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The effect of theophylline on acetic acid induced ulcerative colitis in rats
Biomedicine & Pharmacotherapy 90 (2017) 153–159
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Original article
The effect of theophylline on acetic acid induced ulcerative colitis in rats Masoumeh Ghasemi-Pirbalutia , Ehsan Motaghib,* , Ali Najafic, Mohammad Javad Hosseinic a b c
Department of Immunology, Shahrekord University of Medical Science, Shahrekord, Iran Department of Physiology and Pharmacology, School of Medicine, Kurdistan University of Medical Sciences, Sanandaj 6618634683, Iran Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 November 2016 Received in revised form 6 March 2017 Accepted 14 March 2017
Background: Ulcerative colitis is a relapsing inflammatory disorder of the colon. There is a need to explore the new treatments for this disorder. Theophylline, a competitive inhibitor of phosphodiesterase, is shown to have anti-inflammatory properties. However, the effect of theophylline on ulcerative colitis has not yet been investigated. The present study evaluated the effect of theophylline on acetic acid induced ulcerative colitis in rats. Materials and methods: Colitis was induced by instillation of 2 ml of acetic acid solution (3%). Colon samples were evaluated grossly and microscopically and assayed for myeloperoxidase (MPO) activity and proinflammatory cytokine concentrations. Results: Treatment with theophylline at the doses of 20 and 50 mg/kg attenuated acetic acid induced ulcerative colitis as shown by improvement in body weight loss, macroscopic score, ulcer area, hematocrit and histopathological score. Theophylline treatment also reduced MPO activity and tumor necrosis factor-a (TNF-a), interleukin 1b (IL-1 b) and interleukin 6 (IL-6) concentrations in inflamed colon. Conclusion: Theophylline has a protective effect in acetic acid-induced ulcerative colitis which might be due to its anti-inflammatory activities. Therefore, theophylline has the potential to be used for successful treatment of ulcerative colitis. © 2017 Elsevier Masson SAS. All rights reserved.
Keywords: Theophylline Ulcerative colitis Cytokine Ulceration Myeloperoxidase
1. Introduction Ulcerative colitis is an inflammatory disease affecting the mucosal layer of the distal colon and rectum [1]. Proinflammatory cytokines such as tumor necrosis factor a (TNF-a), interleukin 1b (IL-1 b) and interleukin-6 (IL-6) are involved in the immunopathogenesis of ulcerative colitis [2,3]. Thus, the therapies which reduce the proinflammatory cytokines could be beneficial for the treatment of ulcerative colitis. Although 5-aminosalicylic acid, glucocorticoids and immunosuppressive drugs are effective to some extent, some patients are still refractory or experience serious side effects, so new treatments are needed [4]. Various works have shown that phosphodiesterase (PDE) inhibitors have cell protecting properties [5] and are effective in improvement of different inflammatory conditions such as
* Corresponding author. E-mail address: [email protected] (E. Motaghi). http://dx.doi.org/10.1016/j.biopha.2017.03.038 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
arthritis [6] and autoimmune encephalitis [7]. Inhibition of PDE elevates the level of intracellular cyclic adenosine monophosphate (cAMP) in white blood cells which then reduce proinflammatory cytokines [6,8,9] and inhibits reactive oxygen species [10] and nitric oxide generation [6]. Theophylline as a non-specific PDE inhibitor is widely used worldwide as a bronchodilator in patients with asthma and chronic obstructive pulmonary disease (COPD) for over 60 years [11]. Several studies have shown that the positive effects of theophylline in patient with asthma are not entirely explainable by bronchodilatory effects [12]. Theophylline inhibited lung carcinogenesis in animal model [13]. Moreover, theophylline showed anti-inflammatory activity both in vitro [6,8,9] and in vivo. In vivo studies showed that theophylline attenuates the response to allergen [14] and reduces bronchial mucosal eosinophils in patients with mild asthma [15]. It has been also shown that theophylline may exert various effects on tissues other than lung such as cartilage [6], hearth [16], kidney [17] and esophagus [18]. However, the effect of theophylline on inflamed colon was not yet evaluated. Therefore,
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current study investigated the effect of theophylline on acetic acid induced ulcerative colitis, which is a chemically induced model of colitis that shows various pathophysiological similarity to the ulcerative colitis in human [19]. 2. Materials and methods 2.1. Reagents Dexamethasone and theophylline were gifted from Raha Pharmaceutical Co. (Isfahan, Iran). Hexadecyltrimethylammonium bromide (HTAB) and O-dianisidine dihydrochloride were procured from Sigma–Aldrich (St. Louis, USA). Acetic acid was bought from Merck-Schuchardt (Hohenbrunn, Germany). Enzyme-linked immunosorbent assay (ELISA) kits for rat TNF-a, IL-1b and IL-6 were all purchased from Boster Co. (Pleasanton, CA, USA).
