Activation of the Phase II Enzymes for Neuroprotection by Ginger

May 28, 2014 - ABSTRACT: The cellular endogenous antioxidant system plays pivotal roles in counteracting or retarding the pathogenesis of...
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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

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INTRODUCTION The cellular endogenous antioxidant system is crucial for counteracting reactive oxygen species (ROS)-mediated oxidative stress and preventing cell death. A well-elucidated universal pathway to induce intrinsic antioxidant defense involves transcriptional regulation through activation of the antioxidant-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 transcription 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 © 2014 American Chemical Society

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 prevention 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 sulforaphane,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: Revised: Accepted: Published: 5507

December 11, 2013 May 26, 2014 May 28, 2014 May 28, 2014 dx.doi.org/10.1021/jf405553v | J. Agric. Food Chem. 2014, 62, 5507−5518

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Scheme 1. Synthesis of 6-DG, M-6-DG, and DH-6-DGa

a The active motif of 6-DG is highlighted in circles. Reagents and conditions: (a) NaH, Et2O, 64%; (b) boron trioxide, 78%; (c) boron trioxide, 71%; (d) 10% Pd−C, H2, 70%.

molecules,29−32 we determined herein the antioxidant activity of 6-DG in vitro and employed a neuron-like rat pheochromocytoma cell line, PC12, to assess the molecular mechanisms responsible for the neuroprotective effect of 6-DG. 6-DG, prepared from the readily available starting materials in two steps, efficiently scavenges free radicals and prevents erythrocytes from free radical-induced hemolysis. Importantly, 6-DG activates the Nrf2-ARE signaling pathway, leading to upregulation of phase II enzyme expression and remarkable protection of PC12 cells from hydrogen peroxide (H2O2)- or 6hydroxydopamine (6-OHDA)-induced neurotoxicity. We clarified that the hydroxyl group and the α,β-unsaturated ketone moiety are determinants of the biological function of 6-DG. Our results demonstrate that 6-DG is a novel small molecule activator of Nrf2-ARE pathway and suggest that 6-DG might be a potential candidate for the prevention of neurodegeneration. Targeting the Nrf2-ARE signaling pathway by 6-DG thus discloses a previously unrecognized mechanism underlying the biological action of 6-DG and provides a deep insight into understanding the neuroprotective effects of 6-DG.

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literature with minor modifications. Briefly, acetone reacts with ethyl hexanoate under basic condition (Claisen condensation) and gives 2,4nonanedione in 64% yield. The dione reacting with vanillin and methylvanillin via Claisen−Schmidt condensation affords 6-DG and M-6-DG in 71 and 78% yields, respectively. Hydrogenation of 6-DG catalyzed by palladium on carbon furnished DH-6-DG in 70% yield. All compounds were fully characterized by NMR and MS. 2,4-Nonanedione.33 To a stirred mixture of sodium hydride (1.2 g, 50 mmol) and ethyl hexanoate (7.2 g, 50 mmol) was added acetone (1.51 g, 26 mmol) in 7 mL of dry diethyl ether over a period of 30 min. The temperature of the reaction mixture was kept at 30−40 °C by occasional cooling with ice water. After the acetone was added, the reaction mixture was stirred at 40−50 °C for 1 h. Diethyl ether was continuously added to maintain a fluid reaction mixture. After continuous stirring at room temperature overnight, the reaction was quenched by sequential addition of 8 mL of ethanol and 80 mL of 0.75 M HCl. Extraction with diethyl ether (50 mL × 3) and removal of the solvent under vacuum gave a pale yellow oil, which was added to a hot solution of CuSO4·5H2O (12.4 g, 40 mmol) in 60 mL of water. After cooling, crystals were collected and washed with 100 mL of hexane and further dissolved in 50 mL of 10% H2SO4. The resulting solution was extracted with diethyl ether and dried over Na2SO4. After removal of the solvent, the crude product purified by column chromatography on silica gel gave 2,4-nonanedione (64%). 1H NMR (400 MHz, CDCl3), δ 5.49 (s, 1H), 2.23−2.27 (t, 2H), 2.05 (s, 3H), 1.58−1.62 (m, 2H), 1.30−1.32 (m, 4H), 0.88−0.91 (t, 3H); ESI-MS (M + 1)+, 157.2. 6-DG.34 Vanillin (6.3 g, 42 mmol), 2,4-nonanedione (19.3 g, 124 mmol), and boron trioxide (11.5 g, 165 mmol) were mixed with 50 mL of N,N-dimethylformamide (DMF) and warmed to 90 °C. A solution of isobutylamine (40 mg, 0.4 mmol) in 40 mL of DMF was added dropwise for 2 h. After stirring at 85−90 °C for 1 h, ∼60 mL of the solvent was evaporated in vacuo, and 250 mL of water was added. The resulting mixture was stirred at 60 °C for 1 h and at room temperature for 12 h. The reaction mixture was extracted with ethyl acetate (50 mL × 3), and the solvents were evaporated. The crude product was purified by column chromatography on silica gel to afford 6-DG (71%). mp, 84 °C; 1H NMR (400 MHz, CDCl3), δ 7.51−7.55 (d, J = 16 Hz, 1H), 7.09−7.11 (d, J = 8 Hz, 1H), 7.03 (s, 1H), 6.92− 6.94 (d, J = 8 Hz, 1H), 6.33−6.37 (d, J = 16 Hz, 1H), 5.83 (s, 1H), 5.63 (s, 1H), 3.95 (s, 3H), 2.36−2.40 (t, 2H), 1.63−1.70 (m, 2H), 1.33−1.36 (m, 4H), 0.90−0.93 (t, 3H); ESI-MS (M + 1)+, 291.2. M-6-DG. The synthesis of M-6-HG exactly followed the procedure described for 6-DG except for the use of methylvanillin (6.9 g, 42 mmol) instead of vanillin. Yield, 78%; mp, 45 °C; 1H NMR (400 MHz, CDCl3), δ 7.53−7.57 (d, J = 16 Hz, 1H), 7.11−7.13 (m, 1H), 7.06 (s, 1H), 6.87−6.89 (d, J = 8 Hz, 1H), 6.35−6.39 (d, J = 16 Hz, 1H), 5.64 (s, 1H), 3.89 (s, 6H), 2.37−2.41(t, 2H), 1.26−1.34 (m, 6H), 0.9−0.93 (t, 3H); ESI-MS (M + 1)+, 305.2.

