Licofelone Attenuates Acetic Acid-Induced Colitis in Rats Through Suppression of the Inflammatory Mediators

Licofelone is a dual Cyclooxygenase 1,2 (COX1,2)/5-lipoxygenase) 5-LOX (inhibitor with analgesic and anti-inflammatory effects with possible functions on inflammatory bowel disease (IBD), which is a chronic recurrent condition with no particular treatment. This study evaluated the anti-inflammatory effects of licofelone on acetic acid-induced colitis in rats. Ten groups of male Wistar rats (n = 6) were used. Sham, control group, licofelone at doses of 2.5, 5, and 10 mg/kg, L-NG-nitro arginine methyl ester (L-NAME) (10 mg/kg, i.p.), aminoguanidine (AG) (100 mg/kg, i.p.), 30 min before using licofelone (10 mg/kg). Also, three groups received L-NAME, aminoguanidine, or dexamethasone. Macroscopic, microscopic, and biochemical analysis of myeloperoxidase (MPO), and nuclear factor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β), superoxide dismutase (SOD), reactive oxygen species (ROS), and Toll-like receptor 4 (TLR-4) were assessed in colon tissue. Licofelone at a dose of 10 mg/kg attenuated colitis, increased SOD activity, and significantly reduced colonic levels of the abovementioned inflammatory factors. In addition, licofelone improved macroscopic and microscopic symptoms in the acetic acid-induced colitis model. Moreover, the concurrent use of nitric oxide synthase (NOS) inhibitors with 10 mg/kg of licofelone reversed the observed positive effects, demonstrating the function of nitric oxide in IBD pathogenesis and the probable mechanism for licofelone in the healing process of induced colitis. A reduced level of inflammatory factors confirmed the anti-inflammatory activity of licofelone as a dual COX1,2/5-LOX inhibitor. Furthermore, outcomes revealed the protective role of licofelone in treating experimental colitis. The findings are suggestive of the potential use of licofelone in IBD.

Abstract-Licofelone is a dual Cyclooxygenase 1,2 (COX1,2)/5-lipoxygenase) 5-LOX (inhibitor with analgesic and anti-inflammatory effects with possible functions on inflammatory bowel disease (IBD), which is a chronic recurrent condition with no particular treatment.This study evaluated the anti-inflammatory effects of licofelone on acetic acid-induced colitis in rats.Ten groups of male Wistar rats (n = 6) were used.Sham, control group, licofelone at doses of 2.5, 5, and 10 mg/kg, L-NG-nitro arginine methyl ester (L-NAME) (10 mg/kg, i.p.), aminoguanidine (AG) (100 mg/kg, i.p.), 30 min before using licofelone (10 mg/kg).Also, three groups received L-NAME, aminoguanidine, or dexamethasone.Macroscopic, microscopic, and biochemical analysis of myeloperoxidase (MPO), and nuclear factor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β), superoxide dismutase (SOD), reactive oxygen species (ROS), and Toll-like receptor 4 (TLR-4) were assessed in colon tissue.Licofelone at a dose of 10 mg/ kg attenuated colitis, increased SOD activity, and significantly reduced colonic levels of the abovementioned inflammatory factors.In addition, licofelone improved macroscopic and microscopic symptoms in the acetic acid-induced colitis model.Moreover, the concurrent use of nitric oxide synthase (NOS) inhibitors with 10 mg/kg of licofelone reversed the observed positive effects, demonstrating the function of nitric oxide in IBD pathogenesis and the probable mechanism for licofelone in the healing process of induced colitis.A reduced level of inflammatory factors confirmed the anti-inflammatory activity of licofelone as a dual COX1,2/5-LOX inhibitor.Furthermore, outcomes revealed the protective role of licofelone in treating experimental colitis.The findings are suggestive of the potential use of licofelone in IBD.

