The neuropathology of Parkinson′s disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc), as a result of many contributing factors, including oxidative stress-mediated, mitochondrial dysfunction, environmental toxins and inflammation, ultimately leading to the death of neuronal cells [1
]. Accordingly, we selected 6-hydroxydopamine (6-OHDA)-induced neurotoxicity and oxidative stress to mimic the neuropathological symptoms of PD in an in vitro cellular model [2
]. Oxidative stress is associated with PD dementia and dementia with Lewy bodies, and thus is considered to be responsible for significant central events in the process of PD pathogenesis [3
Thus, pharmacological or nutritional interventions to reduce oxidative stress may potentially ameliorate oxidative stress-related neurodegenerative diseases, including Alzheimer′s disease (AD), amyotrophic lateral sclerosis (ALS) and PD [3
]. Oxidative stress can be defined as an imbalance between cellular antioxidant defense systems and the endogenous or exogenous pro-oxidant burden [6
]. Excessive production and the accumulation of reactive oxygen species (ROS) induced by 6-OHDA can lead to DNA cleavage, protein oxidation and lipid peroxidation, ultimately resulting in cellular dysfunction and apoptosis [7
]. Controlling ROS production and scavenging may be therapeutically beneficial in the management of PD [8
As part of our ongoing studies to explore structurally and pharmacologically-active, unique natural products from diverse, natural sources [9
], we have recently focused on the identification of neuroprotective compounds [13
]. Recently, daidzein-7-α
-L-rhamnopyranoside, identified from the fungus-growing termite-associated Streptomyces
sp. RB1, was found to prevent glutamate-induced hippocampal neuronal cell death via its blocking of intracellular ROS accumulation in HT-22 cells [13
]. In the present study, we investigated Acer tegmentosum
Maxim (Aceraceae), which has been used in Korean traditional medicine to treat liver-related disorders, including hepatitis, liver cancer and hepatic cirrhosis, as well as to detoxify the liver. [14
]. Previous phytochemical investigations of A. tegmentosum
have revealed that its extracts contain various pharmacological metabolites, including flavonoids, lignans, and phenolic compounds [15
]. In a recent study, we successfully isolated and identified a new phenolic compound, isoamericanoic acid B, and nine phenolic compounds from the bark of A. tegmentosum
]. Intriguingly, we observed that a coumarin glycoside, fraxin, isolated from A. tegmentosum
, displayed potent hepatoprotective effects via the nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant enzyme system [18
Nrf2 is a transcription factor that, when activated, translocates to the nucleus to activate homeostatic cell defenses in response to oxidative stress, initiates phase II enzyme expression, such as heme oxygenase-1 (HO-1) and NADPH quinone oxidoreductase 1 (NQO1), which are antioxidant response elements (ARE), and mitigates the pathogenesis of PD [19
]. In PD, Nrf2 is localized in the nucleus; however, insufficient Nrf2 activation has been observed in the brains of PD patients, compared to healthy individuals [20
]. In animal models, Nrf2-deficient mice are highly susceptible to neurotoxin-induced dopaminergic degeneration [21
]. Among the various known cytoprotective enzymes, HO-1 has received considerable attention [23
]. Indeed, several recent studies have demonstrated that an activation of the Nrf2-dependent HO-1 pathway ameliorates the neurotoxicity in oxidative stress-induced neuronal cells [25
]. Moreover, induced phase II enzymes, such as HO-1, have the potential to improve cellular antioxidant capacity and protect against oxidative injury; thus, the induction of HO-1 is a common feature in several neurodegenerative diseases, such as AD and PD [27
]. Consequently, the activation of Nrf-2-mediated HO-1 induction is considered a potential therapeutic target in the management of PD [30
In line with this evidence, we investigated potential neuroprotective metabolites associated with Nrf2-mediated antioxidants, such as HO-1 from an extract of A. tegmentosum. Column chromatographic separation of the EtOAc-soluble fraction of the extract led to the isolation of 15 compounds, followed by high-performance liquid chromatography (HPLC) purification. In this study, cell-based protection screening was first performed to evaluate the neuroprotective effects of the isolated compounds 1–15 using human dopaminergic neuroblastoma SH-SY5Y cells.