MATERIALS AND METHODS

Chemicals and Enzymes. Dulbecco’s modified Eagle’s medium (DMEM), N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNA), dimethyl sulfoxide (DMSO), yeast glutathione reductase (GR), Hoechst 33342, 2′,7′-dichlorfluorescein diacetate (DCFH-DA), 2,2′azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), 2,2-diphenyl1-picrylhydrazyl (DPPH), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), 2,6-dichlorophenol-indophenol (DCPIP), NADH, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), penicillin, and streptomycin were obtained from Sigma-Aldrich (St. Louis, MO, USA). NADPH was obtained from Roche (Mannheim, Germany). Fetal bovine serum (FBS) was obtained from HyClone. 6-Hydroxydopamine (6-OHDA) and antibodies against TrxR1, Trx1, actin, and Nrf2 were from Santa Cruz Biotechnology. Antibodies against HO-1 and NQO1 were from Sangon Biotech (Shanghai, China). The shRNA plasmids targeting coding regions of the rat Nrf2 gene (shNrf2) and the control nontargeting shRNA (shNT) were purchased from GenePharma Co, Ltd. (Shanghai, China). GeneTran III transfection reagent was obtained from Biomiga (San Diego, CA, USA). Bovine serum albumin (BSA), phenylmethanesulfonyl fluoride (PMSF), and sodium orthovanadate (Na3VO4) were from Beyotime (Nantong, China). All other reagents were of analytical grade. Synthesis of 6-DG, Methylated 6-DG (M-6-DG), and Hydrogenated 6-DG (DH-6-DG). 6-DG, M-6-DG, and DH-6-DG described in this study were prepared using a straightforward chemistry (Scheme 1) by following the method described in the 5508

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followed by the addition of 600 μM H2O2 for an additional 12 h or 200 μM 6-OHDA for an additional 24 h. The leakage of LDH from cultured cells was quantified by measuring LDH activity in culture media. Briefly, 50 μL of culture medium was mixed with 110 μL of 100 mM Tris-HCl (pH 7.4) containing 20 μL of 2 mM NADH and 20 μL of 20 mM pyruvate, and the A340 nm change was monitored every 20 s for 5 min. The content of LDH release was calculated by normalizing the LDH activity of samples to that measured in culture medium from the control cells. Hoechst 33342 Staining and Assessment of the Intracellular ROS.29,30 PC12 cells (2 × 105 cells/well) were seeded into 12-well plates. After 24 h, the cells were treated with 6-DG for another 24 h followed by replacement with fresh medium containing 600 μM H2O2 for 12 h or 200 μM 6-OHDA for 24 h. Hoechst 33342 was subsequently added to a final concentration of 5 μg/mL to stain the nuclei. The cells were visualized and photographed under a Leica inverted fluorescent microscope. For assaying the ROS, the medium was removed, and DCFH-DA in fresh FBS-free medium was added (10 μM); incubation was continued for 30 min at 37 °C in the dark. The cells were visualized and photographed under fluorescent microscopy. Measurement of Caspase-3 Activity. PC12 cells (1 × 106 cells/ well) were seeded in 60 mm dishes for 24 h and then treated with different concentrations of 6-DG for another 24 h. After replacement with fresh medium containing 600 μM H2O2 or 200 μM 6-OHDA for 24 h, the cells were lysed with RIPA buffer. The protein content was quantified using the Bradford procedure. The caspase-3 activity in the lystae was determined by following the published procedure.38 Briefly, a cell extract containing 15 μg of total proteins was incubated with the assay mixture (50 mM Hepes, 2 mM EDTA, 5% glycerol, 10 mM DTT, 0.1% CHAPS, 0.2 mM Ac-DEVD-pNA, pH 7.5) for 4 h at 37 °C in a final volume of 100 μL. The absorbance at 405 nm was measured using a microplate reader. The same amounts of DMSO were added to the control experiments, and the activity was expressed as the percentage of the control. Measurement of Total Glutathione, NQO1 Activity, Trx Activity, and TrxR Activity. PC12 cells (1 × 106 cells/well) were seeded in 60 mm dishes for 24 h and treated with different concentrations of 6-DG for another 24 h. After replacement with fresh medium containing H2O2 (600 μM) for 12 h or 6-OHDA (200 μM) for 24 h, cells were collected and resuspended in extraction buffer containing 0.1% Triton X-100 and 0.6% sulfosalicyclic acid in 0.1 M PBS with 5 mM EDTA, pH 7.5 (KPE buffer). The suspension was sonicated on ice for 2−3 min with vortexing every 30 s. The cells were centrifuged at 3000g for 4 min at 4 °C, and the supernatant was ready for the total GSH assay by enzymatic detection as described in the published protocols.39 A 120 μL solution containing 0.33 mg/mL DTNB and 1.66 units/mL glutathione reductase was added to each sample (20 μL). β-NADPH (60 μL of 0.66 mg/mL) was then added quickly, and the absorbance at 412 nm was measured every 15 s for 2 min. The rate of chromophore production at 412 nm is proportional to the concentration of glutathione within the sample. The total glutathione content in the samples was determined by comparison with the predetermined glutathione standard curve. For measuring NQO1, Trx, and TrxR activity, the cells were lysed in RIPA buffer. The total protein content was quantified using the Bradford procedure. The NQO1 activity was determined spectrophotometrically by monitoring the reduction of the electron acceptor, DCPIP, at 600 nm.40 The enzymatic reaction was initiated by the addition of cell lysate (5 μg of total protein) to the reaction mixture (20 mM TrisHCl, pH 7.4, 200 μM NADH, and 40 μM DCPIP), and the decrease in absorbance at 600 nm was measured every 8 s for 2 min at room temperature in the presence or absence of 20 μM dicoumarol. The dicoumarol-inhibitable part of DCPIP’s reduction was used to calculate the NQO1 activity. The same amounts of DMSO were added to the control experiments, and the activity was expressed as the percentage of the control. The Trx and TrxR activities in the lysate were determined by following the published protocols.41 To determine the activity of TrxR, the cell extract containing 20 μg of total proteins was incubated in a final reaction volume of 50 μL containing 100 mM

