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Gastrodin and Isorhynchophylline Synergistically Inhibit MPP+induced Oxidative Stress in SH-SY5Y Cells by Targeting ERK1/2 and GSK-3# Pathways: Involvement of Nrf2 Nuclear Translocation Qiang Li, Chengu Niu, Xiaojie Zhang, and Miaoxian Dong ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00247 • Publication Date (Web): 08 Nov 2017 Downloaded from http://pubs.acs.org on November 10, 2017

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ACS Chemical Neuroscience

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Gastrodin and Isorhynchophylline Synergistically Inhibit MPP+-induced

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Oxidative Stress in SH-SY5Y Cells by Targeting ERK1/2 and GSK-3β Pathways:

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Involvement of Nrf2 Nuclear Translocation

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Authors: Qiang Li,† Chengu Niu,§ Xiaojie Zhang,†

，

‡

and Miaoxian Dong*, †

，

‡

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Authors’ Affiliations: † The Institute of Medicine, Qiqihar Medical University, Qiqihar

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161006, China. §Department of Hematology, the First Affiliated Hospital, Harbin

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Medical University, Harbin 150001, China.

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‡

M.X. Dong and X.J. Zhang shared senior authorship

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Running title: GAS/IRN facilitates Nrf2 nuclear accumulation

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ABSTRACT

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Recently, the pathogenesis of Parkinson’s disease (PD) is multifactorial event.

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Combination therapies might be more effective in controlling the disease. Thus, the

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studies reported were designed to test the hypothesis that gastrodin (GAS)-induced de

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novo synthesis of nuclear factor E2-related factor 2 (Nrf2) and isorhynchophylline

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(IRN) inhibition of Nrf2 nuclear export contribute to their additive or synergistic

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neuroprotective effect. Here, we have demonstrated that the combination of GAS and

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IRN (GAS/IRN) protect SH-SY5Y cells against 1-methyl-4-phenylpyridinium (MPP+)

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toxicity in a synergistic manner. Concomitantly, GAS/IRN led to a statistically

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significant reduction of oxidative stress, as assessed by reactive oxygen species (ROS)

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and lipid hydroperoxides (LPO), and enhancement of both glutathione (GSH) and

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thioredoxin (Trx) systems compared with treatment with either agent alone in

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MPP+-challenged SH-SY5Y cells. Interestingly, GAS but not IRN activated

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extracellular signal-regulated kinases 1 and 2 (ERK1/2), leading to a increase in de

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novo synthesis of Nrf2 and nuclear import of Nrf2. Simultaneously, IRN but not GAS

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suppressed both constitutive glycogen synthase kinase (GSK)-3β and Fyn activation,

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which inhibited nuclear export of Nrf2. Importantly, simultaneous inhibition of

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GSK-3β pathway by IRN and activation of ERK1/2 pathway by GAS synergistically

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induced accumulation of Nrf2 in the nucleus in SH-SY5Y cells challenged with MPP+.

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Furthermore, the activation of the ERK1/2 pathway and inhibition of GSK-3β

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pathway by GAS/IRN are mediated by independent mechanisms. Collectively, these

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novel findings suggest an in vitro model of synergism between IRN and GAS in the

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induction of neuroprotection warrant further investigations in vivo.

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KEYWORDS:

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Gastrodin; Isorhynchophylline; Synergism; Parkinson’s disease; Nuclear factor

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E2-related factor 2

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INTRODUCTION

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Parkinson’s disease (PD) is a neurodegenerative disease with the major pathology

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being the progressive loss of dopaminergic neurons in the substantia nigra resulting in

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dopamine deficiency in the striatum [1]. Pharmacological remedies targeting the

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dopaminergic network are relatively effective at ameliorating symptoms [2]. Despite

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the increasing knowledge regarding the pathomechanism [3], no effective

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disease-modifying therapy is yet available to combat PD [4]. In this context, the need

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to find novel strategies to counteract neurodegenerative progression is becoming

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increasingly clear [5].

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Although several biochemical and genetic defects have been identified [6], oxidative

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stress is widely considered as a central event in the progression of the pathology,

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either as a cause or effect of PD [7]. Excessive reactive oxygen species (ROS) can

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lead to dopaminergic neuron vulnerability and eventual death [8]. Antioxidant

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remedies, particularly herbal antioxidants, show promise to delay or prevent the

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disease [9]. Nuclear factor E2-related factor 2 (Nrf2) plays a critical role in regulating

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cellular defense against oxidative stress by activating the expression of an array of

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antioxidant response element (ARE)-dependent genes [10]. Studies demonstrate that

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Nrf2 knockout mice are hypersensitive to PD-causing toxins [11], whereas Nrf2

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over-expression either through pharmacological activation or by genetic means

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renders a neuroprotective response [12, 13].

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Even though information of phosphorylation at the exact residue(s) of Nrf2 protein by

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activated extracellular signal-regulated kinases 1 and 2 (ERK1/2) is still lacking [14], 4


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unequivocal evidence indicates that ERK1/2 is a positive regulator of Nrf2 nuclear

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accumulation stimulating ARE activation [15]. On the contrary, Fyn kinase can

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directly phosphorylate Nrf2 protein at Tyr-568 and promote its nuclear exclusion, and

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consequently contributing to the suppression of ARE-mediated gene expression [16].

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Glycogen synthase kinase (GSK)-3β acts as an upstream kinase of Fyn that

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contributes to Nrf2 phosphorylation [17].