To elucidate the underlying neuroprotection mechanism, we studied the mechanism of action of the most active compound 5, hyperoside (quercetin 3-O-galactoside), on 6-OHDA-induced oxidative stress in human SH-SY5Y dopaminergic neurons. We also investigated whether hyperoside activates Nrf2-dependent signaling, which induces HO-1 upregulation if it is downstream of the ARE-responsive proteins. Here, we describe, in detail, the neuroprotective effects of hyperoside via the activation of Nrf2/HO-1 signaling in human dopaminergic neuroblastoma SH-SY5Y cells.
Here we evaluated the neuroprotective effects of hyperoside, the most potent active compound isolated from A. tegmentosum, on 6-OHDA-induced oxidative stress, as an in vitro cellular PD model, using human dopaminergic SH-SY5Y cells. We used 6-OHDA to induce oxidative stress, and subsequently, cell death in these dopaminergic neurons. Our findings indicate that hyperoside inhibits 6-OHDA-induced neurotoxicity, most likely by preventing oxidative stress and activating Nrf2-mediated HO-1 signaling.
Hyperoside (quercetin 3-O
-galactoside), a phenolic compound present in the bark of A. tegmentosum
, has displayed multiple biological protective effects against chronic diseases through its antioxidant [47
], anti-inflammatory [48
] and anticancer [49
] activities; moreover, it effectively prevents amyloid beta-induced neurotoxicity [50
], oxygen-glucose deprivation-reperfusion-induced cortical neuron injury [51
] and chronic stress-exposed depressive-like behavior [52
] in vivo. There is growing evidence of the effects of hyperoside on neurodegenerative diseases; however, the mechanisms underlying these effects of hyperoside in PD have not yet been addressed. Moreover, there have been no direct studies demonstrating the molecular mechanisms underlying the antioxidant activities of hyperoside in 6-OHDA-induced neurotoxicity in human dopaminergic SH-SY5Y cells. To our knowledge, this is the first report to demonstrate the neuroprotective effect of hyperoside against 6-OHDA-induced oxidative damage in human dopaminergic SH-SY5Y cells via the Nrf2-mediated HO-1 signaling pathway in a cellular PD model.
As previously mentioned, PD is a progressive condition characterized by selective dopaminergic neuron loss in the substantia nigra pars compacta (SNpc) [53
]. Although the etiology and pathology of PD are not entirely understood, many studies have suggested that, in its pathogenesis, oxidative stress plays an essential role in the loss of dopaminergic neurons [7
]. It is generally recognized that oxidative stress from excessive accumulation of ROS can injure neuronal cells, as well as trigger mitochondrial deterioration and apoptotic cascade events [54
]. In particular, SNpc is considerably more vulnerable to oxidative stress, as it contains inherently fewer antioxidant enzymes than other brain regions [56
]. Moreover, PD is primarily caused by ROS production induced by auto-oxidation and mitochondrial membrane dysfunction in dopaminergic neurons [57
]. Thus, it is essential to improve and enhance the antioxidant defense system to control oxidative stress at the cellular level.
There is a large body of evidence suggesting that oxidative stress is a major contributor to mitochondrial-mediated apoptotic neuronal cell death, and that the preventive effects of antioxidants on these causative cellular events can effectively block cell death [6
]. Besides, abnormal intracellular ROS accumulation is associated with PD, and ultimately, leads to dopaminergic neuronal cell death. Many studies have demonstrated that neuronal injury is an essential source of ROS in the brain; thus, the suppression of excessive ROS production might be an alternative strategy to protect neurons [58
]. Conversely, mitochondrial membrane dysfunction is well established as an associated event in many forms of apoptotic and necrotic cell death, with evidence showing that both modes of cell death may play a role in PD [59
]. Mitochondrial dysfunction may result in apoptotic cell death via a reduction in the mitochondrial membrane potential (MMP), leading to an apoptotic signaling pathway cascade [60
]. Therefore, we tested the neuroprotective effect of hyperoside on 6-OHDA-induced excess ROS production and mitochondrial membrane dysfunction.
