NEURAL REGENERATION RESEARCH Volume 6, Issue 18, June 2011 www.nrronline.org

Cite this article as: Neural Regen Res. 2011;6(18):1375-1379.

Neuroprotective effect of Eleutheroside B on 1-methyl-4-phenylpyridinium ion-induced apoptosis in PC12 cells****☆

Fang Lu1, 2, Yang Dong1, Laijun Deng1, Shumin Liu1, 2, Shihui Zhou1, Lifeng An3, Bo Tang1

1Institute of Traditional Chinese Medicine, Heilongjiang University of Chinese Medicine, Harbin 150040, Heilongjiang Province, China 2Key Laboratory of Chinese Materia Medica (Heilongjiang University of Chinese Medicine), Ministry of Education, Harbin 150040, Heilongjiang Province, China 3Jiamusi College, Heilongjiang University of Traditional Chinese Medicine, Jiamusi 154007, Heilongjiang Province, China

Abstract Apoptosis and viability of PC12 cells following 1-methyl-4-phenylpyridinium ion (MPP+)-induced Fang Lu☆, Ph.D., Institute of injury were monitored by flow cytometry, following Annexin V-propidium iodide double labeling, and Traditional Chinese Medicine, Heilongjiang 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, respectively. The release of University of Chinese lactate dehydrogenase, superoxide dismutase activity and levels of malondialdehyde were Medicine, Harbin 150040, determined by UV spectrophotometry. The changes in mitochondrial membrane potential and the Heilongjiang Province, intracellular concentration of calcium were determined by flow cytometry, and the activity of China; Key Laboratory of Chinese Materia Medica caspase-3 was monitored by western blot. According to cell viability and apoptosis studies, (Heilongjiang University of MPP+-induced apoptosis in PC12 cells was inhibited in the presence of 10 μg/mL of Eleutheroside B. Chinese Medicine), Ministry Our results indicate that the neuroprotective effect of Eleutheroside B, following MPP+-induced of Education, Harbin 150040, Heilongjiang Province, China apoptosis in PC12 cells, involves increasing the anti-oxidative stress capacity of cells, maintaining the high-energy state of mitochondrial membrane potential, reducing intracellular calcium Corresponding author: concentration and inhibiting caspase-3 activity. Shumin Liu, Ph.D., Doctoral Key Words: Eleutheroside B; PC12 cells; apoptosis; 1-methyl-4-phenylpyridinium ion; mitochondria; supervisor, Institute of Traditional Chinese Parkinson’s disease Medicine, Heilongjiang University of Chinese Medicine, Harbin 150040, mental strain[6]. Moreover, this medicine has Heilongjiang Province, China; Key Laboratory of INTRODUCTION been used as a crude drug to treat Chinese Materia Medica stress-induced physiological changes, (Heilongjiang University of Chinese Medicine), Ministry The pathogenesis of Parkinson’s disease various allergic conditions, inflammation and of Education, Harbin 150040, (PD) remains unclear. However, many immunodeficiency[7-10]. Acanthopanax Heilongjiang Province, China [email protected] studies have demonstrated that the senticosus Harms has been reported to occurrence and progression of this disease ameliorate the clinical symptoms of PD[11] Supported by: the Major Projects of National Science is directly related to apoptosis or and has been shown to affect the levels of and Technology, No. [1-3] [12] 2009ZX09103-329*; the degenerative cell death . The neurotoxin dopamine . The main components of National Natural Science + 1-methyl-4-phenylpyridinium ion (MPP ) is Acanthopanax are phenolic glycosides, Foundation for Distinguished Young Scholars of China, No. an active metabolite of 1-methyl-4-phenyl- which are the major biologically active 30901974*; Outstanding 1,2,3,6- tetrahydropyridine, which is ingredient. Eleutheroside B (Figure 1; Youth Science Fund Program of Heilongjiang Province, No. selectively toxic to dopaminergic neurons. C17H24O9, molecular weight: 372.367) is a JC200705*; "Spring PC12 cells are derived from a rat adrenal type of phenolic glycoside separated and Sunshine" Plan of Ministry of Education, No. 2006* pheochromocytoma, which possess purified from the dry root and rhizome of intracellular substrates for the synthesis, Acanthopanax senticosus. Increasing Received: 2011-02-04 Accepted: 2011-05-15 metabolism and transportation of evidence has revealed that oxidative (N20101020001/WLM) dopamine[4]. In addition, MPP+-induced damage may occur in the Parkinsonian Lu F, Dong Y, Deng LJ, Liu apoptosis in PC12 cells has been widely brain and lead to neuronal apoptosis and SM, Zhou SH, An LF, Tang B. used as an ideal model for studying necrosis. This neuronal death is a significant Neuroprotective effect of Eleutheroside B on 1-methyl- [5] neuronopathies and drug action . cause of nervous system degenerative 4-phenylpyridinium ion-induced apoptosis in Acanthopanax senticosus Harms (Rupr. Et diseases and plays an important role in the PC12 cells. Neural Regen Maxim) belongs to the Araliaceae family. In process of PD. The current study assumed Res. 2011;6(18):1375-1379.

