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Honokiol alleviates oxidative stressinduced neurotoxicity via activation of Nrf2 Yanan Hou, Shoujiao Peng, Xinming Li, Juan Yao, Jianqiang Xu, and Jianguo Fang ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00290 • Publication Date (Web): 10 Jul 2018 Downloaded from http://pubs.acs.org on July 11, 2018

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ACS Chemical Neuroscience

Honokiol alleviates oxidative stress-induced neurotoxicity via activation of Nrf2

Yanan Hou1, Shoujiao Peng1, Xinming Li1, Juan Yao1, Jianqiang Xu2 and Jianguo Fang1*

1

State Key Laboratory of Applied Organic Chemistry and College of Chemistry and Chemical

Engineering, Lanzhou University, Lanzhou 730000, China 2

School of Life Science and Medicine, Dalian University of Technology, Panjin Campus, Panjin

124221, China.

*Corresponding author, E-mail: [email protected]; Fax: +86 931 8915557.

1

ACS Paragon Plus Environment

ACS Chemical Neuroscience 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

ABSTRACT Honokiol (Hon), a polyphenol and main active ingredient from the bark of Magnolia officinalis, has been documented having multiple pharmacological functions, including neuroprotection. However, the mechanisms underlying its neuroprotective effects are not well defined. In the present study, we reported that Hon attenuates the H2O2- or 6-hydroxydopamine (6-OHDA)-induced apoptosis of PC12 cells via elevation of glutathione level and upregulation of a multitude of cytoprotective proteins, including heme oxygenase 1, NAD(P)H: quinone oxidoreductase 1, thioredoxin 1 and thioredoxin reductase 1. Further studies reveal that Hon promotes the transcription factor Nrf2 nuclear translocation and activation. Moreover, the cytoprotection of Hon was antagonized by silence of Nrf2 expression, highlighting that Nrf2 is critically engaged in the cellular functions of Hon. Taken together, our study identified that Hon is an effective agonist of Nrf2 in neuronal system and displays potent neuroprotection against oxidative stress-mediated PC12 cell damages. These findings indicate that Hon is promising for further development to be a therapeutic drug against the oxidative stress-related neurodegenerative disorders.

Keywords: Honokiol, Nrf2, Oxidative stress, Antioxidant, Neuroprotection

Table of Contents Graphic (1.373 inches x 3.307 inches)

INTRODUCTION 2


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Reactive oxygen species (ROS) are produced from metabolic pathways and various enzymatic reactions, and eliminated by the cellular antioxidant defense system.1, 2 The intracellular production and removal of ROS exists in homeostasis, and ROS at physiological levels are important signaling molecules in regulating diverse cellular redox events.3 However, excessive ROS lead to oxidative stress, which causes deleterious effects to lipids, DNA and proteins. The brain contains a high content of polyunsaturated fatty acids and consumes a large amount of oxygen, and thus is especially vulnerable to oxidative damage.4, 5 As such, neurodegenerative diseases, such as Parkinson's disease, Alzheimer's disease and Huntington's disease, are closely associated with oxidative stress.6, 7 In line with these observations, strategies to upregulate the cellular antioxidant defense system have shown promises in prevention or treatment of neurodegeneration.7, 8 A set of stress-induced cytoprotective species, such as glutathione (GSH), thioredoxin (Trx) and thioredoxin reductase (TrxR) proteins, glutathione S-transferase family enzymes, heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1), are critical in responding to oxidative injuries and electrophilic assaults.9-12 The expression of these proteins is predominantly regulated by the nuclear factor (erythroid-derived 2)-like 2 (Nrf2), an evolutionally conserved transcription factor in mammalian cells. With the aid of small Maf proteins, Nrf2 binds to the consensus sequences of the antioxidant response element (ARE), which is located in the promoter regions of the genes encoding these cytoprotective species.9, 10, 13, 14 Under physiological conditions, Nrf2 is anchored in the cytosol by its inhibitory partner Kelch-like ECH-associated protein 1 (Keap1) to facilitate the ubiquitination and concomitant proteolysis of Nrf2, leading to a low homeostasis of Nrf2 in multiple types of cells. Under electrophilic/oxidative stress conditions, the cysteine residues in Keap1 are readily modified. As some of these cysteine residues are essential for the association of Keap1 with Nrf2, this modification may lead to a separation of Nrf2 from Keap1.9, 12, 14, 15 This dissociation enables Nrf2 to escape the proteasome degradation, thus elevating the cellular Nrf2 level and further promoting its translocation into nuclei. With the accumulation in nuclei and formation of a heterodimer complex with small Maf proteins, Nrf2 binds to ARE and initiates the transcription of a set of antioxidant genes.9, 13 As Nrf2 is a master regulator of the cellular antioxidant response, approaches to activate Nrf2 have gained increasing interests for treatment of neurodegenerative diseases.8, 12, 16-21 Honokiol (Hon) is a phenolic compound and main ingredient of the bark of the Magnolia 3