2.5. Measurement of proinflammatory cytokines The concentrations of IL-1b, IL-6 and TNF-a in the colon of the rats with ulcerative colitis were measured using ELISA kits in accordance with the manufacturer's instructions (Boster Co., Pleasanton, CA, USA). ELISA was performed in triplicate. 2.6. Histopathological evaluation Colon samples were fixed in formalin solution (10%). Then, tissues were sectioned, deparaffinized and stained with hematoxylin and eosin (H&E). Histopathological evaluation of colon was done as described previously [23] and by a pathologist, who was blinded to experimental groups. Total colitis index was the summation of inflammation extent, inflammation severity and crypt damage scores. 2.7. Myeloperoxidase (MPO) activity assay
2.2. Animals and grouping Adult male Wistar rats (210–240 g) were obtained from the animal house of Kurdistan University of Medical Sciences. The animals were housed three per cage in a regulated environment (23–25 C and 12/12 h light/dark cycle) with free access to standard chow and water. After one week of adaptation, the rats were treated intraperitoneally once daily with 10, 20 or 50 mg/kg of theophylline (treatment), or dexamethasone as a reference drug 24 h before acetic acid instillation and for the subsequent 3 days. Sham and colitis control animals received vehicle (normal saline, 1 ml/kg, i.p.). Each group composed of 6 rats. The doses of theophylline were chosen based on our pilot study and a previous report [6]. 2.3. Colitis induction All rats were fasted for 24 h with free access to water. After light anesthesia with ether, colitis was induced by instillation of 2 ml acid acetic solution (3% in normal saline) using a polyethylene tube which was introduced into the anus to a distance of 8 cm. The rats were then maintained in the head-down position for 30 s to prevent leakage of the acetic acid [20]. Sham group received intracolonic normal saline, instead of acetic acid. All experiments were done according to the protocol of the Ethical Committee of Kurdistan University of Medical Sciences. Body weight was measured each day during the experiment. 2.4. Hematocrit measurement and macroscopic evaluation of colon damage Blood was sampled in a heparin coated capillary tube for hematocrit evaluation 3 days after acetic acid instillation. On the same day, the rat was sacrificed under ether anesthesia. The abdomen was opened via a midline incision and the distal 8 cm of the colon was removed, freed of adherent adipose tissue, opened longitudinally, washed with normal saline to remove any fecal material and was scored macroscopically as follow: 0: no damage; 1: mucosal erythema only; 2: mild mucosal edema, slight bleeding or small erosions; 3: moderate edema, bleeding, erosions or ulcers; 4: severe ulceration, erosions, edema or tissue necrosis [21]. The colon was photographed by a Canon camera (Powershot G9, 12 megapixel, Japan) and the ulcer area (%) was measured using Fiji-win 32 software (NIH Image for the Macintosh) [22]. The colon was then sectioned into 3 portions, frozen in liquid nitrogen and stored at 70 oC for further examinations (ELISA analysis, MPO assay, and histopathological study).
One unit of MPO activity is defined as that required to degrade 1 mM of hydrogen peroxide at 25 oC. MPO activity was measured according to previous method with some changes [24]. Briefly, colon sample was homogenized in sodium phosphate buffer (50 mM) containing HTAB (0.5%) and EDTA (5 mM, pH 7.4) in an ice-water bath using a polytron homogenizer. More buffer was added to the homogenate to reach the concentration of 50 mg tissue per milliliter. Then, the homogenate was sonicated in an ice bath for 10 s, and freeze–thawed three times. Then homogenate was sonicated again for 10 s. After centrifugation at 10,000 g for 15 min at 4 oC, a 100 mL of the resultant pellet was mixed with 2.9 ml of 50 mM phosphate buffer (pH = 6) containing 0.167 mg/ml O-dianisidine dihydrochloride and 0.0005% hydrogen peroxide. The activity of MPO was measured using a UV/Vis spectrophotometer (LSI Model Alfa-1502) at 460 nm. MPO assay were performed in duplicate for each sample. 2.8. Statistical analysis Data are presented as mean S.D. for parametric data and median (range) for non-parametric data. Non-parametric statistical analysis was done using Kruskal–Wallis test followed by Dunn's multiple-comparison or Mann–Whitney U test while, parametric data analysis was performed by one-way ANOVA followed by Tukey test. A P < 0.05 was considered statistically significant. Data were analyzed using GraphPad software (Prism ver. 6.01, San Diego, CA). 3. Results 3.1. The effect of theophylline on body weight loss Body weight was significantly reduced after acetic acid instillation. This reduction was more severe in the colitis control group than in the theophylline (20 and 50 mg) and dexamethasone groups (at least P < 0.05) (Fig. 1). 3.2. The effect of theophylline on macroscopic changes Intra-rectal administration of acetic acid induced severe colitis with extensive epithelial necrosis (Fig. 2B). Intraperitoneal administration of theophylline (20 and 50 mg/kg) produced a significant reduction in macroscopic colon damage score in comparison with colitis control group (P < 0.05) (Fig. 2C and D and Table 1). The colitis control group presented significantly more extensive ulcer area and colon weight compared to Sham group (P < 0.001)
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Fig. 1. Effects of different doses of theophylline (10, 20, 50 mg/kg) and dexamethasone (1 mg/kg) on body weight loss in rats with acetic acid-induced colitis. Data are expressed as the means S.D. *P < 0.05, **P < 0.01 ***P < 0.001 vs. colitis control.
Fig. 2. Effect of theophylline on macroscopic damage in acetic acid-induced colitis in rats, n = 6 per group. (A) Sham group showing normal appearance of colon. (B) Colitis control group showing extensive necrosis, and ulceration, hyperemia and edema in the colon mucosa. (C, D) Theophylline (20 and 50 mg/kg) and (E) dexamethasone groups showing improvement in mucosal damage.
Fig. 3. Effects of different doses of theophylline (10, 20, 50 mg/kg) and dexamethasone (1 mg/kg) on (A) colon ulcer area, (B) colon weight and (C) hematocrit level in rats with acetic acid-induced colitis (acetic acid, 2 ml, 3%). Data are expressed as the means S.D. *P < 0.05, **P < 0.01, ***P < 0.001 vs. colitis control.
Table 1 Effect of theophylline (10, 20, and 50 mg/kg, i.p.) on macroscopic colonic damage score and pathologic parameters of acetic acid induced colitis in rats. Groups
Macroscopic colitis score (0–4) Microscopic score
Sham Colitis control Theophylline 10 mg/kg Theophylline 20 mg/kg Theophylline 50 mg/kg Dexamethasone 1 mg/kg
0 4 3 2 1 1
Inflammation severity (0–3) Inflammation extent (0–3) Crypt damage (0–4) Microscopic colitis index (0–0)b (3–4) (3–4) (0–2)b (1–2)b (1–2)b
0 (0–0)b 3 (2–3) 2 (1–3) 2 (0–2)a 1.5 (1–2)a 1 (0–1)b
0 (0–0)b 3 (2–3) 2 (1–3) 2 (0–2)a 2 (1–2)a 0.5 (0–1)b
The values are expressed as median (range) for scoring data. Microscopic colitis index is presented as mean S.D. a P < 0.05. b P < 0.01. c P < 0.001 compared to colitis control (n = 6).