In this study, we found that the loss of mitochondrial metabolic activity and membrane integrity in response to 6-OHDA was significantly prevented by pretreatment with hyperoside, compared to NAC (10 mM); moreover, even 0.5–2 μM hyperoside was sufficient to exhibit a potent antioxidant effect that benefits clinical drug development. Furthermore, we found that hyperoside significantly suppressed intracellular ROS accumulation and mitochondrial membrane dysfunction in SH-SY5Y cells, which is in agreement with previous observations by other groups who reported it to be an effective protective agent against oxidative stress-mediated cell death [47
]. Based upon our findings, we aimed to elucidate the underlying molecular mechanisms responsible for the neuroprotective effects of hyperoside in the cellular PD model.
The transcription factor Nrf2 plays a key role in the adaptive response to oxidative stress by exerting neuroprotective effects via the induction of a series of phase II detoxification enzymes. Of late, specific Nrf2 inducers have been studied clinically, and Nrf2 has been considered an emerging therapeutic target [62
]. Despite the neuroprotective abilities of compounds with Nrf2 activity, their use in PD is uncertain due to a lack of information regarding their ability to pass through the central nervous system. Thus, in the search for compounds that activate Nrf2 signaling in the brain, those that quickly pass through the blood–brain barrier and can be taken up by neuronal cells representing potential drug targets [63
]. Therefore, we hypothesized that the neuroprotective actions of hyperoside were affected by the activated Nrf2-mediated induction of the HO-1 transcriptional signaling pathway. To better define the neuroprotective role of hyperoside, we examined its effects on the nuclear translocation of Nrf2, as well as the activation of its downstream signaling HO-1, a key protein transcriptional regulated by Nrf2. We found that hyperoside activated Nrf2 nuclear translocation in neuronal cells, demonstrating activation of neuroprotective pathways. Our finding suggests that nuclear translocation of Nrf2 and Nrf2-mediated ARE activation has a pivotal role in the increase in HO-1 induction by hyperoside. Nrf2 activation leads to the induction of HO-1 transcription, which, consequently, results in potent anti-inflammatory and antioxidant effects [65
]. A large body of evidence suggests that HO-1, a critical neuroprotective enzyme, plays a crucial role in maintaining antioxidant homeostasis during cellular stress [67
]. The induction of HO-1 provides neuroprotection; thus, elevating the HO-1 expression by using a pharmacologic modulator may represent a valid strategy for therapeutic intervention [68
]. To obtain direct evidence of the role of Nrf2-mediated HO-1 signaling in hyperoside-induced neuroprotective activities, we examined the effects of Nrf2-siRNA transfection on hyperoside neuroprotection using multiple analyses of human dopaminergic SH-SY5Y cells. Our study demonstrated that hyperoside induces HO-1 expression levels and that Nrf2 knockdown by small interfering RNA (siRNA) blocked hyperoside-induced neuroprotective effects, indicating that Nrf2/HO-1 plays an essential role in mediating the neuroprotective effects of hyperoside. This study validates the neuroprotective effect of hyperoside against 6-OHDA-induced oxidative stress and neurotoxicity via the alleviation of intracellular ROS accumulation, mitochondrial dysfunction and eventually, apoptotic cell death. Collectively, the results of this study demonstrate that hyperoside exhibits remarkable neuroprotective effects against 6-OHDA-induced oxidative stress and neurotoxicity by activating the Nrf2-mediated HO-1 transcriptional signaling pathway.
