Is Coenzyme Q₁₀ Effective in Protection against Ulcerative Colitis? An Experimental Study in Rats

Abstract

Coenzyme Q₁₀ (Co-Q₁₀) is a vitamin-like supplement which appears to be safe, with minimal side effects and low drug interaction potential. Co-Q₁₀ is used in the treatment of a variety of disorders related primarily to suboptimal cellular energy metabolism and oxidative injury. Studies supporting the efficacy of Co-Q₁₀ appear most promising for a variety of diseases, including ulcerative colitis (UC). The present investigation aims to elucidate the possible protective effects of Co-Q₁₀ against UC, as induced by the administration of iodoacetamide to adult male albino rats. In our study, Co-Q₁₀ showed potent anti-oxidant and anti-inflammatory activities through a significant increase in catalase activity and glutathione content. In addition, it significantly decreased myeloperoxidase activity, malondialdehyde content and nitrate/nitrite production. These results suggest that Co-Q₁₀ protects against UC in rats via anti-oxidant and anti-inflammatory potentials, and therefore seems promising for use in further clinical trials.

Ulcerative colitis (UC), is a common, chronic inflammatory disease which affects the gastrointestinal tract especially colon and rectum.¹⁾ It is usually mildly active, but it can be life-threatening during severe attacks due to systemic and colonic complications. In addition, long-term UC patients are at an increased risk of colorectal cancer.²⁾

The exact cause of UC remains unknown, but possible etiologic factors, including genetic, immunologic and environmental factors may be involved.³⁾ In addition, oxidative stress has been involved in the pathogenesis of ulcerative colitis in experimental animals and in humans.^4,5)

It is known that the oxidant/anti-oxidant balance in the intestinal mucosa is seriously impaired in UC patients compared to normal patients, where intestinal inflammation is associated with excessive production of reactive oxygen and nitrogen metabolites.⁶⁾

Treatment of UC aimed to reduce symptoms and mucosal inflammation. Most of the current therapies for UC involve corticosteroids and 5-aminosalicylic acid. Unfortunately, these drugs have serious side effects, which limit their use.⁷⁾ On the other hand, anti-oxidant therapy has shown beneficial effects in several experimental models of rat colitis.⁸⁾

Coenzyme Q₁₀ (Co-Q₁₀) is a lipid-soluble benzoquinone which is considered as a key component of the mitochondrial respiratory chain for adenosine triphosphate synthesis.⁹⁾ It is recognized as an intracellular anti-oxidant that protects membrane phospholipids, mitochondrial membrane protein and low density lipoprotein (LDL) from free radical-induced oxidative damage.¹⁰⁾

Among the recent data, Co-Q₁₀ was found to improve endothelium-bound extracellular superoxide dismutase (SOD).¹¹⁾ In this investigation, the anti-oxidant effect of Co-Q₁₀ was studied compared with sulfasalazine against iodoacetamide (IA)-induced UC in rats.

MATERIALS AND METHODS

Drugs and Chemicals

Sulfasalazine was provided as a gift from Acdima International Company (Egypt), whereas Co-Q₁₀ was obtained as a kind gift from MEPACO Pharmaceutical Company (Egypt). 1,1-3,3-Tetramethoxypropane, 5,5′-dithiobis-2-nitrobenzoic acid (DTNB), iodoacetamide, glutathione (GSH) powder, horseradish peroxidase (HRP), N-(1-naphthyl)-ethylenediamine dihydrochloride (NNED), o-dianisidine hydrochloride, thiobarbituric acid (TBA), malondialdehyde (MDA), Tris-hydroxymethylamino methane, hexadecyltrimethylammonium bromide (HTAB) and sulfanilamide were purchased from Sigma-Aldrich (U.S.A.). Vanadium chloride was obtained from Across (Belgium). All other chemicals and solvents used were of analytical grade.

