Attenuating effects of coenzyme Q10 and amlodipine ...

http://informahealthcare.com/ipi ISSN: 0892-3973 (print), 1532-2513 (electronic) Immunopharmacol Immunotoxicol, Early Online: 1–8 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/08923973.2015.1021357

RESEARCH ARTICLE

Attenuating effects of coenzyme Q10 and amlodipine in ulcerative colitis model in rats Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by 197.35.59.193 on 03/10/15 For personal use only.

Engy M. El Morsy1, Rehab Kamel1, and Maha A. E. Ahmed2 1

Department of Pharmacology and Toxicology, Faculty of Pharmacy, Helwan University, Ein Helwan, Cairo, Egypt and 2Department of Pharmacology and Toxicology, Faculty of Pharmacy, Misr University for Science and Technology (MUST), 6th of October City, Giza, Egypt Abstract

Keywords

Context: Ulcerative colitis is a chronic inflammatory bowel disease. Recent studies reported a pivotal role of elevated intracellular calcium in this disorder. Coenzyme Q10 (CoQ10) and amlodipine are known to maintain cellular energy, decrease intracellular calcium concentration in addition to their antioxidant and anti-inflammatory properties. Objective: The aim of this study was to evaluate the possible protective effects of CoQ10, amlodipine and their combination on ulcerative colitis. Materials and methods: Colitis was induced in rats by intracolonic injection of 3% acetic acid. CoQ10 (10 mg/kg), amlodipine (3 mg/kg) and their combination were administered for 8 consecutive days before induction of colitis. Results: Our results showed that administration of CoQ10, amlodipine and their combination decreased colon tissue malondialdehyde (MDA), tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b), prostaglandin E2 (PGE2), myeloperoxidase (MPO) and heat shock protein (HSP70) levels induced by intracolonic injection of acetic acid and restored many of the colon structure in histological examination. On the other hand, they increased superoxide dismutase (SOD) activity, adenosine-50 -triphosphate (ATP) and interleukin-10 (IL-10) colonic contents. Discussion and conclusion: Administration of either CoQ10 or amlodipine was found to protect against acetic acid-induced colitis. Moreover, their combination was more effective than individual administration of either of them. The protective effect of CoQ10 and amlodipine may be in part via their antioxidant, anti-inflammatory and energy restoration properties.

Amlodipine, CoQ10, inflammation, oxidative stress, ulcerative colitis

Introduction Ulcerative colitis is one of the major subtypes of inflammatory bowel disease. It is characterized by bloody and purulent diarrhea, abdominal pain and recurrent attacks1. The observed intestinal inflammation and mucosal ulceration result from the infiltration of neutrophils, lymphocytes, macrophages and mast cells2. The exact pathophysiology of ulcerative colitis is still not yet fully elucidated. However, it has been generally believed that this disease is strongly correlated with the massive generation of reactive oxygen species and excessive expression of inflammatory cytokines such as TNF-a3,4. Acetic acid-induced colitis is well adopted as a model of ulcerative colitis. This model is characterized by increased inflammatory cytokines, lipid peroxidation and alterations in the mucosal antioxidant defense mechanisms2,5. Experimentally, many compounds with antioxidant and antiinflammatory properties have been appreciated for their Address for correspondence: Dr Engy Mohamed El Morsy, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Helwan University, Cairo, Egypt. Tel: +201151681653. E-mail: [email protected]

History Received 17 October 2014 Revised 19 December 2014 Accepted 17 February 2015 Published online 10 March 2015

capabilities to repair the imbalance between antioxidant and pro-oxidant mechanisms in ulcerative colitis and to attenuate the severity of the disease5,6. Coenzyme Q10 (CoQ10), ubiquinone or ubidecarenone, is a well-known cofactor in the mitochondrial electron transport chain required for ATP production. It is synthesized endogenously in humans and other species, and can also be obtained from exogenous food sources7. In addition to its crucial bioenergetic role, the reduced form of CoQ10 exerts potent lipophilic antioxidant effects, either directly via protecting cellular components from free radicals or indirectly through regeneration of other endogenous antioxidants8,9. In experimental studies, CoQ10 has been appreciated for its antioxidant, anti-inflammatory and ATP-regenerative properties in several models of gastric ulcer, osteoarthritis and diabetes10–12. It seems there is no previous study handling the effect of CoQ10 on acetic acid-induced colitis in rats. Amlodipine is a calcium channel blocker that is used in the treatment of hypertension13. Apart from its well-established cardiovascular effects, experimental studies have shown that amlodipine can inhibit inflammatory cytokines and enhance antioxidant defenses14–16. Recently, Patil et al.17 reported that

