The Chinese herbal formula Liuwei dihuang protects dopaminergic neurons against Parkinson's toxin through enhancing antioxidative defense and preventing apoptotic death

Phytomedicine 21 (2014) 724–733

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The Chinese herbal formula Liuwei dihuang protects dopaminergic neurons against Parkinson’s toxin through enhancing antioxidative defense and preventing apoptotic death Yu-Ting Tseng a , Fang-Rong Chang a , Yi-Ching Lo a,b,∗ a b

Graduate Institute of Natural Products, School of Pharmacy, Kaohsiung Medical University, Kaohsiung 80708, Taiwan Department of Pharmacology, School of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan

a r t i c l e

i n f o

Article history: Received 9 October 2013 Accepted 14 November 2013 Keywords: Liuwei dihuang Traditional Chinese medicine Neuroprotection Dopaminergic neurons 1-Methyl-4-phenyl-1,2,3,6tetrahydropyridine

a b s t r a c t Liuwei dihuang (LWDH), a widely used traditional Chinese medicine (TCM), has been employed as an anti-aging prescription to improve declined function. Parkinson’s disease (PD) is a common adultonset neurodegenerative disorder characterized by the degeneration of dopaminergic nigrostriatal neurons with complex pathological mechanisms, including oxidative stress. Increasing evidence indicate that TCM has the potential to be neuroprotective drugs because of their antioxidant characteristics. The aim of this study is to investigate the mechanisms of LWDH-mediated protection in Parkinson’s toxin-induced dopaminergic neurodegeneration by evaluating water extract of LWDH (LWDH-WE) in 1-methyl-4-phenylpyridinium (MPP+ )-treated primary mesencephalic neurons and 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP)-treated C57BL/6 mice. In the present study, chemical profiling and quantitative analysis of LWDH-WE were revealed using 3D-HPLC technique, and were confirmed by the data of three batches of LWDH-WE. In primary mesencephalic neuronal cultures, LWDH-WE decreased MPP+ -induced loss of tyrosine hydroxylase (TH)-positive neurons and increase of Annexin V-positive neurons. LWDH-WE reduced MPP+ -induced oxidative damage via increasing antioxidant defense (SOD, GSH), decreasing ROS production, and down-regulating NADPH oxidases (Nox2 and Nox4). Also, LWDH-WE inhibited neuronal apoptosis by improving mitochondrial membrane potential, increasing antiapoptotic protein Bcl-2 expression, and down-regulating apoptotic signaling (Bax, cytochrome c, cleaved-caspase3) in MPP+ -treated neurons. In MPTP-treated C57BL/6 mice, LWDH-WE attenuated TH-positive neuronal loss in substantia nigra pars compacta (SNpc), and improved locomotor activity of mice. In conclusion, the present results reveal that LWDH-WE possesses protection on dopaminergic neurons through enhancing antioxidant defense and decreasing apoptotic death, suggesting the potential benefits of LWDH-WE for PD treatment. © 2013 Elsevier GmbH. All rights reserved.

Introduction Liuwei dihuang (LWDH) is a Chinese medicinal formula composed of dihuang (Rehmannia glutinosa), shanyao (Dioscorea opposite), shanzhuyu (Cornus officinalis), zexie (Alisma orientalis), hoelen (Poria cocos) and mudanpi (Paeonia suffruticosa). It has been used clinically in the treatment of diabetics and neurosis, and to improve declined function related aging process and geriatric diseases. Recently, the potential neuroprotective

∗ Corresponding author at: Department of Pharmacology, School of Medicine, College of Medicine, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung 80708, Taiwan. Tel.: +886 7 3234686; fax: +886 7 3234686. E-mail address: [email protected] (Y.-C. Lo). 0944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2013.11.001

effects of LWDH have been noticed and investigated in various experimental models. In scopolamine, p-chloroamphetamine, and cycloheximide-induced amnesia rat models, LWDH activates peripheral cholinergic neuronal system, and modulates the central nervous system by increasing central cholinergic and GABAergic system, and decreasing serotonergic neuronal activity (Hsieh et al. 2003; Wu et al. 2007). Moreover, LWDH attenuates ␤-amyloid toxicity through reducing reactive oxygen species (ROS) and upregulation of heat shock protein and antioxidant activity in C. elegans (Sangha et al. 2012). LWDH also improves learning and memory function in senescence accelerated mice by inhibiting voltage-dependent calcium channels and promoting the function of NMDA receptor (Huang et al. 2012). Parkinson’s disease (PD) is the second most common neurodegenerative disorder characterized by progressive degeneration of