Animals

Adult male Wistar rats, weighing 180–200 g, were used in the current study. Animals were obtained from the Modern Veterinary Office for Laboratory Animals, Cairo, Egypt. The rats were kept under standard conditions of temperature (25±0.5°C) and relative humidity (55±1) with 12-h light/dark cycles for one week for adaptation before being subjected to laboratory experiments and were allowed free access to standard forage and drinking water ad libitum. All animal housing and handling were conducted in compliance with the Beni-Suef University guidelines and in accordance with the research protocols established by the Animal Care Committee of the National Research Center (Cairo, Egypt) which followed the recommendations of the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985).

Induction of Experimental Colitis in Rats

Ulcerative colitis was induced in rats by intra-colonic instillation of 0.1 mL of a 3% IA solution in 1% carboxymethylcellulose (CMC)/1% tween 80 solution in normal saline using medical grade canal for enteral feeding (diameter 2 mm), where the tip of canal was inserted to 8 cm proximal to the anus verge after which fluid was withdrawn. Animals were allowed to hang in air by holding their tails for 1–2 min to prevent spillage of solution from rectum. Rats were left for 7 d to induce colitis.¹²⁾

Experimental Design

Thirty-two rats were randomly assigned to four groups, each of 8 rats. The first group was kept as normal control group. The other three groups received 0.1 mL of 3% IA once rectally together with either the vehicle (10 mL/kg/d, per os (p.o.); UC control group), sulfasalazine (500 mg/kg/d, p.o.; standard treatment group)¹³⁾ or Co-Q₁₀ (30 mg/kg/d, p.o.). All treatments were given orally on a once daily basis after IA administration for 7 consecutive days. Drugs were suspended in 1% CMC/1% tween 80 solution in normal saline.

Selection of the dose of Co-Q₁₀ was performed based on the pilot trial guided with the published literature.¹⁴⁾ We performed a pilot trial using 3 oral doses of Co-Q₁₀, namely 20, 30 and 40 mg/kg/d once daily and presented a graph of inflammatory scoring. The dose of 30 mg/kg showed better results compared with that of 20 mg/kg. Upon increasing the dose to 40 mg/kg, the beneficial anti-inflammatory effect of Co-Q₁₀ was stabilized or decreased (Fig. 1). Accordingly, we selected the dose of 30 mg/kg/d to complete the study.

Fig. 1. Effect of One Week Daily Oral Treatment with Different Doses of Co-Q₁₀ on Inflammatory Scoring for Selection the Best Dose of Co-Q₁₀ To Be Used Experimentally

a; Significantly different from UC control value. ab; Significantly different from Co-Q₁₀ (20 mg/kg/d).

Assessment of Colitis

Determination of Body Weight

Daily estimation of animal body weight was performed using a digital balance (Adam PW 214 made in England).

Colon Weight/Length Ratio

At the end of the experiment, animals were sacrificed with the abdominal cavities opened. The distal 9 cm portion of the colon was removed and cut longitudinally, slightly cleaned in ice cold saline to remove fecal residues then dried with filter papers and weighed. Colon was subjected to microscopical and histopathological examinations to assess the degree of colon edema which reflected the severity of colitis.¹⁵⁾

Macroscopic Scoring

Macroscopic appearance of the colonic mucosa was scored according to a scale ranging from 0 to 4, where (0) means no macroscopic changes, (1) means mucosal erythema only, (2) means mild mucosal oedema, slight bleeding or small erosions, (3) means moderate oedema, bleeding ulcers or erosions and (4) means severe ulceration, erosions, oedema and tissue necrosis.¹⁶⁾ Examination of colon sections macroscopically was performed by an experienced pathologist blinded to the study.

Histopathological Study

Colon samples were fixed in 10% formalin solution in normal saline for histological examination. Briefly, colon samples were embedded in paraffin, cut into sections using slide microtome, dried with descending grades of alcohol and stained with haematoxylin and eosin for histological evaluation of colonic damage using light microscope attached to a digital camera.

A scoring system was set to hitopathological sections, where (0) means normal histologic appearance with absence of colon ulcers or inflammation, (1) means mild inflammatory evidence without ulceration, (2) means appearance of few scattered superficial ulcers, (3) means numerous ulcers in group forms with involvement of lamina propria but not submucosa, (4) means massive ulceration with diffuse inflammatory reaction of both lamina propria and submucosa. Again examination of histopathologic sections was performed by an experienced pathologist blinded to the study.