officinalis, a traditional Chinese medicine commonly known as Houpo (Chinese: 厚朴 ). This molecule has been documented showing multiple pharmacological functions, including anti-inflammatory,22, 23 antioxidant,24, 25 and anti-bacterial abilities.26 In addition, Hon and the crude extract of Magnolia officinalis have been demonstrated its beneficial effects on neurodegenerative disorders in the past years.27-31 However, the underlying mechanisms have not well defined. We disclosed herein that Hon is a potent agonist of Nrf2, and confers a remarkable protection against the H2O2- or 6-hydroxydopamine (6-OHDA)-mediated apoptosis of PC12 cells. Hon promotes the Nrf2 nuclear translocation and elicits the induction of a panel of Nrf2-driven antioxidant enzymes. Transfection of the interfering RNA to silence the expression of Nrf2 abolishes the protection, indicating the essential role of Nrf2 for the cellular function of Hon.

RESULTS AND DISCUSSION Protection of H2O2- or 6-OHDA-induced cell damage We first studied the cytotoxicity of Hon to PC12 cells in order to choose a suitable dosage for the following experiments. As shown in Fig. 1B, Hon showed moderate toxicity and a significant cytotoxicity was observed when the concentrations are more than 20 µM. Thus, the highest concentration used for the following experiments is no more than 10 µM. Then we employed the H2O2 and 6-OHDA injury models to explore the cytoprotective effects of Hon. H2O2 is an endogenous oxidant, and is generally used to generate an oxidative stress model. 6-OHDA is a specific neurotoxin targeting the dopaminergic and noradrenergic neurons, and has been widely applied to induce an experimental model of Parkinsonism. As shown in Fig. 1C & D, treatment of PC12 cells with H2O2 (500 µM) or 6-OHDA (200 µM) for 12 h leads to ~60% death of PC12 cells. However, pretreatment of PC12 cells with low concentrations of Hon (1, 5 and 10 µM) dose-dependently protects PC12 cells from the H2O2- or 6-OHDA-induced cell death. To confirm the MTT results, we then determined the LDH content in the culture medium. LDH is released into the medium once the cell membrane integrity is damaged. Consistent with the results from the MTT assay, pretreatment with Hon significantly decreased the LDH leakage elicited by H2O2 or 6-OHDA (Fig. 1E & F). Taken together, Hon at non-toxic concentrations prevents H2O2- or 6-OHDA-induced neurotoxicity of PC12 cells. 4