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dopaminergic neurons in substantia nigra pars compacta (SNpc) (Dawson and Dawson 2003). PD is a multi-factorial disease triggered by multiple pathogenic factors, including mitochondrial dysfunction, oxidative stress, and neuronal loss (Dardiotis et al. 2013). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is the most common Parkinson’s toxin widely used to establish animal models of PD (Bove and Perier 2011; Liu et al. 2012; Lo et al. 2012). MPTP can cross the blood–brain barrier due to its high lipophilicity and then it rapidly converts to its active metabolite 1-methyl-4-phenylpyridinium (MPP+ ) (Tipton and Singer 1993). The accumulation of MPP+ in dopaminergic neurons will increase ROS production (Anantharam et al. 2007). MPP+ /MPTP-induced ROS overproduction causes mitochondrial dysfunction and NADPH oxidase (Nox) activation, leading to apoptotic death of dopaminergic neurons (Fiskum et al. 2003; Zawada et al. 2011). Therefore, traditional Chinese medicines (TCM) targeting on stabilizing mitochondrial function, inhibiting Nox activation, and promoting antioxidant defense would be candidates for protection of dopaminergic neurons (Lo et al. 2012; Zhang et al. 2008; Zhu et al. 2013). In this study, we investigated the protective effects and mechanisms of the water extract of LWDH (LWDH-WE) in experimental models of PD using MPP+ -treated primary mesencephalic neuronal cultures and MPTP-treated mice. Our results demonstrated LWDHWE protected dopaminergic neurons against Parkinson’s toxin through its anti-oxidative and anti-apoptotic effects. The present results also demonstrated that LWDH improved motor activity of PD-like mice, suggesting the therapeutic benefits of LWDH for PD.

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Liuwei dihuang water extract (LWDH-WE) preparation and analysis LWDH-WE was prepared from the commercial and standardized product Liuwei dihuang concentrated granules manufactured by the Chuang Song Zong Pharmaceutical Co., Ltd. (Pingtung, Taiwan, ROC). The formula of product is Rehmanniae Praeparata Radix 8 g (prepared root of Rehmannia glutinosa), Corni Fructus 4 g (fruit of Cornus officinalis), Dioscoreae Rhizoma 4 g (rhizome of Dioscorea opposita), Alismatis Rhizoma 4 g (rhizome of Alisma orientalis), Moutan Cortex 3 g (root bark of Paeonia suffruticosa) and Poria 3 g (sclerotia of Poria cocos). The LWDH extract was prepared as follows: the crude drugs (1.3 kg) were mixing and extracted three times using 10-fold volume of purified water with boiling refluxing. After finishing the extraction, the materials were filtered to yield three extraction solutions. Three hot solutions were combined and then concentrated under reduced pressure at 50 ◦ C to obtain the semisolid form of LWDH (yield: 47.4%). LWDH-WE was further to perform working solution for highperformance liquid chromatography (HPLC). Working conditions were as following: column: C18 2.1 mm × 100 mm; the gradient program of mobile phases: from 90% of 0.03% Phosphoric acid aqueous solution and 10% of acetonitrile to 50% of 0.03% phosphoric acid and 50% of acetonitrile for 12 min; detection: UV at 237 nm; flow rate: 0.3 mL/min. Standardization of LWDH-WE was done using HPLC fingerprinting with chemical standards including 5hydroxymethyl-2-furaldehyde (Sigma–Aldrich, USA), morroniside (Must, China), loganin (Scientific Pharmaceutical Elite Company, Taiwan), paeoniflorin (Nacalai Tesgue, Japan), verbascoside (Scientific Pharmaceutical Elite Company, Taiwan), and paeonol (Nacalai Tesgue, Japan).