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amlodipine could reduce the ulcer index and gastric pH in rats with gastric ulcer. However, the effect of amlodipine on ulcerative colitis has not yet been studied. This study was carried out to investigate the effect of either amlodipine or (CoQ10) alone and their combination on acetic acid-induced colitis in rats.

Methods

Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by 197.35.59.193 on 03/10/15 For personal use only.

Chemicals and drugs Amlodipine was supplied by Pfizer (Cairo, Egypt). Coenzyme Q10 (CoQ10) was supplied by the Arab Company for Pharmaceuticals & Medicinal Plants (Cairo, Egypt). Acetic acid was supplied by Merck chemicals (Darmstadt, Germany). All the chemicals were of analytical grade and purchased from Sigma-Aldrich Co. (St. Louis, MO).

Immunopharmacol Immunotoxicol, Early Online: 1–8

saline solution (3% v/v) was instilled. Then rats were maintained in a supine Trendelenburg position for 30 s to prevent early leakage of the intracolonic instillate20. Macroscopic scoring Mucosal damage was assessed macroscopically by the scoring system of Millar et al.20 Oxidative stress markers Determination of lipid peroxides level Lipid peroxidation was assessed by measuring malondialdehyde (MDA) level in the colon homogenates supernatants according to Mihara and Uchiyama21. Determination of superoxide dismutase activity

Animals preparation and design Fifty male Sprague–Dawley rats weighing 180–200 g were housed in cages at a temperature-controlled (25 ± 1 C) environment and provided free access to pelleted food and purified drinking water ad libitum. The animal experiments described later were approved by the Ethics Committee, Faculty of Pharmacy, Helwan University, Cairo, Egypt. The animals were randomly divided into five groups, 10 per each group, as follows: Group I: Normal control group received 10% Tween 80 solution orally by gavage for 8 consecutive days. Group II: Colitis control group received 10% Tween 80 solution orally by gavage for 8 consecutive days prior to induction of colitis. Group III: CoQ10 group received 10 mg/kg of CoQ10 orally by gavage suspended in 10% Tween 80 solution18 for 8 consecutive days prior to induction of colitis. Group IV: Amlodipine group received 3 mg/kg of amlodipine orally by gavage suspended in 10% Tween 80 solution19 for 8 consecutive days prior to induction of colitis. Group V: Combination group received 10 mg/kg of CoQ10 followed by 3 mg/kg of amlodipine half an hour later, orally by gavage for 8 consecutive days prior to induction of colitis. At day 8 after 1 h of the aforementioned treatment, rats of all groups were subjected to colon instillation of acetic acid (or saline for group I). After 24 h, all animals were sacrificed by cervical dislocation. Colonic segments were excised, freed of adherent adipose tissue, rinsed with cold normal saline and a portion was used for macroscopic scoring. Another portion of the colonic segments was homogenized in phosphate buffer saline (0.1 M PBS, pH 7.4). The homogenates were centrifuged at 10 000 rpm for 30 min at 4 C, and supernatants were stored at 70 C until biochemical and cytokine assays could be performed. A third portion of colonic segments was stored in 10% neutral buffered formalin for histopathological study.

