Activation of the Phase II Enzymes for Neuroprotection by Ginger Active Constituent 6‑Dehydrogingerdione in PC12 Cells Juan Yao, Chunpo Ge, Dongzhu Duan, Baoxin Zhang, Xuemei Cui, Shoujiao Peng, Yaping Liu, and Jianguo Fang* State Key Laboratory of Applied Organic Chemistry and College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, Gansu 730000, China ABSTRACT: The cellular endogenous antioxidant system plays pivotal roles in counteracting or retarding the pathogenesis of many neurodegenerative diseases. Molecules with the ability to enhance the antioxidant defense thus are promising candidates for neuroprotective drugs. 6-Dehydrogingerdione (6-DG), one of the major components of dietary ginger, has received increasing attention due to its multiple pharmacological activities. However, how this pleiotropic molecule works on the neuronal system has not been studied. This paper reports that 6-DG efficiently scavenges various free radicals in vitro and displays remarkable cytoprotection against oxidative stress-induced neuronal cell damage in the neuron-like rat pheochromocytoma cell line, PC12 cells. Pretreatment of PC12 cells with 6-DG significantly up-regulates a panel of phase II genes as well as the corresponding gene products, such as glutathione, heme oxygenase, NAD(P)H:quinone oxidoreductase, and thioredoxin reductase. Mechanistic study indicates that activation of the Keap1-Nrf2-ARE pathway is the molecular basis for the cytoprotection of 6-DG. This is the first revelation of this novel mechanism of 6-DG as an Nrf2 activator against oxidative injury, providing the potential therapeutic use of 6-DG as neuroprotective agent. KEYWORDS: 6-dehydrogingerdione, Nrf2, oxidative stress, neuroprotection, antioxidant ■ INTRODUCTION The cellular endogenous antioxidant system is crucial for counteracting reactive oxygen species (ROS)-mediated oxida- tive stress and preventing cell death. A well-elucidated universal pathway to induce intrinsic antioxidant defense involves transcriptional regulation through activation of the antiox- idant-responsive element (ARE). 1,2 Under this circumstance, stimulants induce a set of genes encoding antioxidant and detoxifying enzymes (“phase II” enzymes), including heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO1), thioredoxin reductase 1 (TrxR1), thioredoxin (Trx), glutathione (GSH), γ-glutamylcysteine synthetase (γGCS), and γ-glutamyl cysteine ligase (γ-GCL), which provide efficient cytoprotection against oxidative stress and electrophilic assault. 3,4 The transcription factor NF-E2-related factor 2 (Nrf2) mediates transcription of phase II genes by binding to the ARE within nuclear DNA and subsequently initiates antioxidant genes transcription. Under basal conditions, the cytosolic regulatory protein Kelch-like ECH-associated protein 1 (Keap1) binds tightly to Nrf2, retaining it in the cytoplasm. 2 Molecules, such as electrophiles or oxidants, which could modify the critical cysteine residue(s) in the regulatory protein Keap1, would cause the release of Nrf2 and facilitate its translocattion into the nucleus and thus activate the tran- scription of phase II genes. 4-7 In healthy cells, the level of ROS is tightly regulated by the antioxidant defense system. However, upon environmental stress or cellular damage, cells cannot readily detoxify the ROS generated and may thereby suffer from oxidative stress. As neuronal cells are particularly sensitive to oxidative stress, an increasing amount of experimental evidence has indicated that oxidative stress is a causal, or at least an ancillary, factor in the neuropathology of several adult neurodegenerative disorders such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). 8,9 Besides the counteraction of various ROS by exogenous small molecule antioxidants, targeting the preven- tion of oxidative stress could be best achieved by stimulation of endogenous cytoprotective molecules known to serve this purpose because their actions are more sustained and are amplified by transcription-mediated signaling pathways. In this sense, activation of the Nrf2-ARE pathway is a promising therapeutic approach in neurodegenerative diseases. 3,10-12 Thus, the past years have witnessed expanding endeavors in identifying naturally occurring and synthetic neuroprotective small molecule activators of the Nrf2-ARE pathway, 13,14 including carnosic acid, 15 resveratrol, 16 curcumin, 17 sulfor- aphane, 18 quercetin, 19 epicatechin, 20 plumbagin, 21 luteolin, 22 and tert-butylhydroquinone. 18 Ginger (rhizome of the dietary plant Zingiber officinale) has been commonly used as a popular spice or food supplement and has been equally reputed for its medicinal properties for centuries. 23,24 6-Dehydrogingerdione (6-DG), one of the major components of dietary ginger, has received extensive attention due to its multiple pharmacological activities, such as inhibition of lipid peroxidation, 25 enhancement of skin cell proliferation, 26 and induction of cancer cell apoptosis. 27,28 However, how this pleiotropic molecule works on the neuronal system has not been elucidated. As part of our continuing effort in the discovery and development of novel redox active small Received: December 11, 2013 Revised: May 26, 2014 Accepted: May 28, 2014 Published: May 28, 2014 Article pubs.acs.org/JAFC © 2014 American Chemical Society 5507 dx.doi.org/10.1021/jf405553v | J. Agric. Food Chem. 2014, 62, 5507-5518