INTRODUCTION
There are two main types of inflammatory bowel disease (IBD): ulcerative colitis (UC) and Crohn's disease (CD).The etiology of IBD is unknown; however, various factors, such as age, sex, environmental, immunological, pharmacological, etc., are involved in its pathogenesis.Moreover, poor mucosal immunity, oxidative stress, smoking, appendectomy, pregnancy, oral contraceptives, specific dietary habits, measles infection, and vaccination may also affect the rate of incidence and progression of IBD [1,2].The pathogenesis of such diseases is greatly affected by inflammation.The inflammatory pathways are very diverse, and many pathways such as the mechanistic target of rapamycin (mTOR), Toll-like receptor (TLR), signal transducer and activator of transcription (STAT), nuclear factor-kappa B (NF-κB), etc. have shown to induce IBD [3].These pathways are completely interconnected, and the activation of each stimulates the other inflammatory pathways, leading to a synergistic effect.The expression of the TLR gene is an influential inflammatory factor in the innate immune system and progression of inflammatory processes in IBD [4].Inadequate response of T helper cells and extreme expression of cytokines, i.e., interleukin (IL)-12, IL-6, IL-1β, tumor necrosis factor-α (TNF-α), and interferon-gamma (IFN-γ), have been observed immunomodulator IBD patients.Oxidative stress is one of the most critical factor in IBD development due to the destructive effects of active radicals, which exacerbate and induce inflammation [5].Significant decrease in the antioxidant levels, and elevation in reactive oxygen species (ROS) level have been reported in patients with IBD.Inflammation of the joints (arthritis) has been observed in 10% of patients with ulcerative colitis [5].Toxic megacolon ulceration, coronary adenocarcinoma, and the risk of colonic malignancy are of the most common hallmarks of UC due to the inflammatory process.In general, IBD develops in patients between 15 and 30, with 25% of patients being children or adolescents [6,7].Current treatments aim to reduce abnormal inflammation and symptoms such as diarrhea, bleeding, and pain.For instance, corticosteroids are used to reduce inflammation, while immunosuppressants and aminosalicylates are other IBD medications now with fewer therapeutic effects and have various side effects [8,9].An NSAID, licofelone, inhibits the COX/LOX pathway on a dual basis.Licofelone inhibits certain enzymes that produce prostaglandin (PGEs) and leukotrienes and has recently been approved for treating osteoarthritis.Several studies have reported analgesic, anti-inflammatory, and neuroprotective effects of licofelone (especially in the central nervous system).Studies have demonstrated that licofelone has beneficial effects on inflammatory diseases, which are mainly due to its ability to reduce inflammation [10].Therefore, in this study, we investigated the probable anti-inflammatory and antioxidant effects of licofelone in animal of IBD.

Animals
Male Wistar rats (180-200 g) were provided by the animal laboratory of the Medicinal Plants Research Center, Institute of Medicinal Plants (IMP), Karaj, Iran.Sixty rats were allocated for 3 days prior to study to adapt to new condition in the animal house under standard conditions (12:12 h light: dark cycle, humidity (55±7%), and room temperature (23±2 °C)), with access to standard diet and water.This study was approved by the ethical committee of Iran Islamic Azad University (ethics code: IR.IAU.PS.REC.1399.296).

Preparation of Licofelone
Licofelone was dissolved in slightly alkaline water, sonicated, and used for intraperitoneal (i.p.) injection into the rats [11].
Forty-eight hours after induction of colitis, animals were euthanized by CO 2 .The abdomen was immediately opened, and the last 8 cm of the colon was removed and cleaned with normal saline.The colon segments were divided into two parts; one part was kept in formalin 10% for microscopic evaluation.Another part was weighed and stored at −80 °C until biomarker analysis.

Induction of Colitis by Acetic Acid
Initially, the rats were anesthetized by 50 mg/kg pentobarbital sodium (intraperitoneal injection).The rats were positioned on their right sides and were given 1 ml acetic acid 4% by injection through a polyurethane conduit for enteral feeding.Thereafter, the rats were held in supine Trendelenburg position because of preventing anal leakage of acetic acid [12][13][14].

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) [15].

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.Lastly, light microscopy was performed for microscopic evaluation [16].

