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Neuroprotective Effects of a Novel Antioxidant Mixture Twendee X in Mouse Stroke Model
ARTICLE IN PRESS
Neuroprotective Effects of a Novel Antioxidant Mixture Twendee X in Mouse Stroke Model Momoko Kusaki, BSc,* Yasuyuki Ohta, MD, PhD,* Haruhiko Inufusa, MD, PhD,† Toru Yamashita, MD, PhD,* Ryuta Morihara, MD, PhD,* Yumiko Nakano, MD, PhD,* Xia Liu, MD,* Jingwei Shang, MD, PhD,* Feng Tian, BSc,* Yusuke Fukui, MSc,* Kota Sato, MD, PhD,* Mami Takemoto, MD, PhD,* Nozomi Hishikawa, MD, PhD,* and Koji Abe, MD, PhD*
Background: Oxidative stress and inflammation are important aggravating factors in acute ischemic stroke. Methods: In the present study, the neuroprotective effects of a novel antioxidant mixture Twendee X containing multiple antioxidative ingredients, such as coenzyme Q10, ascorbic acid, and cystine, were evaluated. After the pretreatment of a vehicle or Twendee X (20 mg/kg/d) for 14 days, mice were subjected to transient middle cerebral artery occlusion for 60 minutes and further treated with vehicle or Twendee X for 1 or 5 days. Results: Twendee X administration reduced the infarct size, and reduced oxidative stress markers such as 8-hydroxy-2′-deoxyguanosine, 4-hydroxy-2-nonenal, and Nε-(carboxymethyl) lysine (one of advanced glycation end products), as well as inflammatory markers such as ionized calcium binding adapter molecule-1, tumor necrosis factor-α, and monocyte chemotactic protein-1. Conclusions: In the present study, the neuroprotective effects of Twendee X were shown on transient middle cerebral artery occlusion mice via antioxidative and anti-inflammatory pathways, providing a potential of Twendee X as one preventive and therapeutic treatment. Key Words: Ischemic stroke—middle cerebral artery occlusion—mouse—antioxidative—anti-inflammatory. © 2017 National Stroke Association. Published by Elsevier Inc. All rights reserved.
From the *Department of Neurology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan; and †Division of Anti-Oxidant Research, Life Science Research Center, Gifu University, Gifu, Japan. Received December 12, 2016; revision received December 29, 2016; accepted January 7, 2017. Grant support: This work was partly supported by Grants-inAid for Scientific Research (B, 25293202 and C, 15K09316), Challenging Research (15K15527), and Young Research (15K21181), and by Grantsin-Aid from the Research Committees (Mizusawa H, Nakashima K, Nishizawa M, Sasaki H, and Aoki M) from the Ministry of Health, Labour and Welfare, Japan. Address correspondence to Koji Abe, MD, PhD, Department of Neurology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan. E-mail: [email protected]. 1052-3057/$ - see front matter © 2017 National Stroke Association. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2017.01.003
Introduction Ischemic stroke is a leading cause of mortality and neurologic impairments worldwide.1 Therapeutic strategies against stroke remain limited, and further novel therapies are required in daily clinical practice.2 Oxidative stress and inflammation are important aggravating factors in acute ischemic stroke.3,4 Reactive oxygen species (ROS) is gradually generated during cerebral ischemia, then excessively increased after reperfusion notably in the periischemic area with oxidation of cellular DNA, lipids, and proteins,5-8 and then often focused as a medical treatable target.9 We originally showed the strong neuroprotective effects of a free radical scavenger edaravone.8,10,11 Furthermore, we reported that dietary supplements such as ginkgo extract,12 platinum nanoparticle species,13 and antioxidative
Journal of Stroke and Cerebrovascular Diseases, Vol. ■■, No. ■■ (■■), 2017: pp ■■–■■
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nutrient-rich enteral diet showed neuroprotective effects on the ischemic brains of mice. Dietary supplements did not show the strong effects on diseases as medicinal chemicals. However, they are safe and valuable for the prevention and treatment of various diseases.15 Twendee X (TwX) is an anti-aging supplement containing multiple antioxidants and a patented composition.16 TwX has strong antioxidant effects, and increases superoxide dismutase and cell protection effects.17 In the present study, we validated whether TwX could be helpful in ameliorating mouse brain damages and oxidative stress following experimental transient middle cerebral artery occlusion (tMCAO).
Materials and Methods
Figure 1. The 4 experimental mice groups showing V-1d group (saline, sacrificed 1 day after tMCAO), TwX-1d group (TwX, sacrificed 1 day after tMCAO), V-5d group (saline, sacrificed 5 days after tMCAO), and TwX5d group (TwX, sacrificed 5 days after tMCAO). Filled black arrows indicate intraperitoneal injection of saline or TwX. Open white triangles indicate blood sampling. Abbreviation: tMCAO, transient middle cerebral artery occlusion.