Materials and methods

Animal preparation and MPTP animal model

Materials

The use of all the animals in this study was approved by the Animal Care and Use Committee at the Kaohsiung Medical University (approval ID: 98096). Pregnant Sprague-Dawley (SD) rats and male C57BL/6 mice (7–8 weeks old, 20–25 g) were purchased from the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan), and then fed in the Animal Center of Kaohsiung Medical University under constant temperature and 12 h light-dark cycle. For PD animal model, C57BL/6 mice were pre-treated with LWDHWE (15 mg/kg/d or 30 mg/kg/d, i.p.) for 1 week or equal volume of saline as control. Mice were then received four intraperitoneal (i.p.) injections of MPTP (20 mg/kg each, at 2 h interval) to establish experimental PD model. After 7 days, MPTP-treated mice were undergoing locomotor activity assay, and the mice brain were used for TH-staining.

Bovine serum albumin (BSA), arabinoside, dimethyl sulfoxide (DMSO), poly-l-lysine, Triton X-100, 2 ,7 -dichlorodihydrofluorescein diacetate (H2 DCF-DA), 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), MPP+ , MPTP, and mouse antibody against ␤-actin and iNOS were obtained from Sigma–Aldrich (St. Louis, MO, USA). 5,5 ,6,6 -tetrachloro-1,3 ,3,3 tetraethyl benzimidazolyl carbocyanine iodide (JC-1), Minimum essential medium (MEM), fetal bovine serum (FBS), horse serum (HS), glutamine, B27, nonessential amino acids, sodium pyruvate, penicillin, amphotericin B, streptomycin and Alexa Fluor® 488 goat anti-rabbit IgG (H + L) were obtained from Invitrogen (Carlsbad, CA, USA). Mouse antibodies against Bcl-2, Bax, and cytochrome c, goat antibody against Nox4, and all horseradish peroxidase-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse antibody against Nox2 was obtained from BD Bioscience (San Jose, CA, USA). Mouse antibody against caspase-3 was obtained from Cell signaling (Danvers, MA, USA). Rabbit antibody against tyrosine hydroxylase (TH), enhanced chemiluminescence reagent, and polyvinylidene difluoride (PVDF) membrane were obtained from Millipore (Bedford, MA, USA). All materials for SDS-PAGE were obtained from Bio-Rad (Hercules, CA, USA). LDH (lactate dehydrogenase) cytotoxicity assay kit was purchased from G-Biosciences (St. Louis, MO, USA). Annexin V-FITC assay kit was purchased from BioVision Foundation (Mountain View, CA, USA). Simple stain mouse MAX PO was purchased from Nichirei Biosciences (Nichirei, Tokyo, Japan). Diaminobenzidine (DAB) kit was obtained from BioGenex (San Ramon, CA, USA).

Primary cultures of mesencephalic neurons Primary mesencephalic neuronal cultures were prepared from ventral mesencephalon dissected from 15-day-old embryos (E15) of SD rats. After triturating, cells were plated onto plates wells coated with poly-l-lysine previously and were cultured in MEM medium containing 10% FBS, 10% HS, 100 U/ml penicillin, 100 ␮g/ml streptomycin, and 0.25 ␮g/ml amphotericin B at 37 ◦ C in a humidified atmosphere containing 5% CO2 . After 24 h, culture medium of neurons would be replaced by MEM supplemented with 2% B27 and 10 ␮M cytosine arabinoside. After 48 h incubation, cells were then treated with LWDH-WE (0.01, 0.1, 1, 10 ␮g/ml) 1 h before MPP+ (100 ␮M) treatment for 48 h.

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MTT and LDH assay Cell viability was determined by MTT assay and LDH assay. In MTT assay, neurons were treated with 0.5 mg/ml MTT for 3 h in 37 ◦ C, and then the formazan crystals in the cells were solved with 100 ␮l DMSO. Absorbance was read at 560 nm using a microplate reader. In LDH assay, culture medium was collected to measure LDH release using a cytotoxicity detection kit. The tetrazolium salts produced in LDH-induced enzymatic reaction was then reduced to red formazan, thereby allowing a colorimetric detection by a microplate reader at 490 nm. Immunocytochemistry and immunohistochemistry TH immunoreactive neurons were represented as dopaminergic neurons in primary mesencephalic neuron cultures and the brain section of mice. Immunocytochemistry was performed in primary mesencephalic neurons. After drugs treatment, cells were fixed with 4% paraformaldehyde and washed with PBS incubated in 0.2% Triton X-100. Cells were then incubated with 2%