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Inhibition of ROS accumulation DCFH-DA, a fluorescent dye for detecting cellular ROS, was used to investigate whether Hon alleviates ROS accumulation in PC12 cells. Results showed that intracellular ROS burst after PC12 cells were stimulated with either H2O2 or 6-OHDA. However, pretreatment with Hon significantly inhibits the ROS level in a concentration-dependent manner (Fig. 2A & B). These results indicated that Hon could antagonize H2O2- or 6-OHDA-induced ROS accumulation in PC12 cells. Alleviation of H2O2- or 6-OHDA-induced apoptosis Appearance of condensed nucleus and activation of caspase-3 are two characters of apoptosis. In line with the previous reports that H2O2 or 6-OHDA causes cytotoxicity to PC12 cells mainly through induction of apoptosis.32 H2O2- or 6-OHDA-treatment elicits nucleus condensation (indicated by arrows in Fig. 3A & B) and caspase-3 activation (Fig. 3C & D), two typical characters of apoptosis. Pretreatment of the cells with Hon significantly rescues the cells from apoptosis evidenced by the decreased population of the condensed nuclei as well as inhibition of the caspase-3 activation. Upregulation of antioxidant species in PC12 cells Since Hon displays capacity to alleviate the oxidative stress-mediated cell damage, we thus hypothesized that Hon might activate the transcription factor Nrf2, a master player that regulates the cellular antioxidant response. Then we determined the expression of the Nrf2-driven antioxidative genes, including HO-1, NQO1, Trx1, TrxR1, GCLC, and GCLM. As shown in Fig. 4A, treatment of the cells with Hon (10 µM) significantly upregulates these genes’ expression with varying extents. We further examined the expression of the products encoded by these genes. As shown in Fig. 4B & C, treatment of PC12 cells with Hon remarkably upregulated NQO1, Trx1, TrxR1, and HO-1 proteins expression. GSH, a ubiquitous tripeptide thiol, plays a critical role in maintaining the cellular redox homeostasis. GCLC and GCLM are two subunits of the glutamate cysteine ligase, a heterodimeric holoenzyme catalyzing the rate-limiting step in the synthesis of the GSH.33 The total GSH level increases ~1.6-fold when the cells were treated with Hon (10 µM) for 12 h (Fig. 5D). In addition, the activities of NQO1 (Fig. 5A), TrxR (Fig. 5B), and Trx (Fig. 5C) increases by ~1.6-, ~1.5-, and ~1.3-fold, respectively. The activity of TrxR was also determined by TRFS-Green, a selective fluorescent probe of TrxR (Fig. 5E). It gives consistent results as those shown in Fig. 5B. 5



Taken together, these results support that Hon may upregulate a panel of endogenous antioxidant molecules. Rescue of cellular antioxidant defense system The H2O2 or 6-OHDA treatment caused a serious decline of GSH level and activities of NQO1, TrxR, and Trx (Fig. 6). Pretreatment of the cells with Hon (10 µM) completely restores the total GSH level to that of the control group (Fig. 6D). Other antioxidant enzymes, such as NQO1 (Fig. 6A), TrxR (Fig. 6B), and Trx (Fig. 6C) were also rescued if the cells were preincubated with Hon followed by a H2O2 or 6-OHDA assault. Induction of Nrf2 nuclear translocation The translocation of Nrf2 to nuclei from cytosol is prerequisite for the induction of antioxidant genes expression. We next asked whether Hon induced Nrf2 nuclear translocation. After preparation of different subcellular fractions, i. e., the total protein extract, the cytosolic protein extract and the nuclear protein extract, we determined the kinetic of Nrf2 homeostasis by Western blots. After Hon treatment, the nuclear Nrf2 increases (Fig. 7A), while the cytosolic Nrf2 decreases (Fig. 7A). Hon upregulates the total Nrf2 level slightly but significantly (Fig. 7A). The quantitative results were shown in Fig. 7B, C & D. These results indicate that Hon promotes the translocation of Nrf2 to nuclei, which facilitates Nrf2 to bind to ARE for the transcription process. Involvement of Nrf2 for the protection of Hon The above results demonstrated that Hon promotes Nrf2 accumulation in nuclei and upregulates a panel of Nrf2-driven antioxidant molecules. To further examine the contribution of Nrf2 to the cellular action of Hon, we turned to compare the effects of Hon in PC12-shNrf2 cells and the control cells (PC12-shNT cells). The Nrf2 shRNA transfection decreased the Nrf2 protein expression down to 30% of the control (Fig. 8A & B). Next, we evaluated the protective effect of Hon against the H2O2- or 6-OHDA-induced cell death. As shown in Fig. 8C & D, Hon alleviates the H2O2- or 6-OHDA-induced cytotoxicity to the PC12-shNT cells, while this protective effect was almost abolished in the PC12-shNrf2 cells. These results demonstrated that Nrf2 is essential for the cytoprotection of Hon in PC12 cells. Medicinal chemical properties of Hon The ability to pass the blood-brain barrier (BBB) is of great importance for a central nervous 6