Animals and Focal Cerebral Ischemia All experimental procedures were approved by the Animal Committee of the Okayama University Graduate School of Medicine (OKU-2015573). Adult male C57BL/ 6JJcl mice (23-27 g, 8 weeks old) were obtained from CLEA Japan (Tokyo, Japan). The mice were maintained in a temperature-regulated room (23-25°C) on a 12-hour light– dark cycle and allowed free access to food and water. From 9 weeks of age, the mice received vehicle (physiological saline, ip, n = 15) or TwX (20 mg/kg per day, ip, n = 16) for 14 days, and then were subjected to tMCAO for 60 minutes. TwX is a mixture consisting of coenzyme Q10 (3.6 wt%; AQUA Q10 P40-NF, Nissin Pharmaceutical, Tokyo, Japan), niacin amid (.7 wt%), L-cystine (18.2 wt%), ascorbic acid (34.2 wt%), succinic acid (3.6 wt%), fumaric acid (3.6 wt%), L-glutamine (34.6 wt%), and riboflavin (1.5 wt%; Bislase inj; Toa Eiyo, Tokyo, Japan). The TwX mixture was dissolved in saline (Otsuka Pharmaceutical Factory, Tokushima, Japan) and stored at 4°C until use. The TwX solution (20 mg/kg) was intraperitoneally injected to the mice with 500 μL volume. After 14 days of intraperitoneal administration of the vehicle or TwX (11 weeks old), the mice were anesthetized with a mixture of nitrous oxide : oxygen : isoflurane (69%:30%:1%) during surgery with an inhalation mask, and tMCAO was induced using the intraluminal filament technique.18 Body temperature was monitored and maintained at 37 ± 0.3°C by placing the animals on a heating pad (BWT-100; Bio Research Center, Aichi, Japan). After the right common carotid artery was exposed, a 7-0 nylon thread with a silicon-coated tip was inserted into the right middle cerebral artery. After 60 minutes of tMCAO, the siliconcoated thread was pulled out to restore blood flow of MCA. After this tMCAO, each mice group (vehicle or TwX) was further treated with the same vehicle or TwX for 1 (n = 7 each) or 5 (n = 8 or 9) days. The mice received the injection of vehicle or TwX once a day over a period of 1 day (V-1d, TwX-1d) or 5 days (V-5d, TwX-5d) (Fig 1). One day or 5 days after tMCAO, blood was collected from the mice via the retro-orbital puncture 12 hours after
the final administration. For histologic analysis, the mice were then deeply anesthetized by intraperitoneal injection of pentobarbital (40 mg/kg), and then transcardially perfused with chilled phosphate-buffered saline (PBS, pH 7.4), followed by 4% paraformaldehyde in PBS. The whole brain was removed and immersed in the same fixative solution overnight at 4°C. Serum samples were separated by centrifugation (1940 g, 15 minutes, 4°C) and stored at −80°C. After washing with PBS, the fixed brains were incubated in 20% (wt/vol) sucrose in PBS for 24 hours at 4°C. The tissues were frozen in liquid nitrogen and stored at −80°C. Coronal brain section (10 μm thickness) was cut on a cryostat at −20°C and mounted on silane-coated glass slides.
Serum Measurement of Reactive Oxygen Metabolite Levels and Antioxidant Capacity The ROS was examined by d-ROMs test (Diacron International, Italy). The reactive oxygen metabolite levels are expressed as arbitrary “Carratelli units” (CARR U), with 1 CARR U corresponding to .08 mg per 100 mL of H2O2. The total serum antioxidant capacity was assayed by OXY-Adsorbent test (Diacron International, Grosseto, Italy). The d-ROMs test and OXY-Adsorbent test were performed using a spectrophotometer.19
Histology and Immunohistochemistry For quantification of infarct volume, the brain sections (10 μm) were stained with cresyl violet as Nissl staining and examined by microscopy (SZX-12; Olympus Optical, Tokyo, Japan). The brain sections were prepared at a .8-mm interval each, between .8 mm anterior and 2.4 mm posterior to the bregma. The infarct volumes were measured in the 5 sections by pixel counting using a computer program for Photoshop CC (Adobe, San Jose, CA, USA) and then calculated by summation of the 5 serial infarct areas. For immunohistochemistry of Nε-(carboxymethyl) lysine (CML), ionized calcium-binding adapter molecule-1 (Iba-
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1), tumor necrosis factor-α (TNFα), and monocyte chemotactic protein-1 (MCP-1), the frozen sections (10 μm) were incubated in .3% H2O2/methanol for 20 minutes to block endogenous peroxidase activity and to block in 5% bovine serum albumin for 1 hour. The sections were incubated at 4°C overnight with the following primary antibodies: mouse anti-CML antibody (1:100; AGE-M01, Cosmo Bio, Tokyo, Japan); rabbit anti-Iba-1 antibody (1:1000; #019-19741, Wako, Osaka, Japan); rabbit anti-TNFα antibody (1:200; ab6671, Abcam, Cambridge, UK); and rabbit anti-MCP-1 antibody (1:100; ab7202, Abcam). The sections were then washed in PBS and incubated with biotinylated anti-mouse or anti-rabbit IgG secondary antibodies (Vector Laboratories, Burlingame, CA, USA) diluted at 1:500 for 2.5 hours at room temperature. The sections were then incubated with the avidin–biotin–peroxidase complex (VECTASTAIN Elite ABC Kit; Vector Laboratories) for 30 minutes and visualized with 3,3′diaminobenzidine (DAB). Immunohistochemistry for 8-hydroxy-2′-deoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (4-HNE), and sirtuin-1 (Sirt-1) was performed with mouse on mouse (M.O.M) immunodetection kit (Vector Laboratories) according to the manufacturer’s directions. Briefly, the frozen sections were incubated in .3% H2O2/methanol and then blocked in a mouse IgG blocking solution. The sections were incubated at 4°C with the following primary antibodies: mouse anti-8-OHdG (5 μg/mL; MOG-100P, JaICA, Shizuoka, Japan); mouse anti-4-HNE (25 μg/mL; MHN100P, JaICA); and mouse anti-Sirt-1 (1:500; ab50517, Abcam). Immunoreactivity was visualized by a biotinylated secondary antibody, avidin–biotin–peroxidase complex, and DAB. In immunostaining for all markers in this study, a set of sections was also stained in a similar way but without the primary antibody, and served as the negative control.