Preparation of Tissue Homogenate

A portion of the colon was homogenized with 10 volumes of isotonic ice-cooled normal saline using a homogenizer (Ultra-Turrax T₂₅, IKA Labortechnik, Germany) to prepare 10% homogenate. The homogenate was centrifuged at 4000×g for 15 min. The obtained supernatant was used for the measurement of myeloperoxidase (MPO) activity, and nitrate/nitrite (NOx) production as inflammatory markers, as well as MDA production, GSH stores and catalase (CAT) activity as oxidative stress markers.

Determination of Colon MPO Activity

Colon MPO activity served as quantitative index of neutrophil infiltration and inflammation in several tissues, including the intestine.¹⁷⁾ MPO activity was determined in colon homogenate spectrophotometrically according to the method of Krawisz et al.¹⁸⁾

Determination of Colon NOx Production

Colon NOx production, considered as an end product of NO metabolism, can be estimated in UC as an indication to enhanced inducible nitric oxide synthase (iNOS) activity.^19,20) NOx production in the colon was measured spectrophotometrically at 540 nm as previously described.²¹⁾

Determination of Colon MDA Production

Lipid peroxidation, estimated by the determination of the level of thiobarbituric acid reactive species (TBARS), was measured as MDA in the colon homogenates spectrophotometrically at 520–535 nm as previously described.²²⁾

Determination of Colon GSH Stores

Glutathione was measured in colon homogenate according to the method described by Sedlak and Lindsay.²³⁾ The principle of the method depends on the reduction of 1,1-3,3-tetramethoxypropane, 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) by the sulfhydryl group of GSH. The formed product was measured calorimetrically at 412 nm.

Determination of Colon CAT Activity

Catalase activity was measured in the colonic tissues according to Claiborne.²⁴⁾ The principle of this method depends on the decomposition of hydrogen peroxide by CAT activity, where the change in absorbance of a colored product is estimated at 240 nm.

Statistical Analysis

All data were expressed as the mean±standard error (S.E.) of 8 rats per experimental group. Statistical analysis was performed using one-way ANOVA test followed by Student–Newman–Keuls multiple comparisons test by the aid of Graphpad prism5 and Graphpad instant2 computer softwares (San Diego, U.S.A.), with values of p<0.05 considered statistically significant.

RESULTS

Determination of Body Weight

In normal control group, rats’ mean weight at the beginning of the experiment was 179±4.33 g, while at the end of the experiment the mean weight of the normal control rats increased to about 108% of the starting weight.

On the other hand, the mean animal weight of UC control rats decreased in the last day to show a value of 180.67±7.358 g after a strating value of 187.67±8.130. While treatment of rats with sulfasalazine or Co-Q₁₀ showed no significant change (Fig. 2).

Fig. 2. Effect of One Week Daily Oral Treatment with Co-Q₁₀ (30 mg·kg⁻¹·d⁻¹) on Animal’s Weight as Compared to Sulfasalazine (500 mg·kg⁻¹·d⁻¹) on Iodoacetamide Induced Ulcerative Colitis in Rats

Histopathologic and Macroscopic Assessment of Colitis

On histological examination of rat colon from the normal group, the histological features were typically of a normal structure (Fig. 3A). Intra-colonic administration of 0.1 mL of IA 3% caused extensive macroscopic damage of the colon and the colonic mucosa appeared hemorrhagic and ulcerated and was characterized by loss of surface epithelium and crypts, sub-mucosal oedema with inflammatory reaction in lamina propria and crypt abscess (Fig. 3B). The inflammatory process was associated also with increase of both colonic weight/length ratio (Fig. 4) and colonic ulceration score (Fig. 5).