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system (CNS) drug. A molecule’s medicinal chemical properties could help predict whether it could cross the BBB. The follows are general criteria for a molecule to pass the BBB: the molecular weight (MW) < 450, the polar surface area (PSA) < 70 Å2, and the clog P of 0 to 5.34, 35 Hon has a MW of 266.34, and the PSA value was 40.46 by online calculation. The clog P value of Hon was 3.92, which was obtained by detecting the partition coefficient between n-octanol and water. Thus, it is much likely that Hon has the ability to cross the BBB. Indeed, Lin et al. and Wang et al. documented that Hon could be detected in brain tissues after intravenous injection.36, 37

The Magnolia bark (also known as Houpo in Chinese) is used in the traditional Chinese medicine with a long history. The bark extracts have efficacy to various ailments, such as gastrointestinal disorders and cough, and shows nontoxic in both acute and subchronic studies.38, 39 As a major active and abundant molecule in the bark extracts, Hon has been attracted increasing interests to explore its pharmacological activities.40, 41 Recent studies have reported that Hon could activate Nrf2 in different cell types, including pancreatic β cells,42 hepatocytes43 and osteoblast cells.44 However, as far as we know, it is not clear whether Hon could work on Nrf2 in neuronal system. In this work, we employed the neuroblastic PC12 cells as a model, and disclosed that Hon upregulates the antioxidant defense capacity and attenuates the oxidative stress-mediated neurotoxicity via activating Nrf2. Hon also showed protection against beta-amyloid-induced toxicity in PC12 cells.28, 45 We demonstrated here that Hon, at the concentration as low as 5 µM, significantly rescues the cells from H2O2- or 6-OHDA-induced oxidative damage (Figs. 1-3). Furthermore, genetic knockdown of Nrf2 almost completely abolishes the protective effect (Fig. 8), highlighting that activation of Nrf2 underlying the cellular actions of Hon is of physiological significance. In addition, the medicinal chemical properties of Hon and experimental data indicate that Hon could traverse the BBB, supporting that Hon deserves further development as a CNS drug. Besides the activation of Nrf2 by modulation of Cys residues within the Keap1, there are other mechanisms of Nrf2 activation, such as the autophagy lysosomal pathway46 and synoviolin-Nrf2 pathway.47 Nevertheless, targeting the Cys residue(s) of Keap1 is a well-elucidated mechanism for Nrf2 activation. The Keap1 protein is the major negative regulator of Nrf2 by binding to Nrf2 and directing the degradation of Nrf2, and the sulfhydryl groups in Keap1 is essential for its association 7



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with Nrf2. Two classes of compounds, i. e., polyphenol compounds or molecules with an electrophilic center, are well documented as potent activators of Nrf2.48, 49 Hon is a polyphenol, and its structure facilitates it to form a fairly stable quinone intermediate upon oxidation (The proposed structure was shown in the dashed square in Fig. 9) as proposed for many polyphenol Nrf2 activators.19, 20, 50, 51 This quinone intermediate is reactive and readily modifies the Keap1 sulfhydryl groups leading to the dissociation of Keap1 from Nrf2, which in turn stabilizes Nrf2 and promotes it translocating into nuclei (Fig. 7). With the aid of small Maf proteins in nuclei, Nrf2 subsequently binds to the ARE motif in the upstream promoter region of several cytoprotective genes to drive their transcription and translation (Figs. 4-6). Upregulation of the antioxidant defense system thus provides protection against the oxidative stress-induced neurotoxicity (Figs. 1-3). This is likely the underlying mechanism under the observed neuroprotection by Hon (Fig. 9). In summary, we disclosed that Hon, a major active molecule in the traditional Chinese medicine

Houpo, is efficient in preventing PC12 cells from oxidative damage. This cytoprotective effects are via the activation of Nrf2 and the subsequent upregulation of endogenous antioxidant defense system. Our results reveal that Hon is a novel agonist of the Nrf2-ARE signaling pathway in the neuronal system, and this mechanism of action sheds lights in considering further development of Hon as a potential neuroprotective drug.