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Throughout the history of drug discovery and development, plants have been an

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important source for the discovery of novel therapeutically active compounds for

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neurodegenerative diseases [18]. In previous work, we have demonstrated that

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gastrodin (GAS) prevents motor deficits in 1-methyl-4-phenyl-1,2,3,6-

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tetrahydropyridine (MPTP)-intoxicated mice via ERK1/2-Nrf2 pathway and that

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isorhynchophylline (IRN) blocks 1-methyl-4-phenylpyridinium (MPP+)-induced

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oxidative stress in vitro [19]. The complexity of the molecular-biological mechanisms

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for PD requires multi-target therapeutics [20]. A cocktail of agents is a promising

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method in search for new drugs for PD. Does GAS acts synergistically with IRN to

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promote translocation of Nrf2 into the nucleus and, if so, what would be the signaling

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pathway involved in synergistic action? To address these questions we hypothesized

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that GAS-mediated nuclear import of Nrf2 and IRN inhibition of Nrf2 nuclear

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exclusion contribute to synergistic neuroprotective effect by combinations of IRN and

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GAS, hereafter referred to as GAS/IRN. Results in this report support our hypothesis.

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RESULTS AND DISCUSSION

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GAS/IRN can synergistically protect SH-SY5Y cells against MPP+ toxicity by

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attenuation of oxidative stress

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Consistent with our previous observations, IRN or GAS alone exhibited a

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concentration-dependent neuroprotective action [21, 22]. Combined treatment with

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GAS and IRN at a constant concentration ratio of 10 to 3 substantially improved the

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viability of SH-SY5Y cells challenged with MPP+, compared with the cells treated

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with IRN or GAS alone, as shown by the 3-(4,5-dimethylthiazol-2-yl)-5-

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(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium, inner salt (MTS) assay

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(Figure 1A). Calculation of combination index (CI) with CompuSyn software

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revealed that the interaction of GAS and IRN is highly synergistic (Table 1).

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Specifically, the maximal synergism was found when 0.3 µM IRN was combined with

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1 µM GAS, therefore we selected them for further synergistic mechanism studies.

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Again, cell proliferation ELISA BrdU colorimetric assay showed that combination of

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GAS with IRN synergistically stimulate cell proliferation in MPP+-challenged

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SH-SY5Y cells (Supplemental Table S1 and Supplemental Figure S1, available at

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http://pubs.acs.org as Supporting Information), suggesting that promotion of

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proliferation by GAS/IRN may contribute to their inhibitory action on MPP+-induced

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death of SH-SY5Y cells. Moreover, combination treatment of GAS with IRN showed

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the greatest increase in cell viability compared to monotherapy with GAS in

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SH-SY5Y cells exposed to Aβ25-35, as shown by MTS assay (Supplemental Table S2

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and Supplemental Figure S2, available at http://pubs.acs.org as Supporting

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Information). Similar, inhibitory effects on MPP+-induced death of SH-SY5Y cells

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were produced by Nrf2 activator tertiary butylhydroquinone (tBHQ) (Supplemental 6


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Figure S3, available at http://pubs.acs.org as Supporting Information). Short hairpin

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RNA (shRNA)-mediated knockdown of Nrf2 abolished IRN and/or GAS-mediated

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neuroprotection against MPP+-induced toxicity in SH-SY5Y cells (Figure 1B), hinting

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a role of its antioxidant properties. As shown in Figure 1C and D, treatment with IRN

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or GAS alone resulted in modest reduction in both ROS production and LPO level in

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MPP+-challenged SH-SY5Y cells, and these reductions were further aggravated when

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IRN was combined with GAS, suggesting that antioxidant properties of GAS/IRN

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may contribute to their neuroprotective effects.

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Research into historical literatures have revealed that Uncaria rhynchophylla (UR)

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and Gastrodia elata Blume (GE) are usually used in combination to treat tremor

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syndrome in traditional Chinese medicine through antioxidant mechanisms. IRN and

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GAS are two antioxidant compounds found in UR and GE, respectively [23]. Liu et al.

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reported that UR in combination with GE exerts strong antioxidant activities [24].

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Although our knowledge about their bioavailability and pharmacokinetic interactions

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between IRN and GAS is far from being complete, based on the findings from

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preliminary studies and our previous studes [21, 22], we chose IRN and GAS at a

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constant concentration ratio of 10 to 3. However, we have used IRN at 0.3 µM and

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GAS at 1 µM concentrations in this study that should be physiologically attainable in

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vivo [25, 26].

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In the present study, we chose the SH-SY5Y cell line for specific reasons even though

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primary cells are more physiologically relevant than immortalized cell lines. First,

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primary neurons do not maintain cell division in culture and are in heterogeneous

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states of neurodifferentiation. Second, SH-SY5Y cell are more amenable to high level 7



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transduction than primary neurons. Third, very large amounts of cells in our present

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study are required which is difficult to achieve with primary neurons. However,

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SH-SY5Y cells share the limitations common to in vitro experimental models, namely

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an inability to assess glial-neuronal-endothelial interactions, pharmacokinetic

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properties, and related issues of bioavailability, dose, and bioeffective concentrations.

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All these factors mean that it is difficult to extrapolate relevant in vivo concentrations

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of GAS and/or IRN from in vitro results, and typically, the concentrations required for

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a given effect in vitro will be higher than those required for parallel effects in vivo.