BSA for 1 h, and with the anti-TH primary antibody overnight at 4 ◦ C. Secondary antibody was Alexa Fluor® 488 goat anti-rabbit IgG (H + L). Images were collected under a fluorescence microscope and counted the viable neurons. In immunohistochemistry, brains were first perfusion-fixed with 4% paraformaldehyde, and then post fixed overnight after dissected. The brain tissues were dehydrated and embedded in paraffin wax, and 5 ␮m-thick consecutive coronal sections were collected. Sections were stained with rabbit antibody against TH and washed with PBS. The sections were incubated in simple stain mouse MAX PO by the Universal Immuno-enzyme Polymer (UIP) method, and following incubating in DAB substrate. Then, the sections were dehydrated in graded series of alcohol and xylene. TH positive neurons in SNpc were counted using light microscopy (Nikon, Japan) with bright-field illumination. Western blotting analysis After indicated treatment, cells were collected and lysed to determine the protein expression. Cell membranes and

Fig. 1. The three-dimensional HPLC chromatogram of LWDH-WE (A). HPLC chromatograms of six standard chemicals and three different batches of LWDH-WE (B). Standardization of LWDH-WE was done using HPLC fingerprinting with chemical standards including 5-hydroxymethyl-2-furaldehyde, morroniside, loganin, paeoniflorin, verbascoside, and paeonol.

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Fig. 2. Effects of LWDH-WE on MPP+ -induced cytotoxicity. Cell viability was determined by MTT (A) and LDH (B) assay. Fluorescent image of TH-positive neurons were confirmed by staining with anti-TH antibody (green) (C), and the number of TH-stained cells were counted (D) under a fluorescent microscope. Cells were pretreated with LWDH-WE 1 h prior to MPP+ addition (100 ␮M) for 48 h. Bars represent the mean ± S.E.M. from six independent experiments. ### p < 0.001 vs. control, *p < 0.05, **p < 0.01, ***p < 0.001 vs. MPP+ group. Scale bar = 50 ␮m.

mitochondria/cytosol fractionation were obtained by plasma membrane protein extraction kit and mitochondria/cytosol fractionation kit purchased from BioVision Foundation (Mountain View, CA, USA). Collected protein was used for SDS-polyacrylamide gel electrophoresis, and protein concentration was determined by using Bio-Rad protein assay kit. Equal amounts of protein were separated on polyacrylamide gel and transferred to PVDF membranes. Non-specific binding was blocked with TBST (50 mM Tris–HCl, pH 7.6, 150 mM NaCl, 0.1% Tween 20) containing 5% non-fat milk for 1 h at room temperature. The membranes were then each incubated with indicated primary antibodies by different dilution overnight at 4 ◦ C. Membranes were then washed with TBST and incubated with secondary antibodies for 1 h. Protein bands were detected with the enhanced chemiluminescence reagent after six washes with TBST.

Measurement of apoptotic cells Apoptotic cells were detected using Annexin V-FITC/propidium iodide (PI) double staining. Cells were detached and suspended in binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2 ). Then, cells were stained with Annexin V and PI for 15 min in dark (22–25 ◦ C). Annexin V-positive cells were analyzed by Coulter CyFlow Cytometer (Partec, Germany), which were identified as apoptotic cells. Measurement of mitochondrial membrane potential ( m) and intracellular reactive oxygen species (ROS) The changes of m and the production ROS were examined by JC-1 and H2 DCF-DA fluorescence staining, respectively.

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Fig. 3. Effects of LWDH-WE on the number of Annexin V-positive cells (A) and mitochondrial membrane potential (m) (B) in MPP+ -treated primary mesencephalic neurons. Apoptosis was determined using Annexin V/PI double staining and counting the percentage of apoptotic cells (Annexin V-positive cells). m was measured by JC-1, and the fluorescence ratio between red and green was normalized to control ratio. Apoptosis and m were analyzed by flow cytometry. Cells were pretreated with LWDH-WE 1 h prior to MPP+ addition (100 ␮M) for 48 h. Bars represent the mean ± S.E.M. from six independent experiments. ### p < 0.001 vs. control, *p < 0.05, **p < 0.01, ***p < 0.001 vs. MPP+ group.