4. Materials and Methods
4.1. Cell Culture and Treatment
Human dopaminergic neuroblastoma SH-SY5Y cells were obtained from the American Tissue Culture Collection (ATCC® CRL-2142, Manassas, VA, USA). Cells were maintained in Dulbecco′s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (v/v) and 0.1% penicillin/streptomycin at 37 °C in an atmosphere of 5% CO2 and 95% air. Hyperoside and N-acetylcysteine (NAC) were dissolved in dimethyl sulfoxide (DMSO); the stock solution was added directly to the culture medium. 6-hydroxydopamine (6-OHDA) was prepared as a 20 mM stock solution immediately prior to use, and was diluted in PBS to the indicated final concentration. The final solvent concentration was always < 0.1% (v/v). No significant cytotoxicity was observed in any of the experiments.
4.2. Measurement of Cell Viability
Cells (2.5 × 105 cells/well in 24-well plates) were incubated at 37 °C with 200 μM 6-OHDA for 24 h with or without hyperoside (5) or N-acetylcysteine (NAC) pretreatment, and then treated with 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution (5 mg/mL) for 4 h. The dark-blue formazan crystals formed in viable cells were dissolved in DMSO; subsequently, the absorbance of each reaction product was measured using a microplate reader (SpectraMax 250, Molecular Devices, Sunnyvale, CA, USA) at 540 nm.
4.3. Measurement of Lactate Dehydrogenase (LDH) Release
Extracellular and intracellular Lactate Dehydrogenase (LDH) activities were spectrophoto-metrically measured as cytotoxicity using an LDH cytotoxicity assay kit (Thermo Fisher Scientific Inc., Bartlesville, OK, USA), according to the manufacturer′s protocol. Cells (2.5 × 105 cells/well in 24-well plates) were incubated at 37 °C with 200 μM 6-OHDA for 24 h with or without hyperoside or NAC pretreatment; subsequently, the supernatant was assayed by adding 100 μL of the reaction mixture to each well at room temperature. The absorbance of each sample was read at 490 nm using a microplate reader.
4.4. Terminal Deoxynucleotidyl Transferase-Mediated dUTP-Biotin Nick-End Labeling (TUNEL) Assay
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was detected using the DeadEnd™ Fluorometric TUNEL System (Promega, Madison, WI, USA), according to the manufacturer′s protocol. Briefly, cells (5 × 105 cells/well in 6-well plates) were plated on coverslips coated with poly-d-lysine for 24 h. Then, cells were incubated at 37 °C with 200 μM 6-OHDA for an additional 24 h, with or without hyperoside pretreatment. Finally, the cells were washed with phosphate-buffered Saline (PBS). For the immunocytochemistry assay, cells were immersed in 4% paraformaldehyde for 15 min, washed twice with PBS, and finally, permeabilized with 0.2% Triton X-100 for 5 min. Each glass coverslip was covered with equilibration buffer for 10 min. Then, the buffer was aspirated, and the glass coverslips were incubated with terminal deoxynucleotidyl transferase (TdT) buffer for 1 h at 37 °C. Following incubation, the cells were washed again with PBS and observed under a fluorescence microscope (20×). TUNEL positive cells were quantified by counting the degree of DNA fragmentation in different entire areas of the slide in three random fields of each group.
4.5. Measurement of Intracellular Reactive Oxygen Species (ROS) Accumulation
Cells (2.5 × 105 cells/well in 48-well plates or 2 × 105 cells in 4-well chamber slides) were incubated at 37 °C with 200 μM 6-OHDA for 4 h with or without hyperoside pretreatment. The cells were washed with PBS and incubated with 10 mM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) in the dark. After 30 min incubation at 37 °C, cells were examined using a fluorescence spectrophotometer (SpectraMax 250, Molecular Devices) at an excitation wavelength of 530 nm and an emission wavelength of 480 nm. DCFH-DA fluorescence representative images were examined by fluorescence microscopy (20×) (BX51, Olympus Optical Co., Ltd., Tokyo, Japan).
