Coenzyme Q10 supplementation ameliorates inflammatory markers in patients with multiple sclerosis: a double blind, placebo, controlled randomized clinical trial Meisam Sanoobar 1,2, Shahryar Eghtesadi 1,2, Amirreza Azimi 3, Mohammad Khalili 1,2,4, Behnam Khodadadi 5, Shima Jazayeri 1,2, Mahmood Reza Gohari 6, Nahid Aryaeian1,2 1
Department of Clinical Nutrition, School of Nutritional Sciences & Dietetics, Tehran University of Medical Sciences, Tehran, Iran, 2Department of Nutrition and Biochemistry, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran, 3Brain and Spinal Injury Research Center, Tehran University of Medical Sciences, Tehran, Iran, 4Neurosciences Research Center, Tabriz University of Medical Sciences, Tabriz, Iran, 5 Department of Nutrition Sciences and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran, 6Department of Biostatistics, Hospital Management Research Center, Tehran University of Medical Sciences, Tehran, Iran Objectives: Multiple sclerosis (MS) is an immune-mediated neurodegenerative disease of central nervous system and recent studies show that inflammatory processes are highly associated with neurodegeneration in the brain. The purpose of this study was to investigate the effect of coenzyme Q10 supplementation on inflammatory and anti-inflammatory markers in patients with MS. Methods: This randomized, double-blind, placebo-controlled clinical study was performed among 48 patients with relapsing–remitting MS. Subjects were randomly assigned to a placebo group (n = 24) or coenzyme Q10 (CoQ10)-supplemented group (500 mg/day, n = 24). The intervention was administered for 12 weeks. Peripheral blood samples were collected at baseline and after 12-week intervention, to measure inflammatory (tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and matrix metalloproteinase (MMP)-9) and anti-inflammatory (IL-4 and TGF-β) markers. Results: Forty-five patients completed the study. After 12 weeks of intervention, the TNF-α levels (P = 0.003) decreased significantly in the CoQ10 group. Subjects in the CoQ10 group had significantly lower IL-6 levels (P = 0.037), compared to the placebo group. CoQ10 supplementation also resulted in decreased serum levels of MMP-9 as compared to the placebo group (P = 0.011). However, CoQ10 supplementation did not alter the IL-4 and TGF-β levels (P = 0.16 and P = 0.81, respectively). Discussion: CoQ10 supplementation at a dosage of 500 mg appears to decrease the inflammatory markers (TNF-α, IL-6, and MMP-9) in patients with MS. Keywords: Coenzyme Q10, Cytokines, Inflammation, Multiple sclerosis, Supplementation, Antioxidants, TNF-α, IL-6, MMP-9, TGF-β, Randomized placebo-controlled trial
Introduction Multiple sclerosis (MS) is a chronic neurodegenerative T-cell-mediated inflammatory disease of the central nervous system (CNS), which leads to the myelin loss and axonal and neuronal damage.1,2 The relapsing–remitting phase of the disease is associated with immune-mediated responses, such as microglial activation, white matter inflammation, and massive cellular infiltrates in the CNS.1,3 Infiltrated activated Correspondence to: Shahryar Eghtesadi, Department of Nutrition and Biochemistry, School of Public Health, Tehran University of Medical Sciences, Tehran 14155-6446, Iran. Email: [email protected]
© W. S. Maney & Son Ltd 2015 DOI 10.1179/1476830513Y.0000000106
leukocytes in MS patients express and secrete inflammatory cytokines, soluble factors, reactive oxygen species (ROS), and matrix metalloproteinases (MMPs) that can enhance blood–brain barrier (BBB) permeability, which leads to lesion development in MS.4 There is some evidence that levels of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNFα), interleukin (IL)-6, IL-1 and interferon (IFN)-γ, are elevated in the serum, cerebrospinal fluid (CSF), and in brain lesions of MS patients, although relatively low levels of IL-4 and transforming growth factor-β
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(TGF-β) are observed in these patients.5,6 Therefore, CD4+ T-cells of the T-helper type 1 (Th1) and macrophages are involved in the immunopathogenesis of demyelination, while T cells of the Th2 and Th3 phenotypes have a clear role in induction of remission and in suppression of the disease process. It is demonstrated that reversal of the immune response from Th1 to Th2/Th3 is a common therapeutic strategy both in experimental autoimmune encephalomyelitis (EAE) and in MS.5 Also, it seems that MMPs, particularly MMP-9, are essential for inflammatory cell infiltration to the CNS in MS. Several studies have demonstrated that MMP-9 levels increased in the serum and CSF of MS patients.1 Coenzyme Q10 (CoQ10) or ubiquinone is a potent endogenous lipid-soluble antioxidant, and as an obligatory coenzyme for mitochondrial enzyme complexes, regulates oxidative phosphorylation for the production of adenosine triphosphate.7,8 CoQ10 can be synthesized in the body and can be obtained from the diet; however, its total absorption is always inadequate to provide levels in body required to be useful in pathological conditions.9,10 One of the possible therapeutic mechanisms of action of CoQ10 is its ability to alter the immune response.10 CoQ10, as an antioxidant and free radical scavenger, prevents the activation of inflammatory signaling pathways.11 A study by Matthews et al. showed that maybe CoQ10 is able to cross through BBB in rats.12 In recent studies, it is noted that CoQ10 supplementation has influence on inflammatory markers in cell and animal models.9 In a study by Schmelzer et al., preincubation of human monocytic cell line THP-1 with ubiquinol-10 (QH2) decreased the lipopolysaccharide (LPS)-induced pro-inflammatory cytokines and chemokines.13 In another study by Fouad and Jresat, CoQ10 supplementation against acute liver injury induced by acetaminophen attenuated the production of TNF-α and nitric oxide (NO) and reduced the expression of NF-κB and iNOS in the liver of rats.14 In addition, recently CoQ10 is used as a neuroprotective and therapeutic agent in the treatment of different neurodegenerative disorders such as Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, and Friedreich’s ataxia.15 CoQ10 has a suppressive effect on MMPs activity/expression due to its interaction with ROS mediators.16 A study by Besler et al. showed that antioxidant vitamins are decreased in sera of MS patients, but it is also reported that serum CoQ10 levels and CoQ10/cholesterol ratio were normal.17,18 To date, few clinical studies have evaluated the anti-inflammatory role of CoQ10 in neurodegenerative disease. Therefore, the aim of the current study was to investigate the effect of CoQ10 supplementation (500 mg/day) on inflammatory and anti-inflammatory markers in patients with MS. In
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other part of this study, we reported that CoQ10 supplementation increased superoxide dismutase (SOD) activity and reduced malondialdehyde (MDA) levels in MS patients and this manuscript is an analysis of secondary endpoints.19 Also in other studies by our research group, we investigated anti-inflammatory and antioxidant effects of lipoic acid in MS patients.20,21
Methods Subjects This study was designed as a randomized, doubleblind, placebo-controlled clinical trial. The study was approved by the medical ethics committee of the Tehran University of Medical Sciences. All the patients signed a written informed consent to participate in this study. All patients were recruited from the MS clinic of Sina Hospital, Tehran. A total of 48 subjects (males = 4, females = 44), fulfilling the diagnostic criteria of McDonald et al. 22 for MS, were enrolled in this study. All patients had their diagnosis of MS confirmed by MRI and were clinically classified as to have the relapsing–remitting form of MS (RRMS) according to the criteria described in Lublin and Reingold.23 Subjects with a history of diabetes or other chronic disease, current smoker, disease duration of less than 1 year, or those who were on habitual antioxidant supplementation were excluded from the study. Patients under corticosteroid therapy or those who were in relapse phase of disease were also excluded. Participants were allowed to continue medications of MS disease and related symptoms. Table 1 presents baseline characteristics of the study subjects.
