Uptake and Protective Effects of Ergothioneine in Human Endothelial Cells
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1521-0103/350/3/691–700$25.00 http://dx.doi.org/10.1124/jpet.114.214049 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 350:691–700, September 2014 Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics Uptake and Protective Effects of Ergothioneine in Human Endothelial Cells Rachel W. S. Li, C. Yang, Albert S. M. Sit, Y. W. Kwan, Simon M. Y. Lee, Maggie P. M. Hoi, S. W. Chan, Marvin Hausman, Paul M. Vanhoutte, and George P. H. Leung State Key Laboratory for Pharmaceutical Biotechnologies and Department of Pharmacology and Pharmacy, The University of Hong Kong, Hong Kong (R.W.S.L., A.S.M.S., P.M.V., G.P.H.L.); Ethnic Drug Screening and Pharmacology Center, Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission and Ministry of Education, Yunnan Minzu University, Kunming, China (C.Y.); School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong (Y.W.K.); Institute of Chinese Medical Sciences, University of Macau, Macao, China (S.M.Y.L., M.P.M.H.); State Key Laboratory Downloaded from of Chinese Medicine and Molecular Pharmacology, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hong Kong (S.W.C.); and Entia Biosciences Inc., Sherwood, Oregon (M.H.) Received February 18, 2014; accepted July 20, 2014 ABSTRACT jpet.aspetjournals.org Ergothioneine is a thiourea derivative of histidine found in food, 2-yl)-2,5-diphenyltetrazolium bromide] assay. ROS production and especially mushrooms. Experiments in cell-free systems and cell death induced by pyrogallol, xanthine oxidase plus xanthine, chemical assays identified this compound as a powerful and high glucose were suppressed by ergothioneine. The antioxidant. Experiments were designed to test the ability of antioxidant and cytoprotective effects of ergothioneine were endothelial cells to take up ergothioneine and hence benefit from abolished when OCTN-1 was silenced using siRNA. The ex- protection against oxidative stress. Reverse-transcription poly- pression of NADPH oxidase 1 was decreased, and those of merase chain reaction and Western blotting demonstrated glutathione reductase, catalase, and superoxide dismutase at ASPET Journals on January 27, 2020 transcription and translation of an ergothioneine transporter in enhanced by the compound. In isolated rat basilar arteries, human brain microvascular endothelial cells (HBMECs). Uptake ergothioneine attenuated the reduction in acetylcholine-induced of [3H]ergothioneine occurred by the organic cation transporter relaxation caused by pyrogallol, xanthine oxidase plus xanthine, novel type-1 (OCTN-1), was sodium-dependent, and was re- or incubation in high glucose. Chronic treatment with the duced when expression of OCTN-1 was silenced by small inter- compound improved the response to acetylcholine in arteries of fering RNA (siRNA). The effect of ergothioneine on the production rats with streptozotocin-induced diabetes. In summary, ergo- of reactive oxygen species (ROS) in HBMECs was measured thioneine is taken up by endothelial cells via OCTN-1, where the using dichlorodihydrofluorescein and lucigenin, and the effect on compound then protects against oxidative stress, curtailing cell viability was studied using the MTT [3-(4,5-dimethylthiazol- endothelial dysfunction. Introduction reduce the bioavailability of NO by scavenging it avidly, resulting in the formation of peroxynitrite (Gryglewski et al., Endothelial cells synthesize and release various factors, in 1986; Rubanyi and Vanhoutte, 1986). particular nitric oxide (NO), that regulate vascular tone, Ergothioneine was first discovered in the rye ergot Claviceps angiogenesis, inflammatory responses, hemostasis, and per- purpurea. A variety of foods contain ergothioneine in trace meability. Endothelial dysfunction is associated with a quantities, with the highest amounts found in some mush- number of cardiovascular disease processes, including hyper- room species, including Lentinus edodes (shiitake), Pleurotus tension, atherosclerosis, heart failure, coronary syndrome, ostreatus, and Pleurotus eryngii, while moderate amounts are and stroke (Fèlètou and Vanhoutte, 2006). Oxidative stress is an important pathogenic element in endothelial dysfunction. found in certain meat products, such as kidney (Ey et al., Reactive oxygen species (ROS), such as superoxide anions, 2007). Ergothioneine is a dietary water-soluble sulfur-containing histidine derivative (Fig. 1). Ergothioneine was first identi- fied as an antioxidant in cell-free experiments. It inhibits This work was supported by RGC General Research Grant of Hong Kong (Project Number: 770109). formation of hydroxyl radical (Motohashi and Mori, 