DH-6-DG.31 To a mixture of 10 mg of Pd/C (10%) in 5 mL of ethyl acetate under a hydrogen atmosphere was added 10 mg of 6-DG. The reaction was kept at room temperature for 12 h. After the reaction, the Pd/C was removed by filtration, and the filtrate was concentrated and purified by silica gel column chromatography to afford DH-6-DG (70%). 1H NMR (400 MHz, CDCl3), δ 6.85−6.83 (d, J = 8 Hz, 1H), 6.70−6.68 (d, J = 8 Hz, 2H), 5.51 (s, 1H), 5.46 (s, 1H), 3.87 (s, 3H), 2.89−2.83 (m, 2H), 2.59−2.55 (t, 2H), 2.28−2.24 (t, 2H), 1.32−1.26 (m, 6H), 0.92−0.87 (m, 3H); ESI-MS (M + Na)+, 315.3. Free Radical-Scavenging Capacities. The various concentrations of antioxidants were mixed with the DPPH solution (50 μM), and the mixture was shaken vigorously and left to stand at room temperature for 30 min in the dark. The absorbance of the reaction solution was measured spectrophotometrically at 517 nm. The ABTS•+ scavenging ability was determined according to the published method.35 The ABTS•+ solution, which was prepared from oxidation of ABTS (7 mM) by potassium persulfate (2.5 mM) for 12−16 h in the dark at room temperature, was diluted to an absorbance of 0.700 ± 0.020 at 734 nm. The ABTS•+ solution (2 mL) and the antioxidant solution (100 μL) were mixed, and the absorbance at 734 nm was recorded after 30 min. Assay for Hemolysis of Red Blood Cells (RBCs) and Thiobarbituric Acid Reactive Substances (TBARS). Human RBCs were separated from heparinized blood that was drawn from a healthy donor. The 5% suspension of RBC in PBS (pH 7.4) was incubated under air at 37 °C for 5 min into which a PBS solution of AAPH was added to initiate hemolysis. The extent of hemolysis was determined spectrophotometrically as described previously.36 Briefly, aliquots of the reaction mixture were taken out at appropriate time intervals, diluted with 0.15 M NaCl, and centrifuged at 3000 rpm for 5 min to separate the RBCs. The percentage hemolysis was determined by measuring the absorbance of the supernatant at 540 nm and compared with that of complete hemolysis by treating the same RBC suspension with distilled water. For measurements of TBARS, the erythrocyte ghost was incubated at 37 °C in 0.1 M PBS (pH 7.4) and diluted to a final protein concentration of 0.96 mg/mL. The peroxidation was initiated by the addition of AAPH. The reaction mixture was gently shaken at 37 °C, and aliquots of the reaction mixture were taken out at specific intervals to which a TCA−TBA− HCl stock solution (15% w/v trichloroacetic acid; 0.375% w/v TBA; 0.25 M HCl) was added, together with 0.02% w/v BHT. This amount of BHT completely prevents the formation of any nonspecific TBARS from the decomposition of AAPH during the subsequent boiling. The solution was heated in a boiling water bath for 15 min. After cooling, the precipitate was removed by centrifugation. TBARS in the supernatant were quantified at 532 nm using the extinction coefficient of 1.56 × 105 M−1 cm−1.37 Cell Cultures. The rat adrenal pheochromocytoma cell line, PC12 cells, was obtained from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, and was cultured in DMEM supplemented with 10% FBS, 2 mM glutamine, and 100 units mL−1 penicillin/streptomycin and maintained in an atmosphere of 5% CO2 at 37 °C. MTT Assay and Lactate Dehydrogenase (LDH) Release Assay. To evaluate the cytotoxicity, PC12 cells (1 × 104 cells/well) were seeded in 96-well plates for 1 day followed by incubation with 6DG or other agents for 24 h at 37 °C. For the H2O2 or 6-OHDA injury model, PC12 cells (1 × 104 cells/well) were plated in a 96-well plate and allowed to adhere for 24 h and then treated with the drugs for 24 h. After replacement with fresh medium containing 600 μM H2O2 for 12 h or 200 μM 6-OHDA for 24 h, cell viability was determined by MTT assay. At the end of the treatment, 10 μL of MTT (5 mg/mL) was added to each well and incubated for an additional 4 h at 37 °C. One hundred microliters of extraction buffer (10% SDS, 5% isobutanol, 0.1% HCl) was added, and the cells were incubated overnight at 37 °C. Cells treated with DMSO alone were used as controls. The absorbance was measured at 570 nm on a Multiskan GO (Thermo Scientific). For the LDH release assay, the cells (2 × 105 cells/well) were plated in 12-well plates. The following day, the cells were exposed to various concentrations of 6-DG for 24 h, 5509