Fig. 3. Photomicrographs of Colonic Mucosal Sections Obtained from Rats of Different Groups (H&E Staining)

(A) Normal control rat section showing normal mucosa with intact epithelial surface, (B) UC control rat section showing loss of surface epithelium and crypts (black arrow) with diffuse inflammatory reaction of both lamina propria and submucosa (white arrow). Deformed crypts are observed and muscularis mucosa (MM) is disrupted. (C) Sulfasalazine-treated rat section, the section shows small loss of surface epithelium and crypts (black arrow) with inflammatory reaction in lamina propria (white arrow), sub-mucosal oedema and congested blood vessels (head arrows), (D) Co-Q₁₀ group: the section shows minimal loss of surface epithelium (black arrow) but with inflammatory cells in lamina propria (white arrow) and submucosal oedema.

Fig. 4. Effect of One Week Daily Oral Treatment with Co-Q₁₀ on Colon Weight Length Ratio as Compared to Sulfasalazine on Ulcerative Colitis Induced by Iodoacetamide in Rats

* Significantly different from normal control value at p<0.05. ^@ Significantly different from UC control value at p<0.05.

Fig. 5. Effect of One Week Daily Oral Treatment with Co-Q₁₀ on Colon Ulceration Score as Compared to Sulfasalazine on Ulcerative Colitis Induced by IA in Rats

* Significantly different from normal control value at p<0.05. ^@ Significantly different from UC control value at p<0.05.

Treatment with sulfasalazine or Co-Q₁₀ attenuated the extent and severity of the histological signs of tissue damage in colon tissues (Figs. 3C, D) with significant decrease in colon ulcer score (Fig. 5) and colon weight length ratio (Fig. 4).

Regarding colonic histopathological scoring, UC control rats showed significantly higher scores compared with normal control rats. With sulfasalazine or Co-Q₁₀ treatments, the histopathological scores were restored back to normal level (Fig. 6).

Fig. 6. Effect of One Week Daily Oral Treatment with Co-Q₁₀ on Inflammatory Scoring as Compared to Sulfasalazine on Ulcerative Colitis Induced by IA in Rats

a; Significantly different from normal control value. b; Significantly different from UC control.

Colon MPO Activity

The MPO activity of the colon tissue significantly increased in the UC control group as compared with the normal control group (Fig. 7), while treatment with sulfasalazine or Co-Q₁₀ significantly decreased colon MPO activity (Fig. 7).

Fig. 7. Effect of One Week Daily Oral Treatment with Co-Q₁₀ on Colon MPO Activity as Compared to Sulfasalazine on UC Induced by IA in Rats

* Significantly different from normal control value at p<0.05. ^@ Significantly different from UC control value at p<0.05.

Colon NOx Production

Intra-colonic administration of IA significantly increased colon NOx production as compared with normal control (Fig. 8). On the other hand, treatment with sulfasalazine or Co-Q₁₀ significantly decreased colon NOx production (Fig. 8) as compared with UC control.

Fig. 8. Effect of One Week Daily Oral Treatment with Co-Q₁₀ on Colon NOx Production as Compared to Sulfasalazine on UC Induced by IA in Rats

* Significantly different from normal control value at p<0.05. ^@ Significantly different from UC control value at p<0.05.

Colon MDA Content

Colon MDA content significantly increased in UC control group as compared to normal control (Fig. 9), while significantly decreased after treatment of rats with sulfasalazine or Co-Q₁₀ (Fig. 9).

Fig. 9. Effect of One Week Daily Oral Treatment with Co-Q₁₀ on Colon MDA Content as Compared to Sulfasalazine on UC Induced by IA in Rats

* Significantly different from normal control value at p<0.05. ^@ Significantly different from UC control value at p<0.05.

Colon GSH Content

Compared to normal control group, colon GSH content significantly decreases in UC control rats (Fig. 10). After treatment with sulfasalazine or Co-Q₁₀, a significant increase in colon GSH content was evident (Fig. 10).

Fig. 10. Effect of One Week Daily Oral Treatment with Co-Q₁₀ on Colon GSH Content as Compared to Sulfasalazine on UC Induced by IA in Rats

* Significantly different from normal control value at p<0.05. ^@ Significantly different from UC control value at p<0.05.

Colon CAT Activity

Intra-colonic administration of IA significantly decreased the colon CAT activity as compared to normal control group (Fig. 11). Meanwhile, treatment with sulfasalazine or Co-Q₁₀ significantly increased colon CAT activity (Fig. 11).

