Willow bark extract increases antioxidant enzymes and reduces oxidative stress through activation of Nrf2 in vascular endothelial cells and Caenorhabditis elegans

• Published in: Free radical biology & medicine 2013-12, Vol.65, p.1506-1515
• Main Authors: Ishikado, Atsushi, Sono, Yoko, Matsumoto, Motonobu, Robida-Stubbs, Stacey, Okuno, Aya, Goto, Masashi, King, George L, Keith Blackwell, T, Makino, Taketoshi
• Format: Article
• Language: English
• Subjects: Animals; antioxidant activity; Antioxidant Response Elements - genetics; Antioxidants; bark; Benzyl Alcohols - pharmacology; Caenorhabditis elegans; Caenorhabditis elegans - enzymology; Caenorhabditis elegans Proteins - biosynthesis; Caenorhabditis elegans Proteins - genetics; Carboxylic Ester Hydrolases - metabolism; Cells, Cultured; Cyclooxygenase Inhibitors - pharmacology; cytotoxicity; death; DNA; DNA-Binding Proteins - biosynthesis; DNA-Binding Proteins - genetics; Endothelial Cells - enzymology; Enzyme Activation; Enzyme Inhibitors - pharmacology; fluorescent proteins; gene expression; genes; Glucosides - pharmacology; Glutamate-Cysteine Ligase - biosynthesis; Glutamate-Cysteine Ligase - genetics; glutathione; Glutathione - biosynthesis; Glutathione - genetics; heme oxygenase (biliverdin-producing); Heme Oxygenase-1 - biosynthesis; Heme Oxygenase-1 - genetics; human umbilical vein endothelial cells; Human Umbilical Vein Endothelial Cells - enzymology; Imidazoles - pharmacology; inflammation; messenger RNA; NF-E2-Related Factor 2 - genetics; NF-E2-Related Factor 2 - metabolism; oxidative stress; Oxidative Stress - drug effects; p38 Mitogen-Activated Protein Kinases - antagonists & inhibitors; p38 Mitogen-Activated Protein Kinases - genetics; Plant Bark - chemistry; Plant Extracts - pharmacology; protein subunits; protein synthesis; Proto-Oncogene Proteins c-myc - biosynthesis; Proto-Oncogene Proteins c-myc - genetics; Pyridines - pharmacology; RNA Interference; RNA, Messenger - biosynthesis; RNA, Small Interfering; Salix; Salix - chemistry; small interfering RNA; Transcription Factors - biosynthesis; Transcription Factors - genetics
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2012.12.006

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.