Superoxide dismutase (SOD) activity was determined according to Ukeda et al.22 Determination of adenosine-50 -triphosphate level Adenosine-50 -triphosphate (ATP) level was determined according to the manufacturer’s instruction (Abcam, Cambridge, UK). Inflammatory markers Measurement of cytokines levels Cytokines levels were determined by enzyme linked immunosorbent assay kits specific for rats using a sandwich enzyme immunoassay (Cloud-Clone Corp., Houston, TX) according to the provided instructions. The parameters evaluated were tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b) and interleukin-10 (IL-10). Determination of prostaglandin E2 level Prostaglandin E2 (PGE2) measurement was performed according to the manufacturer’s instruction (Cloud-Clone Corp., Houston, TX). Determination of myeloperoxidase content Myeloperoxidase (MPO) colon content was determined using a sandwich enzyme immunoassay according to the manufacturer’s instructions (Cloud-Clone Corp., Houston, TX). Determination of heat shock protein (HSP70) content Heat shock protein (HSP70) quantitative determination was performed using a sandwich enzyme immunoassay according to the manufacturer’s instruction (Uscn Life Science & Technology Company, Missouri, MN).

Induction of colitis Animals were fasted overnight, with access to water ad libitum, before induction of colitis and were anesthetized by ether inhalation. A polypropylene tube with 2 mm diameter was inserted slowly through the rectum into the colon to a distance of 8 cm. Two milliliters of acetic acid in normal

Histopathological study Paraffin tissue blocks were prepared for sectioning and the obtained sections were stained by hematoxylin and eosin stain for histopathological examination through the light microscope.

Coenzyme Q10 and amlodipine in ulcerative colitis

DOI: 10.3109/08923973.2015.1021357

Statistical analysis Results were expressed as mean ± SEM, and different groups were compared using one way analysis of variance (ANOVA) followed by Tukey–Kramer test for multiple comparisons.

Results


Macroscopic scores Intracolonic administration of acetic acid-induced colonic injury characterized by hyperemia and inflammation with dense and diffuse ulcerations, when compared with saline applied control group. Pre-treatment with CoQ10 or amlodipine significantly attenuated the macroscopic score by 37.5 and 33.33%, respectively, while pre-treatment with their combination significantly reduced acetic acid-induced macroscopic damage by 67.67% as compared to group II (colitis control group). This reduction was significant different from each drug alone (Table 1). Oxidative stress markers and ATP Intracolonic administration of acetic acid induced significant increase in MDA level by 2 folds as compared to group I (normal control group) value. However, pre-treatment with CoQ10 or amlodipine significantly reduced MDA level by 25 and 30%, respectively, as compared to group II (colitis control group). Furthermore, pre-treatment with their combination significantly reduced MDA level by 51% as compared with group II. This reduction was significantly different from each drug alone (Figure 1A). On the other hand, there was a significant decrease in SOD activity with induction of colitis by 73% as compared to group I. Likewise, this reduction was significantly enhanced by preadministration of CoQ10, amlodipine and their combination by 32, 63 and 88%, respectively, as compared to group II. It is worth to mention that the combination effect was significantly different from each drug alone (Figure 1B). Intracolonic administration of acetic acid induced significant decrease in ATP level by 44% as compared to group I, while pre-administration of CoQ10, amlodipine and their combination significantly increased this reduction by 25, 18

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and 43% respectively, as compared to group II. The combination effect was significantly different from each drug alone (Figure 1C). Inflammatory markers and HSP70 Acetic acid induced a significant increase in colon TNF-a and IL-1b by 1.6 and 1.7 folds, respectively, as compared to group I (normal control group). Pre-administration of CoQ10 significantly lowered these levels by 32 and 18%, respectively, while pre-treatment with amlodipine showed significant reduction in their levels by 25 and 26%, respectively, as compared to group II. Furthermore, pre-treatment with CoQ10 plus amlodipine showed a significant drooping in their levels by 43 and 38%, respectively, as compared to group II. The combination effect was significantly different from each drug alone (Figure 2A and B). On the other hand, intracolonic administration of acetic acid induced significant decrease in IL-10 level by 45% as compared to group I (normal control group). Pre-treatment with CoQ10, amlodipine and their combination showed a significant elevation in its level by 23, 33 and 54%, respectively, as compared to group II. Likewise, the effect of the drugs combination was significantly different from each single drug alone (Figure 2C). Administration of acetic acid induced significant increase in PGE2 level and MPO content by 1 and 2.8 folds, respectively, as compared to group I. Pre-administration of CoQ10 significantly lowered these levels by 15 and 29%, respectively, while pre-treatment with amlodipine showed significant reduction in their levels by 15 and 36%, respectively, as compared to group II. On the other hand, pre-treatment with CoQ10 plus amlodipine showed a significant decrease in their levels by 27 and 53%, respectively, as compared to group II. The combination effect was significantly different from each drug alone (Figure 2D and E). Acetic acid induced a significant increase in colon HSP70 content by 1.1 folds as compared to group I. Pre-administration of CoQ10, amlodipine and their combination significantly reduced its content by 17, 20 and 26%, respectively, as compared to group II. It is worth noting that this drooping by pre-administration of the combination was not significantly different from each drug alone (Figure 2F).