Fig. 11. Effect of One Week Daily Oral Treatment with Co-Q₁₀ on Colon Catalase Activity as Compared to Sulfasalazine on UC Induced by IA in Rats

* Significantly different from normal control value at p<0.05. ^@ Significantly different from UC control value at p<0.05.

DISCUSSION

Ulcerative colitis is characterized by recurring episodes of inflammation in the mucosal layer which is limited to the colon and its treatment is still a major challenge.²⁵⁾ The exact cause of UC remains unknown, but possible etiological factors have been involved, including genetic, immunologic and environmental.³⁾

Many experimental models have been developed for UC in a trial to get the exact events that illustrate the disease.^26–28) The most common chemicals used to induce models causing acute inflammation of the intestinal barrier are trinitrobenzene sulphonic acid (TNBS),²⁹⁾ dextran sodium sulphate (DSS)³⁰⁾ and oxazolone.³¹⁾ These models have different advantages and disadvantages that have been greatly reviewed by Hoffmann et al.³²⁾

In the current study, the mucosal inflammation was induced by the sulfhydryl blocker IA. It is an alkylating agent causing mucosal injury by blocking sulfhydryl groups that are very important for the vitality of the intestine.³³⁾

Application of sulfhydryl blockers as chemical inducers of inflammation was rarely applied. However, it was proved that IA increased vascular permeability, caused massive mucosal edema, erosion and ulcers.^33–35) Therefore, IA-induced colitis model was used to reflect the histological characteristics of the diseases and sharing many characteristics with human colitis.

In the present investigation, intra-colonic administration of IA 3% to rats caused severe colon ulceration evidenced macroscopically by extensive macroscopic damage of the colon and significant increase in colon ulceration score. The colonic mucosa appeared hemorrhagic, ulcerated and characterized by loss of surface epithelium, crypts and sub-mucosal edema with inflammatory reaction in lamina propria and crypt abscess. In addition, IA significantly increased colon weight/length ratio and significantly increased colon MPO activity and colon NOx production.

This model is based on the fact that endogenous sulfhydryl compounds, such as GSH, play a crucial role in the protection of gastric mucosa.³⁶⁾ The decreased anti-oxidant defense mechanism leads to increased lipid peroxidation causing obliteration and damage to cell membranes. In this study, IA significantly increased colon MDA content and significantly decreased colon GSH content and colon CAT activity.

Sulfasalazine drug is considered a standard drug for management of UC. It is composed of 5-aminosalicylic acid (5-ASA) with anti-inflammatory activity linked to sulfapyridine with antibacterial activity through a diazo-bond, which is readily cleaved by bacterial azoreductases in the colon.³⁷⁾

The anti-inflammatory effect of sulfasalazine was already confirmed in this study according to the obtained results by improving the inflammatory markers through significant reduction of colonic damage score and also significant attenuation of the extent and severity of the histological signs of cell damage. There were no inflammatory cells in the lamina propria and the epithelium remained intact so it was found that sulfasalazine significantly decreased colon weight/length ratio. This result is in harmony with the work of other investigators who reported similar findings.^38,39)

The anti-inflammatory effect of sulfasalazine was proved also by reducing neutrophil infiltration as evidenced by decreased MPO activity, thus decreasing mucosal ulceration.⁴⁰⁾ The anti-inflammatory properties of sulfasalazine are related to the 5-ASA moiety, because it interferes with arachidonic acid metabolism.⁴¹⁾ In addition, it has a role in inhibition of chemo-taxis of inflammatory cells, inhibition of expression of cytokine such as interleukin (IL)-1, IL-6, and tumor necrosis factor α (TNF-α), in addition to inhibition of lymphocyte proliferation and activation.⁴²⁾

The anti-oxidant properties of sulfasalazine were proved in this study through increasing colon GSH content and colon catalase activity. These findings are in agreement with the results of other investigators who reported similar increase in GSH content⁴²⁾ and similar increase in CAT activity.⁴³⁾ On the other hand, sulfasalazine significantly decreased colon MDA content in agreement with Dirlik et al.⁴⁴⁾ That is, the activity of sulfasalazine in treatment of ulcerative colitis not only related to its anti-inflammatory activity but also related to its anti-oxidant and free radical scavenger properties.⁴⁴⁾ The radical scavenger effect of 5-ASA has also been suggested from observations of its ability to prevent xanthine oxidase-induced de polymerization of hyaluronic acid.⁴⁵⁾