0 (0–0)b 2.5 (2–4) 2 (1–2)a 1.5 (0–2)a 2 (1–3) 0 (0–1)b
0 0c 8 0.68 5.83 0.83 4.16 1.04b 5 0.36a 1.16 0.6c
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(Fig. 3A and B). The ulcer area in theophylline (20 and 50 mg/kg) and dexamethasone groups was significantly lower than that of in the colitis control group (P < 0.01, P < 0.001 and P < 0.01, respectively). Colon weight was significantly reduced after treatment with dexamethasone or medium or higher doses of theophylline (20 and 50 mg/kg) in comparison with colitis control group (P < 0.05).
acid. MPO activity was higher in the colon of colitis control group than that of in the theophylline groups (P < 0.001) (Fig. 5A). However, the difference between colitis control and theophylline groups was significant only for theophylline 20 mg/kg group (P < 0.05). The level of MPO in theophylline group (20 mg/kg) was similar to dexamethasone treatment group, however, did not reach to normal level in Sham group.
3.3. Effect of theophylline on hematocrit changes
3.6. Effect of theophylline on proinflammatory cytokine levels
Administration of acetic acid led to hemorrhage or bleeding in colon as shown by reduced level of hematocrit in colitis control group in comparison with that of in the Sham group. Theophylline at the dose of 20 mg/kg and dexamethasone elevated the hematocrit, compared to that of in the colitis control group (P < 0.05) (Fig. 3C).
Intra-rectal administration of acetic acid significantly increased the concentrations of TNF-a, IL-1b and IL-6 as compared with Sham group (P < 0.001). As expected, pretreatment of rats with dexamethasone significantly reduced the levels of all tested cytokines (P < 0.05) (Fig. 5B–D). Pretreatment of animals with theophylline reduced the colon levels of TNF-a and interleukin 1b. However, the reduction was statistically significant only at medium and higher doses (20 and 50 mg/kg) of theophylline (P < 0.05). Theophylline treatment at 50 mg/kg significantly reduced the production of IL-6 in comparison to colitis control group (P < 0.05). Nevertheless, theophylline treatment did not restore the concentrations of tested cytokines toward normal values in Sham group.
3.4. The effect of theophylline on histopathological changes Microscopic observation of H&E stained sections of colon of Sham group showed intact epithelium with no damage (Fig. 4A and Table 1). Intracolonic administration of acetic acid caused hemorrhage, ulceration and necrosis in the colon of colitis control group. Inflamed colon in colitis control group also showed submucosal edema and infiltration of inflammatory cells mainly neutrophils (Fig. 4B). Pretreatment with theophylline (20 and 50 mg/kg, i.p.) attenuated acetic acid-induced colitis. Rats received 20 or 50 mg/kg theophylline showed mild ulceration, inflammation and edema without necrosis (Fig. 4C and D). Dexamethasone group also showed a protective effect in inflamed colon (Fig. 4E). 3.5. The effect of theophylline on MPO activity MPO activity as an indicator of neutrophils infiltration, increased significantly after intra-rectal administration of acetic
4. Discussion This study evaluated the anti-inflammatory and anti-colitis properties of theophylline, a PDE inhibitor drug, in acetic acid induced ulcerative colitis in rats. Marked reduction of colon inflammation, ulcers, colitis score and body weight loss were seen after administration of theophylline especially at medium (25 mg/ kg) and high (50 mg/kg) doses. Acetic acid administration caused a marked weight loss in rats which is in accordance with previous researches [20]. Weight loss might be as a result of reduced appetite and food intake due to
Fig. 4. Effect of theophylline on microscopic damage in acetic acid induced colitis in rats; n = 6 per group (H&E). (A) Sham group showing intact mucosa. (B) Colitis control group showing sever damage of the mucosa, loss of goblet cells, edema, and extensive infiltration of inflammatory cells. (C) Theophylline (20 mg/kg), and (D) theophylline (50 mg/kg) and (E) dexamethasone (1 mg/kg) groups showing focal damage to epithelium and reduction of infiltration of white blood cells.
