Puerarin protects dopaminergic neurons in Parkinson’s disease models

Neuroscience 280 (2014) 88–98

PUERARIN PROTECTS DOPAMINERGIC NEURONS IN PARKINSON’S DISEASE MODELS X. ZHANG, a  J. XIONG, a  S. LIU, a L. WANG, a J. HUANG, a L. LIU, a J. YANG, a G. ZHANG, a K. GUO, a Z. ZHANG, b P. WU, c D. WANG, c Z. LIN, d,e N. XIONG a* AND T. WANG a*

and decreased the abnormal protein overexpressing in PD animal models. These findings suggest that puerarin may develop into a neuroprotective alternative for patients with PD. Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

a Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hubei 430022, China b Department of Neurology, Renmin Hospital of Wuhan University, Wuhan 430060, China c

Key words: Parkinson’s disease, puerarin, oxidative stress, apoptosis.

Hefeng Central Hospital, Hefeng, Enshi, Hubei 445800, China

d Department of Psychiatry, Harvard Medical School, Division of Alcohol and Drug Abuse, and Mailman Neuroscience Research Center, McLean Hospital, Belmont, MA 02478, USA e

INTRODUCTION

Harvard NeuroDiscovery Center, Boston, MA 02114, USA

Parkinson’s disease (PD) is one of most common neurodegenerative disorders in the elder populations (Deas et al., 2011), the pathological feature of which is the loss of dopamine neurons and the presence of Lewy bodies within survival dopaminergic cells in the substantia nigra. Oxidative stress, reactive oxygen species (ROS) over-generation and cell apoptosis have been implicated as important mechanisms in the pathogenesis of PD (Giasson et al., 2000; Xiong et al., 2011). Currently, the medical therapy for PD, including levodopa, DA receptor agonists, catechol-O-methyltransferase (COMT) inhibitors, monoamine oxidase type B (MAO-B) inhibitor, serotonin agonist, adenosine A2 antagonist and anticholinergic agent (Savitt et al., 2006), are all challenged with therapeutic effects or side effects such as wearing-off, on–off phenomenon and dyskinesia. Up to now, there is still no available medication for effectively controlling both motor and non-motor symptoms, and for reversing the progressive nature of PD. Puerarin [7-hydroxy-3-(4-hydroxyphenyl)-1-benzopyran-4-one-8-(b-D-glucopyranoside), C21H20C9] is an active element and natural product extracted from Chinese traditional medicine pueraria lobata (Chen et al., 2012). It has been widely used for decades in China mainland as a powerful free radical scavenger for the clinical treatment of cardiovascular and cerebrovascular diseases to reduce the cardiomyocyte or neuronal damage. In both cardiovascular and cerebrovascular diseases, free radicals contribute to the cell death (Pung et al., 2013). Similar to that in PD, oxidative stress is related to generating cellular stresses that can feed into pathways to dopaminergic cell death (Higgins et al., 2010). These results demonstrate that puerarin (Pue, PUR) may hold the capability of protecting dopaminergic neurons and slowing down the neurodegenerative process in PD through anti-oxidative pathways. Moreover, puerarin is a type of natural estrogen (Shepherd, 2001). Epidemiological data

Abstract—It has been acknowledged that oxidative stress, resulting in the apoptosis of dopaminergic neurons, is a key mechanism in the pathogenesis of Parkinson’s disease (PD). Puerarin, extracted from the root of pueraria lobata, has been clinically used for ischemic heart disease and cerebrovascular diseases as an oxygen free radical scavenger. In this study, we aimed to explore the effect of puerarin on dopaminergic cell degeneration in vitro and in vivo and its possible underlying mechanisms. In SH-SY5Y cells, the reduction of cell viability, apoptosis rate and average DCFH-DA fluorescence intensity of puerarin-treated (0, 10, 50, 100 and 150 lM) cells were significantly lower than control group. In rotenone-based rodent models, puerarin treatment for 7 days ameliorated apomorphine-induced rotations significantly in Pue-50 and Pue-100 group by 45.65% and 53.06% in the first week, by 44.60% and 48.45% in the second week. Moreover, compared to control group, puerarin increased tyrosine hydroxylase (TH) expression in the substantia nigra by 85.52% and 84.26% in Pue-50 group and Pue-100 group, and upregulated the vesicular monoamine transporter 2 (VMAT2) by 41.24% in Pue-50 group and 35.20% in Pue-100 group, and decreased ubiquitin expression by 47.55% in Pue-50 group and 69.15% in Pue-100 group. These data indicated that puerarin alleviated the oxidative stress and apoptosis in a PD cellular model, protected the dopaminergic neurons against rotenone toxicity