Statistical Analysis Data are expressed as mean ± standard deviation. Statistical analysis was performed using a one-way analysis of variance, followed by a Tukey’s honest significant difference test. In all statistical analyses, significance was accepted at P < .05.
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Figure 2. Serum levels of reactive oxygen metabolites (d-ROMs) and antioxidant capacity (OXY-Adsorbent) showing reducing tendencies of Twendee X on oxidative stress 5 days after transient middle cerebral artery occlusion, but not significant.
319.0 ± 52.4 μmol HClO/mL; TwX-1d, 294.0 ± 33.4 μmol HClO/mL) and 5 days after (V-5d, 337.5 ± 51.3 μmol HClO/ mL; TwX-5d, 292.5 ± 39.1 μmol HClO/mL), but again not significant (Fig 2).
Brain Infarct Volume after tMCAO One day after tMCAO, TwX did not show a difference in the brain infarct volume (V-1d = 23.9 ± 2.8 mm3 versus TwX-1d = 23.2 ± 7.1 mm3), but remarkably reduced the infarct volume (18.0 ± 5.6 mm3, P < .05 versus vehicle) on day 5 compared with the vehicle group (28.0 ± 7.1 mm3, Fig 3).
Oxidative Stress in the Mice Brain
Results Serum Oxidative Stress and the Antioxidative Activities As compared with the vehicle (V-1d, 165 ± 21.3 CARR U), TwX did not reduce oxidative stress (d-ROMs) 1 day after tMCAO (TwX-1d, 164 ± 10.3 CARR U), but showed a slight reduction 5 days after (V-5d = 167.5 ± 27.8 versus TwX-5d = 149.5 ± 14.4 CARR U, not significant) (Fig 2). On the other hand, OXY-Adsorbent showed a reduction tendency in TwX both 1 day after tMCAO (V-1d,
Compared with the vehicle, TwX did not clearly reduce the number of positive cells for 3 oxidative stress markers of 8-OHdG (176.6 ± 24.4 versus 140.6 ± 29.9/mm2), 4-HNE (199.0 ± 12.8 versus 188.8 ± 25.9/mm 2 ), and CML (162.4 ± 12.2 versus 163.0 ± 12.2/mm2) in the mice brain 1 day after tMCAO (Fig 4). However, TwX significantly reduced the number of positive cells for all 3 markers in 8-OHdG (200.2 ± 24.4 versus 117.7 ± 6.2/mm2, P < .01), 4-HNE (227.5 ± 21.0 versus 160.1 ± 7.9/mm2, P < .01), and CML (169.2 ± 6.4 versus 140.8 ± 5.5/mm2, P < .05) 5 days after tMCAO compared with the vehicle (Fig 4).
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Figure 3. Cresyl violet stainings of coronal sections (left) and brain infarct volumes (right). Note the significant reduction of infarct volumes in TwX-5d compared with V-5d group (*P < .05).
Inflammation in the Mice Brain Compared with the vehicle (60.8 ± 7.1/mm2), TwX clearly reduced the number of ionized calcium binding adapter molecule-1 (Iba1) positive cells (49.2 ± 5.7/mm2, P < .05 versus vehicle) even 1 day after tMCAO (Fig 5). Five days after tMCAO, the number of Iba1 positive cells (99.6 ± 11.2/ mm2) increased compared with 1 day in the vehicle group (60.8 ± 7.1/mm2, P < .05 versus 1 day), which was remarkably ameliorated (50.3 ± 8.0/mm2) by TwX (P < .01 versus vehicle). On the other hand, compared with the vehicle (TNFα = 172.8 ± 11.8/mm2, MCP-1 = 184.2 ± 18.1/mm2, and Sirt-1 = 185.0 ± 17.4/mm2), TwX did not clearly reduce the number of positive cells for TNFα, MCP-1, and Sirt-1 (161.2 ± 15.2/mm2, 167.2 ± 15.1/mm2, and 166.2 ± 17.4/ mm2, respectively) in the mice brain 1 day after tMCAO (Fig 5). However, TwX remarkably reduced the number of positive cells for TNFα (153.7 ± 8.7/mm2) and MCP-1
(147.3 ± 8.5/mm2) 5 days after tMCAO compared with the vehicle (TNFα = 193.0 ± 13.1/mm2, MCP-1 = 207.3 ± 20.6/ mm2, P < .01 versus vehicle for TNFα, P < .05 versus vehicle for MCP-1) except for Sirt-1 (V-5d = 227.0 ± 14.2/mm2, TwX5d = 194.1 ± 26.8/mm2) (Fig 5).
Discussion The present study demonstrated that a novel antioxidant mixture TwX showed neuroprotective effects on mice cerebral ischemia by reducing the infarct size (Fig 3), and reducing both oxidative stress markers (Fig 4) and inflammatory markers (Fig 5). ROS is incrementally generated during cerebral ischemia, then explosively increased after reperfusion particularly in the penumbra with oxidation of cellular DNA, lipids, and proteins.5-8 Such oxidative stress is detrimental in the cerebral infarction.3 Our previous studies showed the marked neuroprotective effects
Figure 4. Immunohistochemistry for 8-OHdG, 4-HNE, and CML at the peri-infarct area (left) and quantitative analyses of positive cells (right). Note the significant reductions of positive cells for 3 markers in TwX-5d compared with V-5d group (*P < .05, **P < .01). Abbreviations: 4-HNE, 4-hydroxy2-nonenal; 8-OHdG, 8-hydroxy-2′-deoxyguanosine; CML, Nε-(carboxymethyl) lysine.