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feverish and immobility conditions. This article showed that administration of theophylline attenuated body weight loss due to anti-inflammatory effect of theophylline. It is worth to be noted that some of this attenuation might be due to theophylline analgesic effects as a result of suppression of adenosine receptor [25]. Histopathological studies confirmed macroscopic findings and showed that inflammatory changes including edema, hemorrhage, crypt damage, epithelial cell loss, inflammation extent, inflammation severity and necrosis were markedly reduced after treatment with theophylline. Theophylline acted as anti-inflammatory and anti-colitic agent in acetic acid induced colitis model. Theophylline administration significantly decreased leukocyte infiltration in inflamed colon. The results obtained are in accordance with previous findings where theophylline reduced the number of activated T-lymphocytes in the bronchoalveolar lavages and the lungs of patients with asthma after allergen exposure [14] and decreased epithelial damage [26]. Theophylline also reduced neutrophil infiltration and attenuated histopathological changes of airways in COPD [27]. Previous works have shown that theophylline inhibits production of proinflammatory cytokines in various in vitro and in vivo models [6,8,9]. Overproduction of proinflammatory cytokines plays a key role in the pathogenesis of ulcerative colitis [21]. Thus, we evaluated proinflammatory cytokine levels in the colon tissues and showed that theophylline partially normalized proinflammatory cytokines levels including TNF-a, IL-1b and IL6 in acetic acid induced colitis. Our results are in accordance with previous researches, in which it has been shown that TNF-a is partially decreased after treatment with theophylline [12]. Theophylline also decreased MPO activity in the inflamed colon. This finding is in agreement with a report showing that theophylline reduces neutrophil infiltration due to reduced level of neutrophil chemotactic factors in COPD airways [28]. Our experiments showed that administration of theophylline in rats with colitis could increase the hematocrit and attenuate blood loss. However, highest dose of theophylline (50 mg/kg) did not show a significant effect on hematocrit level possibly because of the suppressive effect of theophylline on erythropoietin secretion [29]. Theophylline with three examined doses exerted anti-colitis effects in the current study. Although the lowest dose (10 mg/kg) was not statistically effective, medium and highest doses of theophylline (20 and 50 mg/kg) showed the maximum antiinflammatory and anti-colitis effects. However, there was no significant difference between efficacy of medium and high doses. So, it can be concluded that theophylline has a limited ceiling effect in prevention and/or therapy of ulcerative colitis. Theophylline is mainly considered to be a bronchodilator in asthma. In addition to bronchodilator properties, it has been reported that theophylline attenuates inflammation in airways [28]. Literature has generally focused on in vitro experiments on anti-inflammatory effects of theophylline at cellular and subcellular levels on airway inflammation [28,30]. However, an in vivo study has shown anti-inflammatory effects of theophylline on arthritis [6]. Moreover, Watanabe et al. showed anti-inflammatory effect of intraperitoneal injection of theophylline in carrageenaninduced paw inflammation [31]. Another work also obtained similar results with caffeine, another PDE inhibitor, and showed that caffeine can ameliorate dextran sulphate sodium (DSS) induced colitis in rats [32]. Precise mechanism of the anti-inflammatory and anti-colitis effects of theophylline cannot be simply determined but some hypotheses can be developed. It is well known that theophylline as a blocker of PDE enzyme, elevates cAMP level and elevated level of the intracellular cAMP in leukocytes leads to a significant
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reduction in the production and release of proinflammatory cytokines and other inflammatory mediators such as TNF-a [6], IL4 [8], IL-5 [9], leukotriene B4, leukotriene C4 [33] and superoxide
Fig. 5. Effects of different doses of theophylline (10, 20, 50 mg/kg) and dexamethasone (1 mg/kg) on colon level of (A) MPO activity, (B) tumor necrosis factor-a (TNF-a), (C) interleukin-1b (IL-1b) and (D) interleukin-6 (IL-6) in rats with acetic acid-induced colitis (acetic acid, 2 ml, 3%). Data are expressed as the means S.D. *P < 0.05, ***P < 0.001 vs. colitis control.
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anion [34] in inflammation site. It has been also suggested that anti-inflammatory effects of theophylline are due to inhibition of reactive oxygen species [10] and nitric oxide generation [6]. Theophylline also induces apoptosis and decreases proliferation of T-lymphocytes [35,36] and expression of adhesion molecules in leukocytes [37–41]. These effects may be due to antagonism of adenosine A2A-receptors [38]. Additionally, theophylline elevates the number of suppressor T cells which can suppress antibody production by other lymphoid cells [42]. Moreover, it has been shown that theophylline enhances the activity of histone deacetylases, an enzyme that keeps the chromatin in a repressive state, for genes encoding proinflammatory cytokines [43]. As mentioned above, theophylline is a potent inhibitor of adenosine receptors [25]. It is established that adenosine induces the release of leukotriene and histamine, so adenosine receptor blockade can ameliorate inflammatory conditions including colitis [44]. Moreover, one research indicated that theophylline even at the 10 mg/kg elevates glucocorticoid production, and this mechanism may be at least partially responsible for the anti-inflammatory and anti-colitis effects of theophylline [31]. Anti-colitis effects of theophylline may also be mediated by other minor mechanisms including stimulation of release of adrenal catecholamine, reduction of calcium level in inflammatory cells, and suppression of prostaglandins activities [15]. However, the exact mechanisms of anti-colitis activity remained to be determined in further studies. It is worth to be noted that theophylline also has antitumor activities [37,45]. Since BCL2 has been found to be related to colon cancer [46], suppression of BCL2 expression and activity by theophylline [37] may be a safe and effective way to prevent colitis-associated cancer. Further research is required to evaluate the effect of theophylline on colitis-associated cancers. In summary, this work showed that intraperitoneal injection of theophylline, a non-specific PDE inhibitor, clearly attenuated acetic acid induced ulcerative colitis which became evident via decrease in proinflammatory cytokine levels, histopathological studies and MPO activity assessments. Our findings suggest potential application of theophylline for effective and inexpensive treatment of ulcerative colitis. Our study provides the basis for future investigations on the use of theophylline for the treatment of ulcerative colitis. Conflict of interest The authors have no conflict of interest. Acknowledgment This research project was fully sponsored by Kurdistan University of Medical Sciences, Sanandaj, Iran with grant number 452879. References [1] Y. Ikeda, F. Akbar, H. Matsui, M. Onji, Characterization of antigen-presenting dendritic cells in the peripheral blood and colonic mucosa of patients with ulcerative colitis, Eur. J. Gastroenterol. Hepatol. 13 (2001) 841–850. [2] M.E. Street, G. De’Angelis, C. Camacho-Hübner, G. Giovannelli, M.A. Ziveri, P.L. Bacchini, S. Bernasconi, G. Sansebastiano, M.O. Savage, Relationships between serum IGF-1, IGFBP-2, interleukin-1beta and interleukin-6 in inflammatory bowel disease, Horm. Res. Paediatr. 61 (2004) 159–164. [3] R. Rahimi, S. Nikfar, M. Abdollahi, Meta-analysis technique confirms the effectiveness of anti-TNF-alpha in the management of active ulcerative colitis when administered in combination with corticosteroids, Med. Sci. Monit. 13 (2007) 13–18. [4] W.Y. Chey, A. Hussain, C. Ryan, G.D. Potter, A. Shah, Infliximab for refractory ulcerative colitis, Am. J. Gastroenterol. 96 (2001) 2373–2381.