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Figure 1. Antioxidant activity of 6-DG in vitro: (A) DPPH and (B) ABTS•+ scavenging abilities measured as described under Materials and Methods; (C) inhibition of TBARS formation during the AAPH-induced peroxidation of erythrocyte ghost by 6-DG; (D) protection of 6-DG against AAPH-induced hemolysis of RBC. Data represent the mean ± SD of three independent experiments. Tris-HCl (pH 7.6), 0.3 mM insulin, 660 μM NADPH, 3 mM EDTA, and 15 μM Escherichia coli Trx for 30 min at 37 °C. To determine the activity of Trx, the cell extract containing 20 μg of total proteins was incubated in a final reaction volume of 50 μL containing 100 mM TrisHCl (pH 7.6), 0.3 mM insulin, 660 μM NADPH, 3 mM EDTA, and 60 nM recombinant rat TrxR1 for 30 min at 37 °C. The reaction was terminated by adding 200 μL of 1 mM DTNB in 6 M guanidine hydrochloride, pH 8.0. A blank sample, containing everything except Trx (for TrxR assay) or TrxR (for Trx assay), was treated in the same manner. The absorbance at 412 nm was measured, and the blank value was subtracted from the corresponding absorbance value of the sample. The same amounts of DMSO were added to the control experiments, and the activity was expressed as the percentage of the control. Real-Time Reverse Transcription-PCR (RT-PCR). PC12 cells (1 × 106 cells/well) were seeded in 60 mm dishes for 24 h and treated with 10 μM 6-DG for 0, 6, 12, and 24 h. Total RNA was isolated from cells using the RNAiso plus (TaKaRa, Dalian, China) according to the manufacturer’s protocol and quantified through 260/280 absorbance. Reverse transcription was performed using Primescript RT reagent kit according to the manufacturer’s protocol (TaKaRa). RT-PCR was performed on an Mx3005P RT-PCR System (Agilent Technologies) using SYBR Green PCR Master Mix (Takara) . The GAPDH was used as an internal control. Target gene expression was measured and was normalized to the GAPDH expression level. PCR primers specific to each gene are as follows: GAPDH, 5′-cagtgccagcctcgtctcat-3′ and 5′aggggccatccacagtcttc-3′; HO-1, 5′-gccctggaagaggagatagag-3′ and 5′tagtgctgtgtggctggtgt-3′; NQO1, 5′-tcaccactctactttgctccaa-3′ and 5′ttttctgctcctcttgaacctc-3′; Trx1, 5′-ccttctttcattccctctgtgac-3′ and 5′cccaaccttttgaccctttttat-3′; TrxR1, 5′-actgctcaatccacaaacagc-3′ and 5′ccacggtctctaagccaatagt-3′; GCLC, 5′-caaggacaagaacacaccatct-3′ and 5′-

cagcactcaaagccataacaat-3′; GCLM, 5′-ggcacaggtaaaacccaatagt-3′ and 5′-ttcaatgtcagggatgctttct-3′. GAPDH was used as an internal control. Target gene expression was measured and was normalized to the GAPDH expression level. Preparation of Different Protein Extracts for Western Blot Analysis.42 For the whole cell protein extraction, PC12 cells were lysed with RIPA buffer. For the cytosolic and nuclear extracts, the cells were rinsed with ice-cold PBS and resolved in 100 μL of buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 μM DTT, and 1 μM protease inhibitor cocktail freshly added to buffer A). After 15 min of incubation, 10 μL of Nonidet-P40 (10%) was added, and the mixture was vortexed for 15 s. Thereafter, the lysate was repeatedly centrifuged for 10 min (1000g, 4 °C) to separate the nuclear from the cytosolic fraction. The pellet was resuspended in 100 μL of buffer B (20 mM Hepes, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 μM DTT, and 1 μM protease inhibitor cocktail were freshly added to buffer B) and incubated on ice for 15 min, and the mixture was vortexed for 10−15 s every 2 min. After a final centrifugation step for 10 min (20000g, 4 °C), the supernatant was collected as the nuclear extract. The band intensity was analyzed and quantified by Image pro plus (IPP) software. Knockdown of Nrf2 Expression Using Short Hairpin RNAs. Three shRNAs (shNrf2-842, shNrf2-184, and shNrf2-136) targeting rat Nrf2 gene and one shRNA with scrambled sequence (shNT) as control were used for Nrf2 knockdown experiments. Exponentially growing cells were transfected with different shRNAs using GeneTran III transfection reagent according to the manufacturer’s instruction. After 48 h of transfection, the cells were maintained in DMEM, 10% FBS, 2 mM glutamine, 100 units/mL penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO 2 and selected by 5510

