthesis kit, cDNA was synthesized by reverse transcription method during the following steps. Each step was carried out on the ice and under sterile conditions under a hood.
Finally, the Real-time PCR technique was performed using specific Forward and Revers primers of IL-1β, TNF-α, and GAPDH genes, which was used as housekeeping or reference gene and according to the kit proposed by Kit. The RT-PCR reaction is performed to amplify IL-1β, TNF-α, and GAPDH genes in a final volume of 20 μl (The sequence of primers used is given in table 1) (Momtaz et al., 2021).
Determination of NF-κB and TLR-4. Colonic levels of NF-κB and TLR-4 were qualified with an enzyme-linked immunosorbent Assay (ELISA kit) (ZellBio Co.) based on the Biotin double antibody sandwich technology. According to the manufacturer’s protocol, samples were prepared and placed into the ELISA kit strips precoated with appropriate monoclonal antibodies. After that, the antibodies labeled with biotin were added to combine with streptavidin-HRP, which forms an immune complex. Then, each well was filled with washing, Chromogen, and stop solution. At last, the OD value read within 10 minutes after adding the stop solution at 450 nm.
Determination of SOD. The SOD activity in tissue samples was measured with a SOD detection Kiazist company kit (Tehran, Iran). This test is based on the fluorometric method, and the production of superoxidase radicals by xanthine oxidase is a fundamental principle. These radicals react with resazurin to produce resorufin dye, which is detectable at Ex/Em=575/585. In brief, according to the manufacturer’s instructions, samples were prepared and then transferred into wells, and at last, the absorbance was measured at Ex/Em=575/585 (Nasiri et al., 2021).
Determination of ROS. The test was performed according to the method described previously (7). The formation of intracellular ROS was measured using the oxidation-sensitive fluorescent dye 2,7-dichlorofluorescein diacetate (DCFH-DA). The assay contained extraction buffer and assay buffer, and in this method, the oxidation rate of DCFH-DA to 2,7-dichlorofluorescein (DCF) can be used as a marker to measure the amount of oxygen-free species like ROS. The advantage of this test is that it measures all active oxygen species, not just those produced by specific pathways.
MPO activity assay. The MPO activity was measured as an indicator of leukocyte infiltration into the gastrointestinal tract. In other words, MPO is an enzyme found in neutrophils, monocytes, and macrophages, and it has been responsible for antimicrobial reactions and the prevalence of acute, chronic inflammatory reactions. So, it is a reliable index for assessing the degree of inflammation of colitis (Matheson et al., 1981).
The measurement of MPO activity was performed according to the method described previously (Ghasemi-Niri et al., 2011). The change in absorbance per min was measured immediately at 460 nm spectrophotometrically.
Total protein. The protein concentration in colon tissues was measured according to the Bradford method using Bovine serum albumin (BSA) as the standard (Bradford, 1976).
Statistical analysis. The biochemical values were shown as mean ± standard error of mean (SEM) and analyzed by one-way ANOVA with post hoc Tukey’s test to evaluate group changes. P-value ˂ 0.05 was regarded as significant.