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Original article
The effect of theophylline on acetic acid induced ulcerative colitis in rats Masoumeh Ghasemi-Pirbalutia , Ehsan Motaghib,* , Ali Najafic, Mohammad Javad Hosseinic a b c
Department of Immunology, Shahrekord University of Medical Science, Shahrekord, Iran Department of Physiology and Pharmacology, School of Medicine, Kurdistan University of Medical Sciences, Sanandaj 6618634683, Iran Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 November 2016 Received in revised form 6 March 2017 Accepted 14 March 2017
Background: Ulcerative colitis is a relapsing inflammatory disorder of the colon. There is a need to explore the new treatments for this disorder. Theophylline, a competitive inhibitor of phosphodiesterase, is shown to have anti-inflammatory properties. However, the effect of theophylline on ulcerative colitis has not yet been investigated. The present study evaluated the effect of theophylline on acetic acid induced ulcerative colitis in rats. Materials and methods: Colitis was induced by instillation of 2 ml of acetic acid solution (3%). Colon samples were evaluated grossly and microscopically and assayed for myeloperoxidase (MPO) activity and proinflammatory cytokine concentrations. Results: Treatment with theophylline at the doses of 20 and 50 mg/kg attenuated acetic acid induced ulcerative colitis as shown by improvement in body weight loss, macroscopic score, ulcer area, hematocrit and histopathological score. Theophylline treatment also reduced MPO activity and tumor necrosis factor-a (TNF-a), interleukin 1b (IL-1 b) and interleukin 6 (IL-6) concentrations in inflamed colon. Conclusion: Theophylline has a protective effect in acetic acid-induced ulcerative colitis which might be due to its anti-inflammatory activities. Therefore, theophylline has the potential to be used for successful treatment of ulcerative colitis. © 2017 Elsevier Masson SAS. All rights reserved.
Keywords: Theophylline Ulcerative colitis Cytokine Ulceration Myeloperoxidase
1. Introduction Ulcerative colitis is an inflammatory disease affecting the mucosal layer of the distal colon and rectum [1]. Proinflammatory cytokines such as tumor necrosis factor a (TNF-a), interleukin 1b (IL-1 b) and interleukin-6 (IL-6) are involved in the immunopathogenesis of ulcerative colitis [2,3]. Thus, the therapies which reduce the proinflammatory cytokines could be beneficial for the treatment of ulcerative colitis. Although 5-aminosalicylic acid, glucocorticoids and immunosuppressive drugs are effective to some extent, some patients are still refractory or experience serious side effects, so new treatments are needed [4]. Various works have shown that phosphodiesterase (PDE) inhibitors have cell protecting properties [5] and are effective in improvement of different inflammatory conditions such as
* Corresponding author. E-mail address: [email protected] (E. Motaghi). http://dx.doi.org/10.1016/j.biopha.2017.03.038 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
arthritis [6] and autoimmune encephalitis [7]. Inhibition of PDE elevates the level of intracellular cyclic adenosine monophosphate (cAMP) in white blood cells which then reduce proinflammatory cytokines [6,8,9] and inhibits reactive oxygen species [10] and nitric oxide generation [6]. Theophylline as a non-specific PDE inhibitor is widely used worldwide as a bronchodilator in patients with asthma and chronic obstructive pulmonary disease (COPD) for over 60 years [11]. Several studies have shown that the positive effects of theophylline in patient with asthma are not entirely explainable by bronchodilatory effects [12]. Theophylline inhibited lung carcinogenesis in animal model [13]. Moreover, theophylline showed anti-inflammatory activity both in vitro [6,8,9] and in vivo. In vivo studies showed that theophylline attenuates the response to allergen [14] and reduces bronchial mucosal eosinophils in patients with mild asthma [15]. It has been also shown that theophylline may exert various effects on tissues other than lung such as cartilage [6], hearth [16], kidney [17] and esophagus [18]. However, the effect of theophylline on inflamed colon was not yet evaluated. Therefore,
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current study investigated the effect of theophylline on acetic acid induced ulcerative colitis, which is a chemically induced model of colitis that shows various pathophysiological similarity to the ulcerative colitis in human [19]. 2. Materials and methods 2.1. Reagents Dexamethasone and theophylline were gifted from Raha Pharmaceutical Co. (Isfahan, Iran). Hexadecyltrimethylammonium bromide (HTAB) and O-dianisidine dihydrochloride were procured from Sigma–Aldrich (St. Louis, USA). Acetic acid was bought from Merck-Schuchardt (Hohenbrunn, Germany). Enzyme-linked immunosorbent assay (ELISA) kits for rat TNF-a, IL-1b and IL-6 were all purchased from Boster Co. (Pleasanton, CA, USA).