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Figure 2. Protection of 6-DG against (A) H2O2- and (B) 6-OHDA-induced PC12 cell damage. PC12 cells (1 × 104 cells/well) were plated in a 96well plate for 1 day and subsequently treated with 6-DG for another 24 h. After replacement of the medium containing H2O2 or 6-OHDA and continued incubation for indicated times, cell viability was measured by MTT assay. The content of LDH in the medium after H2O2 (C) or 6OHDA (D) treatment was determined as described under Materials and Methods. All data represent the mean ± SD of three independent experiments. (∗) P < 0.05 and (∗∗) P < 0.01 versus the vehicle group; (#) P < 0.05 and (##) P < 0.01 versus the H2O2- or 6-OHDA-treated group. supplementation with 0.5 mg/mL of G418. Knockdown of the Nrf2 expression in the cells was analyzed by Western blotting. Statistics. Data are presented as the mean ± standard deviation (SD). Statistical differences between two groups were assessed by Student’s t test. Comparisons among multiple groups were performed using one-way analysis of variance (ANOVA), followed by a post hoc Scheffe test. P < 0.05 was used as the criterion for statistical significance.

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activity in vitro, we next investigated if 6-DG has a protective effect against oxidative stress-induced neuronal cell death. 6-DG shows weak cytotoxicity toward PC12 cells, and significant cytotoxicity was observed only at high concentrations (>50 μM, data not shown). As shown in Figure 2A, compared with the control group, PC12 cells treated with H2O2 for 12 h showed about 60% cell death. However, pretreatment of cells with 6DG at nontoxic concentrations (1−10 μM) for 24 h followed by H2O2 challenge significantly decreased the population of dead cells to about 30%. This protection was observed even at 1 μM 6-DG. However, hydrogenation of 6-DG to DH-6-DG almost completely blocked the protection, indicating the importance of the α,β-unsaturated ketone structure in 6-DG. The neurotoxin 6-OHDA has been widely used to generate an experimental model of PD. 6-OHDA-mediated neurotoxicity is engendered, at least in part, by generation of ROS.43 6-OHDA treatment reduced cell viability to about 60% of control (Figure 2B). Similarly, addition of 6-DG also markedly promoted cell viability to >80% of control. LDH is a soluble cytosolic enzyme that is released into the culture medium following loss of membrane integrity resulting from cell damage. To confirm the neuroprotection of 6-DG, we determined the content of LDH leakage after H2O2 or 6-OHDA insult (Figure 2C,D). Both H2O2 and 6-OHDA cause ∼3-fold increase of LDH release. Pretreatment of PC12 cells with 6-DG significantly reduces the leakage of LDH. Taken together, 6-DG at nontoxic concentration can significantly protect PC12 cells from H2O2and 6-OHDA-induced cell damage. Alleviation of H2O2- and 6-OHDA-Induced PC12 Cell Apoptosis by 6-DG. Apoptosis is a precisely regulated process of cell destruction with specific defining morphological and molecular features. Two known apoptotic signaling

RESULTS

Scavenging of Free Radicals in Vitro. Previous studies have indicated that 6-DG is an effective antioxidant.25 We further investigated herein the potency of 6-DG in scavenging free radicals in vitro. As shown in Figure 1A,B, 6-DG dosedependently neutralizes DPPH and ABTS free radicals. To address the role of the hydroxyl group in scavenging radicals, we prepared M-6-DG, the methylated form of 6-DG. M-6-DG completely lost the ability to quench DPPH, whereas it retained partial capacity to scavenge ABTS. The remaining activity might be due to the 1,3-diketone structure in 6-DG as this moiety could also quench radicals.25 Our results demonstrated the critical role of the hydroxyl group in scavenging free radicals. Thermal decomposition of AAPH in aqueous solution under aerobic condition constantly generates peroxyl radicals,36 which attack membrane lipids to induce lipid peroxidation. We found 6-DG efficiently protects the erythrocyte ghost from AAPH-induced formation of TBARS, a biomarker of lipid peroxidation (Figure 1C). In the model of AAPH-induced hemolysis of intact human RBC, 6-DG remarkably retards the hemolysis with a clear concentration dependence (Figure 1D). Protection of PC12 Cells from H2O2- and 6-OHDAInduced Damage. As 6-DG demonstrates potent antioxidant 5511

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Figure 3. Inhibition of H2O2- or 6-OHDA-induced apoptosis in PC12 cells. PC12 cells were treated as described under Materials and Methods, and the morphological changes of nuclei were determined by Hoechst 33342 staining (A, C). Activation of the cellular caspase-3 was determined by a colorimetric assay as described under Materials and Methods. Data represent the mean ± SD of three independent experiments. (∗∗) P < 0.01 versus the vehicle group; (#) P < 0.05 and (##) P < 0.01 versus the H2O2- or 6-OHDA-treated group.