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Further studies are necessary to evaluate antioxidant properties of GAS and/or IRN in

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vivo in the context of PD-related neurotoxin.

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The complex pathophysiological mechanisms involved in the etiology of PD limit the

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discovery of agents to modify its course [27]. Some findings suggest, although do not

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prove, that GAS and IRN alone have exhibited possible preventive and therapeutic

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effects on PD [28, 29]. The key new finding made in our present study is that GAS

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and IRN exert neuroprotection in a synergistic fashion, suggesting that multi-targeting

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strategy with plant-derived compounds combination achieves a disease-modifying

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effect in PD. Although antioxidant therapy is unlikely to provide a panacea against

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neurodegenerative diseases, wide varieties of antioxidants show promise to delay or

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prevent PD [9]. Plants have their own natural antioxidants to protect their structures

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against ROS produced during photosynthesis [30]. Single plant-derived antioxidant

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often does not enabled to reach the therapeutic threshold [31]. Combination therapy

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has the potential to reduce single-agent therapeutic dose and to increase therapeutic

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efficacy [20]. In the current study, although both GAS and IRN individually exhibit

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antioxidant properties, when combined, they synergistically inhibit oxidative stress 8


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evoked by MPP+. This finding suggests that these two agents may work through

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independent mechanism to protect against MPP⁺-induced oxidative damage. The

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significance of the antioxidant properties reported herein will be substantially clearer

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if it is known whether GAS/IRN, administered in vivo, crosses the blood-brain barrier.

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GAS/IRN enhances thioredoxin and GSH antioxidant systems in

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MPP+-challenged SH-SY5Y cells

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GSH and Trx systems are essential for neuronal antioxidant defense [32]. Thus, the

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next question is whether GSH antioxidant systems are regulated following IRN and/or

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GAS treatment. As depicted in Figure 2A, IRN and/or GAS, as expected, enhanced

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GSH concentration in MPP+-challenged SH-SY5Y cells. GAS/IRN showed the

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greatest efficacy in increasing GSH concentration compared to IRN or GAS alone.

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Again, GSH/GSSG ratio revealed a similar result. As illustrated in Figure 2B, C, and

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D, the data from the western blot and PCR analyses indicated that catalytic subunits

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from glutamate cysteine ligase (GCLc) expression at levels of transcription and

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translation in MPP+-challenged SH-SY5Y cells treated with GAS/IRN was higher

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than those treated with IRN or GAS alone. GSH-Px activity in MPP+-challenged

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SH-SY5Y cells in the response to IRN and/or GAS showed similar trends (Figure 2E),

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whereas the mRNA and protein expression of modulatory subunits from glutamate

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cysteine ligase (GCLm) remained unchanged. Alongside, GAS alone or the GAS/IRN

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significantly increased SOD levels, whereas IRN alone failed to do so (Supplemental

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Figure S4, available at http://pubs.acs.org as Supporting Information).

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A causal role for GSH depletion in PD is supported by both clinical and animal 9



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studies [33, 34]. The present study demonstrates that MPP+ increase oxidative stress

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and result in marginally, if at all, increased GCLc expression, which acts as an

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antioxidant adaptive response to oxidative stress in SH-SY5Y cells. GAS and IRN

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synergistically enhance GSH system that may contribute to GAS/IRN-mediated

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neuroprotection. Poor penetration through the blood brain barrier represents a major

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challenge to be overcome in GSH clinical application [35]. Therefore, induction of de

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novo GSH synthesis in brain may be a potential therapeutic strategy against PD.

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Because the Trx system represents an important target for PD therapy [36], we were

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prompted to investigate whether IRN and/or GAS was also capable of activates Trx

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system. As illustrated in Figure 3A, B and C, TrxR enzymes activity and Trx1 protein

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expression were increased in MPP+-challenged SH-SY5Y cells in response to IRN or

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GAS alone, and this elevation was further enhanced when IRN was combined with

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GAS. Specifically, real-time- PCR of Trx1 and TrxR revealed a similar result (Figure

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3D). IRN and/or GAS markedly attenuated MPP+-induced both reduction of NAD+

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and NAD+/NADH ratio. GAS/IRN seemed to enhance increase of NAD+ and

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NAD+/NADH ratio to a greater extent compared with IRN or GAS alone (Figure 3E).

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Intracellular Trx1 exerts most of its antioxidant properties in the presence of TrxR and

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NADPH through scavenging of ROS [37]. Studies have shown that Trx1 protects

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neurons against the oxidative stress and damage caused by the PD-related neurotoxin

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[38]. The present study demonstrates that MPP+-induced adaptive activation of Trx

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system is enhanced by GAS and/or IRN. Thus, it is possible that GAS and IRN share

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Trx system for its antioxidant properties, but differ in their upstream signals. All of

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these findings support the notion that inhibition of oxidative stress by GAS/IRN in 10


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MPP+-challenged SH-SY5Y cells might be at least partially mediated by the

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activation of Trx system. In addition, our findings must be interpreted in the context

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of certain limitations. This current report focused on the effect of GAS and/or IRN on

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GSH system and Trx system. Additional experiments are necessary to evaluate the

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role of GAS and/or IRN in regulating activity of any other antioxidant enzymes.