Study design Regarding the study of Singh et al. 24 that the standard deviation of MDA was reported 0.35 and significant difference was achieved with 0.65 mg/dl of MDA, we calculate the sample size considering the significant of MDA (0.3 mg/dl) with 80% of power of test, and the final sample size determined 24 patients in each group. There was not any power calculation for the outcomes reported in the current manuscript. Fortyeight subjects were randomly assigned to the following two groups by a pharmacist: a placebo group (n = 24) or a CoQ10-supplemented group (100 mg 5 times per day, i.e. 500 mg/day, n = 24). The study was blinded for physicians and technicians examining the blood. Among the patients, three women (2 in the CoQ10 group and 1 in the placebo group) were excluded, due to relapse occurrence during the study (Fig. 1). The CoQ10 (manufactured by International Agencies, Health Burst Co.) and placebo (starch) capsules were identical and commercially available preparations. The intervention was performed for 12
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Table 1 The basic characteristics of study subjects CoQ10 group (n = 22)
Parameters Mean age (years) Men/women (n) Weight (kg) BMI (kg/cm2) EDSS Mean disease duration (month) Dietary intake Energy (kcal/day) Carbohydrate (gr/day) Protein (gr/day) Fat (gr/day) Vitamin A (μg/day) Vitamin E (mg/day) Vitamin C (mg/day) MS drugs Betaferon (interferon beta-1b) CinnoVex** (interferon beta-1a) Resigen** (interferon beta-1a)
Placebo group (n = 23)
P
33.1 ± 7.6 2/20* 66.5 ± 14.8 24.4 ± 4 1.8 ± 1.2 52.8 ± 47
30.9 ± 7.7 2/21* 63.6 ± 14.2 23.4 ± 4.3 1.9 ± 1.1 60.9 ± 51
0.35 0.96 0.50 0.40 0.84 0.58
1 498 ± 556 183.9 ± 79 57.3 ± 26 62 ± 27 588.1 ± 990 34 ± 21 76.1 ± 82
1 609.7 ± 527 200.9 ± 80 60.5 ± 228 64.5 ± 23 428.6 ± 336 37 ± 18 76.9 ± 78
2*
3*
0.49 0.48 0.69 0.74 0.46 0.61 0.97 0.914 –
13*
13*
–
7*
7*
–
Values are means ± standard deviation. *Values are number. P values were obtained from independent t-tests. BMI, body mass index; EDSS, expanded disability status scale. **Generic names of Iranian brand of interferon drugs.
weeks. Each subject received the capsules at the 1st, 5th and 9th weeks from the time of study entry. To monitor compliance, all patients were controlled through phone call every week. At baseline and the final follow-up at week 12, all patients underwent clinical examination using Expanded Disability Status Scale (EDSS) by the same neurologist. The body height and weight of all subjects were measured and the body mass index (kilogram per squared meter) was calculated.
Sampling Fasting blood samples were obtained from all subjects at baseline and endpoint of intervention. All samples were collected in serum separation vacutainers and allowed to clot for 10 minutes. Serum was then collected by centrifugation at 3000 rpm for 10 minutes and quickly frozen and stored at −80°C until analyzed.
Serum levels of cytokines TNF-α, IL-6, IL-4, MMP-9, and transforming growth factor (TGF)-β serum levels were determined by commercial enzyme-linked immunosorbent assay (ELISA) kits (eBio Science, Bender MedSystem GMBH,
Figure 1 Patient flow diagram of the study. Forty-eight patients enrolled in this study. Three patients were dropped out due to attack (MS relapse). Therefore, final analysis was done on 45 patients.
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Vienna, Austria) according to the manufacturer’s instructions. All assays were carried out at the same time and in the same run by the same operator. The intra-assay coefficients of variation were less than 8.5% for all variables.
Statistical analysis Data were analyzed with SPSS version 16 (SPSS Inc., Chicago, IL, USA). Histogram and Kolmogrov– Smirnov tests were applied, to ensure the normal distribution of variables. Paired t-test was used to analyze the data within each group before and after the intervention. Student’s t-test was used to detect differences between groups. Continuous variables are shown as mean ± standard deviation (S.D.) and discrete variables as numbers and percentages. Results were considered statistically significant at P < 0.05.
Results Forty-five subjects with MS completed the study. Mean age (33.1 ± 7.6 vs. 30.9 ± 7.7 years), body weight (66.5 ± 14.8 vs. 63.6 ± 14.2 kg), and body mass index (24.4 ± 4 vs. 23.4 ± 4.3 kg/m2) showed no significant differences between the two groups. There were no significant differences among groups in mean disease duration and EDSS values at baseline. Dietary intake of energy, carbohydrate, protein, fat, vitamin A, vitamin E, and vitamin C was not significantly different between the two groups, and withingroup differences were also not significant (Table 1). The capsules were well tolerated by patients, but just mild stomach irritation was reported by two patients in the CoQ10 group throughout the study. The TNF-α levels in the CoQ10 group decreased significantly (P = 0.003) compared to the placebo group, and within-group differences in the CoQ10 group revealed a significant decrease of TNF-α levels (P = 0.006, Table 2, Fig. 2A). The IL-6 levels were significantly lower in the CoQ10 group compared to the placebo group (P = 0.037), and within-group differences in the CoQ10 group showed a significant reduction of serum IL-6 levels (−0.87 pg/ml, P = Table 2
0.03, Table 2, Fig. 2B). There was a significant decline in the serum MMP-9 levels in the CoQ10 group compared to the placebo (P = 0.011). The MMP-9 levels were significantly lower than baseline in the CoQ10 group at week 12 (88.1 ± 17 vs. 77.6 ± 18.3 ng/ml, P = 0.007, Table 2, Fig. 2E). CoQ10 supplementation for 12 weeks did not have significant effects on the serum levels of anti-inflammatory cytokines IL-4 and TGF-β (P = 0.16 and P = 0.81, respectively, Table 2, Fig. 2C and D).