4.6. Measurement of Intracellular Mitochondrial Membrane Potential (MMP)
Cells (2.5 × 105
cells/well in 48-well plates or 2 × 105
cells in 4-well chamber slides) were incubated at 37 °C with 200 μM 6-OHDA for 24 h with or without hyperoside pretreatment. The cells were washed with PBS, and 10 μM rhodamine 123 was added. After 30 min incubation at 37 °C, the cells were examined at 530 nm using a fluorescence spectrophotometer at an excitation wavelength of 480 nm [69
]. Rhodamine123 fluorescence representative images were examined under a fluorescence microscope (20×).
4.7. RNA Isolation and Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Cells (1 × 106 cells/well in 6-well plates) were incubated at 37 °C with hyperoside for 0, 1, 2, 4, 6 and 12 h. Total mRNA was isolated using Trizol® reagent (Invitrogen, Carlsbad, CA, USA) and reverse-transcription was performed using the Superscript®-III kit (Invitrogen), according to the manufacturers′ instructions. Amplification of cDNA was carried out using specific primers: HO-1 (forward, 5’- CCAGAAGAGCT GCACCGCAA-3’ and reverse, 5’-GCTGGATGTTGAGCAGGAAC-3’) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, forward, 5’- ACCACAGTCCATGCCATCAC-3’ and reverse, 5’-TCCACCACCCTGTTGCTGTA-3’). The amplified polymerase chain reaction (PCR) products were separated by staining with ethidium bromide (EtBr) on 1.5% agarose gel electrophoresis in Tris borate/EDTA buffer (890 mM Tris-Base, 890 mM boric acid, 20 mM EDTA, pH 8.3, alkali) for 30 min at 100 V. Specific bands were compared after visualization and imaging using a UV transilluminator. The mRNA bands were quantified by densitometric analysis using ImageJ software (NIH Image in the public domain, NIH, Bethesda, MD, USA).
4.8. Transient Transfection with siRNA
Transfection of cells with siRNAs was performed using the Lipofectamine®
2000 transfection reagent (invitrogen) according to the manufacturer′s instructions. Commercially-available human Nrf2-specific small interfering RNA (siRNA) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and negative control siRNAs (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used for transfection. To knockdown Nrf2 expression, Lipofectamine®
2000 transfection reagent (10 μL) was added to 200 μL of serum-free Opti-MEM®
, containing 10 μM each siRNA, followed by 20 min incubation at room temperature. Gene silencing was performed for 24 h; subsequently, transfected cells were treated with 2 μM hyperoside for 4 h in the presence or absence of 200 μM 6-OHDA for 24 h. Cell viability, LDH, TUNEL assays, and Western blot analysis were performed as described in Section 4.2
, Section 4.3
, Section 4.4
, and Section 4.10
4.9. Cytosolic and Nuclear Lysate Preparation
Nuclear and cytosolic fractions were prepared with a commercial kit according to the manufacturer′s instructions (Thermo Scientific Inc., Rockford, IL, USA). All steps were carried out on ice, unless stated otherwise. Protease and phosphatase inhibitor cocktail tablets (Complete Mini and Phospho STOP, Roche Applied Science, Indianapolis, IN, USA, respectively) were added to each buffer just prior to use. Briefly, cells (5 × 106 cells/well in 100 mm2 cell culture dishes) were treated with hyperoside for 0, 1, 2, 4, 6 or 12 h at 37 °C. Protein concentrations in the samples were determined using a bicinchoninic acid assay (BCA assay) protein assay kit (Thermo Scientific Inc., Rockford, IL, USA). Nrf2 levels were assessed using western blot analysis, as described below.