*Corresponding authors. Address: Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1277 Jiefang Road, Wuhan 430022, Hubei, China. Fax: +86-27-85776343 (T. Wang), +86-27-85726670 (N. Xiong). E-mail addresses: [email protected] (N. Xiong), [email protected] (T. Wang).   These authors equally contributed to the work. Abbreviations: MPP+, 1-methyl-4-pehenylpyridinium; 6-OHDA, 6-hydroxydopamine; BBB, blood–brain barrier; PD, Parkinson’s disease; ROS, reactive oxygen species; SNc, substantia nigra compacta; TH, tyrosine hydroxylase; VMAT2, vesicular monoamine transporter 2. http://dx.doi.org/10.1016/j.neuroscience.2014.08.052 0306-4522/Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved. 88

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suggested that the mobility of PD in male was higher than that in female (Wooten et al., 2004). The follow-up laboratory study proved that the estrogen could protect the DA neurons (Horstink et al., 2003). The results from these studies further support that puerarin might be protective for dopamine neurons. A previous study indicated that puerarin was neuroprotective against 1-methyl-4-pehenylpyridinium (MPP+) and 6-hydroxydopamine (6-OHDA)induced toxicity by preventing the loss of tyrosine hydroxylase (TH) positive neurons (Horstink et al., 2003). However, the detailed mechanisms underlying the neuroprotective effects of puerarin have not been completely understood. Rotenone, the most important component of rotenoids, is extracted from Leguminosa plants (Bove et al., 2005). It is capable to cross the blood–brain barrier (BBB) and induce ROS production by inhibiting the mitochondrial respiratory chain complex I (Li et al., 2003), which contributes to cell apoptosis. Besides, rotenone also inhibits the formation of microtubules, which participates in dopaminergic neurodegeneration (Brinkley et al., 1974; Marshall and Himes, 1978). In addition, aggregation of a-Synuclein (SNCA) and polyubiquitin, nitrative stress-increased nitric oxide and malondialdehyde levels, activation of astrocytes and microglia, inflammatory reaction, glutamate excitotoxicity and neuron apoptosis are all implicated in the mechanisms of rotenone-evoked toxicity (Xiong et al., 2012). In this study, we aimed to examine the effect of puerarin on rotenone-treated SH-SY5Y cells and animals. The ROS generation and apoptosis rate in SH-SY5Y cells, and APO-induced rotation and the relevant proteins expression in the rat model were assessed to explore the effects of puerarin on the PD models and its potential underlying mechanisms.

EXPERIMENTAL PROCEDURES Cell culture Human neuroblastoma SH-SY5Y cells were cultured in DMEM/F12 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 7% fetal bovine serum (Invitrogen) at 37 °C in a 5% CO2 incubator. The culture medium was changed every 2 to 3 days, and the cells digested (0.5% trypsin) and passaged when reaching 70–80% confluence at the bottom of the culture flasks. MTT assay There were seven groups of treatments in 96-well plates: Con-group (cells without puerarin and rotenone treatment), Rot-group (Rot-group, without puerarin), and in another five puerarin + rotenone groups (Pue-groups, 10, 50, 100, 150 and 200 lM). Rotenone (3 lM) was given for 24 h in all groups to induce cell damage after puerarin had been treated for 24 h. Cells in these groups were assessed by MTT colorimetric assay. MTT (20 lL) was added to each well and incubated at 37 °C for 3–4 h. DMSO (150 lL, Amresco, Solon, OH, USA) was then added into each well and lysate spectrophotometrically measured for absorption at k 570 nm.

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Detection of ROS in SH-SY5Y cells DCFH-DA staining was employed to detect the intracellular ROS level. The treated groups were divided into five groups: Con-group, Rot-group, Pue10-group, Pue50-group, Pue100-group and Pue150-group. Rotenone was also given for 24 h to induce the SH-SY5Y cell damage after puerarin had been treated for 12 h. The harvested cells were suspended in 1 mL culture medium without fetal bovine serum, then added with DCFH-DA (20 lM, Sigma, St. Louis, MO, USA), and incubated at 37 °C in a 5% CO2 incubator for 20–30 min. Finally, all samples were examined by using the flow cytometry (BD, New Jersey, USA). Moreover, the cells were directly fluorescence stained with DCFHDA (20 lM) at 37 °C for 30 min, and was observed under a fluorescence microscope.