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GAS/IRN activates the Nrf2-ARE antioxidant system in SH-SY5Y cells

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challenged with MPP+

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Emerging evidence indicates that transcription of a variety of antioxidant genes

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through cis-acting sequence known as ARE plays a crucial role in providing

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cytoprotection against pro-oxidant stimuli [39]. Thus, we tested if IRN and/or GAS

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induce activation of the ARE. As shown in Figure 4A, GAS/IRN yielded significantly

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greater ARE-mediated luciferase activity than IRN or GAS alone in MPP+-challenged

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SH-SY5Y cells. In parallel, we tested whether IRN and/or GAS stimulates

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translocation of Nrf2 into the nucleus and found that ability of nuclear translocation of

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Nrf2 also significantly potentiated by the combination treatment when compared with

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IRN or GAS alone (Figure 4B and C). These findings were also confirmed by

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ELISA-based TransAM™ Nrf2 (Supplemental Figure S5, available at

20

http://pubs.acs.org as Supporting Information). However, our PCR results, which are

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not consistent with the results of western blot, were contrary to our initial expectation.

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We observed that GAS alone or in combination with IRN up-regulated Nrf2 mRNA

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expression in MPP+-challenged SH-SY5Y cells, while IRN alone have little effect

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(Figure 4D). Given that Bach1 competes with Nrf2 for binding to the ARE in the

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phase II antioxidant enzymes promoter [40], and Keap1 can assemble with the Cul3 11



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protein to form a Cullin-RING E3 ligase complex for the degradation of Nrf2 [41], we

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examined whether IRN and/or GAS might modulate Bach1 and Keap1 protein

3

expression. However, a slight but not significant alteration of both Bach1 and Keap1

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expression due to IRN and/or GAS could also be observed in MPP+-challenged

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SH-SY5Y cells (Figure 4E).

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GAS but not IRN activates ERK1/2, an upstream regulator of Nrf2 nuclear

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translocation, in MPP+-challenged SH-SY5Y cells

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Increasing evidence suggests that MAPKs are implicated in the regulation of

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ARE-driven gene transcription, in which ERK1/2 and JNK are positive regulators

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while p38MAPK is a negative regulator [42]. This led us to hypothesize that GAS

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and/or IRN-mediated ERK1/2 activation stimulates translocation of Nrf2 into the

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nucleus. Using an ELISA-based technique, nuclear extracts from the SH-SY5Y cells

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were assessed for the ability of host transcription factors to bind to their respective

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DNA sequences. Figure 5A depicts a marginal increase in ATF-2, c-Jun, and c-Myc

17

levels in response to MPP+. GAS but not IRN dramatically increases ATF-2, c-Jun,

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and c-Myc levels in MPP+-challenged SH-SY5Y cells, but did not significantly affect

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the levels of MEF2 and STAT1. These results suggest that GAS increases MAPK

20

activation in MPP+-challenged SH-SY5Y cells. These observations were verified by

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western blotting analyses and found that GAS but not IRN induced phosphorylation

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of ERK1/2, but not of p38MAPK or JNK, which support our hypotheses (Figure 5B and

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C). Noteworthy, quantitative analysis of ERK1/2 phosphorylation showed a trend but

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not a statistically significant elevation in IRN/GAS-treated treatment versus GAS

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alone-treatment in MPP+-challenged SH-SY5Y cells, possibly because of higher 12


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variability. In contrast to the complete block of Nrf2 mRNA expression, a dominant

2

negative (DN) mutant of ERK2 only partially attenuated GAS/IRN-induced nuclear

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localization of Nrf2 (Figure 5D, E, and F). Blocking GSK-3β by LiCl partially

4

attenuated GAS/IRN-mediated nuclear localization of Nrf2, whereas the mRNA

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levels of Nrf2 were unaffected by LiCl. These data hint that nuclear export

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mechanism involved in nuclear accumulation of Nrf2 mediated by GAS/IRN.

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Considering the complexity of the antioxidant system, it seems reasonable to consider

9

that the induction of endogenous protective pathways, such as the Nrf2-ARE pathway,

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is a viable strategy for inhibiting oxidative stress [43]. Recent evidence suggests that

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direct phosphorylation of Nrf2 by ERK1/2 has only a slight effect on Nrf2

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translocation. ERK1/2 activation also can lead to phosphorylation of Bach1 blocking

13

the Nrf2 activity [44]. It is more effective in controlling oxidative stress to

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simultaneously engage nuclear import and nuclear export of Nrf2 through

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combination therapies [45]. In the current study, GAS but not IRN was shown to

16

increase the phosphorylation of ERK1/2, and then Nrf2 are de novo synthesized and

17

imported in the nucleus. However, no regulatory effect of GAS/IRN on Bach1 and

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Keap1, two negative regulators of Nrf2, could be observed. Our results also found that

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the ERK1/2, but not JNK and p38MAPK, plays a major role in the GAS/IRN- mediated

20

nuclear accumulation of Nrf2. These data suggest that ERK1/2 is essential, but not

21

sufficient, for nuclear accumulation of Nrf2.