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Figure 5. Immunohistochemistry for Iba-1, TNFα, MCP-1, and Sirt-1 at the peri-infarct area (left) and quantitative analyses of positive cells (right). The number of Iba1 positive cells increased on day 5 (V-5d group) than on day 1 (V-1d group, #P < .05), and the number of Iba-1, TNFα, and MCP-1 positive cells significantly reduced in TwX-5d than V-5d (*P < .05, **P < .01). Abbreviations: Iba1, ionized calcium binding adapter molecule-1; TNFα, tumor necrosis factor-α; MCP-1, monocyte chemotactic protein-1; Sirt-1, sirtuin-1.
of a free radical scavenger edaravone.8,10,11 Other free radical scavengers, ginkgo extract,12,20 platinum nanoparticle species,13 ferulic acid,21 and antioxidative nutrient-rich enteral diet14 also showed neuroprotective effects on the ischemic brains of mice. With the present d-ROMs and OXY-Adsorbent tests, oxidative stress and the antioxidative activities can be easily examined with a small amount of serum samples (25 μL for each mouse). In previous studies, these methods were applied in patients with various diseases, such as acute stroke,22 acute myocardial infarction,23 and Alzheimer’s disease.24 The d-ROMs test was also evaluated in experimental animal models such as tMCAO models25,26 and UCP-1 knockout mice.27 The present data suggested a reduced tendency of serum ROS and reactive oxygen metabolites with TwX (Fig 2). Brain sections showed a reduction of oxidative stress in DNA (8-OHdG), lipid (4-HNE), and advanced glycation end products (CML) (Fig 4). Since TwX is a mixture containing multiple antioxidants such as coenzyme Q10, ascorbic acid, and cystine, it reduced oxidative stress in the present ischemic models (Fig 4). A previous study showed an amelioration of oxidative stress by ascorbic acid in both tMCAO and diabetic models.25 Moreover, TwX significantly reduced oxidative stress in the dose of one-third of ascorbic acid in irradiation mouse model, suggesting synergistic effects by multiple antioxidants.16 Coenzyme Q10 was effective for the treatment of neurologic diseases by remedying mitochondrial dysfunction and oxidative damages.28 A cystine-based antioxidant at-
tenuated oxidative stress in obese mice.29 Inflammation is another pathologic key factor in the ischemic stroke.4 Inflammatory cytokines such as MCP-1 and TNFα activate microglia with Iba-1 potentiation, and finally exert neurotoxic effects.30,31 TwX ameliorated such inflammatory responses especially 5 days after tMCAO (Fig 5). In summary, we presented the neuroprotective effects of TwX on tMCAO mice in reducing infarct size and both oxidative stress and inflammatory markers (Figs 3-5). The present study provides a potential of TwX as one preventive and therapeutic treatment against ischemic stroke. Acknowledgments: We are grateful to Dr. Maruyama T of the Department of Preventive Dentistry at Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, especially for their technical assistance in d-ROMs tests and OXY-Adsorbent tests.
References 1. Lopez AD, Mathers CD, Ezzati M, et al. Global and regional burden of disease and risk factors, 2001: systematic analysis of population health data. Lancet 2006;367:1747-1757. 2. Lakhan SE, Kirchgessner A, Hofer M. Inflammatory mechanisms in ischemic stroke: therapeutic approaches. J Transl Med 2009;7:97. 3. Abe K, Aoki M, Kawagoe J, et al. Ischemic delayed neuronal death. Stroke 1995;26:1478-1489. 4. Dirnagl U, Iadecola C, Moskowitz MA. Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci 1999;22:391-397.
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6 5. Chan PH. Role of oxidants in ischemic brain damage. Stroke 1996;27:1124-1129. 6. Morimoto T, Globus MY, Busto R, et al. Simultaneous measurement of salicylate hydroxylation and glutamate release in the penumbral cortex following transient middle cerebral artery occlusion in rats. J Cereb Blood Flow Metab 1996;16:92-99. 7. Abe K. Therapeutic potential of neurotrophic factors and neural stem cells against ischemic brain injury. J Cereb Blood Flow Metab 2000;20:1393-1408. 8. Zhang W, Sato K, Hayashi T, et al. Extension of ischemic therapeutic time window by a free radical scavenger, Edaravone, reperfused with tPA in rat brain. Neurol Res 2004;26:342-348. 9. Peters O, Back T, Lindauer U, et al. Increased formation of reactive oxygen species after permanent and reversible middle cerebral artery occlusion in the rat. J Cereb Blood Flow Metab 1998;18:196-205. 10. Abe K, Yuki S, Kogure K. Strong attenuation of ischemic and postischemic brain edema in rats by a novel free radical scavenger. Stroke 1988;19:1478-1489. 11. Yamashita T, Kamiya T, Deguchi K, et al. Dissociation and protection of the neurovascular unit after thrombolysis and reperfusion in ischemic rat brain. J Cereb Blood Flow Metab 2009;29:715-725. 12. Zhang WR, Hayashi T, Kitagawa H, et al. Protective effect of ginkgo extract on rat brain with transient middle cerebral artery occlusion. Neurol Res 2000;22:517-521. 