Ulcerative colitis is characterized by intense oxidative stress which means an imbalance between the oxidative free radical molecules and the defense provided by anti-oxidative defense barriers like GSH and CAT.⁴⁶⁾ Therefore it is commonly accepted that oxidative stress is an important determinant of UC pathophysiology.⁴⁷⁾ Accordingly, it is thought that anti-oxidants have a role in the treatment of UC especially after proving serious side effects of drugs which are already used to treat UC like sulfasalazine, which makes their use is limited.⁷⁾ The current study therefore aimed to reveal the benefit and mechanistic role of Co-Q₁₀ in treatment of UC.

Co-Q₁₀ is an important member of the anti-oxidant defense system which is a naturally occurring and widely distributed throughout the human body, and it is also known as ubiquinone.⁴⁸⁾ The role of Co-Q₁₀ in protection against neurodegenerative diseases, aging, as well as other diseases such as diabetes and cardiovascular impairments, is well established.^49,50) However, limited literature exists about its efficacy in protection or treatment of UC.

In this study, Co-Q₁₀ improved the internal defense mechanisms of colon tissue, where it significantly increased colon GSH content and CAT activity. Co-Q₁₀ acts as a powerful anti-oxidant which scavenges free radicals and prevents the initiation and propagation of lipid peroxidation in cellular bio-membranes, thus protecting the stability of the cell membranes and DNA from free radical-induced oxidative damage. It is also known to help regeneration other anti-oxidants, such as tocopherol and ascorbate.^51,52)

Co-Q₁₀ significantly decreased colon MDA content in harmony with the result reported by Modi et al.⁵³⁾ who studied beneficial effects of Co-Q₁₀ in streptozotocin-induced type I diabetic rats, and by Sawicka et al.⁵⁴⁾ who reported the same effect of Co-Q₁₀ during studying the effects of Co-Q₁₀ in cisplatin-induced lipid peroxidation. It is thought that Co-Q₁₀ is capable of preventing programmed cell death or apoptosis.⁵⁵⁾ The protective effect is extended to lipids, proteins and DNA mainly because of its close localization to the oxidative events and the effective regeneration by continuous reduction at all locations.⁵²⁾

It is generally thought that the mechanism of scavenges of free radical resulted from its characteristic structural and physiological function; where the lipophilic character of Co-Q₁₀ enables it to diffuse into the membrane phospholipid bilayer. It can also limit the production of reactive oxygen species (ROS) by blunting the expression of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase,⁵⁶⁾ so it regulates oxidative phosphorylation and prevents lipid peroxidation.⁵³⁾

The anti-inflammatory activity of Co-Q₁₀ was proved in this study by significant decrease of colonic MPO production, NOx production and colon weight/length ratio. This comes in agreement with previous reports regarding the role of Co-Q₁₀ in ameliorating nitrosative stress and inflammatory outcomes.⁵⁷⁾

CONCLUSION

Conclusively, our results reveal a beneficial role of Co-Q₁₀ in the management of IA-induced UC in rats. Concerning our study, Co-Q₁₀ is suggested to offer its benefit through anti-oxidant potential, suppression of lipid peroxidation, support of endogenous anti-oxidant defense, amelioration of nitrosative stress and NOx production, and inhibition of inflammatory infiltration. These results seem promising for further clinical trials concerning the application of safe agents like Co-Q₁₀, at least in conjunction with standard UC therapy to decrease dosage of drugs and to minimize their adverse effects.

Acknowledgments

All the authors wish to thank Dr. Mohamed Sadek Abd-Elbaky who was involved in drafting the manuscript and revising it. Great thanks are giving to Dr. Samraa Hussein for carrying out the histopathological study and interpretation of data.

Conflict of Interest

The authors declare no conflict of interest.

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