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IRN but not GAS inhibits constitutive phosphorylation of GSK-3β and Fyn in

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MPP+-challenged SH-SY5Y cells

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Mounting evidence suggests that Fyn kinase can directly phosphorylate Nrf2 at

2

Tyr-568 and promote its nuclear exclusion and degradation [46]. Thus, we assumed

3

that IRN and/or GAS might decrease the level of Fyn phosphorylation, inhibiting

4

nuclear exclusion of Nrf2. Western blot experiments presented in Figure 6A and B

5

indicate that IRN, but not GAS, inhibits Fyn phosphorylation in MPP+-challenged

6

SH-SY5Y cells. Pretreatment with GSK-3βinhibitor LiCl abrogated

7

GAS/IRN-mediated Fyn phosphorylation. GSK-3β acts as an upstream kinase of Fyn

8

that promotes Nrf2 nuclear exclusion [47]. In our experimental paradigm, we were

9

interested in ascertaining whether GSK-3β inactivation occurred in response to IRN

10

and/or GAS. As shown by western blotting analyses, IRN, but not GAS, suppresses

11

GSK-3β phosphorylation at Tyr-297 (that is, inactivation), while GAS was slightly

12

but not significantly enhanced GSK-3β phosphorylation in MPP+-challenged

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SH-SY5Y cells. Importantly, IRN and GAS/IRN produced similar inhibitory effects

14

on GSK-3β phosphorylation at Tyr-297 in MPP+-challenged SH-SY5Y cells. Fyn

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shRNA did not change GSK-3β phosphorylation by GAS/IRN suggest, although do

16

not prove, that GSK-3β is an upstream kinase for threonine phosphorylation of Fyn

17

(Figure 6C and D). Notably, Akt phosphorylation is apparently not affected by GAS

18

or/and IRN in MPP+-challenged SH-SY5Y cells, indicating that the

19

GAS/IRN-mediated GSK-3β dephosphorylation at Tyr-297 do not result from Akt

20

activation (Supplemental Figure S6A and S6B, available at http://pubs.acs.org as

21

Supporting Information).

22 23

Fyn phosphorylates Nrf2 protein at tyrosine 568 and promotes its nuclear export and

24

degradation [16]. GSK-3β acts upstream of Fyn, and activates its phosphorylation and

25

resulting in its nuclear localization [17]. Having accumulated in the nucleus Fyn 14


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kinase directly phosphorylate Nrf2 [48]. Our results demonstrated for the first time

2

evidence that in SH-SY5Y cells, IRN, or GAS/IRN, but not GAS, can suppress

3

GSK-3β-dependent Fyn phosphorylation, which, in turn, resulted in a marked

4

reduction nuclear exclusion of Nrf2. Levels of phospho-GSK-3β at Tyr-297 and

5

phospho-Fyn correlate with their activation status. Another important finding of this

6

study is that GSK-3β acts as an upstream kinase of Fyn, as supported by the

7

observation that GSK-3β inhibition by LiCl blocked the ability of GAS/IRN to oppose

8

Fyn phosphorylation.

9 10

Activation of the ERK1/2 and inactivation of GSK-3β by GAS/IRN is mediated

11

by independent mechanisms

12 13

The available evidence, albeit limited, is highly suggestive that crosstalk between

14

ERK1/2 and GSK-3β signaling pathways regulate ARE-driven gene transcription [49].

15

As depicted in Figure 7A, inhibition of either ERK1/2 or GSK-3β only partially

16

reversed

17

simultaneous inhibition of these two pathways almost completely abrogated ARE

18

activation induced by GAS/IRN. This additive effect suggested that ERK1/2 and

19

GSK-3β might be involved in the parallel ARE activation pathway rather than in the

20

same pathways. An ERK kinase assay presented in Figure 7B and C indicate that

21

phosphorylate Elk-1, a major substrate of phospho-ERK, was marginally increased by

22

MPP+ alone treatment, strongly increased by co-treatment with MPP+ and GAS/IRN.

23

These results suggest that ERK1/2 activation by GAS/IRN are independent of

24

GSK-3β pathway, as phosphorylation of Elk-1 is parallel to ERK1/2 activation. As

25

shown in the western blot analysis in Figure 7D and E, GAS/IRN-mediated Fyn

the

GAS/IRN-induced

ARE-dependent

luciferase

15


activity,

while


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

dephosphorylation was not altered by ERK1/2 inhibitor PD 98059, indicating that

2

ERK1/2 and Fyn is two separate and independent pathways involving Nrf2 nuclear

3

accumulation. Blocking ERK1/2 by dominant-negative mutation, shRNA-based Fyn

4

knockdown, or shRNA-based GSK-3β knockdown significantly reduced, while

5

shRNA-based Nrf2 knockdown almost completely block, the ROS-scavenging effect

6

of GAS/IRN in MPP+-challenged SH-SY5Y cells (Figure 7F).

7 8

We showed that inhibition of both the ERK1/2 or GSK-3β pathways partially reversed

9

the GAS/IRN-induced accumulation of Nrf2 in the nucleus, which is completely

10

reversed by the simultaneous inhibition of these two pathways. Inhibition of GSK-3β

11

by LiCl failed to alter GAS/IRN-induced ERK1/2 phosphorylation. PD98059 also had

12

no effect on GAS/IRN-induced GSK-3β phosphorylation. This finding suggests that

13

ERK1/2 and GSK-3β is independent pathways regulating Nrf2 nuclear accumulation.