METHODS Materials Dulbecco's modified Eagle’s medium (DMEM), glutathione reductase (GR) from yeast, dimethyl

sulfoxide

(DMSO),

2’,7’-dichlorfluorescein

diacetate

(DCFH-DA),

2,6-dichlorophenol-indophenol (DCPIP), Hoechst 33342, N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNA), NADH, 6-hydroxydopamine (6-OHDA), 5,5’-dithiobis-2-nitrobenzoic acid (DTNB),

penicillin,

streptomycin

and

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide (MTT), were purchased from Sigma-Aldrich (St. Louis, USA). Hon (>98% pure) was obtained from Chengdu Must Bio-Technology (Chengdu, China). NADPH was acquired from Roche (Mannheim, Germany). Fetal bovine serum (FBS) was purchased from HyClone. Primary antibodies against TrxR1 and HO-1 were purchased from Santa Cruz Biotechnology (CA, USA) 8


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and Proteintech (IL, USA), respectively. Primary antibodies against Trx1, NQO1, Nrf2 and actin were from Sangon Biotech (Shanghai, China). Horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology. The shRNA plasmids targeting the rat

Nrf2 gene (shNrf2) and the control nontargeting shRNA (shNT) were products of GenePharma Co, Ltd (Shanghai, China). Phenylmethylsulfonyl fluoride (PMSF), sodium orthovanadate (Na3VO4) and bovine serum albumin (BSA) were acquired from Beyotime (Nantong, China). GeneTran III transfection reagent was purchased from Biomiga (CA, USA). The RNA extraction kit RNAiso plus, the reverse-transcription kit PrimescriptTM RT and Power SYBR Green PCR Master Mix were from TaKaRa (Dalian, China). The recombinant protein of E. coli Trx was expressed and purified as described in our published work.52 The recombinant protein of rat TrxR1 was provided by Prof. Arne Holmgren (Karolinska Institute, Sweden). Other chemicals and reagents are of analytical grade. Cell Culture PC12 cells (rat adrenal pheochromocytoma cells) were purchased from Shanghai Institute of Biochemistry and Cell Biology, and grown at 37 °C in DMEM containing 10% FBS, 100 units/mL penicillin/streptomycin and 2 mM glutamine in a humidified incubator with 5% CO2 and 95% air. PC12 cells in exponential growth phase were employed in all experiments. The stock solution of Hon (100 mM) was prepared by dissolving it in DMSO. The final DMSO concentrations are no more than 0.4% in all experiments. MTT assay The stock solution of MTT (5 mg/mL), dissolved in phosphate buffered saline (PBS), was stored at -20 ºC. PC12 cells (1×104 cells/well) were grown in 96-well plates for one day. Then, the cells were treated with Hon for 24 h. The cell viability was determined by the MTT assay as described in our previous publications.53, 54 To measure the neuroprotective effect of Hon against H2O2- or 6-OHDA-induced cell death, PC12 cells (1×104 cells/well) were seeded in 96-well plates for one day and then incubated with various concentrations of Hon for 12 h. The cells were further exposed to H2O2 (500 µM) or 6-OHDA (200 µM) in fresh medium for additional 12 h, and the MTT assay was performed to measure the cell viability. 9



Lactate dehydrogenase (LDH) assay The cytosolic protein LDH is leaked into the culture medium when the cell membrane is damaged. Detection of LDH in the medium is a convenient way to assay the cell damage. For this experiment, PC12 cells were seeded in 12-well plates (2×105 cells/well) for 24 h. Then the cells were exposed to Hon for 12 h. After the cells were further cultured in the fresh medium containing H2O2 (500 µM) or 6-OHDA (200 µM) for another 12 h, the leakage of LDH was evaluated by measuring its activity in the culture medium according to the method described in previous publications.51, 55 Determination of ROS in cells The redox sensitive fluorescent probe DCFH-DA was employed to determine the accumulation of ROS in PC12 cells. After the cells were seeded in a 12-well plate (2×105 cells/well) and grown for 24 h, the cells were then treated with Hon for another 12 h. The culture medium was discarded and the fresh medium containing H2O2 (500 µM) or 6-OHDA (200 µM) was supplied, and the cells were continued to incubate for 5 h. The culture medium was replaced by the fresh FBS-free medium with DCFH-DA (10 µM), and the cells were maintained at 37 °C for 30 min. Cell images were acquired by an inverted fluorescent microscopy (Leica DM4000) under the green channel. Hoechst 33342 staining assay Hoechst 33342 is a cell membrane permeable dye. It binds to DNA and gives a strong blue fluorescence. PC12 cells were seeded in a 12-well plate (2×105 cells/well) for 24 h, and then treated with Hon for 12 h. After replacing the culture medium with the fresh one containing H2O2 (500 µM) or 6-OHDA (200 µM) and continuing growth for 5 h, the cells were stained with Hoechst 33342 (5 µg/mL) in fresh medium without FBS for 15 min at 37 °C. Cell images were recorded by an inverted fluorescent microscopy (Leica DM4000) under the blue channel. Caspase-3 activity assay The caspase-3 activity was determined according to our published procedure.51, 55 In brief, the treated PC12 cells were collected, washed with ice-cold PBS, and lysed with RIPA buffer. The total proteins of the extract were quantified by the Bradford procedure. Then the protein extract from the cells were incubated with Ac-DEVD-рNA for 4 h at 37 °C. The absorbance at 405 nm, due to the release of the p-nitroanilide from the Ac-DEVD-рNA under caspase-3 catalyzation, was measured. qRT-PCR 10