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 3500 g for 30 min; 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)
RNA was extracted using RNA extraction kit (Bio-FACT company, Korea, BR123-R10K) 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 RNAcontaining solution diluted to 100 µl with DEPC-treated water, and as mentioned earlier, its optical absorption was read by the spectrophotometer at 260 and 280 nm.
Then, using cDNA synthesis kit (BioFACT company, Korea, BR631-096), cDNA was synthesized by reverse transcription method following the instruction of the kit.
The Real-time PCR technique was performed on LightCycler 96 using the comparative Ct quantitation method by using specific Forward and Revers primers of IL-1β, and TNF-α.GAPDH was used as housekeeping or reference gene.The RT-PCR reaction was 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).Ct values of GAPDH were subtracted from Ct values of the target genes (ΔCt).ΔCt values of licofelonetreated animals were compared with ΔCt values of untreated animals.Relative changes in gene expression were determined using the 2 -ΔΔCt method compared with reference GAPDH mRNA level [17].

Determination of NF-κB and TLR-4
Colonic levels of NF-κB and TLR-4 were quantified with an enzyme-linked immunosorbent assay ELISA kit (ZellBio GmbH Company, Germany, ZB-10287C-R9648 and ZB-10080C-R9648) based on the Biotin double antibody sandwich technology.According to the manufacturer's protocol, samples were prepared and placed into the ELISA kit strips pre-coated with appropriate monoclonal antibodies.Then, the antibodies labelled with biotin were added to combine with streptavidin-HRP, forming an immune complex.Then, each well was filled with stop solution.Finally, the absorbance was measured within 10 min after adding the stop solution at 450 nm.

Determination of SOD
The SOD activity in tissue samples was measured with a SOD detection kit (Kiazist Company, Iran, KSOD96).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 lastly, the absorbance was measured at Ex/Em = 575/585 [18].

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.

MPO Activity Assay
Measurement of MPO activity is indicative of leukocytes infiltration into the gastrointestinal tract.MPO is an enzyme found in neutrophils, monocytes, and macrophages, and it is associated with antimicrobial reactions, as well as acute and chronic inflammation.Thus, it is a reliable index for assessing the degree of inflammation of colitis [19].The measurement of MPO activity was performed according to the method described previously [20].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 [21].

Statistical Analysis
An analysis of all data was conducted using SPSS V24 (SPSS Inc., Chicago, IL, USA) as the statistical package.The results were recorded 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.

Macroscopic Evaluation
Morphological observations of the colon were graded into five scales according to Table 2.The macroscopic assessment of the colon in control group showed no epithelium damage, whereas the induction of colitis led to the severe mucosal injury, wall thickening, ulceration, edema and necrosis in the colon of the acetic acid group.In contrast, the administration of dexamethasone (1 mg/kg) and licofelone at doses of 2.5, 5, and 10 mg/ kg ameliorated the macroscopic lesions compared with the acetic acid group (Fig. 1 and Table 2).However, licofelone at 10 mg/kg showed the highest recovery of colitis compared to the control (P < 0.001).Treatment with L-NAME and AG reversed the protective effects of licofelone and increased the intensity of inflammation (P < 0.001).

Effect on Tissue Histopathology Assay
Histological assessment of the colon indicates that induction of colitis in the control group caused inflammation, necrosis, ulceration, adhesions, and wall thickening (P < 0.001).In contrast, histological assessment of the colon in sham group showed normal mucosa, submucosal, muscularis propria, and serosa without inflammation and necrosis.The administration of dexamethasone (1 mg/kg) and licofelone at doses of 2.5, 5, and 10 mg/kg led to the reduction of acetic acid-induced colonic damage.However, licofelone at 10 mg/kg showed the highest recovery of colitis compared to the control (P < 0.001).Treatment with L-NAME and AG reversed the protective effects of licofelone and increased the intensity of inflammation (P < 0.001) (Fig. 2).

Effect on mRNA Expression of IL-1β
As it is depicted in Fig. 3, the administration of acetic acid caused a significant increase in the mRNA levels of IL-1β in the control group in comparison with the sham group (P < 0.001).Administration of dexamethasone (1 mg/kg), and licofelone (10 mg/kg) significantly lowered the mRNA expression of IL-1β in comparison with the control group (P < 0.01).There was no significant change in mRNA expression of IL-1β between licofelone groups at 5 and 10 mg/kg and the dexamethasone group.Treatment with L-NAME and AG reversed the protective effects of licofelone, and as shown in Fig. 3, the mRNA expression of IL-1β was significantly higher.