After indicated treatment, cells were stained with 2 ␮M JC-1 or 10 ␮M H2 DCF-DA with 30 min incubation at 37 ◦ C, and then detached with PBS. DCF fluorescence was analyzed by Coulter CyFlow Cytometer and was determined by an excitation of 495 nm and emission of 520 nm. Normally, JC-1 accumulates in mitochondria and fluoresces red, but remains in cytoplasm and fluoresces green while apoptosis. Therefore, the changes of JC-1 fluoresces ratio between red and green was used for measuring m. Red JC1 fluoresce were detected by excitation/emission at 540/570 nm, and green JC-1 fluoresce were detected by excitation/emission at 495/520 nm. Superoxide dismutase (SOD) activity assay SOD activity was measured by a SOD activity kit purchased from Assay Designs (Ann Arbor, MI, USA). This method was assayed by xanthine oxidase and conversion of WST-1 to WST-1 formazan. After indicated treatment, neurons were detached and cytosolic protein was extracted. SOD activity was determined by adding the master mix and xanthine solution. Absorbance was measured by

ELISA reader at 450 nm for 10 min at 1-min interval. Protein concentration was quantified by Bio-Rad protein assay kit. SOD activity was calculated versus a SOD standard curve and normalized to the protein concentration.

Glutathione (GSH) quantification GSH detection kit was purchased from Enzo Life Sciences (Farmingdale, NY, USA). The principle of this kit is based on the production of yellow colored 5-thio-2-nitrobenzoic acid (TNB) from GSH and DTNB. After indicated treatment, neurons were trypsinized and collected the pellets. Then, suspended pellets in 5% metaphosphoric acid after brief sonication, collect the supernatants by centrifuging at 14,000 × g for 5 min. Then reaction Mix and GSH reductase supplied in the kit were added, following incubation and measurement by ELISA reader at 405 nm for 10 min at 1-min interval. The total amount of GSH was determined by means of a calibration curve and normalized to the protein concentration, which was quantified by Bio-Rad protein assay kit.

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Fig. 4. Effects of LWDH-WE on expression of Bax (A), Bcl-2 (B), cytosolic cytochrome c (C), and cleaved-caspase-3 (D) in MPP+ -treated primary mesencephalic neurons. Densitometry analyses are presented as the relative ratio of protein/␤-actin protein, and are represented as percentages of MPP+ group. Cells were pretreated with LWDHWE 1 h prior to MPP+ (100 ␮M) addition for 48 h. Bars represent the mean ± S.E.M. from six independent experiments. ### p < 0.001 vs. control, *p < 0.05, **p < 0.01, ***p < 0.001 vs. MPP+ group.

Locomotor activity To perform locomotor activity, mice were placed in a chamber (50 cm × 50 cm × 25 cm) for 5-min followed by automatic measurement by a digiscan analyzer, which transmitted the activity data to a computer. The activity data presented was total distance (mm) of movement and mean velocity (mm/s) within the 5-min recording period. Statistical analysis All data were expressed as means ± S.E.M., and statistical significance was analyzed using one-way ANOVA followed by Dunnett’s test for all pair comparisons. A value of p < 0.05 or less were considered statistically significant. Statistical analysis was performed using InStat version 3.0 (Graph Pad Software, San Diego, CA, USA). Results Analysis of chemical components of LWDH-WE Standardized LWDH-WE were obtained from a GMP pharmaceutical factory. The HPLC profile of LWDH-WE was showed by a three-dimensional plot (Fig. 1A), and the content of chemical standards in LWDH-WE were as follow: 5-hydroxymethyl2-furaldehyde 3.19 ± 0.07 mg/g, morroniside 5.25 ± 0.03 mg/g, loganin 2.37 ± 0.04 mg/g, paeoniflorin 2.12 ± 0.02 mg/g, verbascoside 0.10 ± 0.00 mg/g and paeonol 1.14 ± 0.01 mg/g. We further reconfirmed the composition of other two batches of LWDH-WE under the same condition by HPLC. The three different batches LWDH-WE were showed in Fig. 1B. The standardized formula extraction was subjected to following bio-tests.