Table 1. Effect of CoQ10, amlodipine and their combination on score lesion in rats colons with acetic acid-induced ulcerative colitis. Groups

Gross lesion

% Reduction

Group Group Group Group Group

– 4.00 ± 0.00a 2.50 ± 0.22a,b 2.66 ± 0.21a,b 1.33 ± 0.21a,b,c,d

– 0 37.50 ± 5.59b 33.33 ± 5.27b 67.67 ± 5.27b,c,d

I II II IV V

Group I: Normal control group received 10% Tween 80 solution orally by gavage for 8 consecutive days. Group II: Colitis control group received 10% Tween 80 solution orally by gavage for 8 consecutive days prior to induction of colitis. Group III: received 10 mg/kg of CoQ10. Group IV: received 3 mg/kg of amlodipine. Group V: received 10 mg/kg of CoQ10 followed by 3 mg/kg of amlodipine half an hour later. All drugs were administered for 8 consecutive days prior to induction of colitis by acetic acid. Data are presented as means ± SEM (n ¼ 10). a,b,c,d Significantly different from group I, group II, group III and group IV, respectively, at p50.001.

Histopathological examination Histopathological examination of colon sections from group I (normal control group) revealed normal structure of the mucosa, glandular structure in the lamina propria, submucosa, muscularis and serosa (Figure 3A). Induction of colitis by intracolonic administration of acetic acid caused ulceration, necrosis and hemorrhage in the mucosa associated with edema, inflammatory cells infiltration, hemorrhage and congestion of the blood vessels in the submucosa as well as in the muscularis (Figure 3B). Pre-treatment with CoQ10, amlodipine and their combination restored many of the colon structure showing edema with few inflammatory cells infiltration in the lamina propria of the mucosal layer and congestion in the blood vessels were detected of the submucosa (Figure 3C–E).


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Figure 1. Effect of CoQ10, amlodipine and their combination on lipid peroxidation (MDA) level (A), superoxide dismutase (SOD) activity (B) and adenosine-50 -triphosphate (ATP) level (C) measured in rats colons with acetic acid-induced ulcerative colitis. Group I: Normal control group received 10% Tween 80 solution orally by gavage for 8 consecutive days. Group II: Colitis control group received 10% Tween 80 solution orally by gavage for 8 consecutive days prior to induction of colitis. Group III: received 10 mg/kg of CoQ10. Group IV: received 3 mg/kg of amlodipine. Group V: received 10 mg/kg of CoQ10 followed by 3 mg/kg of amlodipine half an hour later. All drugs were administered for 8 consecutive days prior to induction of colitis by acetic acid. Data are presented as means ± SEM (n ¼ 10) in each group. (a, b, c, d) Significantly different from group I, group II, group III and group IV, respectively, at p50.05.

Discussion Acetic acid-induced colitis is an experimental model of inflammatory bowel disease (IBD) characterized by oxidative damage23. This may lead to membrane lipid peroxidation and severe cell damage that plays an important role in the pathogenesis of the disease20,24. In consistency with the above findings, this study shows a significant increase in MDA level accompanied by a significant decrease in SOD activity. SOD is a key enzyme which inactivates superoxide ion by transforming it into more stable metabolite, hydrogen peroxide25. Accordingly, SOD restrains lipid peroxidation in colon by eliminating free-radicals. A significant body of research has indicated that decreasing SOD activity in colon tissues leads to mucosal injury due to reduced ability of oxidative radicals scavenging26. In addition, there was a significant depletion of ATP level in colitis group as compared to normal control group. This is consistent with a previous study showing that mitochondrial functions can be altered by oxidative stress, which inhibits mitochondrial respiration and ATP synthesis27. Reactive oxygen species induce the opening of mitochondrial permeability transition pores, resulting in mitochondrial swelling, release of matrix death factors and cell apoptosis28. In addition, accumulation of mitochondrial calcium due to free radicals leads to further damage and necrosis29.