Apoptosis analysis The cells were harvested and suspended in 100 lL binding-buffer solution. The density of cells was adjusted to 5  105/mL. All the samples were given 10 lL Annexin V-FITC (Bender Medsystem, Vienna, Austria) incubated at 37 °C in darkness for 20 min, then examined by using flow cytometry (BD). Five minutes before examination, 5 lL PI and 400 lL binding-buffer were added to the samples. The whole process was performed in the darkness.

Animal treatment The animal experiment in this study was approved by the Ethics Committee on Animal Experimentation of Tongji Medical College, Huazhong University of Science and Technology, China. 37 eight-week-old male Sprague– Dawley rats (220 ± 20 g) from the center of Experimental Animals, Tongji Medical College, Huazhong University of Science and Technology were used. The temperature and the air humidity were maintained at 25 °C and 60 ± 5%. Rotenone (1 lL, 6 lg/lL in DMSO) was administrated by using a stereotaxic frame (RWD Life Science, Shenzhen, China) according to the methods of our previous study (Xiong et al., 2009). Briefly, the SD rats were anesthetized with 10% Chloral Hydrate (3.0 mL/kg, in 0.9% NaCl, intraperitoneal injection), and fixed on a flat. We injected rotenone to two sites, the right ventral tegmental area (VTA; anteroposterior 5.0 mm, lateral 1.0 mm, dorsoventral 7.8 mm) and substantia nigra compacta (SNc) (anteroposterior 5.0 mm, lateral 2.0 mm, dorsoventral 7.8 mm) with a rate of 0.2 lL/min. The needle remained for 5 min, then was withdrawal slowly for 5 min. In this experiment, the rotenone-treated rats were divided into three groups: Rotenone group (Rot-group), rotenone + Pue 50 mg/kg-group (Pue-50 group), and rotenone + Pue 100 mg/kg-group (Pue-100 group) Prasain et al., 2004; Zhu et al., 2010; Li et al., 2010, puerarin were intraperitoneally administrated to the rats daily for 7 days.

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APO-induced rotation The rats were put on a platform for adapting to the environment for 5 min Apomorphine was subcutaneously injected (APO, 2 mg/kg, Sigma) at three days after the surgery and the next every week until the fourth week. The rotation number of the rats was recorded over 10 min. Immunohistostaining The animals were sacrificed after the last APO-induced behavior test. The brain tissue was kept in the 4% paraformaldehyde for 24 h and then sliced by a sledge microtome (Letiz wetzlar, Wetzlar, GER). The midbrain was coronal sectioned 1.2 mm caudal to the bregma while the SNc was cut from 4.5 to 6.2 mm caudal to the bregma (Xiong et al., 2009). The sections (5 lm) were dewaxed in dimethylbenzene, washed in different concentrations of ethyl alcohol, then immersed in the 0.3% H2O2 for 30 min. After that, the sections were treated with 0.1% Triton-X100 for 30 min and with 5% bovine serum albumin (BSA, Amresco, Solon, OH, USA) for another 1 h. At last, the sections were incubated overnight with anti-tyrosine hydroxylase (TH, 1:500 dilution, sheep polyclonal antibody, Abcam, Cambridge, UK), anti-vesicular monoamine transporter 2 (VMAT2) (1:50 dilution, rabbit polyclonal antibody, Santa Cruz, CA, USA), Ubiquitin antibody (Ub, 1:50 dilution, rat polyclonal antibody, Santa Cruz) at 4 °C. After washing with PBS, the sections were incubated with the secondary FITC-conjugated donkey-antisheep IgG (1:200, California, USA) or FITC-conjugated donkey-anti-rabbit IgG (1:100, California, USA) diluted by 10 lg/mL Hoechst 33,342 for 1 h at 37 °C. At last, the sections were examined by using a fibered confocal fluorescence microscopy (FCFM, Zeiss, Oberkochen, Germany). DA determination The striatal tissue was taken out from the rat brain of different groups. The brain tissue was quickly frozen at 80 °C, then dissolved in the 0.01 MHClO4 solution with 0.01% EDTA (Zhou et al., 2004) and homogenized. The supernatant was collected after centrifuging at 12,000 rpm for10 min and detected with an HPLC system equipped with a fluorescence detector (Waters, MA). The Lichrosorb Column (C18, 10 lm, 25 cm  4.6 mm, Waters, MA) was employed, and the mobile phase consisted of trisodium citrate (0.02 M), sodium dihydrogen phosphate (0.05 M), methanol (40%), EDTA (0.028 g/L), and SDS (0.15 g/L). The solution was adjusted to pH 3.0 with 98% H2SO4, filtered through a 0.45-lm membrane, and degassed. The flow rate was set to 1.0 mL/min, and the column temperature was set at 40 °C. After separation, dopamine levels were detected at the excitation wavelength of 280 nm and an emission wavelength of 315 nm (Xiong et al., 2009). Statistical analysis The statistical analysis used a one-way ANOVA, a twoway ANOVA and/or Student T tests. The data were

presented as mean ± the standard deviation (SD). And the P value of significant difference was considered as P < 0.05.