Discussion A large body of evidence suggests that inflammation is an important contributor to axonal injury and neuronal cytotoxicity in MS in which levels of proinflammatory cytokines are increased, while antiinflammatory cytokines are decreased in CSF and serum of MS patients.5,6,25 The results of this study demonstrated that 12 weeks of CoQ10 supplementation at a dosage of 500 mg/day decreased the serum levels of pro-inflammatory cytokines IL6, TNF-α, and MMP-9 in MS patients. However, the serum levels of anti-inflammatory cytokines TGF-β and IL-4 were not affected after the CoQ10 supplementation. The anti-inflammatory effects of CoQ10 have been studied in cells by Schmelzer et al. who showed that CoQ10 decreases the release of TNF-α in monocytic cell lines (human monocytic cell line THP1 and murine monocytic cell line RAW 264.7).13 Our findings are in agreement with those reported by Fuller et al., who have shown that CoQ10 suppressed the increased levels of inflammatory mediators such as IL-6, prostaglandin E2, and MMP-1 in human dermal fibroblasts.11 Muzio et al. reported that the serum levels of TNF-α are significantly higher in patients with MS that may play an important role in the pathogenesis of MS.26 The expression of TNF-α mRNA correlates with fragmentation of DNA in oligodendrocytes within active demyelinating lesions.26 Increased level of TNF-α in CSF of MS patients is cytotoxic for oligodendrocytes
Baseline and end values of cytokine profiles of participants in CoQ10 and placebo groups CoQ10 group (n = 22)
Variables* TNF-α (pg/ml) IL-6 (pg/ml) TGF-β (ng/ml) IL-4 (pg/ml) MMP-9 (ng/ml) EDSS
Baseline†
End
6.2 ± 0.97 1.52 ± 2.4 2.2 ± 1.5 32.9 ± 75.4 88.1 ± 17.7 1.88 ± 1.2
0.87 ± 0.18 0.65 ± 1.5 2.2 ± 1.4 23.9 ± 54.6 77.6 ± 18.3 1.79 ± 1.2
Change Mean (95% CI) −0.39 (−0.12, −0.65) −0.87 (−0.06, −1.6) 0.13 (−0.71, 0.73) 9.01 (−3.1, 21) −10.3 (−3.1, −17.6) −0.09 ± 0.4
Placebo group (n = 23) P**
Baseline***
End
Change Mean (95% CI)
P**
P****
0.006 0.035 0.9 0.13 0.007 0.35
6.09 ± 0.92 1.53 ± 2.2 2.14 ± 1.44 31.39 ± 67.8 85.9 ± 16.6 1.95 ± 1.1
1.4 ± 2.1 1.4 ± 2.1 2.03 ± 1.2 30.4 ± 65.6 86.1 ± 17.7 1.95 ± 1.1
0.03 (0.1, 0.08) 0.05 (0.02, 0.13) 1.08 (3.7, 5.9) 0.97 (0.81, 2.7) 0.16 (3.5, 3.2) 0
0.53 0.17 0.64 0.27 0.9 –
0.003 0.037 0.81 0.16 0.011 0.34
*Values are means ± standard deviation. **P values were for test difference within groups. ***Baseline values of variables between groups did not have significant difference. ****P values were for test change between two groups.
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Figure 2 Changes in the concentration of different inflammatory and anti-inflammatory markers after a 12-week period of intervention. (A) TNF-α level in serum (pg/ml). (B) IL-6 level in serum (pg/ml). (C) TGF-β level in serum (pg/ml). (D) IL-4 level in serum (pg/ml). (E) MMP-9 level in serum (ng/ml). Points (circles and asterisks) outside the plots represent values outside the 2 (3) SD interval. TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; TGF-β, transforming growth factor-β; IL-4, interleukin-4; MMP-9, matrix metalloproteinase-9.
and seems to correlate with disease severity.27 In the present study, CoQ10 supplementation significantly decreased the serum levels of TNF-α in patients with MS (Fig. 1). The results are in agreement with those of Kumar et al. who reported that 12 weeks supplementation with 270 mg/day of Q10 along with oral carnitine significantly decreased TNF-α and IL6 in patients with heart failure.10 In this line, Bessler et al. showed that CoQ10 reduces the secretion of
TNF-α and IL-2 by human peripheral blood mononuclear cells due to its ability to modulate human immune function.28 In accordance with our results, Carmona et al. showed that i.p. injection of CoQ10 modulates the expression of TNF-α mRNA in adipose tissue in ob/ob mice.29 In another study by Fouad et al., CoQ10 treatment significantly decreased the elevation of TNF-α in mice with acute cisplatin nephrotoxicity.30
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Among inflammatory mediators, TNF-α and IL-6 play a crucial role in the inflammatory cascade as effector or regulatory molecules.31 Some studies demonstrated that IL-6 levels are elevated in the serum and CSF of MS patients. IL-6, in MS patients, modulates T-cell proliferation and differentiation and enhances VCAM-1 expression which enables T-cell infiltration into the nervous system.6 We observed that IL-6 concentration decreased significantly in the CoQ10 treated group, compared to the placebo group. In a similar study, Lee et al. have demonstrated that CoQ10 supplementation at a dosage of 150 mg/ day for 12 weeks decreases the inflammatory marker IL-6 in patients with coronary artery disease.9 In other study by Sohet et al., CoQ10 supplementation for 8 weeks in a mouse model of obesity tends to decrease hepatic mRNA expression of inflammatory markers IL-6 and TNF-α.32 Inui et al. reported that CoQ10 treatment attenuated UVB-induced production of IL-6 in normal human keratinocytes.33 In a study by Premkumar et al., co-administration of CoQ10 (100 mg/day) and B vitamins (riboflavin 10 mg/day and niacin 50 mg/day) for 90 days reduced the serum levels of cytokines, including IL6, IL-8, IL-1β and TNF-α, in patients with breast cancer.34 On the contrary, in a study by Gökbel et al., CoQ10 treatment at a dose of 100 mg/day for 8 weeks had no effect on inflammatory markers (IL6 and TNF-α) in healthy subjects. These results may be due to the fact that either healthy subjects do not have high levels of inflammation or the dose of the supplement was insufficient to exert an anti-inflammatory effect.35 Since there are few studies investigating anti-inflammatory effects of CoQ10, its exact molecular mechanisms remained unknown. However, it seems that the anti-inflammatory