In addition to oxidative stress, intracolonic acetic acid induces acute intestinal inflammation23. Our results showed a significant increase in the levels of TNF-a, IL-1b and PGE2 after induction of colitis. This was in harmony with Dogan et al.30 IL-1b and TNF-a are key immunoregulatory cytokines that amplify the inflammatory response by activating a cascade of immune cells such as neutrophils31. Following their activation, neutrophils secrete MPO enzyme abundantly32. Indeed, our results demonstrated that the level of colonic MPO was significantly increased in acetic acid group. Moreover, Eigler et al.33 reported that IL-1b liberates prostaglandins, and also augments hydrogen peroxide, bradykinin and histamine induction leading to stimulation of tissue damage. In this study, colon tissue damage was confirmed by macroscopic score, and histopathological study which revealed epithelial cell necrosis, edema and neutrophil infiltration, in acetic acid group as compared to normal control group. On the other hand, the current results showed a significant decrease of colon IL-10 level in acetic acid group as compared to normal control group. IL-10 is a cytokine with potent antiinflammatory and immune regulatory activity34. It deactivates macrophages and inhibits the production of inflammatory cytokines, as well as reactive oxygen species35,36. A wide variety of stresses, such as oxidative stress and inflammation, are accompanied by an increase in the


DOI: 10.3109/08923973.2015.1021357


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Figure 2. Effect of CoQ10, amlodipine and their combination on inflammatory markers measured in rats colons with acetic acid-induced ulcerative colitis: (A) tumor necrosis factor-a (TNF-a), (B) interleukin-1b (IL-1b), (C)interleukin-10 (IL-10), (D) prostaglandin E2 (PGE2) levels, (E) myeloperoxidase (MPO) and (F) heat shock protein (HSP70) contents. Group I: Normal control group received 10% Tween 80 solution orally by gavage for 8 consecutive days. Group II: Colitis control group received 10% Tween 80 solution orally by gavage for 8 consecutive days prior to induction of colitis. Group III: received 10 mg/kg of CoQ10. Group IV: received 3 mg/kg of amlodipine. Group V: received 10 mg/kg of CoQ10 followed by 3 mg/kg of amlodipine half an hour later. All drugs were administered for 8 consecutive days prior to induction of colitis by acetic acid. Data are presented as means ± SEM (n ¼ 10) in each group. (a, b, c, d) Significantly different from group I, group II, group III and group IV, respectively, at p50.05.

expression of HSP7037. The mechanism of cytoprotection by HSP70 is generally explained by the notion that HSP70 binds to damaged and misfolded proteins and helps refolding. It facilitates restoration of the function of the denatured or severely damaged proteins38. HSP70 activity is known to be ATP-dependent39. Our results showed a significant increase in colon HSP70 level after rectal administration of acetic acid as compared to control group, and this was accompanied by significant ATP depletion. The important finding of this study is that CoQ10, amlodipine and their combination ameliorated the macroscopic and biochemical changes induced by intracolonic acetic acid administration. It is noteworthy that the combination of drugs afforded a higher protection than either drug

alone, acting in an additive manner in almost all the parameters. The obtained results revealed that administration of CoQ10, amlodipine and their combination significantly decreased the gross lesion scores, suppressed lipid peroxidation, prevented the reduction of SOD activity and restored ATP level as compared to colitis group. The protective effect of CoQ10 can be attributed to its ability to inhibit the generation of reactive oxygen species40, and to scavenge lipid peroxidation products41, either by acting as a chain breaking antioxidant, or indirectly by recycling vitamin E42. In addition, CoQ10 had the ability to preserve mitochondrial integrity, as CoQ10 is an essential cofactor of the respiratory chain43 and a blocker of ATP depletion44. Likewise, Bonakdar and Guarneri45 proved that CoQ10