RESULTS Puerarin protected SH-SY5Y cells from rotenone toxicity We first studied whether or not puerarin had an effect on cell survival of rotenone-treated SH-SY5Y by MTT assay. In all groups, we have divided the SH-SY5Y cells into Con-group (SH-SY5Y cells only), Rot-group (cells treated with rotenone) and Pue-group (cells with puerarin pre-treated then added with rotenone). Compared with Con-group, all the treated groups including Rot-group showed a significant decrease in cell viability (P < 0.05). The viability of Rot-group declined more than 50% compared with Con-group. Compared with Rot-group, the viability of Pue-group cells were significantly elevated in the dose range of 10–150 lM (P < 0.05) (Fig. 1). For the apoptosis rate assessment, the data showed a significant decrease in apoptosis rate in Puerarin-treated groups compared with Rot-group (P < 0.05), in accordance with the cell viability results. The apoptosis rate also decreased in the dose range from 10 to 150 lM by 38.87%, 36.50%, 41.72% and 39.65% respectively, compared with Rotgroup (Fig. 2B). Puerarin mitigated intracellular oxidative stress in rotenone-treated SH-SY5Y cells The DCFH staining was employed to verify whether puerarin could reduce the intracellular ROS level. The average ROS level in the rotenone group was 346.15% higher than that in the Con-group. Pre-treatment with puerarin alleviated the ROS overproduction by 26.76%, 25.76%, 18.03%, 17.34% in Pue-10, -50, -100 and 150 groups (Fig. 3B). Furthermore, from the fluorescence images, the average fluorescence intensity in puerarintreated cells was decreased by 42.35%, 43.03% and 4.13% in Pue 10-, Pue 50- and Pue 100-group, respectively (Fig. 3H). These data indicated that puerarin alleviated the rotenone-toxin-induced oxidative damage in SH-SY5Y cells. Puerarin alleviated behavioral defects of rotenoneinduced parkinsonian animals The rotenone-treated rats (6 lg rotenone in 1 lL DMSO) showed hogback, face-washing behavior, hypokinesia or bradykinesia, eating disorders, and decreased ability to clean up leading the gross turned from white into yellow. In the Con-group (DMSO injected only), the rats still lived in a healthy condition (self-eating, self-habit observed) without any hogback, hypokinesia or bradykinesia. All the rotenone-treated rats were injected with different puerarin concentration. 3 days, 1, 2, 3 and 4 weeks after stereotaxic surgery, the apomorphineinduced rotation number of rats in Pue-treated groups were significantly less than that in Rot-group. Compared to Rot-group, the rotation number in Pue-50 and

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Fig. 1. Puerarin protected the rotenone-induced cell viability reduction. We used MTT to test the cell viability in different groups. This figure showed the effect of Puerarin pre-treatment in different concentrations on the basis of rotenone 24-h-treated cells, puerarin increased the viability of cells (⁄P < 0.05, compared with cell control group, #P < 0.05, compared with Rot-group).

Fig. 2. Puerarin prohibited the apoptosis of rotenone-induced cells. (A) Scatter diagram of Annexin V/PI staining from Con-group, Rot-group, Rot + PUR 10, Rot + PUR 50, Rot + PUR 100, and Rot + PUR 150, respectively. (B) We tested the apoptosis rate of cells by flow cytometry. The figures showed the statistical analysis in six groups for the apoptosis rate (⁄P < 0.05, compared with Rot-group. #P < 0.05, compared with control group).

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Fig. 3. Effect of puerarin on rotenone-induced oxidative stress damage in cells. (A) Scatter diagram of DCFH-DA staining from Con-group, Rotgroup, Rot + PUR 10, Rot + PUR 50, Rot + PUR 100, and Rot + PUR 150, respectively. (B) We also tested the oxidative stress level by flow cytometry. The figures showed the statistical analysis of six groups for the average fluorescent intensity tested by the FCM. (C–G) ROS production observed under fluorescence microscope in Rot-group, Rot + PUR 10, Rot + PUR 50, Rot + PUR 100, and Rot + PUR 150. (H) Statistical analysis of six groups for the average fluorescent intensity under fluorescence microscope (⁄P < 0.05, compared with Rot-group; # P < 0.05, compared with control group).