14 15

The available evidence in humans, albeit limited, is highly suggestive that two key

16

amino acid resiudes (Ser40 and Tyr568) may regulate Nrf2 localization through

17

phosphorylation [50]. Alteration of Nrf2 phosphorylation at Ser40 and/or Tyr568 in

18

the response to GAS and/or IRN should hence be investigated when proper tools such

19

as monoclonal antibodies will be available to establish their possible role in GAS

20

and/or IRN-induced Nrf2 nuclear translocation. Although the methodology we used to

21

determine the GSK-3β activation has certain limitations, it is conceivable that the

22

inhibiting Nrf2 nuclear exclusion can be attributed to the IRN inhibition of

23

constitutive GSK-3β activation.

24 25

The key findings made in our present study are that GAS and IRN exert synergistic 16


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1

neuroprotective effect in an in vitro models induced by PD-related neurotoxin.

2

GAS/IRN-mediated synergistic neuroprotection is most likely to be attributable to its

3

antioxidant properties by dual-promoting Trx and GSH systems. This may be

4

mediated through two complementary pathways: (i) ERK1/2-dependent de novo

5

synthesis of Nrf2, and (ii) GSK-3β-dependent suppression of Nrf2 nuclear exclusion.

6

Our findings lend strong support to our hypothesis as expected.

7 8

Based on our observations, a schematic diagram illustrating putative mechanism

9

whereby GAS/IRN protects against MPP+-induced SH-SY5Y cells oxidative stress is

10

presented in Figure 8. IRN blocks nuclear exclusion of Nrf2 by reducing Fyn

11

phosphorylation, which is mediated by inhibition of GSK-3β activation. In addition,

12

GAS activated ERK1/2, which also enhanced de novo synthesis of Nrf2 and its

13

nuclear translocation. GAS/IRN-mediated Nrf2 nuclear accumulation induces

14

ARE-driven gene transcription and attenuates oxidative stress in vitro. Nevertheless,

15

the underlying mechanisms are certainly more complex than it is described here.

16

Whether both de novo synthesis of Nrf2 and inhibition of its nuclear export are

17

sufficient to explain synergistic neuroprotection by GAS/IRN remains unclear.

18

Therefore, these data suggest that an in vitro model of synergism between IRN and

19

GAS in the induction of neuroprotection warrant further investigations in vivo.

20 21 22 23 24 25

17



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1

METHODS

2 3

Cell culture and treatment paradigm

4 5

Human neuroblastoma cells (SH-SY5Y) were purchased from the Shanghai Institute

6

of Cell Biology (Shanghai, China) and grown in Dulbecco’s Modified Eagle’s

7

Medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum

8

and 1% penicillin-streptomycin maintained at 37 °C in a humidified atmosphere of 5%

9

CO2 and 95% air. SH-SY5Y cells were never cultivated beyond passage 20. For

10

neuronal differentiation of SH-SY5Y cells, retinoic acid (RA) was added a day after

11

plating at final concentration 10 ߤM and maintained for 3 days as previously

12

described [51]. SH-SY5Y cells were treated with 1 µM GAS (98% purity by HPLC;

13

Jingzhu Bio-Technology Co., Ltd, Nanjing, China), 0.3 µM IRN (98% purity by

14

HPLC; Jingzhu), GAS plus IRN, or tBHQ (98% purity by HPLC; Sigma-Aldrich, St.

15

Louis, MO) for 1 h prior to the challenge with MPP+ (Sigma-Aldrich) at 500 µM or

16

β-amyloid peptide (fragment 25-35; Sigma-Aldrich) in cultured hippocampal neurons.

17

for additional 24 h. For inhibitor studies, the cells were pretreated with or without

18

PD98059 (Sigma), DPI (Sigma), or/and LiCl (Sigma) for 30 min before treatment

19

with or without GAS or/and IRN in the presence or absence of MPP+.

20 21

Analyses of cell viability and proliferation

22 23

A MTS/PMS reagent based CellTiter 96® AQueous Non-Radioactive Cell Proliferation

24

Assay kit (Promega, Madison, WI) was used to quantify the number of viable cells.

25

Cell proliferation was assessed by using a commercially available chemiluminescent 18


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1

cell proliferation ELISA BrdU kit (Roche Applied Science, Mannheim, Germany)

2

according to the protocol provided by the manufacturer.

3 4

Measurement of oxidative stress

5 6

Intracellular ROS levels were determined by oxidative conversion of cell permeable

7

2',7'-dichlorfluorescein-diacetate (DCFH-DA) to DCF using OxiSelect™ Intracellular

8

ROS Assay Kit obtained from Cell Biolabs Inc. (San Diego, CA). The measurement

9

of lipid hydroperoxides (LPO) were performed by using Lipid Hydroperoxide Assay

10

kit, which measures LPO directly utilizing the redox reactions with ferrous ions,

11

purchased from Cayman Chemical Company (Ann Arbor, MI). Glutathione

12

peroxidase (GSH-Px) activity was measured indirectly by a coupled reaction with

13

GSH reductase using Glutathione Peroxidase Assay Kit (Cayman Chemical, MI), and

14

enzyme activity was expressed as the nmol/min/mg of protein. Thioredoxin reductase

15

(TrxR) activity was measured using Thioredoxin Reductase Activity Colorimetric

16

Assay Kit (BioVision Inc, Milpitas, CA). Total intracellular nicotinamide adenine

17

dinucleotide diaphorase (NADH) and NAD+ were measured using the NAD/NADH

18

Quantification Colorimetric Kit (BioVision), and total GSH and oxidized GSH (GSSG)

19

were measured using the GSH/GSSG-Glo™ Assay Kit (Promega). NAD+⁄ NADH

20

ratio and the GSH⁄GSSG ratio were then calculated based on levels of NAD, NADH,

21

GSH and GSSG. Superoxide dismutase (SOD) activity was measured using

22

Superoxide Dismutase Assay Kit (Cayman Chemical). All assays were performed as

23

per the corresponding manufacturer’s protocol.