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After PC12 cells (1×106 cells/well) were seeded in 60-mm dishes and allowed to grow for 24 h, Hon (10 µM) was added and incubated for the indicated times. The total cellular RNA was prepared with the RNAiso plus by following the manufacturer’s instructions. The cDNA was prepared by reverse-transcription from 50 nanograms of total RNA, which was performed by using the PrimescriptTM RT reagent kit. The qRT-PCR was performed on the Agilent Mx3005P RT-PCR System by using the Power SYBR Green PCR Master Mix. Samples were run in triplicate and the relative expression of different genes was measured by normalizing the expression of each target mRNA to that of the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The relative gene expression was calculated with the 2-△△Ct method. The primers were ordered form Invitrogen (Shanghai, China), and the sequences were as the follows: 5’-gccctggaagaggagatagag-3’ and 5’-tagtgctgtgtggctggtgt-3’ for HO-1; 5’-ccttctttcattccctctgtgac-3’ and 5’-cccaaccttttgaccctttttat-3’ for

Trx1;

5’-actgctcaatccacaaacagc-3’

and

5’-ccacggtctctaagccaatagt-3’

for

TrxR1;

5’-tcaccactctactttgctccaa-3’ and 5’-ttttctgctcctcttgaacctc-3’ for NQO1; 5′-caaggacaagaacacaccatct-3′ and 5′-cagcactcaaagccataacaat-3′ for the catalytic subunit of glutamate cysteine ligase catalytic subunit (GCLC); 5′-ggcacaggtaaaacccaatagt-3′ and 5′-ttcaatgtcagggatgctttct-3′ for the modifier subunit

of

glutamate

cysteine

ligase

(GCLM);

5’-cagtgccagcctcgtctcat-3’

and

5’-aggggccatccacagtcttc-3’ for GAPDH. Western blot analysis For western blot, the whole cell extracts, nuclear protein extracts and cytosolic protein fraction were prepared from the treated cells as described previously.53 An equal amount of total protein samples (30 µg of total protein per lane) from each group were separated by SDS-PAGE, and then transferred onto the methanol-activated PVDF membranes (Millipore, USA). The membranes were blocked with 5% nonfat milk for 1 h at room temperature before they were incubated with different primary antibodies (1:1000) overnight at 4 °C. After that, they were further incubated with the corresponding secondary antibodies (1:2000). Then, the membranes were incubated with the enhanced chemiluminescence kit (GE Healthcare Life Sciences), and the signals were acquired by the Imagequant LAS4000 (GE Healthcare). The relative band intensities were quantified by using the ImageJ software. Assay of total glutathione, and activities of NQO1, Trx and TrxR 11



After the cells were treated with Hon for 12 h, they were harvested and lysed in KPE buffer (0.1 M PBS, pH 7.5, with 5 mM EDTA, 0.6% sulfosalicylic acid and 0.1% TritonX-100). The total proteins of the cell extract were quantified by the Bradford procedure, and used for the enzymatic measurement of total GSH as described in the published protocols.56, 57 To measure the activities of NQO1, TrxR and Trx, the cells were harvested and lysed in RIPA buffer. Then the total protein of the extract was quantified by the Bradford procedure. The activities of NQO1,51, 58 TrxR and Trx59, 60 were measured according to our published protocols. Image-based TrxR activity in live PC12 cells TRFS-Green, a fluorescent probe for TrxR, is designed and synthesized in our group.61 TrxR catalyzes the reduction of TRFS-Green to emit the green fluorescence. The cells (2×105 cells/well) were grown in 12-well plates for one day. Then the cells were treated with Hon for 8 h, followed by addition of TRFS-Green (10 µM) and incubation for another 4 h at 37 °C in dark. Cell images were acquired by an inverted fluorescent microscopy. The fluorescence intensity represents the relative TrxR activity in cells.