Pue-100 group decreased by 17.47% and 25.00% for the first week, by 24.53% and 43.56% for the second week, by 36.58% and 53.08% for the third week and by 60.85% and 63.96% for the fourth week (Fig. 4). Especially in the second, third and fourth weeks, the rotation number showed significant differences between

Fig. 4. Effects of puerarin on apomorphine (APO)-evoked rotational behavior in parkinsonian rats. We evaluated the change in rat behavior by injecting APO to rats. The figures showed the effects of Puerarin on APO-evoked rotations 3 days, 1, 2, 3 and 4 weeks after surgery. Puerarin decreased APO-evoked rotations in parkinsonian rats, especially at the second, third and fourth weeks (⁄P < 0.05, compared with Rot-group).

Rot-group and Pue-group. These results indicated that puerarin prevented the deterioration of behavioral defects induced by rotenone, especially in the advanced stage. Puerarin up-regulated TH and VMAT2 expression and dopamine levels in dopaminergic cells In all four groups (Con-group, Pue-50 group and Pue100 group), immuno-fluorescence stain of brain tissue indicated that rotenone toxicity decreased fluorescence intensity of TH in dopaminergic neurons. The decrease in TH average fluorescence intensity was prohibited in the substantia nigra of Pue-50 group and Pue-100 group by 45.81% and 69.97% compared to Rot-group (Fig. 5Q). These data indicated that puerarin rescued dopaminergic neurons from rotenone-induced damage. We also assessed another dopaminergic maker (VMAT2) in the substantia nigra. Compared with Congroup, VMAT2 expression was decreased by 46.92% in Rot-group, by 25.03% in Pue-50 group and by 28.24% in Pue-100 group. Puerarin up-regulated the VMAT2 expression by 41.24% in Pue-50 group and by 35.20% in Pue-100 group (Fig. 5R). VMAT2 expression in the substantia nigra displayed no difference between Pue-50 and Pue-100 groups. Immunohistostaining of

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Fig. 5. TH, VMAT 2, and UB immunoreactivity in SNc Puerarin prevented loss of TH protein immunoreactivity in the substantia nigra, increased protective protein VMAT2 expression and decreased Ub protein expression. (A–D) TH protein, (E–H) VMAT protein and (I–L) Ub protein observed under fibered confocal fluorescence microscopy (FCFM) in Con-group, Rot-group, Rot + PUR 50 and Rot + PUR 100 group, respectively. (Q, R, T) Statistical analysis of TH, VMAT2 and Ub expression in four groups for the average fluorescent intensity under FCFM. (⁄P < 0.05, compared with Rot-group. #P < 0.05, compared with control group).

ubiquitin protein showed that rotenone alone upregulated ubiquitin expression by 201.12% compared with that in the Con-group. Compared to Rot-group, puerarin decreased ubiquitin expression by 47.55% and 69.15%, in Pue-50 and Pue-100 groups respectively (Fig. 5T). As the expression level of ubiquitin protein represents the activity of ubiquitin-modification on abnormal protein, puerarin might be involved in the decrease of the paraprotein expression in DA neurons. We also investigated the levels of dopamine in striatum and midbrain. The levels of dopamine in the striatum were assessed as well. The results indicated that puerarin inhibited rotenone-induced loss of DA. Specifically, compared to Rot-group, puerarin increased DA level by 49.50%, and 115.62% in Rot + Pue50 mg/ kg and +Pue100 mg/kg groups, respectively (Fig. 6). The results were in agreement with the information on the fluorescence intensity of TH that marked dopaminergic cell as observed by fluorescence microscope.