effect of CoQ10 can be attributed to either its free radical scavenging activity or its ability to inhibit the activation on NFκB signaling pathway.30 TGF-β is an anti-inflammatory cytokine with immune suppressive effects on T-cells which has a protective role in EAE.5 TGF-β, produced by T-cells of the Th2 phenotype, is able to suppress T-cells proliferation, cytotoxic lymphocytes maturation, and macrophage activity.36 The relative low levels and abnormalities of TGF-β production have been reported in MS.5,37 Moreover, previous studies have shown altered TGF-β signaling pathway and its decreased production in MS patients. These abnormalities have several implications in pathogenesis of MS. Decrease in TGF-β production and signaling pathway have a negative impact on immune regulation which contribute to CNS inflammation.37 Our results suggest that CoQ10 supplementation for 12 weeks (500 mg/day) do not have important impact on serum levels of TGF-β in MS patients. It is reported
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that interferon beta increases protein and mRNA levels of TGF-β in patients with MS.2 In a study by Losy et al., interferon beta significantly increased TGF-β production and normalized its level in RRMS patients after 6 months of therapy, but as inflammation arose, TGF-β levels declined again.38 While interferon therapeutic protocols reported a temporary enhancement in TGF-β levels, but CoQ10 supplementation did not affect this cytokine in MS patients, and it seems that CoQ10 may exert its antiinflammatory effects via inhibiting inflammatory cytokines, rather than increasing of anti-inflammatory cytokines. We did not find any significant effect of CoQ10 supplementation on serum level of IL-4 as compared to the placebo group. In contrast to our finding, in a study by Kandhare et al., administration of CoQ10 (50 and 100 mg/day) for 10 weeks in alcoholinduced neuropathic pain rats led to a significant reduction of the IL-4 level as compared to the control group.39 IL-4, produced by Th2 cells, inhibits the activation of Th1 cells and subsequently decreases the production of IL-1 and TNF-α.36 Studies have shown that IL-4 treatment in EAE leads to inhibition of the synthesis of inflammatory cytokines, induction of TGF-β-producing Th2/Th3 and diminished CNS demyelination.5 Hohnoki et al. have reported the elevation of serum IL-4 levels in MS.40 Moreover, in both acute and chronic active MS lesions, the highlevel expression of IL-4 was found.36 Furlan et al. found increased serum levels of IL-4 mRNA in MS patients during INF-β treatment.2 It seems that upregulation of IL-4 may reduce the severity of EAE, but in its absence, other Th2 cytokines may compensate for its function and therefore may inhibit any change in the course of MS disease.36 In the present study, despite no significant effect of CoQ10 on the serum level of IL-4 in MS patients, it seems that CoQ10 may affect other Th2 cytokines such as IL-5, IL-10, and IL-13. However, more extensive research is needed to confirm these findings. The current study showed that CoQ10 supplementation for 12 weeks resulted in a significant decline in serum MMP-9 levels, compared to the placebo group. In a similar study, consumption of Mediterranean diet supplemented with CoQ10 for 4 weeks in elderly subjects produced a lower postprandial MMP-9 and IL-1β gene expression, compared with control diets.41 Inui et al. showed that UVBinduced MMP-1 production of normal keratinocyte decreased in the presence of CoQ10.33 In another study, Bahar et al. reported that incubation of CoQ10 reduced MMP-2 activity in MCF-7 cell line as a breast cancer cellular model at a dose proportionate manner.16 Several studies have shown that serum levels of MMP-9 are significantly higher in MS
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patients, compared to control subjects. There is accumulating evidence that MMP-9 upregulation contributes to increased loss of BBB integrity and sustained proteolytic stress by MMP overproduction may be an important factor for the development of neural loss in MS patients.42 ROS, particularly H2O2, is considered as one of the major routes of MMP regulation and expression.16 Moreover, cytokines such as TNF-α and IL-6 modulate the regulation and expression of MMPs.42 CoQ10 is the most important antioxidant against H2O2 and also exerts a quelling role on the expression of cytokines and MMP mRNAs.16 Thus, the modulatory effect of CoQ10 on MMP-9 levels in this study might be due to its interaction with ROS mediators and its ability to decrease inflammatory cytokines in MS patients. Our study has several limitations. First, the intervention duration was not long enough to observe long-term effects of CoQ10 on anti-inflammatory cytokines. Furthermore, the number of participants was small. Second, due to budget limitations, we were unable to perform magnetic resonance imaging analysis at endpoint of study and we could not measure serum levels of CoQ10 in patients. Third, the clinical relevance of the findings is uncertain. The current study is an analysis of secondary outcomes from our study in MS patients.19 In conclusion, CoQ10 supplementation for 12 weeks in MS patients resulted in decreased serum levels of TNF-α, IL-6, and MMP-9 as compared to the placebo group, but did not affect TGF-β and IL-4 cytokines. Further studies need to elucidate the exact mechanisms of CoQ10 supplementation in MS patients and to confirm the findings of this study.
Acknowledgements None of the authors had any personal or financial conflict of interest. The present study was supported by a grant (No. 13295) from the Vice-Chancellor for Research, Tehran University of Medical Sciences, Iran. The authors would like to thank the staff of Sina Hospital (Tehran, Iran) for their assistance in this project.