24 25

Luciferase activity assay 19



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1 2

SH-SY5Y cells were transiently transfected with ARE-containing luciferase reporter

3

plasmid pGL4.37[luc2P/ARE/ Hygro] (Promega) by using FuGENE® 6 Transfection

4

Reagent (Promega), following the protocol provided by the manufacturer. The renilla

5

luciferase reporter plasmid pRL-TK (Promega) was used for standardization of

6

transfection efficiency. At 24 h post-transfection, SH-SY5Y cells were treated as

7

indicated for additional 24 h. After treatment, the cells were lysed and the luciferase

8

activity was measured using a Dual-Glo® Luciferase Assay System (Promega)

9

according to the manufacturer’s instructions. ARE-mediated luciferase activity was

10

expressed as firefly luciferase values normalized to renilla luciferase values.

11 12

Transcription factor activation assay

13 14

The mitogen-activated protein kinase (MAPK) regulated transcription factors ATF-2,

15

c-Myc, STAT1, c-Jun, and MEF2 in the nuclear extracts were measured using an

16

enzyme-linked immunosorbent assay (ELISA)-based TransAM™ MAPK Family

17

Transcription Factor Assay kits (Active Motif Corp., CA) per the manufacturer’s

18

instructions. The color developed, proportional to the amount of specific transcription

19

factor, after adding the HRP substrate was read as optical density units at 450 nm.

20 21

Real-time RT-PCR

22 23

Total RNA was isolated with TRIzol reagent (Invitrogene, Carlsbad, CA). RNA was

24

converted to cDNA by primeScript™ RT reagent kit (Takara Biotechnology, Dalian,

25

China). Real-time PCR was performed using a SYBR® premix Ex Taq™ (Takara) 20


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1

with the following primers: Nrf2, forward primer, 5’-CTT GGC CTC AGT GAT TCT

2

GAA GTG-3’, reverse primer, 5’-CCT GAG ATGG TGA CAA GGG TTG TA-3’;

3

catalytic subunits from glutamate cysteine ligase (GCLc), forward primer, 5’-GAA

4

GTG GAT GTG GAC ACC AGA TG-3’, reverse primer, 5’-TTG TAG TCA GGA

5

TGG TTT GCG ATA A-3’; GCLm, forward primer, 5’-GGA GTT CCC AAA TCA

6

ACC CAG A-3’, reverse primer, 5’-TGC ATG AGA TAC AGT GCA TTC CAA-3’;

7

Trx1, forward primer, 5’-TTG GAC GCT GCA GGT GAT AAA C-3’, reverse primer,

8

5’-GGC ATG GCA TGC ATT TGA CTT CAC ACT C-3’; TxR, forward primer,

9

5’-TAT CAG GAG GGC AGA CTT CAA-3’, reverse primer, 5’- GAC CAT CAC

10

CTT CTT GCC ATA-3’; glyceraldehyde-3-phosphate dehydrogenase (GAPDH),

11

forward primer, 5’-GAA GGT GAA GGT CGG ATG C-3’, reverse primer, 5’-GAA

12

GAT GGT GAT GGG ATT TC-3’. Data were analyzed using the comparative

13

threshold cycle (2-∆∆Ct) method.

14 15

Western blotting analyses

16 17

Nuclear and Cytoplasmic Protein Extraction kit (Beyotime Biotechnology, Haimen,

18

China) was used to fractionate nuclear and cytosolic fractions according to the

19

manufacturer’s instruction. Proteins were separated by SDS-PAGE and transferred

20

onto nitrocellulose membranes, and blotted with antibodies against BTB and CNC

21

homology 1 (Bach1), Nrf2, Keap1, p- c-Jun N-terminal kinases (JNK), p-Fyn, Akt,

22

and p-Akt, which were obtained from Santa Cruz Biotechnology (Santa Cruz, CA,

23

USA), antibodies to Trx1, p-ERK1/2, and p-p38MAPK, which were obtained from Cell

24

Signaling Technology (Beverly, MA, USA), as well as antibodies to GCLc, GCLm,

25

and p-GSK-3β, which were obtained from Abcam Inc. (Cambridge, MA, USA). After 21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

incubation with peroxidase-conjugated secondary antibodies, membranes were

2

developed with a SuperSignal™ West Pico (Thermo, MA). Equal protein loading was

3

controlled by immunoblot of GAPDH, Lamin B or the corresponding

4

nonphosphorylated species.

5 6

ERK1/2 kinase assay

7 8

ERK1/2 kinase activity assays were performed using nonradioactive kit obtained from

9

Cell Signaling Technology (Beverly, MA) as per the manufacturer’s protocol. Briefly,

10

cell lysates were immunoprecipitated using a monoclonal phospho-ERK1/, then

11

incubated with an Elk-1fusion protein, which allowed precipitated phospho-ERK to

12

phosphorylate Elk-1, a major substrate of p-ERK1/2.