2.5. Measurement of proinflammatory cytokines The concentrations of IL-1b, IL-6 and TNF-a in the colon of the rats with ulcerative colitis were measured using ELISA kits in accordance with the manufacturer's instructions (Boster Co., Pleasanton, CA, USA). ELISA was performed in triplicate. 2.6. Histopathological evaluation Colon samples were fixed in formalin solution (10%). Then, tissues were sectioned, deparaffinized and stained with hematoxylin and eosin (H&E). Histopathological evaluation of colon was done as described previously [23] and by a pathologist, who was blinded to experimental groups. Total colitis index was the summation of inflammation extent, inflammation severity and crypt damage scores. 2.7. Myeloperoxidase (MPO) activity assay
2.2. Animals and grouping Adult male Wistar rats (210–240 g) were obtained from the animal house of Kurdistan University of Medical Sciences. The animals were housed three per cage in a regulated environment (23–25 C and 12/12 h light/dark cycle) with free access to standard chow and water. After one week of adaptation, the rats were treated intraperitoneally once daily with 10, 20 or 50 mg/kg of theophylline (treatment), or dexamethasone as a reference drug 24 h before acetic acid instillation and for the subsequent 3 days. Sham and colitis control animals received vehicle (normal saline, 1 ml/kg, i.p.). Each group composed of 6 rats. The doses of theophylline were chosen based on our pilot study and a previous report [6]. 2.3. Colitis induction All rats were fasted for 24 h with free access to water. After light anesthesia with ether, colitis was induced by instillation of 2 ml acid acetic solution (3% in normal saline) using a polyethylene tube which was introduced into the anus to a distance of 8 cm. The rats were then maintained in the head-down position for 30 s to prevent leakage of the acetic acid [20]. Sham group received intracolonic normal saline, instead of acetic acid. All experiments were done according to the protocol of the Ethical Committee of Kurdistan University of Medical Sciences. Body weight was measured each day during the experiment. 2.4. Hematocrit measurement and macroscopic evaluation of colon damage Blood was sampled in a heparin coated capillary tube for hematocrit evaluation 3 days after acetic acid instillation. On the same day, the rat was sacrificed under ether anesthesia. The abdomen was opened via a midline incision and the distal 8 cm of the colon was removed, freed of adherent adipose tissue, opened longitudinally, washed with normal saline to remove any fecal material and was scored macroscopically as follow: 0: no damage; 1: mucosal erythema only; 2: mild mucosal edema, slight bleeding or small erosions; 3: moderate edema, bleeding, erosions or ulcers; 4: severe ulceration, erosions, edema or tissue necrosis [21]. The colon was photographed by a Canon camera (Powershot G9, 12 megapixel, Japan) and the ulcer area (%) was measured using Fiji-win 32 software (NIH Image for the Macintosh) [22]. The colon was then sectioned into 3 portions, frozen in liquid nitrogen and stored at 70 oC for further examinations (ELISA analysis, MPO assay, and histopathological study).
One unit of MPO activity is defined as that required to degrade 1 mM of hydrogen peroxide at 25 oC. MPO activity was measured according to previous method with some changes [24]. Briefly, colon sample was homogenized in sodium phosphate buffer (50 mM) containing HTAB (0.5%) and EDTA (5 mM, pH 7.4) in an ice-water bath using a polytron homogenizer. More buffer was added to the homogenate to reach the concentration of 50 mg tissue per milliliter. Then, the homogenate was sonicated in an ice bath for 10 s, and freeze–thawed three times. Then homogenate was sonicated again for 10 s. After centrifugation at 10,000 g for 15 min at 4 oC, a 100 mL of the resultant pellet was mixed with 2.9 ml of 50 mM phosphate buffer (pH = 6) containing 0.167 mg/ml O-dianisidine dihydrochloride and 0.0005% hydrogen peroxide. The activity of MPO was measured using a UV/Vis spectrophotometer (LSI Model Alfa-1502) at 460 nm. MPO assay were performed in duplicate for each sample. 2.8. Statistical analysis Data are presented as mean S.D. for parametric data and median (range) for non-parametric data. Non-parametric statistical analysis was done using Kruskal–Wallis test followed by Dunn's multiple-comparison or Mann–Whitney U test while, parametric data analysis was performed by one-way ANOVA followed by Tukey test. A P < 0.05 was considered statistically significant. Data were analyzed using GraphPad software (Prism ver. 6.01, San Diego, CA). 3. Results 3.1. The effect of theophylline on body weight loss Body weight was significantly reduced after acetic acid instillation. This reduction was more severe in the colitis control group than in the theophylline (20 and 50 mg) and dexamethasone groups (at least P < 0.05) (Fig. 1). 3.2. The effect of theophylline on macroscopic changes Intra-rectal administration of acetic acid induced severe colitis with extensive epithelial necrosis (Fig. 2B). Intraperitoneal administration of theophylline (20 and 50 mg/kg) produced a significant reduction in macroscopic colon damage score in comparison with colitis control group (P < 0.05) (Fig. 2C and D and Table 1). The colitis control group presented significantly more extensive ulcer area and colon weight compared to Sham group (P < 0.001)
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Fig. 1. Effects of different doses of theophylline (10, 20, 50 mg/kg) and dexamethasone (1 mg/kg) on body weight loss in rats with acetic acid-induced colitis. Data are expressed as the means S.D. *P < 0.05, **P < 0.01 ***P < 0.001 vs. colitis control.
Fig. 2. Effect of theophylline on macroscopic damage in acetic acid-induced colitis in rats, n = 6 per group. (A) Sham group showing normal appearance of colon. (B) Colitis control group showing extensive necrosis, and ulceration, hyperemia and edema in the colon mucosa. (C, D) Theophylline (20 and 50 mg/kg) and (E) dexamethasone groups showing improvement in mucosal damage.
Fig. 3. Effects of different doses of theophylline (10, 20, 50 mg/kg) and dexamethasone (1 mg/kg) on (A) colon ulcer area, (B) colon weight and (C) hematocrit level in rats with acetic acid-induced colitis (acetic acid, 2 ml, 3%). Data are expressed as the means S.D. *P < 0.05, **P < 0.01, ***P < 0.001 vs. colitis control.
Table 1 Effect of theophylline (10, 20, and 50 mg/kg, i.p.) on macroscopic colonic damage score and pathologic parameters of acetic acid induced colitis in rats. Groups
Macroscopic colitis score (0–4) Microscopic score
Sham Colitis control Theophylline 10 mg/kg Theophylline 20 mg/kg Theophylline 50 mg/kg Dexamethasone 1 mg/kg
0 4 3 2 1 1
Inflammation severity (0–3) Inflammation extent (0–3) Crypt damage (0–4) Microscopic colitis index (0–0)b (3–4) (3–4) (0–2)b (1–2)b (1–2)b
0 (0–0)b 3 (2–3) 2 (1–3) 2 (0–2)a 1.5 (1–2)a 1 (0–1)b
0 (0–0)b 3 (2–3) 2 (1–3) 2 (0–2)a 2 (1–2)a 0.5 (0–1)b
The values are expressed as median (range) for scoring data. Microscopic colitis index is presented as mean S.D. a P < 0.05. b P < 0.01. c P < 0.001 compared to colitis control (n = 6).