13. Takamiya M, Miyamoto Y, Yamashita T, et al. Strong neuroprotection with a novel platinum nanoparticle against ischemic stroke- and tissue plasminogen activatorrelated brain damages in mice. Neuroscience 2012;221:4755. 14. Yunoki T, Deguchi K, Omote Y, et al. Anti-oxidative nutrient rich diet protects against acute ischemic brain damage in rats. Brain Res 1587;2014:33-39. 15. Janson M. Orthomolecular medicine: the therapeutic use of dietary supplements for anti-aging. Clin Interv Aging 2006;1:261-265. 16. Inufusa H. Composition for protection against celldamaging effects, US Patent: 9089548 B2. issued date July 28th, 2015. 17. Inufusa H. Characterization of cell protection effects of Twendee X by oxidative stress. J World Mitochondria Soc 2016;2:42. 18. Yamashita T, Ninomiya M, Hernández Acosta P, et al. Subventricular zone-derived neuroblasts migrate and
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differentiate into mature neurons in the post-stroke adult striatum. J Neurosci 2006;26:6627-6636. Tamaki N, Tomofuji T, Ekuni D, et al. Periodontal treatment decreases plasma oxidized LDL level and oxidative stress. Clin Oral Investig 2011;15:953-958. Nada SE, Shah ZA. Preconditioning with Ginkgo biloba (EGb 761®) provides neuroprotection through HO1 and CRMP2. Neurobiol Dis 2012;46:180-189. Cheng C-Y, Su S-Y, Tang N-Y, et al. Ferulic acid provides neuroprotection against oxidative stress-related apoptosis after cerebral ischemia/reperfusion injury by inhibiting ICAM-1 mRNA expression in rats. Brain Res 2008;1209:136-150. Assenza G, Zappasodi F, Squitti R, et al. Neuronal functionality assessed by magnetoencephalography is related to oxidative stress system in acute ischemic stroke. Neuroimage 2009;44:1267-1273. Gasparetto C, Malinverno A, Culacciati D, et al. Antioxidant vitamins reduce oxidative stress and ventricular remodeling in patients with acute myocardial infarction. Int J Immunopathol Pharmacol 2005;18:487-496. Squitti R, Rossini PM, Cassetta E, et al. d-penicillamine reduces serum oxidative stress in Alzheimer’s disease patients. Eur J Clin Invest 2002;32:51-59. Iwata N, Okazaki M, Kamiuchi S, et al. Protective effects of oral administrated ascorbic acid against oxidative stress and neuronal damage after cerebral ischemia/reperfusion in diabetic rats. J Heal Sci 2010;56:20-30. Sato K, Kameda M, Yasuhara T, et al. Neuroprotective effects of liraglutide for stroke model of rats. Int J Mol Sci 2013;14:21513-21524. Stier A, Bize P, Habold C, et al. Mitochondrial uncoupling prevents cold-induced oxidative stress: a case study using UCP1 knockout mice. J Exp Biol 2014;217:624-630. Beal MF. Mitochondrial dysfunction and oxidative damage in Alzheimer’s and Parkinson’s diseases and coenzyme Q10 as a potential treatment. J Bioenerg Biomembr 2004;36:381-386. Sinha-Hikim I, Sinha-Hikim AP, Shen R, et al. A novel cystine based antioxidant attenuates oxidative stress and hepatic steatosis in diet-induced obese mice. Exp Mol Pathol 2011;91:419-428. Iadecola C, Anrather J. The immunology of stroke: from mechanisms to translation. Nat Med 2011;17:796-808. Hinojosa AE, Garcia-Bueno B, Leza JC, et al. CCL2/ MCP-1 modulation of microglial activation and proliferation. J Neuroinflammation 2011;8:77.
Neuroprotective Effects of a Novel Antioxidant Mixture Twendee X in Mouse Stroke Model Momoko Kusaki, BSc,* Yasuyuki Ohta, MD, PhD,* Haruhiko Inufusa, MD, PhD,† Toru Yamashita, MD, PhD,* Ryuta Morihara, MD, PhD,* Yumiko Nakano, MD, PhD,* Xia Liu, MD,* Jingwei Shang, MD, PhD,* Feng Tian, BSc,* Yusuke Fukui, MSc,* Kota Sato, MD, PhD,* Mami Takemoto, MD, PhD,* Nozomi Hishikawa, MD, PhD,* and Koji Abe, MD, PhD*
Background: Oxidative stress and inflammation are important aggravating factors in acute ischemic stroke. Methods: In the present study, the neuroprotective effects of a novel antioxidant mixture Twendee X containing multiple antioxidative ingredients, such as coenzyme Q10, ascorbic acid, and cystine, were evaluated. After the pretreatment of a vehicle or Twendee X (20 mg/kg/d) for 14 days, mice were subjected to transient middle cerebral artery occlusion for 60 minutes and further treated with vehicle or Twendee X for 1 or 5 days. Results: Twendee X administration reduced the infarct size, and reduced oxidative stress markers such as 8-hydroxy-2′-deoxyguanosine, 4-hydroxy-2-nonenal, and Nε-(carboxymethyl) lysine (one of advanced glycation end products), as well as inflammatory markers such as ionized calcium binding adapter molecule-1, tumor necrosis factor-α, and monocyte chemotactic protein-1. Conclusions: In the present study, the neuroprotective effects of Twendee X were shown on transient middle cerebral artery occlusion mice via antioxidative and anti-inflammatory pathways, providing a potential of Twendee X as one preventive and therapeutic treatment. Key Words: Ischemic stroke—middle cerebral artery occlusion—mouse—antioxidative—anti-inflammatory. © 2017 National Stroke Association. Published by Elsevier Inc. All rights reserved.