LWDH-WE attenuates MPP+ -induced cytotoxicity and loss of TH-positive neurons in primary mesencephalic neuron cultures The cytoprotective effects of LWDH-WE on MPP+ -induced neurotoxicity were determined by MTT and LDH assay. Primary mesencephalic neurons were treated with LWDH-WE (0.01–10 ␮g/ml) 1 h prior to MPP+ (100 ␮М) addition for 48 h. Results indicated that LWDH-WE significantly attenuated cytotoxicity (Fig. 2A and B) in MPP+ -treated neuronal cells. To further examine the protective effects of LWDH-WE on dopaminergic neurons in primary mesencephalic neurons cultures, the number of dopaminergic neurons were counted and identified by TH immunocytochemistry. Results indicated the number of TH-positive neurons in MPP+ -treated cultures was significantly lower than that observed in control (without any treatment). However, LWDH-WE (0.1–10 ␮g/ml) significantly decreased loss of TH-positive neurons in MPP+ -treated cultures (Fig. 2C and D). LWDH-WE attenuates MPP+ -induced neuronal apoptosis and mitochondrial dysfunction To further ascertain the protective effects of LWDH-WE on MPP+ -induced cell death, Annexin V/PI staining was performed to measure neuronal apoptosis. Results indicated that MPP+ increased number of Annexin V-positive cells, and LWDH-WE treatment could attenuate MPP+ -induced apoptotic death (Fig. 3A). While MPP+ -induced cell apoptosis has been linked to mitochondrialdependent apoptotic signaling, we next examined the effects of LWDH-WE on MPP+ -induced changes of m, which was determined by measuring the ratio of JC-1 red and green fluorescence. Results indicated that LWDH-WE significantly increased m in MPP+ -treated neuronal cultures, suggesting LWDHWE could modulate mitochondrial function by improvement of m (Fig. 3B). Furthermore, the effects of LWDH-WE on

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SOD activity (U/mg protein)

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L W D H -W E ( μ g /m L ) M P P + (1 0 0 μ M ) Fig. 6. Effects of LWDH-WE on SOD activity (A), and GSH level (B) in MPP+ -treated primary mesencephalic neurons. SOD and GSH were determined by commercial kits. Cells were pretreated with LWDH-WE 1 h prior to MPP+ (100 ␮M) addition for 48 h. Bars represent the mean ± S.E.M. from six independent experiments. ### p < 0.001 vs. control, **p < 0.01, ***p < 0.001 vs. MPP+ group.

Fig. 5. Effects of LWDH-WE on ROS production (A) and expression of Nox2 (B) and Nox4 (C) in MPP+ -treated primary mesencephalic neurons. ROS was determined by H2 DCF-DA staining and analyzed by flow cytometer. Densitometry analyses are presented as the relative ratio of protein/␤-actin protein, and are represented as percentages of MPP+ group. Cells were pretreated with LWDH-WE 1 h prior to MPP+ (100 ␮M) addition for 48 h. Bars represent the mean ± S.E.M. from six independent experiments. ## p < 0.01, ### p < 0.001 vs. control, *p < 0.05, **p < 0.01, ***p < 0.001 vs. MPP+ group.

MPP+ -induced apoptosis-related proteins in primary mesencephalic neuron cultures were also examined. As shown in Fig. 4A and B, LWDH-WE down-regulated pro-apoptotic protein Bax, and upregulated anti-apoptotic protein Bcl-2. Moreover, LWDH-WE decreased expression of cytosolic cytochrome c (Fig. 4C) and the cleaved-caspase-3 protein (Fig. 4D) of MPP+ -treated neuron cultures.