DISCUSSION In this study, our data showed that puerarin displayed protective effects on rotenone-based cell and animal models for PD. Our main findings include: puerarin (1) prohibited the decrease of SH-SY5Y cell viability induced by rotenone, (2) mitigated the rotenone-induced cell apoptosis, (3) attenuated the excess of ROS

Fig. 6. Puerarin inhibited the loss of dopamine in neurons. We used HPLC system to test the dopamine levels in different groups. This figure showed the effect of puerarin pre-treatment with different concentrations (50 mg/kg, 100 mg/kg) on rotenone-induced rat models. Puerarin decreased the loss of dopamine. (⁄P < 0.05, compared with Rot group, #P < 0.05, compared with control group).

production in SH-SY5Y cells and an animal model, (4) alleviated the characteristics of rotenone-induced Parkinsonism in the rat model, and (5) up-regulated TH and VMAT2 expressions to protect dopaminergic cells in the substantia nigra while decreased the ubiquitin expression. The rate of TH or VMAT2 expressional up-regulation by puerarin had no apparent difference

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between Pue-50 and Pue-100 groups, but was increased compared with Rot-group. Rotenone models were employed in the present study. Rotenone, one of most commonly used component of agriculture chemicals, can lead to systemic inhibition of complex I and elevated expression of ubiquitin and a-synuclein (Fan et al., 1984), and mimic the progressive nature of human PD (Meredith et al., 2008). There were several studies on the protection of puerarin in Parkinsonism models. One of these previous studies found that puerarin protects neurons in Parkinsonism model induced by 6-OHDA through activating the Nrf2/ARE signaling pathway (Li et al., 2013). Other study demonstrated that puerarin protected dopaminergic neurons by activating the PI3K/Akt signaling pathway in MPP+-induced cell model (Zhu et al., 2012). It was also demonstrated that Bcl-2/Bax was a protective target of puerarin in MPP+ and 6-OHDA-induced Parkinsonism models (Zhu et al., 2010; Zhu et al., 2012). We have not found any other reported experiments to study the effect and mechanism of puerarin on rotenone-induced cellular or animal models. We chose rotenone to induce dopaminergic neuron damage because of these three reasons: (1) the toxin of 6-OHDA is supervirulent, easier to induce acute toxicity (Blandini et al., 2008); (2) no a-synuclein overexpression was observed in the mostly used 6-OHDA models (Sachs and Jonsson, 1975; Vercammen et al., 2006); (3) the neurotoxin MPTP has species selectivity as it is more effective to induce parkinsonian symptoms in monkey and mouse than in rats (Hunot et al., 1997; Schmidt and Ferger, 2001; Xiong et al., 2009; Xiong et al., 2012). Puerarin is a natural product and traditional Chinese medicine that has been widely used for the treatment of cardiovascular, cerebrovascular diseases and alcohol toxicity for many years (Fan et al., 1984; Overstreet et al., 2003; Xu et al., 2005). Epidemiologic studies indicated that the risk of women suffering from PD was lower than men. Injecting estrogen for women before their menopause can decrease the mobility of PD Shepherd (2001). These results suggest that lower estrogen level may be relevant to PD onset. Puerarin, a naturally occurring isoflavone, is also known as a natural estrogen. It was reported that the protective effect of puerarin was mediated by decreasing ubiquitin expression and inhibiting mitochondria-dependent apoptosis in 6-OHDA and MPP+-induced PD models (Anglade et al., 1997; Li et al., 2003; Cheng et al., 2009; Zhu et al., 2010; Zhang et al., 2010). Here, we further confirmed the neuroprotective effect of puerarin on dopaminergic neurons in rotenone-induced parkinsonian cellular and animal models. Oxidative process is a central event for the development of clinical disease as the environment factors (MPTP, rotenone) are the risk factors for generating reactive intermediates inhibiting complex I of the mitochondrial transport chain (Ischiropoulos and Beckman, 2003; Zhu et al., 2014). Oxidative stress may lead to the neuronal death and increase in cell apoptosis (Sawada and Shimohama, 2000). Previous studies reported that puerarin could clear the oxygen radicals as its ability of antioxidant stress and antiapoptotic

