Evaluation of anti-inflammatory effect of anti-platelet agent-clopidogrel in experimentally induced inflammatory bowel disease.

AIM: To evaluate the anti-inflammatory effect of antiplatelet agent, clopidogrel, in experimentally induced inflammatory bowel disease (IBD).
MATERIALS AND METHODS: TNBS induced Crohn's disease model and oxazolone induced ulcerative colitis model were used to evaluate the role of clopidogrel in IBD. Spargue Dawley female and Wistar male rats were used respectively. The colitis was induced by a single intra-colonic application of TNBS (0.25 ml, 120 mg/ml in 50% ethanol) and oxazolone (450 μl 5% oxazolone in 50% ethanol). Rats were divided into four groups (n=6) in each model namely normal control, sham control, test and standard group. Drug treatment was carried out for 21 days. After 21 days, animals were sacrificed and evaluated for weight change, colon mucosal damage index (CMDI), disease activity Index (DAI) and myeloperoxidase (MPO) activity.
RESULTS: Results showed that clopidogrel provided significant protection against mucosal damage in both the models of IBD. It significantly reduced (P<0.05) the decrease in body weight and CMDI, DAI and MPO scores.
CONCLUSION: The results indicate that clopidogrel may be effective in treatment of Crohn's disease and ulcerative colitis. Platelet inhibition may be one of the mechanism for effectiveness of clopidogrel in the treatment of IBD.

Introduction

Inflammatory bowel disease (IBD) is a non specific inflammatory disorders of the gastrointestinal (GI) tract. The causes of IBD remain unknown.[1] The term IBD describes two major chronic diseases which include ulcerative colitis (UC) and Crohn's disease (CD). UC is usually limited to the colon and rectum[2] while CD may affect any part of the GI tract from mouth to anus.[2] Common symptoms of active UC and CD are diarrhoea, with stools containing blood and mucous, and abdominal pain.

Early work revealed that the incidence of IBD is much higher in North America, northern Europe and the United Kingdom as compared to Africa, Asia, Latin America and southern Europe.[3] A limited medication is availabe for treatment of IBD. A meta-analysis showed a significant increase in risk for lymphoma in IBD patients treated with azathioprine or 6-mercaptopurine. Mesalamine and sulfasalazine are generally considered to be the mainstay in the treatment of UC but may be ineffective for maintaining remission in CD patients.[4] Corticosteroids are also similarly ineffective in sustaining the medically induced remission.[5]

Although population-based studies found a lifetime need of steroids in 50% of the patients,[6] IBD patients frequently develop severe complications despite the correct administration of conventional medical therapy.[7] It is thus, important to find new therapeutic agents for use in IBD in humans.

Several studies suggest that platelets play an important role in exacerbations of CD and UC. There is an increase in circulating platelets in IBD patients.[8] This is often associated with a reduced platelet life span and reduction in mean platelet volume.[9] A role for platelets in mediating leukocyte recruitment to the inflamed colon is also likely, since platelet P-selectin and RANTES are also detected in IBD[10]

It therefore, appears that platelet dysfunction may contribute to the pathogenesis of the mucosal lesion in IBD. This study was designed to evaluate the pharmacological role of antiplatelet agent, clopidogrel, in inflammatory bowel disease.

Materials and Methods

Drugs and Chemicals

Clopidogrel, prednisolone (Cadilapharmaceuticals, Ahmedabad), sulfasalazine (Wallacepharmaceuticals), 4-Ethoxymethylene 2-phenyl 2-oxazoline 5- one (oxazolone) and Trinitrobenzenesulphonic acid (TNBS) (Sigma Aldhrich, USA) were used. All other reagents and chemicals were of pure grade and obtained from Merck Bioscience Ltd.