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and neuroprotection in multiple sclerosis and other neurodegenerative diseases. J Neuroimmunol. 2006;176(1):198–215. Muzio L, Martino G, Furlan R. Multifaceted aspects of inflammation in multiple sclerosis: the role of microglia. J Neuroimmunol 2007;191(1):39–44. Silber E, Sharief MK. Axonal degeneration in the pathogenesis of multiple sclerosis. J Neurol Sci 1999;170(1):11–18. Bessler H, Bergman M, Blumberger N, Djaldetti M, Salman H. Coenzyme Q10 secreases TNF-ALPHA. and IL-2 secretion by human peripheral blood mononuclear cells. J Nutr Sci Vitaminol 2010;56(1):77–81. Carmona MC, Lefebvre P, Lefebvre B, Galinier A, Bénani A, Jeanson Y, et al. Coadministration of coenzyme Q prevents rosiglitazone-induced adipogenesis in ob/ob mice. Int J Obes 2009; 33(2):204–11. Fouad AA, Al-Sultan AI, Refaie SM, Yacoubi MT. Coenzyme Q10 treatment ameliorates acute cisplatin nephrotoxicity in mice. Toxicology 2010;274(1):49–56. Martino G, Furlan R, Brambilla E, Bergami A, Ruffini F, Gironi M, et al. Cytokines and immunity in multiple sclerosis: the dual signal hypothesis. J Neuroimmunol 2000; 109(1):3–9. Sohet FM, Neyrinck AM, Pachikian BD, de Backer FC, Bindels LB, Niklowitz P, et al. Coenzyme Q10 supplementation lowers hepatic oxidative stress and inflammation associated with dietinduced obesity in mice. Biochem Pharmacol 2009;78(11): 1391–400. Inui M, Ooe M, Fujii K, Matsunaka H, Yoshida M, Ichihashi M. Mechanisms of inhibitory effects of CoQ10 on UVBinduced wrinkle formation in vitro and in vivo. Biofactors 2008;32(1–4):237–43. Premkumar VG, Yuvaraj S, Sathish S, Shanthi P, Sachdanandam P. Anti-angiogenic potential of Coenzyme Q10, riboflavin and niacin in breast cancer patients
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undergoing tamoxifen therapy. Vasc Pharmacol 2008;48(4): 191–201. Gokbel H, Gergerlioglu HS, Okudan N, Gul I, Buyukbas S, Belviranli M. Effects of coenzyme Q10 supplementation on plasma adiponectin, interleukin-6, and tumor necrosis factoralpha levels in men. J Med Food 2010;13(1):216–18. Imitola J, Chitnis T, Khoury SJ. Cytokines in multiple sclerosis: from bench to bedside. Pharmacol Therapeutics 2005;106(2): 163–77. Meoli EM, Oh U, Grant CW, Jacobson S. TGF-beta signaling is altered in the peripheral blood of subjects with multiple sclerosis. J Neuroimmunol 2011;230(1–2):164–8. Losy J, Michałowska-Wender G. In vivo effect of interferon-β 1a on interleukin-12 and TGF-β1 cytokines in patients with relapsing–remitting multiple sclerosis. Acta Neurol Scand 2002; 106(1):44–6. Kandhare AD, Ghosh P, Ghule AE, Bodhankar SL. Elucidation of molecular mechanism involved in neuroprotective effect of Coenzyme Q10 in alcohol-induced neuropathic pain. Fundam Clin Pharmacol 2013;27(6):603–22. Hohnoki K, Inoue A, Koh CS. Elevated serum levels of IFNgamma, IL-4 and TNF-alpha/unelevated serum levels of IL-10 in patients with demyelinating diseases during the acute stage. J Neuroimmunol 1998;87(1–2):27–32. Yubero-Serrano EM, Gonzalez-Guardia L, Rangel-Zuniga O, Delgado-Lista J, Gutierrez-Mariscal FM, Perez-Martinez P, et al. Mediterranean diet supplemented with coenzyme Q10 modifies the expression of proinflammatory and endoplasmic reticulum stress-related genes in elderly men and women. J Gerontol A Biol Sci Med Sci 2012;67(1):3–10. Leppert D, Lindberg RL, Kappos L, Leib SL. Matrix metalloproteinases: multifunctional effectors of inflammation in multiple sclerosis and bacterial meningitis. Brain Res Brain Res Rev 2001;36(2–3):249–57.
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Department of Clinical Nutrition, School of Nutritional Sciences & Dietetics, Tehran University of Medical Sciences, Tehran, Iran, 2Department of Nutrition and Biochemistry, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran, 3Brain and Spinal Injury Research Center, Tehran University of Medical Sciences, Tehran, Iran, 4Neurosciences Research Center, Tabriz University of Medical Sciences, Tabriz, Iran, 5 Department of Nutrition Sciences and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran, 6Department of Biostatistics, Hospital Management Research Center, Tehran University of Medical Sciences, Tehran, Iran Objectives: Multiple sclerosis (MS) is an immune-mediated neurodegenerative disease of central nervous system and recent studies show that inflammatory processes are highly associated with neurodegeneration in the brain. The purpose of this study was to investigate the effect of coenzyme Q10 supplementation on inflammatory and anti-inflammatory markers in patients with MS. Methods: This randomized, double-blind, placebo-controlled clinical study was performed among 48 patients with relapsing–remitting MS. Subjects were randomly assigned to a placebo group (n = 24) or coenzyme Q10 (CoQ10)-supplemented group (500 mg/day, n = 24). The intervention was administered for 12 weeks. Peripheral blood samples were collected at baseline and after 12-week intervention, to measure inflammatory (tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and matrix metalloproteinase (MMP)-9) and anti-inflammatory (IL-4 and TGF-β) markers. Results: Forty-five patients completed the study. After 12 weeks of intervention, the TNF-α levels (P = 0.003) decreased significantly in the CoQ10 group. Subjects in the CoQ10 group had significantly lower IL-6 levels (P = 0.037), compared to the placebo group. CoQ10 supplementation also resulted in decreased serum levels of MMP-9 as compared to the placebo group (P = 0.011). However, CoQ10 supplementation did not alter the IL-4 and TGF-β levels (P = 0.16 and P = 0.81, respectively). Discussion: CoQ10 supplementation at a dosage of 500 mg appears to decrease the inflammatory markers (TNF-α, IL-6, and MMP-9) in patients with MS. Keywords: Coenzyme Q10, Cytokines, Inflammation, Multiple sclerosis, Supplementation, Antioxidants, TNF-α, IL-6, MMP-9, TGF-β, Randomized placebo-controlled trial
Introduction Multiple sclerosis (MS) is a chronic neurodegenerative T-cell-mediated inflammatory disease of the central nervous system (CNS), which leads to the myelin loss and axonal and neuronal damage.1,2 The relapsing–remitting phase of the disease is associated with immune-mediated responses, such as microglial activation, white matter inflammation, and massive cellular infiltrates in the CNS.1,3 Infiltrated activated Correspondence to: Shahryar Eghtesadi, Department of Nutrition and Biochemistry, School of Public Health, Tehran University of Medical Sciences, Tehran 14155-6446, Iran. Email: [email protected]
© W. S. Maney & Son Ltd 2015 DOI 10.1179/1476830513Y.0000000106
leukocytes in MS patients express and secrete inflammatory cytokines, soluble factors, reactive oxygen species (ROS), and matrix metalloproteinases (MMPs) that can enhance blood–brain barrier (BBB) permeability, which leads to lesion development in MS.4 There is some evidence that levels of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNFα), interleukin (IL)-6, IL-1 and interferon (IFN)-γ, are elevated in the serum, cerebrospinal fluid (CSF), and in brain lesions of MS patients, although relatively low levels of IL-4 and transforming growth factor-β