Figure 4. Prevention of ROS accumulation in PC12 cells by 6-DG. Pretreatment with 6-DG significantly alleviates ROS accumulation induced by H2O2 (A) or 6-OHDA (B) determined by DCFH-DA staining as described under Materials and Methods. The phase contrast (top panel) and fluorescence (bottom panel) images were acquired by inverted fluorescence microscopy.

pathways, the extrinsic cell surface receptor pathway44 and the intrinsic mitochondrial pathway,45 converge on the caspase activation.46 As shown in Figure 3A,C, both H2O2 and 6OHDA elicit apoptosis in PC12 cells evidenced by the appearance of apoptotic nuclei as highly fluorescent, condensed bodies (indicated by arrows), whereas no apoptotic nuclei were observed in control cells. Pretreatment of PC12 cells with 6DG remarkably lowers the population of apoptotic nuclei. Activation of caspase-3 is a biochemical character in all apoptotic cells.47 We thus further quantified the level of caspase-3 activation. Both H2O2 and 6-OHDA activate the

cellular caspase-3, but with different potencies (Figure 3B,D). Again, 6-DG significantly alleviates the extent of caspase-3 activation in a concentration-dependent manner. Collectively, 6-DG, at nontoxic concentrations, can alleviate H2O2- and 6OHDA-induced apoptosis in PC12 cells. Inhibition of ROS Accumulation in PC12 Cells. DCFHDA is a cell membrane permeable dye. After diffusing into cells, DCFH-DA is deacetylated by cellular esterases to nonfluorescent 2′,7′-dichlorodihydrofluorescin, which is rapidly converted to highly fluorescent 2′,7′-dichlorofluorescein upon reacting with ROS. Stimulation of the cells with H2O2 or 65512

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activities were significantly increased (Figure 6E,H,K). After 6-DG (10 μM) treatment, the total GSH level (Figure 6L), NQO1 activity (Figure 6E), Trx activity (Figure 6H), and TrxR activity (Figure 6K) were increased by ∼1.6-, ∼2-, ∼1.7-, and ∼1.3-fold, respectively. H2O2 and 6-OHDA treatments caused serious GSH depletion (Figure 6M,N), whereas pretreatment with 6-DG gradually restored the total GSH level. Promotion of Nrf2 Nuclear Localization by 6-DG. Induction of antioxidant gene expression via the Nrf2dependent cytoprotective pathway requires translocation of Nrf2 from cytosol to nucleus. We therefore examined whether 6-DG could activate the Nrf2 signaling pathway in PC12 cells. Upon 6-DG treatment, immunoblotting results revealed that the Nrf2 expression slightly increased with peaking at 4 h and gradually decreased to the basal level (Figure 7A). Notably, the cytosolic Nrf2 decreased progressively, in coincidence with its continuous rise in the nucleus (Figure 7B,C), indicating the translocation of Nrf2 from cytosol to nucleus. The accumulation of Nrf2 in nuclei thus facilitates its binding to ARE and subsequently initiates the transcription process. Involvement of Nrf2 in Neuroprotection of 6-DG. To further clarify whether 6-DG protects PC12 cells via the Nrf2 pathway, we conducted Nrf2 knockdown experiments in PC12 cells. The knockdown efficiency of different shRNAs (shNrf2842, shNrf2-184, shNrf2-136, and shNT) was validated by Western blots (Figure 8A). The expression of Nrf2 protein in all cells transfected with shNrf2s was drastically decreased. Next, we evaluated the protective effect of 6-DG against oxidative insult toward the transfected cells. In line with the cytoprotective function of Nrf2, silence of Nrf2 expression slightly increased the cytotoxicity of H2O2 and 6-OHDA (Figure 8B,C). 6-DG displays a similarly protective pattern in PC12-shNT cells as observed in normal PC12 cells. However, this protection drops remarkably in PC12 cells transfected with different shNrf2 plasmids, and the most significant results were observed in PC12-shNrf2-842 cells, where the protection of 6DG is almost completely abolished upon either H2O2- or 6OHDA-induced cell death. In PC12-shNrf2-184 and PC12shNrf2-136 cells, the protection of 6-DG is only partially lost. This could be due to the variation of the silence efficiency in cells. Although the protection of 6-DG declines to variable levels in different Nrf2 knockdown cells, the extents of all the decrease are significant. Taken together, these results indicate that the cytoprotection of 6-DG in PC12 cells was mediated through the action of Nrf2.