Effect on mRNA Expression of TNF-α
The administration of acetic acid significantly increased the mRNA expression of TNF-α in the control group in comparison with the sham group (P < 0.001).The administration of dexamethasone (1 mg/ kg) decreased the expression of TNF-α mRNA level compared with the control (P < 0.01).In addition, the licofelone groups at 5 and 10 mg/kg significantly reduced the TNF-α expression as compared with the control group (P < 0.05 and P < 0.001, respectively).On the other hand, all three treatment groups L-2.5, L-5, and L-10 were not significantly different from the dexamethasonetreated group.Treatment with L-NAME and AG reversed the protective effects of licofelone (Fig. 4).group was significantly higher in comparison with the sham group (P < 0.001).Dexamethasone and licofelone at 10 mg/kg significantly reduced the enzyme levels as compared with the control group (P < 0.001).In addition, licofelone at 5 mg/kg reduced the MPO activity in comparison with the control group (P < 0.01).Among the treated groups, licofelone at 10 mg/kg was found to be the most effective.Treatment with L-NAME and AG reversed the protective effects of licofelone (Fig. 5).

Effect on NF-κB
As shown in Fig. 6, the administration of acetic acid led to a significant elevation of the NF-κB level in the control group as compared with the sham group (P < 0.001).Additionally, L-2.5, 5, 10, and dexamethasone-treated groups showed a significant reduction in NF-κB level in comparison with the control group (P < 0.001).All three treatment groups L-2.5, L-5, and L-10 were not significantly different from the dexamethasone-treated group.Treatment with L-NAME and AG reversed the protective effects of licofelone (Fig. 6).

Effect on TLR-4
Acetic acid treatment caused a significant elevation in TLR-4 concentration in the control group compared with the sham group (P < 0.001).In addition, dexamethasone, L-5, and L-10 reduced the TLR-4 level as compared with the control group (P < 0.001).Treatment with L-NAME and AG reversed the protective effects of licofelone (Fig. 7).

Effect on SOD
The SOD activity was reduced in the acetic acid-induced colonic inflammation rats.The SOD activity was diminished in the control group as compared with the sham group (P < 0.001).Moreover, dexamethasone, L-5, and L-10 groups caused a significant elevation in SOD activity when compared with the control group (P < 0.001).Treatment with L-NAME and AG caused a reduction of the SOD activity as compared with the sham group (Fig. 8).

Effect on ROS
The ROS activity is shown in Fig. 9.In the control group, the ROS level was higher than that of the sham group (P < 0.001).The ROS level was significantly lower in the dexamethasone treated animals compared with that of the control group (P < 0.001).The L-10-treated group exhibited a significant reduction in ROS level compared with that of the control group (P < 0.01).Treatment with L-NAME and AG enhanced the SOD activity, similar to that of the control group.