1986), dx.doi.org/10.1124/jpet.114.214049. and superoxide anions (Cargnoni et al., 1995), singlet oxygen ABBREVIATIONS: COX-1, cyclooxygenase 1; DCHF, dichlorodihydrofluorescein; eNOS, endothelial nitric oxide synthase; HBMECs, human brain microvascular endothelial cells; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NOX-1, NADPH oxidase 1; PBS, phosphate- buffered saline; OCTN-1, organic cation transporter novel type-1; ROS, reactive oxygen species; RT-PCR, reverse-transcription polymerase chain reaction; siRNA, small interfering RNA; SOD, superoxide dismutase; U46619, (Z)-7-[(1S,4R,5R,6S)-5-[(E,3S)-3-hydroxyoct-1-enyl]-3-oxabicyclo [2.2.1]heptan-6-yl]hept-5-enoic acid. 691 692 Li et al. production (Obayashi et al., 2005), lipid peroxidation (Deiana Cell Culture. Human brain microvascular endothelial cells et al., 2004), and peroxynitrite oxidative damage (Aruoma (HBMECs) were purchased from ScienCell Research Laboratories et al., 1997). As opposed to other naturally occurring thiols (Carlsbad, CA) and cultured according to the manufacturer’s pro- (e.g., glutathione, N-acetylcysteine), ergothioneine is pres- cedures. Cells were incubated in 5% CO2/5% air at 37°C. Specific ent in aqueous solutions predominantly as a thione rath- culture medium for HBMECs was purchased from ScienCell and the medium was supplemented with 5% fetal bovine serum, 1% endothe- er than a tautomeric thiol structure. As a consequence, lial cell growth supplement (ScienCell), and 1% penicillin/streptomycin. ergothioneine is a stable antioxidant that is nonautoxidizable The medium was changed every three days. Experiments were per- at physiologic pH (Hartman, 1990). Ergothioneine is positively formed between the second and sixth passage. charged at physiologic pH, and requires the specific carrier RNA Isolation and Reverse-Transcription Polymerase Chain protein organic cation transporter novel type-1 (OCTN-1) to Reaction. Total RNA was isolated from HBMECs using TRIzol cross the cell membrane (Grigat et al., 2007; Nakamura et al., reagent (Invitrogen). Two micrograms of total RNA were used for first 2008). OCTN-1 is mainly found in kidney, trachea, and bone strand cDNA synthesis using random hexamer primers and Super- marrow (Tamai et al., 1997; Koepsell et al., 2007), but its script II RNase Reverse Transcriptase (Invitrogen). The resulting existence in other cell types has not been explored. first strand cDNA was directly used for polymerase chain reaction A number of compounds possess antioxidant properties amplification. The two primers used for amplifying OCTN-1 (acces- sion number AF079117) were 59-CTGGATGCTCCTAATTTACATGG- in vitro but are ineffective in vivo. A typical example is 39 (sense) and 59-AGGAGACTCTCTAGAAATGGTTGG-39 (antisense), flavonoids (Halliwell, 2009). Although in vitro studies demon- which generated a 785-bp fragment. The program was as follows: strate that ergothioneine may act as a cytoprotectant (Aruoma 30 cycles of 94°C for 1 minute, 55°C for 1.5 minutes, and 72°C for Downloaded from et al., 1999; Rahman et al., 2003; Jang et al., 2004; Markova 1.5 minutes. Products were analyzed by agarose gel electrophoresis et al., 2009), few in vivo findings with ergothioneine are and visualized under UV light by staining with ethidium bromide. available. Oral administration of ergothioneine protects fatty Western Blotting. HBMECs were grown to confluence in 10-cm acids against ferric-nitrilotriacetate–induced oxidation in rat culture dishes. The cells were washed three times with ice-cold kidney and liver (Deiana et al., 2004). In vivo treatment of phosphate-buffered saline (PBS), and suspended in 2 ml of 5 mM sodium phosphate (pH 8), and protease inhibitor cocktail (Sigma- rats with ergothioneine also protects intestinal and liver jpet.aspetjournals.org tissues from damage during ischemia/reperfusion injury Aldrich) was added to a 1:100 (v/v) ratio. Cells were sonicated briefly and centrifuged at 3000g for 10 minutes to remove nuclei and (Bedirli et al., 2004; Sakrak et al., 2008). In addition, chronic unbroken cells. The resulting supernatant was resolved on 9% (w/v) administration of ergothioneine inhibits brain lipid peroxida- SDS-PAGE and electrotransferred onto nitrocellulose membranes. tion, protects against cisplatin-induced neuronal injury, and After blocking with 5% (w/v) nonfat dry milk in PBS overnight at 4°C, enhances cognition in the mouse (Song et al., 2010). Endo- nitrocellulose membranes were incubated with the primary anti- thelial cells are exposed to oxidative stress, particularly in bodies [1:100 (v/v) dilution in blocking solution] at room temperature diabetic subjects (Guzik et al., 2002); however, the potential for 2 hours. The membranes were then washed extensively with at ASPET Journals on January