13 14

Adenovirus infection and plasmids transfection

15 16

ERK2 Recombinant Adenovirus (Dominant Negative) were purchased from Cell

17

Biolabs Inc (San Diego, CA). For overexpression studies, SH-SY5Y Cells were plated

18

in 6 well dishes. The virus was added directly to the cells at a final concentration of

19

108 PFU/ml and allowed to incubate for an additional 24 hours, and then treated as

20

described above. Fyn shRNA, GSK-3β shRNA, and Nrf2 shRNA were purchased

21

from Santa Cruz Biotechnology. For gene knockdown studies, SH-SY5Y Cells were

22

transfected with short hairpin RNA (shRNA) plasmid as indicated using FuGENE® 6

23

Transfection Reagent obtained from Promega (Madison, WI) according to the

24

manufacturer’s protocol, allowed to incubate for 24 h, and then treated as described

25

above. Control shRNA Plasmid-A (Santa Cruz Biotechnology, Santa Cruz, CA) was 22


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1

used as a negative control. Knockdown efficiency of ~70% was observed with

2

Dominant Negative mutation or shRNA plasmid, as determined by western blot.

3 4

Statistical analysis

5 6

Data in bar graphs represent means (SD) and means were obtained from average data

7

of at least three independent experiments, if not otherwise stated. An analysis of

8

variance (ANOVA) followed by post hoc Student-Newman-Keuls (SNK) test was

9

carried out for multiple comparisons using SSPS software. Statistical significance was

10

accepted for P values ＜ 0.05.

11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 23



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1 2

ASSOCIATED CONTENT

3 4

Supporting Information

5

Result from ELISA BrdU assay (Table S1 and Figure S1), MTS assay (Table S2 and

6

Figure S2), MTS assay (Figure S3), SOD activity assay (Figure S4), ELISA-based

7

TransAM™ Nrf2 assay (Figure S5), and Western blotting analyses for Akt

8

phosphorylation (Figure S6).

9 10 11

This material is available via the Internet at http://pubs.acs.org.

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 24


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1

AUTHOR INFORMATION

2 3

Corresponding author

4 5

* E-mail address: [email protected]

6 7

Author Contributions

8 9

Miaoxian Dong and Xiaojie Zhang designed the study, and supervised the study.

10

Qiang Li and Miaoxian Dong carried out experiments. Miaoxian Dong and Chengu

11

Niu undertook the statistical analysis, and wrote the first draft of the manuscript.

12 13

Funding

14 15

This work was supported by the National Natural Science Foundation of China grant

16

81373629 awarded to Miaoxian Dong.

17 18

Notes

19 20

The authors declare that they have no conflict of interest.

21 22 23 24 25

25



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1

ACKNOWLEDGMENTS

2 3

We thank Dr. Yingcai Niu for critical comments on the manuscript and Xiaoming Li

4

for secretarial help.

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

26


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33



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1

Table 1

2

exposed to MPP+

Page 34 of 54

Combination index for GAS and IRN on cell viability in SH-SY5Y cells

3

GAS (µM)

IRN (µM)

CI

Fa

Description

0.25

0.075

1.205

0.368

Antagonism

0.5

0.15

0.705

0.414

Synergism

1.0

0.3

0.614

0.447

Synergism

2.0

0.6

0.685

0.471

Synergism

4.0

1.2

0.830

0.492

Synergism

4

Data was analyzed using CompuSyn software. Interpretation of CI values: CI < 0.9

5

means synergism; CI = 0.9 – 1.1 means additive; CI > 1.1 means antagonism. CI,

6

combination index; Fa, fraction affected (that is, absorbance)

7 8 9 10 11 12 13 14 15 16 17 18 19

34


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1

FIGURE LEGENDS

2 3

Figure 1

4 5

IRN in combination with GAS exerts synergetic neuroprotection against MPP+

6

toxicity through a Nrf2-regulated antioxidant mechanisms. SH-SY5Y cells were

7

treated with varying concentrations of IRN and GAS at a fixed ratio (3:10) for 1 h

8

prior to the challenge with MPP+ at 500 µM for additional 24 h. A, cell viability was

9

evaluated by MTS assay. B, after 24 h of transfection with Nrf2 shRNA, SH-SY5Y

10

cells were treated with IRN and/or GAS. Cell viability was evaluated by MTS assay.

11

C, SH-SY5Y cells were treated with IRN and/or GAS, and intracellular ROS

12

generation was assessed by the DCFH-DA method. D, lipid hydroperoxide was

13

assessed by utilizing the redox reactions with ferrous ions. All values are expressed as

14

means ± SD, n = 3; *, p < 0.05 vs. control; §, p < 0.05 vs. MPP+ challenge; ∫, p < 0.05

15

vs. GAS or IRN alone.

16 17

Figure 2

18 19

GAS/IRN enhances GSH system in MPP+-challenged SH-SY5Y cells. A,

20

SH-SY5Y cells were treated with IRN and/or GAS followed by challenge with MPP+

21

for 24 h. GSH and GSSG levels were assessed by fluorometric techniques. B, protein

22

expression of GCLc and GCLm were analyzed by western blot using indicated

23

antibodies. GAPDH served as loading controls. C, bar graph shows quantification of

24

GCLc and GCLm normalized to GAPDH. D, total RNA was extracted by the Trizol

25

method, and the levels of GCLc and GCLm mRNAs were determined by real-time 35



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1

RT-PCR, normalized by GAPDH. E, GSH-Px activity was determined using the

2

spectrophotometric method. All values are expressed as means ± SD, n = 3; *, p

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