(Sawada et al., 1998). It was reported that puerarin may induce the synthesis of antioxidant enzymes through activating nuclear translocation by the Nrf2/ARE signaling pathway (Li et al., 2013). Moreover, puerarin inhibited caspase-3 expression in MPTP-induced SH-SY5Y cell models by suppressing the PI3K/Akt pathway (Wang et al., 2014) and implicated the c-Jun-NH2-terminal kinase pathway against MPP+-induced PC12 cell models (Wang et al., 2011). Puerarin also attenuated the cytochrome c release and inhibited the caspase-9 and caspase-3 expression in MPTP-induced PC12 cell models (Cheng et al., 2009). Taking together our findings and previous results in the cells model, anti-oxidative stress, anti-apoptosis, suppressing ROS overgeneration may take part in the dopaminergic neuroprotection mechanism of puerarin. When the puerarin concentration was 10 lM and 50 lM, the apoptosis rate and ROS production were lower than other concentration. Thus, we deduced that to the SH-SY5Y cells, 10–50 lM may be suitable concentrations for the dopaminergic neuroprotection. Nevertheless, it is worth mentioning that previous study indicated the limitation of using DCFH-DA staining. First, the DCFH-DA probe can be affected by the reductant-oxidant in vivo. In order to exclude this effect, we set blank group which was only cells without puerarin or rotenone administration and the results showed that the intracellular ROS were significantly lower than other groups. Moreover, cytochrome c released from mitochondria during apoptosis has a direct effect on the measurement of DCFH-DA (Kalyanaraman et al., 2012). To overcome the limitation, we also detected cell apoptosis in dopaminergic SH-SY5Y cells. The apoptosis data demonstrated similar tendency to the DCFH-DA staining results. Previous study demonstrated the protective effects of puerarin in the MPTP and 6-OHDA-induced Parkinsonism models (Cheng et al., 2009; Zhu et al., 2010; Li et al., 2013). In previous research, the number of TH-positive cells showed that there is no significant difference between puerarin group and con-group in the substantia nigra of PD mice (Zhou et al., 2014). Our data showed that puerarin mitigated the rotenone-induced toxicity to the dopaminergic neurons by examining TH immunostaining in the substantia nigra and detecting the dopamine levels in the midbrain and striatum, indicating the protective effects of puerarin on neurons. The changing trend of TH expression was consistent with the variation tendency of VMAT2 expression in midbrain. VMAT2 is an important chemical substance adjusting the DA concentration in the cytoplasm of dopaminergic neurons. VMAT2 can recruit DA into the neuron-intracellular vesicles and regulate the release of DA. There were several investigators who assessed VMAT2 function in mice (Takahashi et al., 1997). VMAT2 knocked-out mice cannot live a long time after their birth (Fukui et al., 2007). Previous research indicated that the decrease in VMAT2 expression can be observed both in PD mouse model and PD patients (Wang et al., 1997; Munoz et al., 2012), and gene variation of VMAT2 was involved with PD onset (Lin et al., 2010). Moreover, in a previous study, we demonstrated that VMAT2 expression

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Fig. 7. Working model for protective effect of puerarin on DA neurons. Up-regulation of VMAT2 expression by puerarin reduces cytosolic dopamine concentration and down-regulates apoptosis pathways, making DA neurons healthier. Moreover, anti-apoptotic, anti-oxidative and antiinflammatory effects, inhibition of calcium influx, improvement of microcirculation, increase in glial cell line-derived neurotrophic factor and brainderived neurotrophic factor levels, activation of PI3K/Akt, Nrf2/ARE pathway (Li et al., 2013; Wang et al., 2014), inhibition of nuclear p53 accumulation and the JNK signaling pathway, reducing accumulation of ubiquitin-conjugated proteins by regulating ubiquitin proteasome system are all involved in the neuroprotective effects of puerarin (Wang et al., 2011; Zhu et al., 2012).

decreased after rotenone administrated (Watabe and Nakaki, 2008). The expression of VMAT2 which located to the nerve terminal decreased may relate with tau protein expression and lead the Parkinson disease onset (Wu et al., 2013). In this study, compared to Rot-group, puerarin mitigated the down-regulation of VMAT2 expression. Puerarin may regulate VMAT2 expression thus to adjust the dopaminergic oxidative-toxic effect in cytoplasm, playing an important role in protecting dopaminergic neurons. Ubiquitin is an important component in the ubiquitin– proteasome system and present in Lewy body as well. Its primary function is to eliminate abnormal and improperly folded protein. It was published that the proteasome was involved in the degradation of unfolded a-synuclein (Tofaris et al., 2001). It was suggested that, among the sporadic PD patients, PD-related a-synuclein could be ubiquitin-modified. To support this view, past research had also found the ubiquitinated protein accumulation in Lewy bodies of sporadic PD (Anderson et al., 2006; Lim, 2007). Puerarin was also involving with the protective mechanisms in ubiquitin proteasome system (Cheng et al., 2009). Ubiquitin-positive neuronal inclusions also can be detected by immuno-staining in other neurodegenerative disorders like amyotrophic lateral sclerosis,