Animals

Healthy, male albino Wistar and female Spargue Dawley rats were procured from Zydus Research Centre, Ahmedabad. Rats were fed with standard chew diet and water ad libitum. They were housed in standard conditions of temperature (25 ± 2°C), relative humidity (45-55%) and 12-hour light dark cycle. The experimental protocol was reviewed and approved by the Institutional Animal Ethics Committee (IAEC), S J Thakkar pharmacy college, Rajkot (proposal no. SJT/028-2011). All experiments were carried out according to the guidelines suggested by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

Oxazolone Induced Ulcerative Colitis

Ulcerative colitis was induced inmale rats weighing 150-200 g by intrarectal administration of oxazolone on 1st day.[11] The study comprised of four groups of six animals each and the treatment given is described below:

Normal Control Group: Vehicle only (0.25% w/v sodiumcarboxy methyl cellulose [CMC], p.o.)

Disease Control group: Oxazolone (450 μl of 5% oxazolone in 50% ethanol solution, intrarectally) on first day and vehicle (0.25% w/v sodium CMC, p.o.) for next 21 days

Standard Group: Oxazolone as above +sulphasalazine (360 mg/kg, p.o.) for next 21 days

Test Group: Oxazolone as above +clopidogrel (50 mg/kg,p.o.) fornext 21days.

TNBS Induced Crohn's Disease

Crohn's disease was induced in female Sprague Dawley rats weighing 150-200 g by intrarectal administration of TNBS on 1st day.[12] The study comprised of four groups of six animals each and the treatment given is described below:

Normal Control Group: - Vehicle only (0.25% w/v sodium CMC, p.o.)

Disease Group: -TNBS (0.25 ml of 120 mg/ml TNBS in 50% ethanol solution, intrarectally) on first day and vehicle for next 21 days

Standard Group: -TNBS as above +prednisolone (2 mg/kg, p.o.) for next 21 days

Test Group: -TNBS as above +clopidogrel (50 mg/kg p.o.) for next 21days.

On 22nd day, animals in both models were sacrificed by cervical dislocation and colon was removed. Percent change in body weight, colon mucosal damage index (CMDI), disease activity index (DAI) and myeloperoxidase (MPO) activity were measured as below.

% Change in Body Weight[13]

The body weight of animals was measured at regular time intervals from day 0 to 21 and % change in weight was calculated.

Colon Mucosal Damage Index[14]

Distal 10 cm of colon segment was excised, opened by a midline incision and rinsed with saline. It was observed from luminal side. Macroscopic scoring was done as follows: 0 = normal mucosa, no damage on the mucosal surface, 1 = mild hyperaemia and edema, no erosion or ulcer on the mucosal surface, 2 = moderate hyperaemia and edema, erosion appearing on the mucosal surface, 3 = severe hyperaemia and edema, necrosis and ulcer appearing on the mucosal surface with the major ulcerative area <1 cm and 4 = severe hyperaemia and edema, necrosis and ulcer appearing on the mucosal surface with the major ulcerative area >1 cm.

Disease Activity Index[14]

The tissue samples from colon for histology were fixed overnight in 4% neutral buffer formalin and were processed, sectioned (4 μm thick) and stained with haematoxylin and eosin. Each sample was observed and evaluated. A score was given according to the method described earlier: 1. Infiltration of inflammatory cells (0-none, 1-slight increase, 2-marked increase); 2. Deposition of fibrin protein (0-absent, 1-present); 3. Submucosalneutrophil migratin (0-absent, 1-present); 4. Submucosaledema (0-nil, 1-patchy, 2-confluent); 5. Epithelial necrosis (0-nil, 1-localised, 2-extensive); 6. Epithelial ulceration (0-absent, 1-present).[15]

Myeloperoxidase (MPO) activity[16]

The colon was dissected out and homogenized (50 mg/L) in 50 mmol/L ice cold potassium phosphate buffer (pH 6) containing 0.5% of hexadecyltrimethyl ammonium bromide. The homogenate was frozen and thawed thrice, then centrifuged at 4000 rpm for 20 min at 4°C for measurement of myeloperoxidase activity.