Nrf2-shRNA transfection PC12 cells (2×105 cells/well) were seeded in a 6-well plate. When the cells achieved ~60% confluence, they were transfected with the Nrf2-shRNA (shNrf2-842) or the nontargeting control shRNA (shNT) using the Gene Tran III transfection reagent by following the manufacturer’s protocol. The cells were selected by G418 (0.5 mg/mL) to generate the stably transfected cells, i. e., PC12-shNrf2 and PC12-shNT. The expression of Nrf2 in shNrf2-842 cells and shNT cells were determined by the Western blots. Determination of cLogP and PSA The partition coefficient of Hon between water and n-octanol was determined according to the published method.51 The PSA values were calculated from the website (www.molinspiration.com). Statistical analysis Data were expressed as the mean ± standard deviation (SD). The statistical differences between two groups were analyzed using Student’s t-test. The statistical differences among multiple groups were analyzed using one-way analysis of variance (ANOVA). P-values less than 0.05 were considered to be statistically significant. 12


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AUTHOR CONTRIBUTIONS Y. Hou, S. Peng and J. Fang designed the research; Y. Hou, S. Peng, X. Li and J. Yao performed the research; Y. Hou, J. Xu and J. Fang analyzed and discussed the data; And Y. Hou and J. Fang wrote the paper.

ACKNOWLEDGEMENTS The authors appreciated Prof. Arne Holmgren (Karolinska Institute, Sweden) for the recombinant rat TrxR. This work was supported by the National Natural Science Foundation of China (21572093, 21778028, 31670767), Lanzhou University (the Fundamental Research Funds for the Central Universities, lzujbky-2017-ot02 & lzujbky-2017-sp06), Dalian University of Technology (the Fundamental Research Funds for the Central Universities, DUT17JC36 and DUT17RC(4)27) and the 111 project.

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Figures and Figure Captions

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(1-column width) Fig. 1 Hon confers protection against H2O2- or 6-OHDA-induced cell damage in PC12 cells. (A) The chemical structure of Hon. (B) Cytotoxicity of Hon on PC12 cells. PC12 cells (1×104 cells/well) were plated in 96-well plates for 1 day and then treated with increasing concentrations of Hon for 24 h. Then, the cell viability was measured using the MTT method. (C) & (D) Protective effects of Hon against H2O2- or 6-OHDA-induced PC12 cell injury. PC12 cells (1×104 cells/well) were plated in 96-well plates for 1 day and then treated with increasing concentrations of Hon for 12 h. After incubation with fresh medium containing H2O2 (500 µM) or 6-OHDA (200 µM) for 12 h, the cell viability was measured by the MTT method. (E) & (F) Effects of Hon pretreatment on H2O2- or 6-OHDA-induced LDH release. PC12 cells (2×105 cells/well) were plated in 12-well plates. On the next day, the cells were exposed to Hon for 12 h, followed by addition of H2O2 (500 µM) or 6-OHDA (200 µM) for another 12 h. The LDH activity in the culture medium was measured. Results are from three independent experiments and expressed as means ± SD. *, P < 0.05, and **, P < 0.01 vs. the control group. ^, P < 0.05, and ^^, P < 0.01 vs. the H2O2 or 6-OHDA treatment.

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(1-column width) Fig. 2 Hon alleviates ROS production in PC12 cells. PC12 cells were seeded in 12-well plates and treated with Hon for 12 h, and then exposed to H2O2 (500 µM, A) or 6-OHDA (200 µM, B) for 5 h. ROS production was determined by DCFH-DA staining. Scale bars: 50 µm.