DISCUSSION
This study confirms that licofelone has beneficial effects on inflammatory mediators in IBD animal model, which was clarified via analysis of the inflammatory indicators (i.e.TNF-α, IL-1β, MPO, TLR-4, and NF-κB), and oxidative indicator (i.e.SOD, ROS).It is widely understood that an animal model of acetic acid-induced colitis represents the clinical and histopathological manifestations of IBD, similar to those seen in humans [22].
In this investigation, licofelone was intraperitoneally injected into acetic acid-induced colitis rats at three doses.The compound implied its best function at 10 mg/kg/day by attenuating TNF-α, IL-1β, MPO, ROS, TLR-4, and NF-κB levels, while increasing the SOD activity.The administration of acetic acid induced acute, severe, and transmural inflammation in the colonic tissue, which was significantly improved by licofelone, at all concentrations, in both microscopic and macroscopic examinations.
anti-platelet and anti-inflammatory activities [23].There is no doubt that inflammatory markers, such as TLR-4, play significant roles in developing IBD [24,25].The signaling pathways activated by TLR-4 are responsible for activating several downstream cascades such as the NF-κB pathway, which is crucial for the initiation and progression of IBD.Literature review reveals that anti-inflammatory agents such as dexamethasone [26], or amitriptyline [27] could reduce colonic inflammation by inhibiting the TLR-4 receptor/pathway.Herein, we found that licofelone treatment significantly inhibited TLR-4 expression in acetic acid-induced colitis animals.However, the administration of L-NAME (non-specific NOS inhibitor) and AG (nNOS inhibitor) alone or in concurrent with licofelone could not suppress the TLR-4 expression.
However, the exact mechanism of action of licofelone or its common characteristics in different disorders or cell types has not yet been identified.Inhibition of mitochondrial enzyme complexes, the NF-κB pathway, the NO pathway, etc. may effectively alleviate the colitis pathology.
On the other hand, plenty of studies confirmed that pro-inflammatory and inflammatory cytokines such as ILs, damage the mucosa directly.Extensive mucosal damage can result in increased release of mucosal cytokines such as TNF-α and IL-1β [28]; thereby, both the onset and progression of IBD are affected by cytokines mainly through altering the epithelial integrity, modulating the mucosal immune system, and regulating the neutrophil and macrophage infiltration and activation, resulting in colonic damage.Induction of pro-inflammatory cytokines leads to the NF-κB translocation and activation, which in turn enhances the inflammatory cytokines production in the intestine, thus exacerbating IBD [30,31].Therefore, TNF-α, IL-1β, and their upstream signaling pathways, such as NF-κB, are promising anti-inflammatory targets in managing patients with IBD.Fig. 5 MPO activity in the colon.*** : significant distinction with sham (P < 0.001).### : significant distinction with control (P < 0.001).## : significant distinction with control (P < 0.01).&&& : significant distinction with L-10 (P < 0.001).Values are mean± SEM.
It was shown that licofelone has inhibitory effect on the COX-2 enzyme activity in human mesangial cells and prostaglandin E2 release in response to IL-18.Furthermore, licofelone decreases the 5-LOX enzyme activity and leukotriene release when exposed to IL-18.It has been shown that licofelone reduces the phosphorylation of p38 mitogen-activated protein kinase induced by IL-18 and suppresses the production of monocyte chemotactic protein-1 and IFN-γ [29].
In another study, licofelone (2.5, 5, and 10 mg/ kg/day, i.p.) treatment for 7 days significantly improved locomotor activity, reduced the severity of catatonia and oxidative damage, and improved mitochondrial enzyme complex activity in comparison with MPTP treatment.It was also noted that the administration of licofelone attenuated the expression of the apoptotic factor (caspase-3) and the transcription factor (NF-κB/p65) in comparison with MPTP treatment [32].
In our study, L-2.5, 5, 10, and dexamethasone treatment reduced the level of NF-κB, which was enhanced following IBD induction.Blockage of related NO signaling pathways by L-NAME and AG, diminished the protective effects of licofelone.
In an in vivo study, TNF-α, and IL-1β levels were elevated in the control, L-NAME, and AG-treated groups.In contrast, their levels were reduced in rats that only received licofelone in different doses, suggesting that licofelone can inhibit both COX/LOX, and the related NO signaling pathways to reduce pro-inflammatory markers in inflamed colonic sites [10].This study showed that licofelone possessed anti-inflammatory properties in IBD model, and was able to enhance the antioxidant defense system through a process that involves nitration and lipid peroxidation.In fact, it is well known that dual inhibitors of COX and LOX are clinically more effective than selective inhibitors of COX, as they are able to inhibit both metabolic pathways of arachidonic acid [33].