Pick’s disease and motor neuron diseases (Manetto et al., 1988; Mayer et al., 1996; Mayer, 2003). We showed that puerarin decreased ubiquitin expression significantly and up-regulated the degradation of abnormal protein. The Lewy body formation and a-synuclein ubiquitinmodification may decrease. Thereby, the dopamine neuron damage would be lightened. Puerarin is widely used in clinical management around China. Some researches declared that using glucose and glucose drug conjugates facilitated the drug crossing BBB (Uriel et al., 1996). As a C-glucoside, puerarin can be detected in the rat brain tissue after oral administration. So the previous study suggested that puerarin could cross the BBB (Prasain et al., 2004). As clinical observations, puerarin showed a few possible side effects after several weeks’ therapy, including syndromes such as erythra, edema, pyrexia and intravenous hemolysis. The side effects of puerarin could be observed more frequently in old people (>60 years-old) because of the decreased ability of metabolizing this compound. But the occurrence of side effects has no significant difference between male and female (Yue et al., 2008). As demonstrated, phytoestrogen may decrease the testicle weight of male rats, but there was no significant differences in testosterone levels between control group and phytoestrogen-treated group.

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As one type of phytoestrogen (Lin et al., 2009), puerarin did display some effects on male rats, although the effects were weaker compared to 17aˆ-estradiol (E2) Zhou et al., 2014. Most studies explored the effects of puerarin in immature male rats, with a drug administration period of 14 days or longer. For the clinical use, the treatment period was 7 days, and the patients were usually adults aged 40 or elder. Since the shorter treatment period, and the weaker phytoestrogen activity than classic estrogen, and the long-term clinical observation on cardiovascular and cerebrovascular patients, the side effects of puerarin as a type of estrogen on male should be rarely observed in the clinical therapy. Several studies showed neuroprotective effects of puerarin on Parkinsonism models. A previous study demonstrated that puerarin protected dopaminergic neurons in a Parkinsonism model induced by 6-OHDA through activating the Nrf2/ARE signaling pathway (Li et al., 2013). Another study indicated that puerarin exerted its protective effects by activating the PI3K/Akt signaling pathway in MPP+-infused dopaminergic cells (Zhu et al., 2012). Moreover, Bcl-2/Bax was a protective target of puerarin in MPP+- and 6-OHDA-induced Parkinsonism models (Zhu et al., 2010; Zhu et al., 2012) as well. Our study is the first to show the effects of puerarin on rotenone-induced parkinsonian cellular and animal models and its related mechanisms involving anti-apoptotic and anti-oxidative pathways. In agreement with previous studies and based on the results of this study, the mechanisms of the neuroprotective effects of Puerarin may include the following (Fig. 7): (1) anti-apoptotic effects besides antioxidative effects in cellular and animal models, consistent with its neuroprotection in clinical stroke patients, hyperlipidemia, osteonecrosis and alcohol-induced disorders (Zhou et al., 2014; (2) up-regulating VMAT2 expression which helps to take up cytosolic dopamine and reduce the cytotoxicity of dopamine; (3) inhibiting calcium influx, improving microcirculation and counteracting cell death; (4) increasing the activity of gliocytes and the levels of glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor (Zhu et al., 2010; Li et al., 2013); (5) activating PI3K/Akt, Nrf2/ARE pathway, inhibiting nuclear p53 accumulation and the JNK signaling pathway (Wang et al., 2011; Zhu et al., 2012); (6) reducing accumulation of ubiquitinconjugated proteins by regulating the function of ubiquitin proteasome system (Cheng et al., 2009) and (7) displaying anti-inflammatory functionality (Zhou et al., 2014). In summary, as a medicine used for the treatment of the ischemic heart disease patients, puerarin, may be a new neuroprotective candidate for clinical PD treatment. The potential mechanism may be via antioxidative stress, anti-apoptosis, upregulation of VMAT2 expression and enhancing the degradation of aggregated proteins. Although we have shown that pretreatment of puerarin can protect DA neurons in animal model, it needs further clinical trials to verify whether puerarin plays neuroprotective role in PD patients in the future.

COMPETING INTERESTS We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this study is consistent with those guidelines. None of the authors have any conflict of interest to disclose. Acknowledgments—This work was supported by grants 30870866, 81071021 and 31171211 from the National Natural Science Foundation of China (to TW), grant 81200983 from the National Natural Science Foundation of China (to NX), grant 81100958 from the National Natural Science Foundation of China (to ZTZ), grant 81301082 from the National Natural Science Foundation of China (to JSH), grant 2012B09 from China Medical Foundation (to NX) and grant 0203201343 from Hubei Molecular Imaging Key Laboratory (to NX). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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(Accepted 29 August 2014) (Available online 10 September 2014)