From the *Department of Neurology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan; and †Division of Anti-Oxidant Research, Life Science Research Center, Gifu University, Gifu, Japan. Received December 12, 2016; revision received December 29, 2016; accepted January 7, 2017. Grant support: This work was partly supported by Grants-inAid for Scientific Research (B, 25293202 and C, 15K09316), Challenging Research (15K15527), and Young Research (15K21181), and by Grantsin-Aid from the Research Committees (Mizusawa H, Nakashima K, Nishizawa M, Sasaki H, and Aoki M) from the Ministry of Health, Labour and Welfare, Japan. Address correspondence to Koji Abe, MD, PhD, Department of Neurology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan. E-mail: [email protected]. 1052-3057/$ - see front matter © 2017 National Stroke Association. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2017.01.003
Introduction Ischemic stroke is a leading cause of mortality and neurologic impairments worldwide.1 Therapeutic strategies against stroke remain limited, and further novel therapies are required in daily clinical practice.2 Oxidative stress and inflammation are important aggravating factors in acute ischemic stroke.3,4 Reactive oxygen species (ROS) is gradually generated during cerebral ischemia, then excessively increased after reperfusion notably in the periischemic area with oxidation of cellular DNA, lipids, and proteins,5-8 and then often focused as a medical treatable target.9 We originally showed the strong neuroprotective effects of a free radical scavenger edaravone.8,10,11 Furthermore, we reported that dietary supplements such as ginkgo extract,12 platinum nanoparticle species,13 and antioxidative
Journal of Stroke and Cerebrovascular Diseases, Vol. ■■, No. ■■ (■■), 2017: pp ■■–■■
1
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nutrient-rich enteral diet showed neuroprotective effects on the ischemic brains of mice. Dietary supplements did not show the strong effects on diseases as medicinal chemicals. However, they are safe and valuable for the prevention and treatment of various diseases.15 Twendee X (TwX) is an anti-aging supplement containing multiple antioxidants and a patented composition.16 TwX has strong antioxidant effects, and increases superoxide dismutase and cell protection effects.17 In the present study, we validated whether TwX could be helpful in ameliorating mouse brain damages and oxidative stress following experimental transient middle cerebral artery occlusion (tMCAO).
Materials and Methods
Figure 1. The 4 experimental mice groups showing V-1d group (saline, sacrificed 1 day after tMCAO), TwX-1d group (TwX, sacrificed 1 day after tMCAO), V-5d group (saline, sacrificed 5 days after tMCAO), and TwX5d group (TwX, sacrificed 5 days after tMCAO). Filled black arrows indicate intraperitoneal injection of saline or TwX. Open white triangles indicate blood sampling. Abbreviation: tMCAO, transient middle cerebral artery occlusion.
Animals and Focal Cerebral Ischemia All experimental procedures were approved by the Animal Committee of the Okayama University Graduate School of Medicine (OKU-2015573). Adult male C57BL/ 6JJcl mice (23-27 g, 8 weeks old) were obtained from CLEA Japan (Tokyo, Japan). The mice were maintained in a temperature-regulated room (23-25°C) on a 12-hour light– dark cycle and allowed free access to food and water. From 9 weeks of age, the mice received vehicle (physiological saline, ip, n = 15) or TwX (20 mg/kg per day, ip, n = 16) for 14 days, and then were subjected to tMCAO for 60 minutes. TwX is a mixture consisting of coenzyme Q10 (3.6 wt%; AQUA Q10 P40-NF, Nissin Pharmaceutical, Tokyo, Japan), niacin amid (.7 wt%), L-cystine (18.2 wt%), ascorbic acid (34.2 wt%), succinic acid (3.6 wt%), fumaric acid (3.6 wt%), L-glutamine (34.6 wt%), and riboflavin (1.5 wt%; Bislase inj; Toa Eiyo, Tokyo, Japan). The TwX mixture was dissolved in saline (Otsuka Pharmaceutical Factory, Tokushima, Japan) and stored at 4°C until use. The TwX solution (20 mg/kg) was intraperitoneally injected to the mice with 500 μL volume. After 14 days of intraperitoneal administration of the vehicle or TwX (11 weeks old), the mice were anesthetized with a mixture of nitrous oxide : oxygen : isoflurane (69%:30%:1%) during surgery with an inhalation mask, and tMCAO was induced using the intraluminal filament technique.18 Body temperature was monitored and maintained at 37 ± 0.3°C by placing the animals on a heating pad (BWT-100; Bio Research Center, Aichi, Japan). After the right common carotid artery was exposed, a 7-0 nylon thread with a silicon-coated tip was inserted into the right middle cerebral artery. After 60 minutes of tMCAO, the siliconcoated thread was pulled out to restore blood flow of MCA. After this tMCAO, each mice group (vehicle or TwX) was further treated with the same vehicle or TwX for 1 (n = 7 each) or 5 (n = 8 or 9) days. The mice received the injection of vehicle or TwX once a day over a period of 1 day (V-1d, TwX-1d) or 5 days (V-5d, TwX-5d) (Fig 1). One day or 5 days after tMCAO, blood was collected from the mice via the retro-orbital puncture 12 hours after
the final administration. For histologic analysis, the mice were then deeply anesthetized by intraperitoneal injection of pentobarbital (40 mg/kg), and then transcardially perfused with chilled phosphate-buffered saline (PBS, pH 7.4), followed by 4% paraformaldehyde in PBS. The whole brain was removed and immersed in the same fixative solution overnight at 4°C. Serum samples were separated by centrifugation (1940 g, 15 minutes, 4°C) and stored at −80°C. After washing with PBS, the fixed brains were incubated in 20% (wt/vol) sucrose in PBS for 24 hours at 4°C. The tissues were frozen in liquid nitrogen and stored at −80°C. Coronal brain section (10 μm thickness) was cut on a cryostat at −20°C and mounted on silane-coated glass slides.