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Figure 3. Hematoxilin- and eosin-stained sections showing the effect of CoQ10, amlodipine and their combination on histological rats colons changes after acetic acid-induced ulcerative colitis. Original magnification, 16, (A) Group I: normal control group received 10% Tween 80 solution orally by gavage for 8 consecutive days. (B) Group II: received 10% Tween 80 solution orally by gavage for 8 consecutive days prior to induction of colitis. (C) Group III: received 10 mg/kg of CoQ10. (D) Group IV: received 3 mg/kg of amlodipine. (E) Group V: received 10 mg/kg of CoQ10 followed by 3 mg/kg of amlodipine half an hour later. All drugs were administered orally by gavage for 8 consecutive days prior to induction of colitis by acetic acid. (mu): mucosal epithelium; (g): glandular structure; (sm): submucosal; (ml): muscularis; (s): serosa; (u): ulceration; (n): necrosis; (h): hamorrhage; (o): edema; (m): infiltration; (v): congested blood vessels.

increases ATP production, prevents lipid peroxidation, and is an indirect stabilizer of calcium channels to decrease calcium overload. Recently, it was shown that CoQ10 suppresses voltage-dependant calcium influx in cerebral cortex glutamatergic nerves46. The protective effect of amlodipine could be attributed to the ability of amlodipine to limit the influx of calcium into smooth muscle cells, which lowers intracellular calcium, relaxes blood vessels and increases colon mucosal blood flow47. In addition, amlodipine was shown to exert antioxidative effects in vitro and in vivo by inhibiting the oxidizability of the cell membrane and low-density lipoproteins48. This effect was mediated by quenching free radicals due to its highly lipophilic properties and its chemical structure49. Our results are in harmony with those of Pronobesh et al.50 who reported that amlodipine acts as an antioxidant, regulates membrane fluidity and raises the activities of mitochondrial antioxidant enzymes.

Moreover, it has been reported that amlodipine restored mitochondrial ATP content during experimental myocardial infarction. This may be due to the antioxidant properties of amlodipine as well as its ability to prevent the increase in mitochondrial Ca2+ concentration51. The present results revealed that CoQ10, amlodipine and their combination significantly attenuated the production of TNF-a, IL-1b, PGE2, MPO and HSP70 and increased IL-10 level as compared to colitis group. CoQ10 exhibits anti-inflammatory properties by reducing the release of proinflammatory cytokines and COX-2 expression during inflammatory injury52. Other studies had proven the ability of CoQ10 to hamper neutrophils infiltration, as well as MPO activity53,54. Therefore, it is crystal clear that CoQ10 can decrease HSP70 after decreasing the colon inflammation. In addition, Jin and his co-workers55 reported that CoQ10 promoted the expression of IL-10 in periodontal tissues of experimental periodontitis rats.


DOI: 10.3109/08923973.2015.1021357

It was shown that calcium ions play an important role in the synthesis and release of the chemical mediators of inflammation56. Therefore, a decrease in intracellular calcium levels may prevent or attenuate inflammation57. On the other hand, inhibition of PGE2 may follow the decrease in TNF-a58. The ability of amlodipine to inhibit reactive oxygen species and inflammatory markers in this work, may explain its ability to decrease HSP70 and increase IL-10 colon levels.

13. 14.

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Conclusion Taken together, the results of this study prove the efficacy of CoQ10 and amlodipine in attenuation of acetic acid-induced colitis in rats. Their combination affords a greater protection than each drug individually. This may be due in part to their antioxidant and anti-inflammatory properties as well as their ability to restore ATP and hence energy to cells.

Declaration of interest The authors report no declarations of interest.

Acknowledgements

17. 18. 19. 20. 21. 22.

The authors wish to thank Professor Adel B. Kholoussy (Department of Pathology, Faculty of Veterinary Medicine, Cairo University) for his help in histopathological examination.

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