Involvement of heme oxygenase-1 induction via Nrf2/ARE activation in protection against H2O2-induced PC12 cell death by a metabolite of sesamin contained in sesame seeds

Induction of phase II antioxidant enzymes by activation of Nrf2/ARE (antioxidant response element) signaling has been considered as a promising strategy to combat with oxidative stress-related diseases. In the present study, we tested for potential effects of sesamin, a major lignan contained in sesame seeds, its stereoisomer episesamin, and their metabolites on Nrf2/ARE activation in rat pheochromocytoma PC12 cells. Luciferase reporter assays showed that primary metabolites of sesamin and episesamin, SC-1 and EC-1 were the most potent ARE activators among all tested compounds. SC-1 {(1R,2S,5R,6S)-6-(3,4-dihydroxyphenyl)-2-(3,4-methylenedioxyphenyl)-3,7-dioxabicyclo-[3,3,0]octane} enhanced nuclear translocation of Nrf2 and up-regulated expression of phase II antioxidant enzymes including heme oxygenase-1 (HO-1). Treatment with SC-1 resulted in increased phosphorylation of p38 MAP kinase and transient increase in intracellular ROS levels. N-acetylcysteine (NAC) treatment abolished p38 phosphorylation as well as HO-1 induction caused by SC-1, indicating that ROS are upstream signals of p38 in Nrf2/ARE activation by SC-1. Furthermore, preconditioning with SC-1 attenuated H(2)O(2)-induced cell death in a dose-dependent manner. Finally, treatment with a HO-1 inhibitor, Zn-protoporphyrin (ZnPP), and overexpression of a dominant-negative mutant of Nrf2 diminished SC-1-mediated neuroprotection. Our results demonstrate that SC-1 is capable of protecting against oxidative stress-induced neuronal cell death in part through induction of HO-1 via Nrf2/ARE activation, suggesting its potential to reduce oxidative stress and ameliorate oxidative stress-related neurodegenerative diseases.     

A novel kavalactone derivative protects against H2O2-induced PC12 cell death via Nrf2/ARE activation

Oxidative stress is involved in the pathogenesis of neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases. Natural kavalactones isolated from Piper methysticum (Piperaceae) are capable of activating the Nrf2/ARE (antioxidant response element) pathway and thus enhancing the expression of phase II antioxidant enzymes such as heme oxygenase-1 (HO-1). In an attempt to identify kavalactone derivatives that are more potent in Nrf2/ARE activation than natural compounds, we synthesized a series of chemically-modified kavalactones and studied their effects on the ARE enhancer activity in rat pheochromocytoma PC12 cells. Among 81 compounds tested, a kavalactone derivative, 2′,6′-dichloro-5-methoxymethyl-5,6-dehydrokawain [(E)-6-(2′,6′-dichlorostyryl)-4-methoxy-5-(methoxymethyl)-2H-pyran-2-one] (1), exhibited the strongest ARE enhancer activity. The ARE activation and HO-1 protein induction by the compound 1 were higher than those by natural kavalactones. The compound did not affect cell viability and induced expression of various phase II enzymes. Nuclear translocation of Nrf2 after treatment with 1 was preceded by phosphorylation of ERK1/2 and p38. The compound transiently increased intracellular ROS levels. Finally, pretreatment with the compound ameliorated H₂O₂-induced cell death, which was associated with increased expression of HO-1. These results suggest that the compound 1 protects against oxidative stress-induced neuronal cell death via a preconditioning effect on the Nrf2/ARE activation.     

