title: Willow bark extract increases antioxidant enzymes and reduces oxidative stress through activation of Nrf2 in vascular endothelial cells and Caenorhabditis elegans.
abstract: Willow bark extract (WBE) is listed in the European Pharmacopoeia and has been traditionally used for treating fever, pain, and inflammation. Recent studies have demonstrated its clinical usefulness. This study investigated the antioxidative effects of WBE in human umbilical vein endothelial cells (HUVECs) and Caenorhabditis elegans. WBE prevented oxidative-stress-induced cytotoxicity of HUVECs and death of C. elegans. WBE dose-dependently increased mRNA and protein expression levels of the nuclear factor erythroid 2-related factor 2 (Nrf2) target genes heme oxygenase-1, γ-glutamylcysteine ligase modifier and catalytic subunits, and p62 and intracellular glutathione (GSH) in HUVECs. In the nematode C. elegans, WBE increased the expression of the gcs-1::green fluorescent protein reporter, a well-characterized target of the Nrf2 ortholog SKN-1, in a manner that was SKN-1-dependent. WBE increased intranuclear expression and DNA binding of Nrf2 and the activity of an antioxidant response element (ARE) reporter plasmid in HUVECs. WBE-induced expression of Nrf2-regulated genes and increased GSH levels in HUVECs were reduced by Nrf2 and p38 small interfering (si) RNAs and by the p38-specific inhibitor SB203580. Nrf2 siRNA reduced the cytoprotective effect of WBE against oxidative stress in HUVECs. Salicin, a major anti-inflammatory ingredient of WBE, failed to activate ARE-luciferase activity, whereas a salicin-free WBE fraction showed intensive activity. WBE induced antioxidant enzymes and prevented oxidative stress through activation of Nrf2 independent of salicin, providing a new potential explanation for the clinical usefulness of WBE.
authors.affiliation: R&D Department, Sunstar Inc., Osaka 569-1195, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Ishikado
authors.affiliation: R&D Department, Sunstar Inc., Osaka 569-1195, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Sono
authors.affiliation: R&D Department, Sunstar Inc., Osaka 569-1195, Japan.
authors.forename: Motonobu
authors.initials: M
authors.lastname: Matsumoto
authors.affiliation: Section on Islet Cell & Regenerative Biology, Research Division, Joslin Diabetes Center, Harvard Medical School, Boston, MA 02215, USA.
authors.forename: Stacey
authors.initials: S
authors.lastname: Robida-Stubbs
authors.affiliation: R&D Department, Sunstar Inc., Osaka 569-1195, Japan.
authors.forename: Aya
authors.initials: A
authors.lastname: Okuno
authors.affiliation: R&D Department, Sunstar Inc., Osaka 569-1195, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Goto
authors.affiliation: Section on Vascular Cell Biology, Research Division, Joslin Diabetes Center, Harvard Medical School, Boston, MA 02215, USA.
authors.forename: George L
authors.initials: GL
authors.lastname: King
authors.affiliation: Section on Islet Cell & Regenerative Biology, Research Division, Joslin Diabetes Center, Harvard Medical School, Boston, MA 02215, USA. Electronic address: Keith.Blackwell@joslin.harvard.edu.
authors.forename: T
authors.initials: T
authors.lastname: Keith Blackwell
authors.affiliation: R&D Department, Sunstar Inc., Osaka 569-1195, Japan. Electronic address: taketoshi.makino@jp.sunstar.com.
authors.forename: Taketoshi
authors.initials: T
authors.lastname: Makino
mesh_terms: D000818:Animals; D061745:Antioxidant Response Elements / Q000235:genetics; D000975:Antioxidants; D001592:Benzyl Alcohols / Q000494:pharmacology; D017173:Caenorhabditis elegans / Q000201:enzymology*; D029742:Caenorhabditis elegans Proteins / Q000096:biosynthesis / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism; D002478:Cells, Cultured; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D042783:Endothelial Cells / Q000201:enzymology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005960:Glucosides / Q000494:pharmacology; D005721:Glutamate-Cysteine Ligase / Q000096:biosynthesis / Q000235:genetics; D005978:Glutathione / Q000096:biosynthesis / Q000235:genetics; D051547:Heme Oxygenase-1 / Q000096:biosynthesis / Q000235:genetics; D061307:Human Umbilical Vein Endothelial Cells / Q000201:enzymology*; D007093:Imidazoles / Q000494:pharmacology; D051267:NF-E2-Related Factor 2 / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects; D024301:Plant Bark / Q000737:chemistry; D010936:Plant Extracts / Q000494:pharmacology*; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis / Q000235:genetics; D011725:Pyridines / Q000494:pharmacology; D034622:RNA Interference; D012333:RNA, Messenger / Q000096:biosynthesis; D034741:RNA, Small Interfering; D032108:Salix / Q000737:chemistry*; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics
chemical_list: D000975:Antioxidants; D001592:Benzyl Alcohols; D029742:Caenorhabditis elegans Proteins; D016861:Cyclooxygenase Inhibitors; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D005960:Glucosides; D007093:Imidazoles; D051267:NF-E2-Related Factor 2; D010936:Plant Extracts; D016271:Proto-Oncogene Proteins c-myc; D011725:Pyridines; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014157:Transcription Factors; C073702:skn-1 protein, C elegans; C005696:salicin; D051547:Heme Oxygenase-1; D048051:p38 Mitogen-Activated Protein Kinases; D002265:Carboxylic Ester Hydrolases; C019734:arylesterase; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione; C093642:SB 203580
keywords: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; ARE; Antioxidant enzyme; Caenorhabditis elegans; FBS; Free radicals; GCLC; GCLM; GFP; HO-1; HPLC; HUVEC; Keap1; Kelch-like ECH-associated protein 1; MTT; NGM; Nrf2; Oxidative stress; PBS; WBE; Willow bark extract; antioxidant response element; fetal bovine serum; green fluorescent protein; heme oxygenase-1; high-performance liquid chromatography; human umbilical vein endothelial cell; nematode growth medium; nuclear factor erythroid 2-related factor 2; phosphate-buffered saline; tBHP; tert-butylhydroperoxide; willow bark extract; γ-glutamylcysteine ligase, catalytic subunit; γ-glutamylcysteine ligase, modifier subunit