0 (0–0)b 2.5 (2–4) 2 (1–2)a 1.5 (0–2)a 2 (1–3) 0 (0–1)b
0 0c 8 0.68 5.83 0.83 4.16 1.04b 5 0.36a 1.16 0.6c
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(Fig. 3A and B). The ulcer area in theophylline (20 and 50 mg/kg) and dexamethasone groups was significantly lower than that of in the colitis control group (P < 0.01, P < 0.001 and P < 0.01, respectively). Colon weight was significantly reduced after treatment with dexamethasone or medium or higher doses of theophylline (20 and 50 mg/kg) in comparison with colitis control group (P < 0.05).
acid. MPO activity was higher in the colon of colitis control group than that of in the theophylline groups (P < 0.001) (Fig. 5A). However, the difference between colitis control and theophylline groups was significant only for theophylline 20 mg/kg group (P < 0.05). The level of MPO in theophylline group (20 mg/kg) was similar to dexamethasone treatment group, however, did not reach to normal level in Sham group.
3.3. Effect of theophylline on hematocrit changes
3.6. Effect of theophylline on proinflammatory cytokine levels
Administration of acetic acid led to hemorrhage or bleeding in colon as shown by reduced level of hematocrit in colitis control group in comparison with that of in the Sham group. Theophylline at the dose of 20 mg/kg and dexamethasone elevated the hematocrit, compared to that of in the colitis control group (P < 0.05) (Fig. 3C).
Intra-rectal administration of acetic acid significantly increased the concentrations of TNF-a, IL-1b and IL-6 as compared with Sham group (P < 0.001). As expected, pretreatment of rats with dexamethasone significantly reduced the levels of all tested cytokines (P < 0.05) (Fig. 5B–D). Pretreatment of animals with theophylline reduced the colon levels of TNF-a and interleukin 1b. However, the reduction was statistically significant only at medium and higher doses (20 and 50 mg/kg) of theophylline (P < 0.05). Theophylline treatment at 50 mg/kg significantly reduced the production of IL-6 in comparison to colitis control group (P < 0.05). Nevertheless, theophylline treatment did not restore the concentrations of tested cytokines toward normal values in Sham group.
3.4. The effect of theophylline on histopathological changes Microscopic observation of H&E stained sections of colon of Sham group showed intact epithelium with no damage (Fig. 4A and Table 1). Intracolonic administration of acetic acid caused hemorrhage, ulceration and necrosis in the colon of colitis control group. Inflamed colon in colitis control group also showed submucosal edema and infiltration of inflammatory cells mainly neutrophils (Fig. 4B). Pretreatment with theophylline (20 and 50 mg/kg, i.p.) attenuated acetic acid-induced colitis. Rats received 20 or 50 mg/kg theophylline showed mild ulceration, inflammation and edema without necrosis (Fig. 4C and D). Dexamethasone group also showed a protective effect in inflamed colon (Fig. 4E). 3.5. The effect of theophylline on MPO activity MPO activity as an indicator of neutrophils infiltration, increased significantly after intra-rectal administration of acetic
4. Discussion This study evaluated the anti-inflammatory and anti-colitis properties of theophylline, a PDE inhibitor drug, in acetic acid induced ulcerative colitis in rats. Marked reduction of colon inflammation, ulcers, colitis score and body weight loss were seen after administration of theophylline especially at medium (25 mg/ kg) and high (50 mg/kg) doses. Acetic acid administration caused a marked weight loss in rats which is in accordance with previous researches [20]. Weight loss might be as a result of reduced appetite and food intake due to
Fig. 4. Effect of theophylline on microscopic damage in acetic acid induced colitis in rats; n = 6 per group (H&E). (A) Sham group showing intact mucosa. (B) Colitis control group showing sever damage of the mucosa, loss of goblet cells, edema, and extensive infiltration of inflammatory cells. (C) Theophylline (20 mg/kg), and (D) theophylline (50 mg/kg) and (E) dexamethasone (1 mg/kg) groups showing focal damage to epithelium and reduction of infiltration of white blood cells.