OHDA leads to an intracellular burst of ROS, whereas the control cells display negligible ROS level (Figure 4). Pretreatment of the cells with 6-DG dose-dependently reduces the ROS accumulation. As oxidative stress, arising from the overproduction of ROS, has been implicated in a final common pathway for neurotoxicity in a wide variety of acute and chronic neurological disorders, the inhibition of ROS accumulation in neuronal cells might be the molecular basis of the neuroprotective action of 6-DG. Induction of Phase II Gene Expression. Analysis of the chemical structure of 6-DG reveals that there is one Michael acceptor moiety (highlighted in a circle in Scheme 1), which is a core structure of many ARE inducers.5,10,13 In analogy to known ARE activators, we thus hypothesized that 6-DG might activate the cellular ARE response. We measured the expression of Nrf2-driven antioxidant genes (HO-1, NQO1, Trx1, TrxR1, GCLC, and GCLM) after treatment with 6-DG (10 μM) in PC12 cells (Figure 5). The time course studies showed the

Figure 5. Induction of cytoprotective genes in PC12 cells by 6-DG. The cells were treated as described under Materials and Methods, and the mRNA levels of different genes were analyzed and normalized using GAPDH as an internal standard by qRT-PCR as described. Data represent the mean ± SD of three independent experiments. (∗∗) P < 0.01 versus the control group (0 h).

highest induction of all genes at 6 h after stimulation with 6DG. A >10-fold increase of the HO-1 mRNA was observed after 6 h of treatment, consistent with HO-1 being a sensitive marker for oxidative/electrophilic stress. The expression of the genes decreased after 6 h, but was still significantly higher at 12 h compared to the control. After treatment with 6-DG for 24 h, the mRNA levels of HO-1 and TrxR1 were back to the control level, whereas the others remained significantly elevated. Taken together, these results indicate that 6-DG is a potent activator of Nrf2-regulated antioxidant defenses. Up-regulation of the Antioxidant Defense System in PC12 Cells. The induction of phase II detoxifying enzymes is an important defense mechanism against exogenous insult. As a series of phase II genes has been up-regulated at different levels, we then examined the corresponding gene products. 6-DG dose-dependently elevated HO-1, NQO1, Trx1, and TrxR1 protein expression and cellular GSH level in PC12 cells (Figure 6). By adopting the convenient assays, we further determined the cellular activity of NQO1, Trx1, and TrxR1. In line with the up-regulation of protein expression, all of the enzymes’

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DISCUSSION ROS are constantly produced under physiological metabolisms, and the intricate balance between their production and neutralization is controlled dynamically by the cellular antioxidant defense system. Oxidative stress, arising from excessive production of ROS, causes damage to proteins, lipids, and nucleic acids and eventually entails cell death. Neuronal cells in the brain are particularly vulnerable to oxidative stress as they possess high concentrations of oxidizable unsaturated fatty acids and high levels of pro-oxidants (e.g., iron) and show high oxygen consumption and low levels of antioxidant defense capacities.48 Consistent with the above-described sense, there has been increasing evidence underpinning oxidative stress as a major cause in a number of neurodegenerative disorders such as AD and PD.8,9 Treatment through replacing the dying or dead neurons in these diseases has been demonstrated with limited success.49 Therefore, pharmacological interventions aiming at 5513

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Figure 6. Up-regulation of antioxidant enzymes by 6-DG in PC12 cells. (A) Induction of HO-1 expression by 6-DG. (B) Quantification of the band intensity in panel A. (C) Induction of NQO1 expression and enzyme activity (E) by 6-DG. (D) Quantification of the band intensity in panel C. (F) Induction of Trx1 expression and total Trx enzyme activity (H) by 6-DG. (G) Quantification of the band intensity in panel F. (I) Induction of TrxR1 expression and total TrxR enzyme activity (K) by 6-DG. (J) Quantification of the band intensity in panel I. (L) 6-DG induces up-regulation of GSH in PC12 cells. (M, N) Prevention of H2O2- or 6-OHDA-induced GSH depletion by 6-DG. The total intracellular GSH was measured as described under Materials and Methods. Data are expressed as the mean ± SD of three experiments. (∗) P < 0.05 and (∗∗) P < 0.01 versus the vehicle group; (#) P < 0.05 and (##) P < 0.01 versus the H2O2- or 6-OHDA-treated group.

alleviating the oxidative stress are promising to confer protection to neuronal cells, eventually leading to retardation or blockage of the progression of these neurodegenerative diseases. Tactics against oxidative stress include direct antioxidant action by ROS neutralization, as well as indirect

antioxidant defense by awakening the pathway(s) involved in the expression of endogenous cytoprotective molecules. In this regard, accumulating attention has been paid to discovering antioxidants or molecules that can induce endogenous 5514

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Figure 7. Promotion of Nrf2 nuclear accumulation by 6-DG. After the cells were treated with 6-DG (10 μM) for 0, 2, 4, 6, or 8 h, the cells were harvested and different cell extracts were prepared as described. Total Nrf2 (A), cytosolic Nrf2 (B), and nuclear Nrf2 (C) were analyzed by Western blotting with an antibody against Nrf2. Actin was used as a loading control. Quantification of the blots is presented in the right-hand panel. Data are expressed as the mean ± SD of three experiments. (∗) P < 0.05 and (∗∗) P < 0.01 versus the control group (0 h).