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(TGF-β) are observed in these patients.5,6 Therefore, CD4+ T-cells of the T-helper type 1 (Th1) and macrophages are involved in the immunopathogenesis of demyelination, while T cells of the Th2 and Th3 phenotypes have a clear role in induction of remission and in suppression of the disease process. It is demonstrated that reversal of the immune response from Th1 to Th2/Th3 is a common therapeutic strategy both in experimental autoimmune encephalomyelitis (EAE) and in MS.5 Also, it seems that MMPs, particularly MMP-9, are essential for inflammatory cell infiltration to the CNS in MS. Several studies have demonstrated that MMP-9 levels increased in the serum and CSF of MS patients.1 Coenzyme Q10 (CoQ10) or ubiquinone is a potent endogenous lipid-soluble antioxidant, and as an obligatory coenzyme for mitochondrial enzyme complexes, regulates oxidative phosphorylation for the production of adenosine triphosphate.7,8 CoQ10 can be synthesized in the body and can be obtained from the diet; however, its total absorption is always inadequate to provide levels in body required to be useful in pathological conditions.9,10 One of the possible therapeutic mechanisms of action of CoQ10 is its ability to alter the immune response.10 CoQ10, as an antioxidant and free radical scavenger, prevents the activation of inflammatory signaling pathways.11 A study by Matthews et al. showed that maybe CoQ10 is able to cross through BBB in rats.12 In recent studies, it is noted that CoQ10 supplementation has influence on inflammatory markers in cell and animal models.9 In a study by Schmelzer et al., preincubation of human monocytic cell line THP-1 with ubiquinol-10 (QH2) decreased the lipopolysaccharide (LPS)-induced pro-inflammatory cytokines and chemokines.13 In another study by Fouad and Jresat, CoQ10 supplementation against acute liver injury induced by acetaminophen attenuated the production of TNF-α and nitric oxide (NO) and reduced the expression of NF-κB and iNOS in the liver of rats.14 In addition, recently CoQ10 is used as a neuroprotective and therapeutic agent in the treatment of different neurodegenerative disorders such as Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, and Friedreich’s ataxia.15 CoQ10 has a suppressive effect on MMPs activity/expression due to its interaction with ROS mediators.16 A study by Besler et al. showed that antioxidant vitamins are decreased in sera of MS patients, but it is also reported that serum CoQ10 levels and CoQ10/cholesterol ratio were normal.17,18 To date, few clinical studies have evaluated the anti-inflammatory role of CoQ10 in neurodegenerative disease. Therefore, the aim of the current study was to investigate the effect of CoQ10 supplementation (500 mg/day) on inflammatory and anti-inflammatory markers in patients with MS. In
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other part of this study, we reported that CoQ10 supplementation increased superoxide dismutase (SOD) activity and reduced malondialdehyde (MDA) levels in MS patients and this manuscript is an analysis of secondary endpoints.19 Also in other studies by our research group, we investigated anti-inflammatory and antioxidant effects of lipoic acid in MS patients.20,21
Methods Subjects This study was designed as a randomized, doubleblind, placebo-controlled clinical trial. The study was approved by the medical ethics committee of the Tehran University of Medical Sciences. All the patients signed a written informed consent to participate in this study. All patients were recruited from the MS clinic of Sina Hospital, Tehran. A total of 48 subjects (males = 4, females = 44), fulfilling the diagnostic criteria of McDonald et al. 22 for MS, were enrolled in this study. All patients had their diagnosis of MS confirmed by MRI and were clinically classified as to have the relapsing–remitting form of MS (RRMS) according to the criteria described in Lublin and Reingold.23 Subjects with a history of diabetes or other chronic disease, current smoker, disease duration of less than 1 year, or those who were on habitual antioxidant supplementation were excluded from the study. Patients under corticosteroid therapy or those who were in relapse phase of disease were also excluded. Participants were allowed to continue medications of MS disease and related symptoms. Table 1 presents baseline characteristics of the study subjects.
Study design Regarding the study of Singh et al. 24 that the standard deviation of MDA was reported 0.35 and significant difference was achieved with 0.65 mg/dl of MDA, we calculate the sample size considering the significant of MDA (0.3 mg/dl) with 80% of power of test, and the final sample size determined 24 patients in each group. There was not any power calculation for the outcomes reported in the current manuscript. Fortyeight subjects were randomly assigned to the following two groups by a pharmacist: a placebo group (n = 24) or a CoQ10-supplemented group (100 mg 5 times per day, i.e. 500 mg/day, n = 24). The study was blinded for physicians and technicians examining the blood. Among the patients, three women (2 in the CoQ10 group and 1 in the placebo group) were excluded, due to relapse occurrence during the study (Fig. 1). The CoQ10 (manufactured by International Agencies, Health Burst Co.) and placebo (starch) capsules were identical and commercially available preparations. The intervention was performed for 12
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Table 1 The basic characteristics of study subjects CoQ10 group (n = 22)
Parameters Mean age (years) Men/women (n) Weight (kg) BMI (kg/cm2) EDSS Mean disease duration (month) Dietary intake Energy (kcal/day) Carbohydrate (gr/day) Protein (gr/day) Fat (gr/day) Vitamin A (μg/day) Vitamin E (mg/day) Vitamin C (mg/day) MS drugs Betaferon (interferon beta-1b) CinnoVex** (interferon beta-1a) Resigen** (interferon beta-1a)
Placebo group (n = 23)
P
33.1 ± 7.6 2/20* 66.5 ± 14.8 24.4 ± 4 1.8 ± 1.2 52.8 ± 47
30.9 ± 7.7 2/21* 63.6 ± 14.2 23.4 ± 4.3 1.9 ± 1.1 60.9 ± 51
0.35 0.96 0.50 0.40 0.84 0.58
1 498 ± 556 183.9 ± 79 57.3 ± 26 62 ± 27 588.1 ± 990 34 ± 21 76.1 ± 82
1 609.7 ± 527 200.9 ± 80 60.5 ± 228 64.5 ± 23 428.6 ± 336 37 ± 18 76.9 ± 78
2*
3*
0.49 0.48 0.69 0.74 0.46 0.61 0.97 0.914 –
13*
13*
–
7*
7*
–
Values are means ± standard deviation. *Values are number. P values were obtained from independent t-tests. BMI, body mass index; EDSS, expanded disability status scale. **Generic names of Iranian brand of interferon drugs.
weeks. Each subject received the capsules at the 1st, 5th and 9th weeks from the time of study entry. To monitor compliance, all patients were controlled through phone call every week. At baseline and the final follow-up at week 12, all patients underwent clinical examination using Expanded Disability Status Scale (EDSS) by the same neurologist. The body height and weight of all subjects were measured and the body mass index (kilogram per squared meter) was calculated.
Sampling Fasting blood samples were obtained from all subjects at baseline and endpoint of intervention. All samples were collected in serum separation vacutainers and allowed to clot for 10 minutes. Serum was then collected by centrifugation at 3000 rpm for 10 minutes and quickly frozen and stored at −80°C until analyzed.
Serum levels of cytokines TNF-α, IL-6, IL-4, MMP-9, and transforming growth factor (TGF)-β serum levels were determined by commercial enzyme-linked immunosorbent assay (ELISA) kits (eBio Science, Bender MedSystem GMBH,
Figure 1 Patient flow diagram of the study. Forty-eight patients enrolled in this study. Three patients were dropped out due to attack (MS relapse). Therefore, final analysis was done on 45 patients.
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Vienna, Austria) according to the manufacturer’s instructions. All assays were carried out at the same time and in the same run by the same operator. The intra-assay coefficients of variation were less than 8.5% for all variables.