Anindicator dye, O-dianisidine (50 mg), was prepared in 3 ml of methanol. One ml of this solution was mixed in 100 ml of potassium phosphate buffer and 16.7 μl of 3% hydrogen peroxide (H2O2) was added as substrate. To perform the assay, 0.1 ml of sample homogenate was added to a cuvette containing 2.9 ml of the substrate- dye solution in the sample chamber of a double beam spectrophotometer. The change in absorbance was recorded at 15 seconds intervals over 1 minute at room temperature at 460 nm. One unit of MPO activity was defined as that degrading one micromole of peroxidise per minute at 25°C. This calculation was done by using following formula:[17]

Where, X= weight of tissue taken for homogenate x volume of supernatant taken for final reaction

Statistical Analysis

All values were expressed as mean ± SEM. The statistical significance was tested by analysis of variance (ANOVA) followed by tukey's multiple range test. P< 0.05 was considered as significant. Graph pad Prism (version 5.0) software was used for this purpose.

Results

(i) Changes of Sign and Symptoms

A mucous thin stool was seen after 4-5 days and it worsened gradually. After 2 weeks, the stools contained more of pus and blood. At the end of treatment period, these symptoms improved to a great extent.

(ii) Body Weight Change

The increase in body weight was observed to be significantly (P<0.001) lower in both the experimental models as compared to the normal control group. Body weight increase in clopidogrel treated group was close to the normal control group as the end of 21 days [Tables 1 and 2].

Table 1

Effect of clopidogrel on body weight, CMDI, DAI in oxazolone induced ulcerative colitis

Table 2

Effect of clopidogrel on body weight, CMDI, DAI in TNBS induced Crohn's disease in rats

(iii) Inflammatory Changes in Colonic Mucosa

The main parameters, used to assesse the degree of colonic inflammation were CMDI, DAI and MPO activities.

Colon Mucosal Damage Index (CMDI)

The colon tissue damage score in disease control group was significantly higher in comparison to normal control group [Tables 1 and 2] in both the models (P<0.001). Clopidogrel treated group showed significant reduction in CMDI score as compared to disease control group (P<0.05), which indicated reduction of the macroscopical lesions in both models of IBD.

Disease Activity Index (DAI)

As a parameter for microscopical assessment of inflammation, a histological score called DAI was measured. Disease control group showed more severe symptoms and thus higher DAI score [Tables 1 and 2] as compared to normal control group in both models (P<0.001). In case of clopidogrel treated group results showed [Figures 1 and 2] a significant reductionin DAI score (P< 0.01) as compared to disease control group [Tables 1 and 2] in both the models.

Figure 1

Effect of test drugs on microscopic damage (DAI) in oxazolone induced colitis in rats (n= 6 per group), (a)normal control group showing normal archite-cture, (b) disease control group showing total distortion of mucosa with marked infiltration of neutrophils, (c) prednisolone group revealed intact mucosal membrane with normal structural architecture, (d) clopidogrel treated group also showed intact mucosa with mild infiltration of neutrophils

Figure 2

Effect of test drugs on microscopic damage (DAI) in TNBS induced IBD, n= 6, (a) normal control group showing normal architecture, (b) disease control group showing necrotic damage with severe infiltration of neutrophils, (c) prednisolone group revealed intact mucosal membrane with normal architecture, (d) clopidogrel group also showed intact mucosa with mild infiltration of neutrophils

Measurement of Myeloperoxidase(MPO) Activity

As an index of neutrophil migration, MPO activity was measured and it was found to be significantly higher (P< 0.001) in disease control group as compared to normalin both the models. In case of clopidogrel treated group, the MPO activity was significantly reduced (P< 0.01) as compared to the disease control group [Figures 3 and 4].

Figure 3

Effect of clopidogrel on myeloperoxidase (MPO) activity in oxazolone induced IBD model Mean ± SEM (n=6) has been shown, # P< 0.001, as compared to control group, ** P< 0.01, as compared to disease control group, *** P<0.001, as compared to disease control group, (ANOVA test followed by Tukey's multiple comparison test)

Figure 4

Effect of clopidogrel on myeloperoxidase (MPO) activity in TNBS induced IBD model Mean ± SEM (n=6) has been shown, # P< 0.001, as compared to control group,** P< 0.01, as compared to disease control group, *** P< 0.001, as compared to disease control group, (ANOVA test followed by Tukey's multiple comparison test)

Discussion

Ulcerative colitis and Crohn's disease are chronic inflammatory disorders of the gastrointestinal tract and are broadly categorized as inflammatory bowel disease (IBD). Since IBD-like diseases do not occur naturally in animals, different types of exogenous irritants are used to induce inflammatory reactions of the gut.[18] Trinitrobenzene sulfonic acid (TNBS) induced Crohn's disease model and oxazolone induced ulcerative colitis model are used for the investigation of inflammatory reactions in the colon.