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feverish and immobility conditions. This article showed that administration of theophylline attenuated body weight loss due to anti-inflammatory effect of theophylline. It is worth to be noted that some of this attenuation might be due to theophylline analgesic effects as a result of suppression of adenosine receptor [25]. Histopathological studies confirmed macroscopic findings and showed that inflammatory changes including edema, hemorrhage, crypt damage, epithelial cell loss, inflammation extent, inflammation severity and necrosis were markedly reduced after treatment with theophylline. Theophylline acted as anti-inflammatory and anti-colitic agent in acetic acid induced colitis model. Theophylline administration significantly decreased leukocyte infiltration in inflamed colon. The results obtained are in accordance with previous findings where theophylline reduced the number of activated T-lymphocytes in the bronchoalveolar lavages and the lungs of patients with asthma after allergen exposure [14] and decreased epithelial damage [26]. Theophylline also reduced neutrophil infiltration and attenuated histopathological changes of airways in COPD [27]. Previous works have shown that theophylline inhibits production of proinflammatory cytokines in various in vitro and in vivo models [6,8,9]. Overproduction of proinflammatory cytokines plays a key role in the pathogenesis of ulcerative colitis [21]. Thus, we evaluated proinflammatory cytokine levels in the colon tissues and showed that theophylline partially normalized proinflammatory cytokines levels including TNF-a, IL-1b and IL6 in acetic acid induced colitis. Our results are in accordance with previous researches, in which it has been shown that TNF-a is partially decreased after treatment with theophylline [12]. Theophylline also decreased MPO activity in the inflamed colon. This finding is in agreement with a report showing that theophylline reduces neutrophil infiltration due to reduced level of neutrophil chemotactic factors in COPD airways [28]. Our experiments showed that administration of theophylline in rats with colitis could increase the hematocrit and attenuate blood loss. However, highest dose of theophylline (50 mg/kg) did not show a significant effect on hematocrit level possibly because of the suppressive effect of theophylline on erythropoietin secretion [29]. Theophylline with three examined doses exerted anti-colitis effects in the current study. Although the lowest dose (10 mg/kg) was not statistically effective, medium and highest doses of theophylline (20 and 50 mg/kg) showed the maximum antiinflammatory and anti-colitis effects. However, there was no significant difference between efficacy of medium and high doses. So, it can be concluded that theophylline has a limited ceiling effect in prevention and/or therapy of ulcerative colitis. Theophylline is mainly considered to be a bronchodilator in asthma. In addition to bronchodilator properties, it has been reported that theophylline attenuates inflammation in airways [28]. Literature has generally focused on in vitro experiments on anti-inflammatory effects of theophylline at cellular and subcellular levels on airway inflammation [28,30]. However, an in vivo study has shown anti-inflammatory effects of theophylline on arthritis [6]. Moreover, Watanabe et al. showed anti-inflammatory effect of intraperitoneal injection of theophylline in carrageenaninduced paw inflammation [31]. Another work also obtained similar results with caffeine, another PDE inhibitor, and showed that caffeine can ameliorate dextran sulphate sodium (DSS) induced colitis in rats [32]. Precise mechanism of the anti-inflammatory and anti-colitis effects of theophylline cannot be simply determined but some hypotheses can be developed. It is well known that theophylline as a blocker of PDE enzyme, elevates cAMP level and elevated level of the intracellular cAMP in leukocytes leads to a significant
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reduction in the production and release of proinflammatory cytokines and other inflammatory mediators such as TNF-a [6], IL4 [8], IL-5 [9], leukotriene B4, leukotriene C4 [33] and superoxide
Fig. 5. Effects of different doses of theophylline (10, 20, 50 mg/kg) and dexamethasone (1 mg/kg) on colon level of (A) MPO activity, (B) tumor necrosis factor-a (TNF-a), (C) interleukin-1b (IL-1b) and (D) interleukin-6 (IL-6) in rats with acetic acid-induced colitis (acetic acid, 2 ml, 3%). Data are expressed as the means S.D. *P < 0.05, ***P < 0.001 vs. colitis control.
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anion [34] in inflammation site. It has been also suggested that anti-inflammatory effects of theophylline are due to inhibition of reactive oxygen species [10] and nitric oxide generation [6]. Theophylline also induces apoptosis and decreases proliferation of T-lymphocytes [35,36] and expression of adhesion molecules in leukocytes [37–41]. These effects may be due to antagonism of adenosine A2A-receptors [38]. Additionally, theophylline elevates the number of suppressor T cells which can suppress antibody production by other lymphoid cells [42]. Moreover, it has been shown that theophylline enhances the activity of histone deacetylases, an enzyme that keeps the chromatin in a repressive state, for genes encoding proinflammatory cytokines [43]. As mentioned above, theophylline is a potent inhibitor of adenosine receptors [25]. It is established that adenosine induces the release of leukotriene and histamine, so adenosine receptor blockade can ameliorate inflammatory conditions including colitis [44]. Moreover, one research indicated that theophylline even at the 10 mg/kg elevates glucocorticoid production, and this mechanism may be at least partially responsible for the anti-inflammatory and anti-colitis effects of theophylline [31]. Anti-colitis effects of theophylline may also be mediated by other minor mechanisms including stimulation of release of adrenal catecholamine, reduction of calcium level in inflammatory cells, and suppression of prostaglandins activities [15]. However, the exact mechanisms of anti-colitis activity remained to be determined in further studies. It is worth to be noted that theophylline also has antitumor activities [37,45]. Since BCL2 has been found to be related to colon cancer [46], suppression of BCL2 expression and activity by theophylline [37] may be a safe and effective way to prevent colitis-associated cancer. Further research is required to evaluate the effect of theophylline on colitis-associated cancers. In summary, this work showed that intraperitoneal injection of theophylline, a non-specific PDE inhibitor, clearly attenuated acetic acid induced ulcerative colitis which became evident via decrease in proinflammatory cytokine levels, histopathological studies and MPO activity assessments. Our findings suggest potential application of theophylline for effective and inexpensive treatment of ulcerative colitis. Our study provides the basis for future investigations on the use of theophylline for the treatment of ulcerative colitis. Conflict of interest The authors have no conflict of interest. Acknowledgment This research project was fully sponsored by Kurdistan University of Medical Sciences, Sanandaj, Iran with grant number 452879. References [1] Y. Ikeda, F. Akbar, H. Matsui, M. Onji, Characterization of antigen-presenting dendritic cells in the peripheral blood and colonic mucosa of patients with ulcerative colitis, Eur. J. Gastroenterol. Hepatol. 13 (2001) 841–850. [2] M.E. Street, G. De’Angelis, C. Camacho-Hübner, G. Giovannelli, M.A. Ziveri, P.L. Bacchini, S. Bernasconi, G. Sansebastiano, M.O. Savage, Relationships between serum IGF-1, IGFBP-2, interleukin-1beta and interleukin-6 in inflammatory bowel disease, Horm. Res. Paediatr. 61 (2004) 159–164. [3] R. Rahimi, S. Nikfar, M. Abdollahi, Meta-analysis technique confirms the effectiveness of anti-TNF-alpha in the management of active ulcerative colitis when administered in combination with corticosteroids, Med. Sci. Monit. 13 (2007) 13–18. [4] W.Y. Chey, A. Hussain, C. Ryan, G.D. Potter, A. Shah, Infliximab for refractory ulcerative colitis, Am. J. Gastroenterol. 96 (2001) 2373–2381.
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