Statistical analysis Data were analyzed with SPSS version 16 (SPSS Inc., Chicago, IL, USA). Histogram and Kolmogrov– Smirnov tests were applied, to ensure the normal distribution of variables. Paired t-test was used to analyze the data within each group before and after the intervention. Student’s t-test was used to detect differences between groups. Continuous variables are shown as mean ± standard deviation (S.D.) and discrete variables as numbers and percentages. Results were considered statistically significant at P < 0.05.
Results Forty-five subjects with MS completed the study. Mean age (33.1 ± 7.6 vs. 30.9 ± 7.7 years), body weight (66.5 ± 14.8 vs. 63.6 ± 14.2 kg), and body mass index (24.4 ± 4 vs. 23.4 ± 4.3 kg/m2) showed no significant differences between the two groups. There were no significant differences among groups in mean disease duration and EDSS values at baseline. Dietary intake of energy, carbohydrate, protein, fat, vitamin A, vitamin E, and vitamin C was not significantly different between the two groups, and withingroup differences were also not significant (Table 1). The capsules were well tolerated by patients, but just mild stomach irritation was reported by two patients in the CoQ10 group throughout the study. The TNF-α levels in the CoQ10 group decreased significantly (P = 0.003) compared to the placebo group, and within-group differences in the CoQ10 group revealed a significant decrease of TNF-α levels (P = 0.006, Table 2, Fig. 2A). The IL-6 levels were significantly lower in the CoQ10 group compared to the placebo group (P = 0.037), and within-group differences in the CoQ10 group showed a significant reduction of serum IL-6 levels (−0.87 pg/ml, P = Table 2
0.03, Table 2, Fig. 2B). There was a significant decline in the serum MMP-9 levels in the CoQ10 group compared to the placebo (P = 0.011). The MMP-9 levels were significantly lower than baseline in the CoQ10 group at week 12 (88.1 ± 17 vs. 77.6 ± 18.3 ng/ml, P = 0.007, Table 2, Fig. 2E). CoQ10 supplementation for 12 weeks did not have significant effects on the serum levels of anti-inflammatory cytokines IL-4 and TGF-β (P = 0.16 and P = 0.81, respectively, Table 2, Fig. 2C and D).
Discussion A large body of evidence suggests that inflammation is an important contributor to axonal injury and neuronal cytotoxicity in MS in which levels of proinflammatory cytokines are increased, while antiinflammatory cytokines are decreased in CSF and serum of MS patients.5,6,25 The results of this study demonstrated that 12 weeks of CoQ10 supplementation at a dosage of 500 mg/day decreased the serum levels of pro-inflammatory cytokines IL6, TNF-α, and MMP-9 in MS patients. However, the serum levels of anti-inflammatory cytokines TGF-β and IL-4 were not affected after the CoQ10 supplementation. The anti-inflammatory effects of CoQ10 have been studied in cells by Schmelzer et al. who showed that CoQ10 decreases the release of TNF-α in monocytic cell lines (human monocytic cell line THP1 and murine monocytic cell line RAW 264.7).13 Our findings are in agreement with those reported by Fuller et al., who have shown that CoQ10 suppressed the increased levels of inflammatory mediators such as IL-6, prostaglandin E2, and MMP-1 in human dermal fibroblasts.11 Muzio et al. reported that the serum levels of TNF-α are significantly higher in patients with MS that may play an important role in the pathogenesis of MS.26 The expression of TNF-α mRNA correlates with fragmentation of DNA in oligodendrocytes within active demyelinating lesions.26 Increased level of TNF-α in CSF of MS patients is cytotoxic for oligodendrocytes
Baseline and end values of cytokine profiles of participants in CoQ10 and placebo groups CoQ10 group (n = 22)
Variables* TNF-α (pg/ml) IL-6 (pg/ml) TGF-β (ng/ml) IL-4 (pg/ml) MMP-9 (ng/ml) EDSS
Baseline†
End
6.2 ± 0.97 1.52 ± 2.4 2.2 ± 1.5 32.9 ± 75.4 88.1 ± 17.7 1.88 ± 1.2
0.87 ± 0.18 0.65 ± 1.5 2.2 ± 1.4 23.9 ± 54.6 77.6 ± 18.3 1.79 ± 1.2
Change Mean (95% CI) −0.39 (−0.12, −0.65) −0.87 (−0.06, −1.6) 0.13 (−0.71, 0.73) 9.01 (−3.1, 21) −10.3 (−3.1, −17.6) −0.09 ± 0.4
Placebo group (n = 23) P**
Baseline***
End
Change Mean (95% CI)
P**
P****
0.006 0.035 0.9 0.13 0.007 0.35
6.09 ± 0.92 1.53 ± 2.2 2.14 ± 1.44 31.39 ± 67.8 85.9 ± 16.6 1.95 ± 1.1
1.4 ± 2.1 1.4 ± 2.1 2.03 ± 1.2 30.4 ± 65.6 86.1 ± 17.7 1.95 ± 1.1
0.03 (0.1, 0.08) 0.05 (0.02, 0.13) 1.08 (3.7, 5.9) 0.97 (0.81, 2.7) 0.16 (3.5, 3.2) 0
0.53 0.17 0.64 0.27 0.9 –
0.003 0.037 0.81 0.16 0.011 0.34
*Values are means ± standard deviation. **P values were for test difference within groups. ***Baseline values of variables between groups did not have significant difference. ****P values were for test change between two groups.
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Figure 2 Changes in the concentration of different inflammatory and anti-inflammatory markers after a 12-week period of intervention. (A) TNF-α level in serum (pg/ml). (B) IL-6 level in serum (pg/ml). (C) TGF-β level in serum (pg/ml). (D) IL-4 level in serum (pg/ml). (E) MMP-9 level in serum (ng/ml). Points (circles and asterisks) outside the plots represent values outside the 2 (3) SD interval. TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; TGF-β, transforming growth factor-β; IL-4, interleukin-4; MMP-9, matrix metalloproteinase-9.