neuronal cells using flow cytometer. Results indicated that MPP+ -induced ROS overproduction was attenuated by LWDH-WE (Fig. 5A). In addition, LWDH-WE also attenuated MPP+ -induced overexpression of Nox2 and Nox4, which are major producers of ROS (Fig. 5B and C). Furthermore, LWDH-WE significantly increased enzymatic antioxidant SOD activity (Fig. 6A) and nonenzymatic antioxidant GSH level (Fig. 6B) in primary mesencephalic neuron cultures. LWDH-WE protects dopaminergic neurons and improves locomoter activity in MPTP-treated mouse model of PD Finally, we examined the protective effects of LWDH-WE in MPTP-treated mouse model of PD. As shown in Fig. 7, a markedly loss of dopaminergic neurons (TH-positive neurons) in the SNpc of MPTP-treated mice were observed. However, LWDH-WE pretreatment decreased TH-positive neurons loss of MPTP-treated mice. Moreover, MPTP reduced locomotor activity of mice, which was determined by measuring mean velocity and total distance of movement. However, LWDH-WE pretreatment improved locomotor activity of MPTP-treated mice (Fig. 8). Discussion

LWDH-WE attenuates MPP+ -induced oxidative stress and enhances antioxidant defense in neurons To determine the effects of LWDH-WE on MPP+ -induced oxidative neuronal damage, we first investigated the effects of LWDH-WE on MPP+ -induced ROS generation and Nox activation. ROS generation was determined by measuring H2 -DCFDA-loaded

PD is a neurodegenerative disease characterized by motor dysfunction resulting from loss of dopaminergic neurons in the substantia nigra. The complicated pathogenesis mechanisms lead to the difficulties on the drug development of PD treatment. Recent studies reveal TCM with anti-oxidative characteristics possess the potential protection on PD (Lo et al. 2012; Zhang et al. 2008). The

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Fig. 7. Effects of LWDH-WE on TH-positive neurons in SNpc of MPTP-treated mice. C57BL/6 mice were treated with LWDH-WE (15 mg/kg/d or 30 mg/kg/d, i.p.) for 7 days and then MPTP (20 mg/kg, 4 times, at 2 h interval, i.p.) was injected on 8th day. Mice were sacrificed on the 7th day after MPTP injection. (A) Control group, (B) MPTP group, (C) LWDH-WE (15 mg/kg) + MPTP, (D) LWDH-WE (30 mg/kg) + MPTP. Scale bar = 100 ␮m. (E) Data represented as mean ± S.E.M. from at least six independent experiments. ### p < 0.001 vs. control (saline only), *p < 0.05, ***p < 0.001 vs. MPTP group.

present results demonstrated the novel protection of LWDH-WE on dopaminergic neurons via targeting factors of oxidative stress and apoptosis using in vitro and in vivo models of PD. Some of the used chemical markers to analyze and standardize LWDH-WE have been reported their neuroprotective effects through their anti-oxidative and anti-apoptotic properties. Morroniside and loganin can protect human dopaminergic SH-SY5Y cells against H2 O2 -induced apoptosis through suppressing ROS production and apoptotic signals (Kwon et al. 2011; Wang et al. 2008). Verbascoside attenuates A␤-induced cell death via up-regulation

of HO-1 and activation of ERK and PI3K/Akt pathway (Wang et al. 2012). The neuroprotective effects of paeoniflorin and paeonol were observed in in vitro model of PD (Tseng et al. 2012; Wang et al. 2013). In this study, we further explored the protective effects of LWDH-WE on MPP+ /MPTP models of PD. Oxidative stress and mitochondrial dysfunction constitute key pathogenic events of PD, and have been recognized as key initiators of cell apoptosis (Dawson and Dawson 2003; Jiang et al. 2013). The reduction of membrane potential of mitochondria provides an important clue about the functional loss of mitochondria (Joshi

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Fig. 8. Effects of LWDH-WE on locomotor activity by measuring trace-paths (A), mean velocity (B), and total distance (C) in MPTP-treated mice. Locomotor activity was detected in 5 min and analyzed at the 7th day of MPTP injection. (a) Control, (b) MPTP (c) LWDH-WE (15 mg/kg) + MPTP, (d) LWDH-WE (30 mg/kg) + MPTP. Bars represent the mean ± S.E.M. from six independent experiments. ### p < 0.001 vs. control (saline only), *p < 0.05, ***p < 0.001 vs. MPTP group.