4.10. Western Blot Analysis
Cells (1 × 106 cells/well in 6-well plates) were plated and treated with hyperoside for 0, 1, 2, 4, 6 or 12 h; then the cells were washed and collected with PBS and centrifuged at 400× g for 5 min. The cell pellets were extracted using the T-per tissue protein extraction buffer (Thermo Fisher Scientific Inc., Bartlesville, OK, USA), containing protease and phosphatase inhibitor cocktail tablets (Complete Mini and Phospho STOP, respectively). After centrifugation at 10,000× g for 15 min, the supernatant was separated and stored at −70 °C. The protein concentration in the samples was determined using a BCA protein assay kit. Protein samples were separated on an 8–12% SDS–polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA) that was further blocked with 5% skim milk, containing 0.5 mM Tris-HCl (pH 7.5), 150 mM NaCl and 0.1% Tween-20 for 1 h at room temperature. After blocking for 1 h at room temperature, the membrane was reacted with primary antibodies overnight at 4 °C (each antibody at a dilution of 1:1000; HO-1, laminin B1 and Nrf2, except for β-actin (1:20,000)). After three washes with tris-buffered saline combined with 0.1% polysorbate 20 (Tween-20) (this combination referred to as TBST or TTBS), the blots were incubated with horseradish-peroxidase-conjugated anti-rabbit or anti-mouse secondary antibodies in TBST at a 1:5000 dilution for 1 h at room temperature. Then the blots were washed three times in this TBST buffer. The blots were developed using an enhanced chemiluminescence detection method in a mixture of SuperSignal® West Pico Chemiluminescent Substrate reagents (Thermo Fisher Scientific Inc., Bartlesville, OK, USA). Protein bands were quantified via densitometric analysis using ImageJ software from NIH (Bethesda, MD, USA).
4.11. Transient Transfection and Dual-Luciferase Assay
Luciferase activity was assayed using a dual-luciferase assay kit (Promega, Madison, WI, USA), according to the manufacturer′s instructions. To determine promotor activity, antioxidant response element (ARE) reporter constructs were purchased from SABiosiences (Qiagen, Valencia, CA, USA). Brief, cells were plated in 24-well plates overnight. After overnight transfection, the cells were exposed to hyperoside for indicated times and concentrations. Luminescence in cell lysates was measured using a single tube luminometer (FB12, Berthold Detection Systems GmbH, Pforzheim, Germany).
SH-SY5Y cells (2 × 105 cells/well) were grown on poly-d-lysine coated chamber slides overnight and treated with hyperoside. After washing with PBS, cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton X-100 in PBS for 10 min. After washing with PBS, cells were blocked in 5% BSA solution in PBS for 30 min and incubated overnight with anti-Nrf2 (1:250) at 4 °C. Subsequently, the cells were washed with PBS and reacted with Texas red®-conjugated goat anti-mouse IgG antibodies (1:250) and Hoechst 33258 (5 μg/mL) for 1 h. After washing with PBS, the cells were mounted on glass slides in Permafluor aqueous mounting fluid. The Nrf2 nuclear translocation was observed under a fluorescence microscope (20×); the results are representative of three independent experiments.
4.13. Statistical Analysis
Data were analyzed with Prism 5.0 software (GraphPad Software, Inc., San Diego, CA, USA) and are presented as the means ± the standard error of the mean (SEM). Statistical analyses were performed using a one way analysis of variance (ANOVA), followed by the Newman-Keuls test. Any p values < 0.05 were considered statistically significant.
Our study demonstrates that compounds 1, 2, 3, 5, 6, 7, 9, 11 and 13 isolated from A. tegmentosum showed neuroprotective effects on human dopaminergic neuron cells. Among these active compounds, hyperoside (5) showed the most potent neuroprotective effect against 6-OHDA-induced cell toxicity. In terms of its molecular mechanism, hyperoside significantly inhibited oxidative stress-mediated neuronal cell toxicity induced by 6-OHDA. We also demonstrated that hyperoside induces the activation of Nrf2-dependent cellular antioxidant defenses, leading to the inhibition of 6-OHDA-induced oxidative injuries. Importantly, this is the first report, to our knowledge, demonstrating that Nrf2/HO-1 signaling may contribute to the neuroprotection mediated by hyperoside against 6-OHDA-induced oxidative stress.
Our findings provide direct experimental evidence and a molecular interpretation for the antioxidant properties of hyperoside in neurodegenerative diseases and suggest that A. tegmentosum and its constituents have the potential for development as neuroprotective agents for the prevention of PD progression and related diseases.