Moreover, we found that the levels of colonic MPO were significantly enhanced following acetic acid injury, while licofelone treatment reduced MPO levels.
Our results are in agreement with other studies that have shown that the activity of colonic MPO increases in animals with acetic acid-induced IBD [34][35][36].
Our results indicate that ROS level was significantly lower in dexamethasone, as well as L-5 and L-10 groups than in the control animals.After treatment with L-NAME and AG, the protective effect of licofelone was reversed due to earlier obstruction of the related NO signaling pathways, and the ROS levels were significantly higher.
Kumar et al. [37] demonstrated that oral administration of licofelone (2.5, 5, and 10 mg/kg) caused a significant decrease in lipid peroxidation, and nitrite concentration, while there was an increase in antioxidant enzyme activity (SOD and catalase).However, licofelone (10 mg/kg, oral administration) was not able to alter lipid peroxidation, nitrite concentrations, or the SOD and catalase enzymatic activities in 3-nitropropionic acid-induced Huntington's disease-like symptoms animal model.
In the present study, acetic acid-induced colonic inflammation resulted in a reduction in SOD activity.Dexamethasone, L-5, and L-10 treatment caused a significant increase in SOD activity, whereas treatment with L-NAME and AG reversed the licofelone's protective effects by inhibiting the NO receptors and preventing NOS signaling pathways.
Similar to the findings of the other studies, our results indicated that licofelone increased the SOD activity and inhibited NOs in colitis [38].
Three forms of nitric oxide synthase (NOSs) are responsible for the synthesis of NO from l-arginine, of which two are expressed in the neuronal cells and the other in the endothelial cells, respectively (nNOS and eNOS).Additionally, iNOS is produced in various cells during inflammation.It has been reported that IBD patients have higher levels of NO.NO has been shown to provide protection and to have beneficial effects in treating acute colitis [39][40][41].Fig. 7 TLR-4 level in colon.*** : significant distinction with sham (P < 0.001).### : significant distinction with control (P < 0.001).&&& : significant distinction with L-10 (P < 0.001).Values are mean± SEM.
In our study, utilization of NOS inhibitors such as L-NAME, and AG, exhibited no ameliorating effect on ulcers and inflammation in colonic samples.Licofelone was utterly ineffective in treating macroscopic ulcers and lesions when administered with the NOS inhibitors.The biochemical evaluations revealed an increase in IL-1β and TNF-α levels after induction of colitis.However, the licofelone administration reduced the mRNA expression of IL-1β, and TNF-α.A comparison between the NOS inhibitor groups and the control group showed that the NOS inhibitors were not able to reduce the production of inflammatory cytokines.Intriguingly, injection of NOS inhibitors 30 min before licofelone, neutralized the licofelone's anti-inflammatory effect.Infiltration and expansion of inflammatory cells, destruction of goblet cells, and demolition of crypt structures were some of the histological characterizations that clarified the point that the NOS inhibitors neutralized the protective effects of licofelone.Licofelone reduced necrosis, edema, congestion, and damage severity in the mucosa and submucosa, most notably at a dose of 10 mg/kg.All treatment groups that received AG 100 mg/kg, L-NAME 10 mg/kg, and licofelone (10 mg/kg with AG or L-NAME) displayed transmural inflammation, extensive ulceration, and necrosis.
Taken together, licofelone appears to be a protective compound against inflammation in colitis.In a trial study conducted on healthy volunteers, licofelone exhibited a better safety profile than conventional nonsteroidal anti-inflammatory drugs.This effect was implicated in a lower incidence of ulcers [42].It was shown that licofelone suppressed the development and growth of small intestinal and colonic tumors via inhibition of the COX/LOX pathway in a colon cancer animal model [43].Excessive NO production caused by a high level of inflammation and mucosal injury is one of the most dramatic and consistent findings in experimental IBD models [25].

CONCLUSION
Herein, for the first time we confirmed that licofelone significantly reduced microscopic, and clinical signs of colitis in animal model through modulation of TNF-α, IL-1β, MPO, ROS, TLR-4, and NF-κB levels.Licofelone is known to have anti-inflammatory properties, a representation of its ability to treat diseases caused by inflammation, although further pre-clinical and clinical researches are mandatory.Therefore, we propose that licofelone could prevent inflammation and colon injury, at least in part through the NO pathway, and worth considering for clinical investigation.

Figure 5
Figure 5 represents the colonic MPO activity in the treated animals.The MPO activity in the control

Table 1
Sequences of Primers Licofelone Attenuates Acetic Acid-Induced Colitis in Rats Through Suppression of the Inflammatory Mediators