Serum Measurement of Reactive Oxygen Metabolite Levels and Antioxidant Capacity The ROS was examined by d-ROMs test (Diacron International, Italy). The reactive oxygen metabolite levels are expressed as arbitrary “Carratelli units” (CARR U), with 1 CARR U corresponding to .08 mg per 100 mL of H2O2. The total serum antioxidant capacity was assayed by OXY-Adsorbent test (Diacron International, Grosseto, Italy). The d-ROMs test and OXY-Adsorbent test were performed using a spectrophotometer.19
Histology and Immunohistochemistry For quantification of infarct volume, the brain sections (10 μm) were stained with cresyl violet as Nissl staining and examined by microscopy (SZX-12; Olympus Optical, Tokyo, Japan). The brain sections were prepared at a .8-mm interval each, between .8 mm anterior and 2.4 mm posterior to the bregma. The infarct volumes were measured in the 5 sections by pixel counting using a computer program for Photoshop CC (Adobe, San Jose, CA, USA) and then calculated by summation of the 5 serial infarct areas. For immunohistochemistry of Nε-(carboxymethyl) lysine (CML), ionized calcium-binding adapter molecule-1 (Iba-
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1), tumor necrosis factor-α (TNFα), and monocyte chemotactic protein-1 (MCP-1), the frozen sections (10 μm) were incubated in .3% H2O2/methanol for 20 minutes to block endogenous peroxidase activity and to block in 5% bovine serum albumin for 1 hour. The sections were incubated at 4°C overnight with the following primary antibodies: mouse anti-CML antibody (1:100; AGE-M01, Cosmo Bio, Tokyo, Japan); rabbit anti-Iba-1 antibody (1:1000; #019-19741, Wako, Osaka, Japan); rabbit anti-TNFα antibody (1:200; ab6671, Abcam, Cambridge, UK); and rabbit anti-MCP-1 antibody (1:100; ab7202, Abcam). The sections were then washed in PBS and incubated with biotinylated anti-mouse or anti-rabbit IgG secondary antibodies (Vector Laboratories, Burlingame, CA, USA) diluted at 1:500 for 2.5 hours at room temperature. The sections were then incubated with the avidin–biotin–peroxidase complex (VECTASTAIN Elite ABC Kit; Vector Laboratories) for 30 minutes and visualized with 3,3′diaminobenzidine (DAB). Immunohistochemistry for 8-hydroxy-2′-deoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (4-HNE), and sirtuin-1 (Sirt-1) was performed with mouse on mouse (M.O.M) immunodetection kit (Vector Laboratories) according to the manufacturer’s directions. Briefly, the frozen sections were incubated in .3% H2O2/methanol and then blocked in a mouse IgG blocking solution. The sections were incubated at 4°C with the following primary antibodies: mouse anti-8-OHdG (5 μg/mL; MOG-100P, JaICA, Shizuoka, Japan); mouse anti-4-HNE (25 μg/mL; MHN100P, JaICA); and mouse anti-Sirt-1 (1:500; ab50517, Abcam). Immunoreactivity was visualized by a biotinylated secondary antibody, avidin–biotin–peroxidase complex, and DAB. In immunostaining for all markers in this study, a set of sections was also stained in a similar way but without the primary antibody, and served as the negative control.
Statistical Analysis Data are expressed as mean ± standard deviation. Statistical analysis was performed using a one-way analysis of variance, followed by a Tukey’s honest significant difference test. In all statistical analyses, significance was accepted at P < .05.
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Figure 2. Serum levels of reactive oxygen metabolites (d-ROMs) and antioxidant capacity (OXY-Adsorbent) showing reducing tendencies of Twendee X on oxidative stress 5 days after transient middle cerebral artery occlusion, but not significant.
319.0 ± 52.4 μmol HClO/mL; TwX-1d, 294.0 ± 33.4 μmol HClO/mL) and 5 days after (V-5d, 337.5 ± 51.3 μmol HClO/ mL; TwX-5d, 292.5 ± 39.1 μmol HClO/mL), but again not significant (Fig 2).
Brain Infarct Volume after tMCAO One day after tMCAO, TwX did not show a difference in the brain infarct volume (V-1d = 23.9 ± 2.8 mm3 versus TwX-1d = 23.2 ± 7.1 mm3), but remarkably reduced the infarct volume (18.0 ± 5.6 mm3, P < .05 versus vehicle) on day 5 compared with the vehicle group (28.0 ± 7.1 mm3, Fig 3).
Oxidative Stress in the Mice Brain
Results Serum Oxidative Stress and the Antioxidative Activities As compared with the vehicle (V-1d, 165 ± 21.3 CARR U), TwX did not reduce oxidative stress (d-ROMs) 1 day after tMCAO (TwX-1d, 164 ± 10.3 CARR U), but showed a slight reduction 5 days after (V-5d = 167.5 ± 27.8 versus TwX-5d = 149.5 ± 14.4 CARR U, not significant) (Fig 2). On the other hand, OXY-Adsorbent showed a reduction tendency in TwX both 1 day after tMCAO (V-1d,
Compared with the vehicle, TwX did not clearly reduce the number of positive cells for 3 oxidative stress markers of 8-OHdG (176.6 ± 24.4 versus 140.6 ± 29.9/mm2), 4-HNE (199.0 ± 12.8 versus 188.8 ± 25.9/mm 2 ), and CML (162.4 ± 12.2 versus 163.0 ± 12.2/mm2) in the mice brain 1 day after tMCAO (Fig 4). However, TwX significantly reduced the number of positive cells for all 3 markers in 8-OHdG (200.2 ± 24.4 versus 117.7 ± 6.2/mm2, P < .01), 4-HNE (227.5 ± 21.0 versus 160.1 ± 7.9/mm2, P < .01), and CML (169.2 ± 6.4 versus 140.8 ± 5.5/mm2, P < .05) 5 days after tMCAO compared with the vehicle (Fig 4).
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Figure 3. Cresyl violet stainings of coronal sections (left) and brain infarct volumes (right). Note the significant reduction of infarct volumes in TwX-5d compared with V-5d group (*P < .05).