and Bakowska 2011) and play a critical role in intrinsic apoptotic pathway. Bcl-2 superfamily, including anti-apoptotic Bcl-2 and pro-apoptotic Bax, influences mitochondrial outer membrane permeabilization and apoptotic susceptibility (Kelekar and Thompson 1998). MPP+ can promote membrane insertion of Bax and allowing cytochrome c redistributing from mitochondria to cytosol, leading to further activation of downstream effector caspase-3 (Circu and Aw 2010). The present results indicated that LWDH-WE modulated mitochondrial function against MPP+ -induced changes of m. Moreover, LWDH-WE attenuated MPP+ -induced neuronal apoptotic death, at least in part, by down-regulating apoptotic signals (Bax expression, cytosolic cytochrome c release and

caspase-3 activation) and upregulating anti-apoptotic protein Bcl2, which effects stabilize mitochondrial function and contribute to neuroprotection. The protective effect of LWDH-WE on dopaminergic neurons was also demonstrated by immunofluorescence detection of TH-positive neurons in this study. Noxs, one of the most important biological sources of ROS, plays a critical role in the disease progression of PD (Sorce et al. 2012). Among them, Nox2 as well as Nox4 expressed predominantly in the midbrain of mice (Infanger et al. 2006). In the present study, LWDH-WE inactivated Nox2/Nox4 and attenuated MPP+ induced ROS overproduction in primary mesencephalic neurons cultures. In addition, regulation of antioxidant defense system via

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Fig. 9. Protective mechanisms of Liuwei dihuang water extract (LWDH-WE) on dopaminergic neurons in MPP+ /MPTP models of Parkinson’s disease.

increasing enzymatic SOD or nonenzymatic GSH is known to prevent oxidative-induced neuronal damage (De Vos et al. 2007; Herrero-Mendez et al. 2009). The present results indicated that LWDH-WE not only attenuated MPP+ -induced ROS overproduction and Nox activation, but also increase SOD activity and GSH level. To further confirm LWDH-WE neuroprotection, MPTP mouse model of PD was used. MPTP provoke degeneration of nigralstriatal dopaminergic neurons and cause deficit of locomotor activity, which are important hallmarks of PD (Satpute et al. 2013). Results suggested that LWDH-WE significantly decreased the loss of THpositive neuron in SNpc, and improved locomotor activity of MPTP-treated mice. In conclusion, the present results indicate the neuroprotective effects of LWDH-WE against oxidative stress and neuronal apoptosis (Fig. 9) in models of PD. In the future, TCM that possess anti-oxidative and anti-apoptotic mechanisms might be a valuable study project on therapy strategy of PD. Acknowledgments This study was supported by grant provided from National Science Council of Taiwan to Y.C.L. [grant number: NSC99-2320B-037-023-MY3 and NSC102-2628-B-037-001-MY3]. The authors are grateful to the Chuang Song Zong Pharmaceutical Co., Ltd. (Pingtung, Taiwan, ROC) for kindly providing water extracts of Liuwei dihuang (LWDH-WE) used in this study. References Anantharam, V., Kaul, S., Song, C., Kanthasamy, A., Kanthasamy, A.G., 2007. Pharmacological inhibition of neuronal NADPH oxidase protects against 1methyl-4-phenylpyridinium (MPP+)-induced oxidative stress and apoptosis in mesencephalic dopaminergic neuronal cells. Neurotoxicology 28, 988–997. Bove, J., Perier, C., 2011. Neurotoxin-based models of Parkinson’s disease. Neuroscience 211, 51–76. Circu, M.L., Aw, T.Y., 2010. Reactive oxygen species, cellular redox systems, and apoptosis. Free Radic. Biol. Med. 48, 749–762. Dardiotis, E., Xiromerisiou, G., Hadjichristodoulou, C., Tsatsakis, A.M., Wilks, M.F., Hadjigeorgiou, G.M., 2013. The interplay between environmental and genetic factors in Parkinson’s disease susceptibility: the evidence for pesticides. Toxicology 307, 17–23. Dawson, T.M., Dawson, V.L., 2003. Molecular pathways of neurodegeneration in Parkinson’s disease. Science 302, 819–822.

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