low as 1 μM, could significantly protect neuronal cells from oxidative insult. This concentration could be easily reached by daily intake of ginger or ginger products. The contribution of 6DG to the observed activities of ginger and the physiological significance of the neuroprotective action of 6-DG require further investigation. 6-DG both acts as a direct antioxidant in scavenging free radicals in vitro and confers protective effects against oxidative stress-induced neuronal cell damage by activating the cellular antioxidant defense systems. As a phenolic compound, the direct antioxidant action depends primarily on the ability of 6DG to donate its phenolic H atom to different radicals via a hydrogen abstraction reaction. Our result that M-6-DG almost completely lost free radical-scavenging activity (Figure 1A,B) supports that the hydroxyl group in 6-DG plays a key role in the direct antioxidant activity. More importantly, 6-DG, via

antioxidant defense as potential therapeutic agents for neurodegenerative diseases.13,16,50,51 This study demonstrated for the first time that 6-DG, an active constituent from dietary plant ginger, was a promising neuroprotective agent. Although 6-gingerol and 6-shogaol are two major components of ginger, the amount of 6-DG in ginger is also significantly high (up to 20 mg/100 g ginger).52 Compared to 6-shogaol and 6-DG, 6-gingerol appears to have lower biological activity in different model systems.53−57 6-DG has a larger conjugated system than 6-shogaol and contains one additional active 1,3-diketone moiety, which could explain why 6-DG displays better antioxidant activity than 6-shogaol does.53,54 Furthermore, 6-DG shows lower cytotoxicity than 6-shogaol.55 The high antioxidant activity and low cytotoxicity make 6-DG an ideal molecule for further development as a therapeutic agent. In our present work, we found that 6-DG, as 5515

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Figure 8. Involvement of Nrf2 in the cytoprotection of 6-DG in PC12 cells. (A) Evaluation of Nrf2 knockdown efficiency by different shRNAs. PC12 cells were transfected with shRNAs specifically targeting rat Nrf2 gene (shNrf2s) or nontargeting shRNA (shNT), and the efficiency of silencing Nrf2 was evaluated by Western blots. (B, C) Impairment of cytoprotection of 6-DG in Nrf2-knockdown cells. Different PC12 cells were treated with 6-DG for 24 h followed by treatment with 600 μM H2O2 for 12 h or 200 μM 6-OHDA for 24 h. Cell viability was determined by MTT assay. Results are represented as the mean ± SD (n = 6). (∗∗) P < 0.01 and (∗) P < 0.05.

Figure 9. Dual neuroprotective mode of 6-DG in PC12 cells.

cytoprotection of 6-DG is mediated, at least in part, via Nrf2 activation. Our findings suggest a dual neuroprotective mechanism of 6DG shown schematically in Figure 9. The lipophilic character of 6-DG allows it to easily pass the plasma membrane and accumulate within cells. As a phenolic compound, 6-DG may directly counteract against various ROS. Furthermore, 6-DG, like many other electrophiles,5,10,59 could selectively interact with the Cys residue(s) on the cytosolic protein Keap1, which is an adaptor protein for ubiquitinating Nrf2 and, thus, drives the continuous degradation of Nrf2. The binding of 6-DG to Keap1 perturbs the Keap1-Nrf2 complex and stabilizes Nrf2, causing the liberation of Nrf2 and enabling it to be translocated into nucleus, where it binds to ARE and stimulates the transcription of cytoprotective genes. Thus, 6-DG provides dual protection against oxidative stress-induced neuronal cell damage. In conclusion, we have demonstrated that 6-DG was effective in preventing oxidative stress-induced neuronal cell damage. This neuroprotection may involve its capacity in directly neutralizing free radicals and activating endogenous cellular antioxidant defense. The hydroxyl group is critical for the direct antioxidant activity, whereas the α,β-unsaturated ketone structure is indispensable for activation of the Keap1-Nrf2ARE pathway in cells. Our discovery will provide deep insights in understanding 6-DG effects in vivo, and this protective

activation of the Keap1-Nrf2-ARE pathway, awakens the endogenous antioxidant defense system, including stimulation of GSH synthesis and up-regulation of diverse antioxidant enzyme expression and activity. The nuclear translocation of Nrf2 is a prerequisite for induction of the endogenous antioxidant defense. However, the mechanisms underlying such a process are not well understood but may involve oxidation/alkylation of key thiols in Keap1 and/or phosphorylation of Nrf2.58 Upon exposure to electrophiles or oxidants, the Cys-rich protein Keap1 is modified by forming a covalent adduct or disulfide bonds within the protein, and it is this Cysbased modification of Keap1 that allows dissociation, accumulation, and subsequent nuclear translocation of Nrf2, where it initiates the transcription of cytoprotective genes (phase II genes).58 Analysis of the chemical structure of 6-DG reveals that the molecule contains one α,β-unsaturated ketone moiety (highlighted in Scheme 1), a thiol-reactive electrophilic center that may covalently modify the Cys thiols. Many natural or synthetic molecules bearing such a structure are known to be effective inducers of phase II genes.5,10,59 Thus, we speculated that alkylation of certain Cys residues might be the molecular basis for the activation of the Keap1-Nrf2-ARE pathway by 6DG, which was supported by the observation that removal of the electrophilic center from 6-DG severely blocks the protection (Figure 2A). The observation that knocking down Nrf2 expression abolishes the effect of 6-DG suggests that the 5516

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mechanism could lead to the development of novel small molecules as potential neuroprotective agents.

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AUTHOR INFORMATION

Corresponding Author

*(J.F.) E-mail: [email protected]. Fax: +86 931 8915557. Funding

Financial support from Lanzhou University (Fundamental Research Funds for the Central Universities, lzujbky-2014-56), the National Natural Science Foundation of China for Fostering Talents in Basic Research of the (J1103307), and the 111 project is gratefully acknowledged. Notes

The authors declare no competing financial interest.

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dx.doi.org/10.1021/jf405553v | J. Agric. Food Chem. 2014, 62, 5507−5518