Reduction in body weight is a sign of the generation of inflammatory bowel disease. In oxazolone induced colitis and TNBS induced Crohn's disease model, the disease control group showed less increase in body weight for 21 days. These results were indicating induction of disease in both models. Clopidogrel treatment improved as the rate of increase in body weight in both models of IBD. A further assessment of the disease by macroscopical and microscopical scoring (CMDI and DAI) the disease control group showed bowel wall thickening, adhesion to surrounded tissue as well as erosion, edema and small patches of ulcer in colon. The results of CMDI score suggest that clopidogrel treatment showed significant protection from macroscopic damage of colon in both the models of IBD.

At the same time, the DAI score of disease control groups revealed neutrophil infiltration, fibrin deposition, submucosal neutrophil migration, submucosaledema, epithelial necrosis and epithelial ulceration with loss of epithelial villi. The clopidogrel treated groups showed improvement in the above pathophysiological characteristics of the disease in both models of IBD.

MPO activity helps in the assessment of polymorphonuclear cell (PMN) infiltration as a measure of inflammatory injury of colon. Margination and extravasation of circulatory PMNs contributes markedly to chronic inflammatory injury of colon in IBD.[19] The higher MPO activity was found in disease control groups and the clopidogrel treatment significantly reduced the PMN infiltration (and therefore the PMN induced progression of disease symptoms) and ameliorated the inflammation in IBD.

Platelets can function as inflammatory cells are supported by several lines of evidence. First, platelets produce and store enormous amount of inflammatory mediators; second, platelets simultaneously cross talk with and activate different cells; third, platelets are in turn activated by multiple proinflammatory substances through cognate receptors expressed on their surface.[20] Upon activation, a large number of these substances are promptly released by platelets. Among them, histamine, prostaglandin E and D2, platelet-derived growth factor (PDGF), thromboxane A2 and serotonin control vascular permeability and regulate vasodilation or vasocontraction;[20] adenine nucleotides, PDGF, and PF4 induce neutrophil activation and degranulation; TGF-beta and basic fibroblast growth factor (bFGF) promote fibroblast proliferation and wound repair while vascular endothelial growth factor (VEGF) is a major trophic factor for endothelial cells and promotes angiogenesis.[21] In IBD, the activated platelets often express P-selectin and CD40 ligand which are important for cell adhesion and activation of inflammatory cells.[22] Cell adhesion molecules play an important role in adherence of granulocytes to vascular endothelial cells in intestinal mucosa, followed by the activation and infiltration of mononuclear cells into the inflammatory mucosa. Molecules related to CD40 may be important in activating granulocytes, B cells, and macrophages, which can be compatible with predominant infiltration and activation of granulocytes in UC and macrophage lineage cells forming granulomatous lesions in Crohn's disease. Such activation is often observed in involved mucosa in patients with UC, which may cause specific histopathological features observed in this disease. P-selectin on activated platelets is shed from cell surface to serum and is found in soluble form. This may act as a marker of severity of the mucosal inflammation in ulcerative colitis.[23] It is thus, evident that platelets are an important marker cell in IBD and the inhibition of platelet activation could be one of the useful ways to reduce the IBD symptoms.

This study revealed that clopidogrel potentially inhibits the inflammatory factors in IBD and resolves the symptoms of IBD. It has been suggested earlier that clopidogrel potentiates the inhibition of platelet activation, platelet-leukocyte interaction and P-selectin expression on CD 40 lymphocyte via P2Y12 receptor inhibition,[2425] which may probably be a mechanism that helps to resolve the symptoms of IBD.