and seems to correlate with disease severity.27 In the present study, CoQ10 supplementation significantly decreased the serum levels of TNF-α in patients with MS (Fig. 1). The results are in agreement with those of Kumar et al. who reported that 12 weeks supplementation with 270 mg/day of Q10 along with oral carnitine significantly decreased TNF-α and IL6 in patients with heart failure.10 In this line, Bessler et al. showed that CoQ10 reduces the secretion of
TNF-α and IL-2 by human peripheral blood mononuclear cells due to its ability to modulate human immune function.28 In accordance with our results, Carmona et al. showed that i.p. injection of CoQ10 modulates the expression of TNF-α mRNA in adipose tissue in ob/ob mice.29 In another study by Fouad et al., CoQ10 treatment significantly decreased the elevation of TNF-α in mice with acute cisplatin nephrotoxicity.30
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Among inflammatory mediators, TNF-α and IL-6 play a crucial role in the inflammatory cascade as effector or regulatory molecules.31 Some studies demonstrated that IL-6 levels are elevated in the serum and CSF of MS patients. IL-6, in MS patients, modulates T-cell proliferation and differentiation and enhances VCAM-1 expression which enables T-cell infiltration into the nervous system.6 We observed that IL-6 concentration decreased significantly in the CoQ10 treated group, compared to the placebo group. In a similar study, Lee et al. have demonstrated that CoQ10 supplementation at a dosage of 150 mg/ day for 12 weeks decreases the inflammatory marker IL-6 in patients with coronary artery disease.9 In other study by Sohet et al., CoQ10 supplementation for 8 weeks in a mouse model of obesity tends to decrease hepatic mRNA expression of inflammatory markers IL-6 and TNF-α.32 Inui et al. reported that CoQ10 treatment attenuated UVB-induced production of IL-6 in normal human keratinocytes.33 In a study by Premkumar et al., co-administration of CoQ10 (100 mg/day) and B vitamins (riboflavin 10 mg/day and niacin 50 mg/day) for 90 days reduced the serum levels of cytokines, including IL6, IL-8, IL-1β and TNF-α, in patients with breast cancer.34 On the contrary, in a study by Gökbel et al., CoQ10 treatment at a dose of 100 mg/day for 8 weeks had no effect on inflammatory markers (IL6 and TNF-α) in healthy subjects. These results may be due to the fact that either healthy subjects do not have high levels of inflammation or the dose of the supplement was insufficient to exert an anti-inflammatory effect.35 Since there are few studies investigating anti-inflammatory effects of CoQ10, its exact molecular mechanisms remained unknown. However, it seems that the anti-inflammatory effect of CoQ10 can be attributed to either its free radical scavenging activity or its ability to inhibit the activation on NFκB signaling pathway.30 TGF-β is an anti-inflammatory cytokine with immune suppressive effects on T-cells which has a protective role in EAE.5 TGF-β, produced by T-cells of the Th2 phenotype, is able to suppress T-cells proliferation, cytotoxic lymphocytes maturation, and macrophage activity.36 The relative low levels and abnormalities of TGF-β production have been reported in MS.5,37 Moreover, previous studies have shown altered TGF-β signaling pathway and its decreased production in MS patients. These abnormalities have several implications in pathogenesis of MS. Decrease in TGF-β production and signaling pathway have a negative impact on immune regulation which contribute to CNS inflammation.37 Our results suggest that CoQ10 supplementation for 12 weeks (500 mg/day) do not have important impact on serum levels of TGF-β in MS patients. It is reported
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that interferon beta increases protein and mRNA levels of TGF-β in patients with MS.2 In a study by Losy et al., interferon beta significantly increased TGF-β production and normalized its level in RRMS patients after 6 months of therapy, but as inflammation arose, TGF-β levels declined again.38 While interferon therapeutic protocols reported a temporary enhancement in TGF-β levels, but CoQ10 supplementation did not affect this cytokine in MS patients, and it seems that CoQ10 may exert its antiinflammatory effects via inhibiting inflammatory cytokines, rather than increasing of anti-inflammatory cytokines. We did not find any significant effect of CoQ10 supplementation on serum level of IL-4 as compared to the placebo group. In contrast to our finding, in a study by Kandhare et al., administration of CoQ10 (50 and 100 mg/day) for 10 weeks in alcoholinduced neuropathic pain rats led to a significant reduction of the IL-4 level as compared to the control group.39 IL-4, produced by Th2 cells, inhibits the activation of Th1 cells and subsequently decreases the production of IL-1 and TNF-α.36 Studies have shown that IL-4 treatment in EAE leads to inhibition of the synthesis of inflammatory cytokines, induction of TGF-β-producing Th2/Th3 and diminished CNS demyelination.5 Hohnoki et al. have reported the elevation of serum IL-4 levels in MS.40 Moreover, in both acute and chronic active MS lesions, the highlevel expression of IL-4 was found.36 Furlan et al. found increased serum levels of IL-4 mRNA in MS patients during INF-β treatment.2 It seems that upregulation of IL-4 may reduce the severity of EAE, but in its absence, other Th2 cytokines may compensate for its function and therefore may inhibit any change in the course of MS disease.36 In the present study, despite no significant effect of CoQ10 on the serum level of IL-4 in MS patients, it seems that CoQ10 may affect other Th2 cytokines such as IL-5, IL-10, and IL-13. However, more extensive research is needed to confirm these findings. The current study showed that CoQ10 supplementation for 12 weeks resulted in a significant decline in serum MMP-9 levels, compared to the placebo group. In a similar study, consumption of Mediterranean diet supplemented with CoQ10 for 4 weeks in elderly subjects produced a lower postprandial MMP-9 and IL-1β gene expression, compared with control diets.41 Inui et al. showed that UVBinduced MMP-1 production of normal keratinocyte decreased in the presence of CoQ10.33 In another study, Bahar et al. reported that incubation of CoQ10 reduced MMP-2 activity in MCF-7 cell line as a breast cancer cellular model at a dose proportionate manner.16 Several studies have shown that serum levels of MMP-9 are significantly higher in MS
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patients, compared to control subjects. There is accumulating evidence that MMP-9 upregulation contributes to increased loss of BBB integrity and sustained proteolytic stress by MMP overproduction may be an important factor for the development of neural loss in MS patients.42 ROS, particularly H2O2, is considered as one of the major routes of MMP regulation and expression.16 Moreover, cytokines such as TNF-α and IL-6 modulate the regulation and expression of MMPs.42 CoQ10 is the most important antioxidant against H2O2 and also exerts a quelling role on the expression of cytokines and MMP mRNAs.16 Thus, the modulatory effect of CoQ10 on MMP-9 levels in this study might be due to its interaction with ROS mediators and its ability to decrease inflammatory cytokines in MS patients. Our study has several limitations. First, the intervention duration was not long enough to observe long-term effects of CoQ10 on anti-inflammatory cytokines. Furthermore, the number of participants was small. Second, due to budget limitations, we were unable to perform magnetic resonance imaging analysis at endpoint of study and we could not measure serum levels of CoQ10 in patients. Third, the clinical relevance of the findings is uncertain. The current study is an analysis of secondary outcomes from our study in MS patients.19 In conclusion, CoQ10 supplementation for 12 weeks in MS patients resulted in decreased serum levels of TNF-α, IL-6, and MMP-9 as compared to the placebo group, but did not affect TGF-β and IL-4 cytokines. Further studies need to elucidate the exact mechanisms of CoQ10 supplementation in MS patients and to confirm the findings of this study.
Acknowledgements None of the authors had any personal or financial conflict of interest. The present study was supported by a grant (No. 13295) from the Vice-Chancellor for Research, Tehran University of Medical Sciences, Iran. The authors would like to thank the staff of Sina Hospital (Tehran, Iran) for their assistance in this project.
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