Preconditioning by sesquiterpene lactone enhances H2O2-induced Nrf2/ARE activation

The Nrf2/ARE pathway plays a pivotal role in chemoprevention and neuroprotection. Here, we report that sesquiterpene lactones extracted from Calea urticifolia and feverfew increased enhancer activity of the ARE. ARE activation was dependent on the number of α,β-unsaturated carbonyl groups each compound bears and calealactone A (CL-A) harboring 3 of those was the most potent ARE inducer. At subtoxic doses, CL-A induced expression of heme oxygenase-1 (HO-1) gene, one of ARE target genes, through activation of the Nrf2/ARE pathway involving transient ROS generation and activation of PI3-K/Akt and MAPK pathways. Interestingly, H₂O₂-induced ARE activation and HO-1 induction were potentiated by pretreatment with CL-A at lower concentrations, at which Nrf2/ARE activation by the compound was minimal. These results suggest a possibility that preconditioning by sesquiterpene lactone may enhance activation of the Nrf2/ARE pathway and induction of phase II detoxification/antioxidant enzymes upon oxidative stress, thereby resulting in increased resistance to oxidative damage.     

Nitric oxide stimulates Nrf2 nuclear translocation in vascular endothelium

Vascular endothelial cells respond to nitric oxide by activating MAPK pathways and upregulating stress-activated proteins such as gamma-glutamylcysteine synthetase (gamma-GCS) and heme oxygenase-1 (HO-1). Since consensus sequences for the antioxidant response element (ARE) are found in the promoters of the gamma-GCS and HO-1 genes, we examined nuclear translocation of Nrf2, a CNC-bZIP protein which binds to and activates the ARE. We found a dramatic increase in Nrf2 nuclear translocation 1-8h following the nitric oxide donor spermine NONOate. Translocation was inhibited by pretreatment of cells with N-acetylcysteine suggesting involvement of an oxidative mechanism in this response. Translocation was also blocked by PD 98059 and SB 203580, inhibitors of ERK and p38 pathways, respectively. In addition to effects on Nrf2 subcellular localization, spermine NONOate increased Nrf2 protein levels by a mechanism which was inhibited by PD 98059. Pretreatment with N-acetylcysteine, PD 98059, and SB 203580 decreased HO-1 upregulation in spermine NONOate-treated cells. These results suggest that ERK and p38 pathways may regulate nitric oxide-mediated adaptive responses in vascular endothelium via translocation of Nrf2 and activation of the ARE.     

Induction of glutathione synthesis and heme oxygenase 1 by the flavonoids butein and phloretin is mediated through the ERK/Nrf2 pathway and protects against oxidative stress

Butein and phloretin are chalcones that are members of the flavonoid family of polyphenols. Flavonoids have well-known antioxidant and anti-inflammatory activities. In rat primary hepatocytes, we examined whether butein and phloretin affect tert-butylhydroperoxide (tBHP)-induced oxidative damage and the possible mechanism(s) involved. Treatment with butein and phloretin markedly attenuated tBHP-induced peroxide formation, and this amelioration was reversed by l-buthionine-S-sulfoximine [a glutamate cysteine ligase (GCL) inhibitor] and zinc protoporphyrin [a heme oxygenase 1 (HO-1) inhibitor]. Butein and phloretin induced both HO-1 and GCL protein and mRNA expression and increased intracellular glutathione (GSH) and total GSH content. Butein treatment activated the ERK1/2 signaling pathway and increased Nrf2 nuclear translocation, Nrf2 nuclear protein-DNA binding activity, and ARE-luciferase reporter activity. The roles of the ERK signaling pathway and Nrf2 in butein-induced HO-1 and GCL catalytic subunit (GCLC) expression were determined by using RNA interference directed against ERK2 and Nrf2. Both siERK2 and siNrf2 abolished butein-induced HO-1 and GCLC protein expression. These results suggest the involvement of ERK2 and Nrf2 in the induction of HO-1 and GCLC by butein. In an animal study, phloretin was shown to increase GSH content and HO-1 expression in rat liver and decrease carbon tetrachloride-induced hepatotoxicity. In conclusion, we demonstrate that butein and phloretin up-regulate HO-1 and GCL expression through the ERK2/Nrf2 pathway and protect hepatocytes against oxidative stress.