Inflammation in the Mice Brain Compared with the vehicle (60.8 ± 7.1/mm2), TwX clearly reduced the number of ionized calcium binding adapter molecule-1 (Iba1) positive cells (49.2 ± 5.7/mm2, P < .05 versus vehicle) even 1 day after tMCAO (Fig 5). Five days after tMCAO, the number of Iba1 positive cells (99.6 ± 11.2/ mm2) increased compared with 1 day in the vehicle group (60.8 ± 7.1/mm2, P < .05 versus 1 day), which was remarkably ameliorated (50.3 ± 8.0/mm2) by TwX (P < .01 versus vehicle). On the other hand, compared with the vehicle (TNFα = 172.8 ± 11.8/mm2, MCP-1 = 184.2 ± 18.1/mm2, and Sirt-1 = 185.0 ± 17.4/mm2), TwX did not clearly reduce the number of positive cells for TNFα, MCP-1, and Sirt-1 (161.2 ± 15.2/mm2, 167.2 ± 15.1/mm2, and 166.2 ± 17.4/ mm2, respectively) in the mice brain 1 day after tMCAO (Fig 5). However, TwX remarkably reduced the number of positive cells for TNFα (153.7 ± 8.7/mm2) and MCP-1
(147.3 ± 8.5/mm2) 5 days after tMCAO compared with the vehicle (TNFα = 193.0 ± 13.1/mm2, MCP-1 = 207.3 ± 20.6/ mm2, P < .01 versus vehicle for TNFα, P < .05 versus vehicle for MCP-1) except for Sirt-1 (V-5d = 227.0 ± 14.2/mm2, TwX5d = 194.1 ± 26.8/mm2) (Fig 5).
Discussion The present study demonstrated that a novel antioxidant mixture TwX showed neuroprotective effects on mice cerebral ischemia by reducing the infarct size (Fig 3), and reducing both oxidative stress markers (Fig 4) and inflammatory markers (Fig 5). ROS is incrementally generated during cerebral ischemia, then explosively increased after reperfusion particularly in the penumbra with oxidation of cellular DNA, lipids, and proteins.5-8 Such oxidative stress is detrimental in the cerebral infarction.3 Our previous studies showed the marked neuroprotective effects
Figure 4. Immunohistochemistry for 8-OHdG, 4-HNE, and CML at the peri-infarct area (left) and quantitative analyses of positive cells (right). Note the significant reductions of positive cells for 3 markers in TwX-5d compared with V-5d group (*P < .05, **P < .01). Abbreviations: 4-HNE, 4-hydroxy2-nonenal; 8-OHdG, 8-hydroxy-2′-deoxyguanosine; CML, Nε-(carboxymethyl) lysine.
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Figure 5. Immunohistochemistry for Iba-1, TNFα, MCP-1, and Sirt-1 at the peri-infarct area (left) and quantitative analyses of positive cells (right). The number of Iba1 positive cells increased on day 5 (V-5d group) than on day 1 (V-1d group, #P < .05), and the number of Iba-1, TNFα, and MCP-1 positive cells significantly reduced in TwX-5d than V-5d (*P < .05, **P < .01). Abbreviations: Iba1, ionized calcium binding adapter molecule-1; TNFα, tumor necrosis factor-α; MCP-1, monocyte chemotactic protein-1; Sirt-1, sirtuin-1.
of a free radical scavenger edaravone.8,10,11 Other free radical scavengers, ginkgo extract,12,20 platinum nanoparticle species,13 ferulic acid,21 and antioxidative nutrient-rich enteral diet14 also showed neuroprotective effects on the ischemic brains of mice. With the present d-ROMs and OXY-Adsorbent tests, oxidative stress and the antioxidative activities can be easily examined with a small amount of serum samples (25 μL for each mouse). In previous studies, these methods were applied in patients with various diseases, such as acute stroke,22 acute myocardial infarction,23 and Alzheimer’s disease.24 The d-ROMs test was also evaluated in experimental animal models such as tMCAO models25,26 and UCP-1 knockout mice.27 The present data suggested a reduced tendency of serum ROS and reactive oxygen metabolites with TwX (Fig 2). Brain sections showed a reduction of oxidative stress in DNA (8-OHdG), lipid (4-HNE), and advanced glycation end products (CML) (Fig 4). Since TwX is a mixture containing multiple antioxidants such as coenzyme Q10, ascorbic acid, and cystine, it reduced oxidative stress in the present ischemic models (Fig 4). A previous study showed an amelioration of oxidative stress by ascorbic acid in both tMCAO and diabetic models.25 Moreover, TwX significantly reduced oxidative stress in the dose of one-third of ascorbic acid in irradiation mouse model, suggesting synergistic effects by multiple antioxidants.16 Coenzyme Q10 was effective for the treatment of neurologic diseases by remedying mitochondrial dysfunction and oxidative damages.28 A cystine-based antioxidant at-
tenuated oxidative stress in obese mice.29 Inflammation is another pathologic key factor in the ischemic stroke.4 Inflammatory cytokines such as MCP-1 and TNFα activate microglia with Iba-1 potentiation, and finally exert neurotoxic effects.30,31 TwX ameliorated such inflammatory responses especially 5 days after tMCAO (Fig 5). In summary, we presented the neuroprotective effects of TwX on tMCAO mice in reducing infarct size and both oxidative stress and inflammatory markers (Figs 3-5). The present study provides a potential of TwX as one preventive and therapeutic treatment against ischemic stroke. Acknowledgments: We are grateful to Dr. Maruyama T of the Department of Preventive Dentistry at Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, especially for their technical assistance in d-ROMs tests and OXY-Adsorbent tests.
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