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(1-column width) Fig. 3 Hon attenuates H2O2- or 6-OHDA-induced apoptosis of PC12 cells. (A) & (B) PC12 cells were seeded in 6-well plates and treated with Hon or vehicle for 12 h, and then, incubated with fresh medium containing 500 µM H2O2 (A) or 200 µM 6-OHDA (B) for 5 h. Apoptotic cells were measured by Hoechst 33342 staining. Scale bars: 100 µm. (C) & (D) PC12 cells were plated in 60-mm dishes and treated with Hon or vehicle for 12 h. Then, cells were incubated with fresh medium containing H2O2 (500 µM, C) or 6-OHDA (200 µM, D) for 12 h. Activity of cellular caspase-3 was determined. Results from three independent experiments were expressed as means ± SD. *, P < 0.05, and **, P < 0.01 vs. the control group. ^, P < 0.05, and ^^, P < 0.01 vs. H2O2 or 6-OHDA treatment.

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(1-column width) Fig. 4 Hon upregulates antioxidant defense system in PC12 cells. (A) Induction of phase II genes. PC12 cells treated with Hon (10 µM) were harvested at indicated times, and total RNA was prepared. The samples were analyzed for the mRNA levels of NQO1, TrxR1, Trx1, GCLC, GCLM, and HO-1. (B) & (C) Induction of phase II proteins expression. Protein expression levels of NQO1, Trx1, TrxR1, and HO-1 in PC12 cells treated with Hon for 12 h were determined by Western blots. Actin was used to ensure equal protein loading. Representative Western blot results (B), and quantification of the blots (C) were shown. Results from three times independent experiments were expressed as means ± SD. *, P < 0.05, and **, P < 0.01 vs. the control group.

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(1-column width) Fig. 5 Hon upregulates activity of antioxidant molecules in PC12 cells. Elevation of NQO1 activity (A), TrxR activity (B), Trx activity (C), and total GSH (D). PC12 cells were seeded in 60-mm dishes and treated with Hon for 12 h. Then, the cells were harvested and the activities were detected. Results from three independent experiments were expressed as means ± SD. *, P < 0.05, and **, P < 0.01 vs. the control group. (E) Detection of the TrxR activity by the fluorescent probe TRFS-Green. PC12 cells were treated with Hon for 8 h, and then TRFS-Green (10 µM) was added and incubated for another 4 h. The relative fluorescence intensity (F. I.) in cells was quantified by the ImageJ, and was given at the bottom of each picture. Scale bar: 50 µm.

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(1-column width) Fig. 6 Hon rescues the antioxidant molecules activity. Hon prevented H2O2- or 6-OHDA-induced decrease of NQO1 activity (A), TrxR activity (B), Trx activity (C), and GSH level (D). PC12 cells were plated in 60-mm dishes and treated with Hon for 12 h followed by incubation with H2O2 (500 µM) or 6-OHDA (200 µM) for another 12 h. The cells were collected and the activities were detected. Results from three independent experiments were expressed as means ± SD. *, P < 0.05, and **, P < 0.01 vs. the control group. ^, P < 0.05, and ^^, P < 0.01 vs. H2O2 or 6-OHDA treatment.

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(1-column width) Fig. 7 Hon promotes Nrf2 nuclear translocation. PC12 cells were treated with 10 µM Hon for the indicated times. The cells were harvested, and cell extracts were prepared. (A) The nuclear Nrf2, cytosolic Nrf2 and total Nrf2 were analyzed by the Western blots. Quantification of the blots was shown in (B), (C) & (D). Results from three independent experiments were expressed as means ± SD. *, P < 0.05, and **, P < 0.01 vs. the control group.

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(1-column width) Fig. 8 Nrf2 is involved in the cytoprotection of Hon. (A) Validation of Nrf2 levels in PC12-shNT and PC12-shNrf2 cells. (B) Quantification of the blots in (A). PC12-shNT and PC12-shNrf2 cells were plated in 96-well plates and incubated with indicated concentrations of Hon for 12 h and then exposed to H2O2 (500 µM, C) or 6-OHDA (200 µM, D) for 12 h. Cell viability was determined by the MTT method. Results from three independent experiments were expressed as means ± SD. *, P < 0.05, and **, P < 0.01 vs. the control group. ^, P < 0.05, and ^^, P < 0.01 vs. H2O2 or 6-OHDA treatment.

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(1-column width) Fig. 9 A schematic model of activation of Keap1/Nrf2/ARE pathway by Hon.

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Honokiol Alleviates Oxidative Stress-Induced ... - ACS Publications

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