Chinese medicine, the dry root and rhizome that Eleutheroside B exerts significant www.crter.cn are used in the nourishment of Qi (the most neuroprotective effects against www.nrronline.org + basic composition of body that helps MPP -induced cytotoxicity in PC12 cells doi:10.3969/j.issn.1673-5374. maintain normal life activity and through inhibiting oxidative stress. 2011.18.003 physiological function) by invigorating the In this study, we evaluated the protective spleen, tonifying the kidney and relieving effect of Eleutheroside B against MPP+-

1375 Lu F, et al. / Neural Regeneration Research. 2011;6(18):1375-1379. induced apoptosis in PC12 cells and its possible Eleutheroside B inhibited lactate dehydrogenase mechanism of action. (LDH) release from PC12 cells The release/leakage of LDH from cells is a sign of impaired plasma membrane integrity[14] and is proportional to the number of damaged PC12 cells. Compared with the control group, LDH release increased in the model group (242.63 ± 43.21 U/L vs. 557.82 ± 67.63 U/L, P < 0.01). Compared with the model group, LDH release decreased following 10 μg/mL Figure 1 Chemical structure of Eleutheroside B. Eleutheroside B pretreatment (557.82 ± 67.63 U/L vs. 407.32 ± 39.75 U/L, P < 0.05; supplementary Table 1 online). RESULTS Eleutheroside B enhanced the anti-oxidant capacity of PC12 cells Optimal concentration of Eleutheroside B The activity of superoxide dismutase (SOD) can reflect Based on our pilot study, PC12 cells treated with endogenous antioxidant capacity. Malondialdehyde 300 μmol/L MPP+ for 48 hours were selected as the (MDA) content can directly reflect the extent of lipid model group[13]. Cell viability was assessed using peroxidation and indirectly reflect the severity of damage 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium caused by free radicals[15]. Compared with the model bromide (MTT) assay and apoptosis was monitored by group, SOD activity significantly increased in flow cytometry following Annexin V-propidium iodide (PI) MPP+-treated PC12 cells following Eleutheroside B double labeling. Compared with the control group, the treatment (P < 0.05), whereas MDA content decreased amount of apoptotic cell death increased following MPP+ (P < 0.01). This result indicated that the antioxidant treatment alone (P < 0.01). However, this cytotoxic effect capacity in PC12 cells was enhanced in the presence of was attenuated in the presence of Eleutheroside B (2.5, Eleutheroside B (Table 2). 5, 10, 20 μg/mL), and when the final concentration reached 5, 10 and 20 μg/mL, cell viability and apoptotic Table 2 Effect of Eleutheroside B on SOD activity and cell death were significantly reduced (P < 0.05). However, MDA levels in PC12 cells exposed to 300 μmol/L 1-methyl- at concentrations of 40 and 80 μg/mL, Eleutheroside B 4-phenylpyridinium ion for 48 hours had cytotoxic effects on cells (Table 1). Group SOD (U/mL) MDA (nmol/L)

Control 23.11±6.41 9.52±2.23 Table 1 Effect of Eleutheroside B at different a b Model 14.23±4.52 17.21±2.84 concentrations on survival and apoptotic rate in PC12 cells Eleutheroside B 18.27±5.27c 11.32±1.98d exposed to 300 μmol/L MPP+ for 48 hours aP < 0.05, bP < 0.01, vs. control group; cP < 0.05, dP < 0.01, vs. Survival rate model group. Data are expressed as the mean ± SEM of three Apoptotic Group Concentration (% control independent experiments. SOD: Superoxide dismutase; MDA: rate (%) group) malondialdehyde. Control 100.00 10.26±5.82b + Model MPP 300 μmol/L 87.51 38.63±14.51 Eleutheroside B increased mitochondrial Eleutheroside B 2.5 μg/mL + 91.70 30.44±8.28 MPP+ 300 μmol/L transmembrane potential (MMP) in PC12 cells 5 μg/mL + 100.00 25.57±7.94a Disruption of MMP induces apoptosis[16]. To characterize + MPP 300 μmol/L changes in mitochondria, MMP was monitored by flow 10 μg/mL + 102.12 24.34±6.76a MPP+ 300 μmol/L cytometry using the fluorescent dye Rhodamine123. 20 μg/mL + 100.00 25.39±7.19a Compared with the control, rhodamine123 fluorescence + MPP 300 μmol/L intensity significantly reduced after PC12 cells were 40 μg/mL + 82.62 29.62±9.23 + MPP+ 300 μmol/L treated with 300 μmol/L MPP (2 335 ± 249 vs. 1 387 ± 80 μg/mL + 80.38 30.81±9.60 219, P < 0.01). However, in the presence of + MPP 300 μmol/L Eleutheroside B, the reduction of MMP in MPP+-injured aP < 0.05, bP < 0.01, vs. model group; data shown are PC12 cells was significantly attenuated compared with representative of 12 independent experiments; data of apoptotic the model group (1 387 ± 219 vs. 1 926 ± 210, P < 0.05; rate are expressed as mean ± SEM. MPP+: 1-methyl-4- phenylpyridinium ion. Figure 2, supplementary Table 2 online). Eleutheroside B reduced intracellular calcium concentrations These results indicate that Eleutheroside B inhibits Intracellular Ca2+ concentrations were detected by flow MPP+-induced apoptosis in PC12 cells, but that the cytometry, where a high fluorescent intensity indicated a efficacy of neuroprotection is dose-dependent. Based on higher concentration of intracellular Ca2+. Compared with these studies, a concentration of 10 μg/mL was chosen untreated control cells, the fluorescent intensity in the as the optimal concentration of Eleutheroside B. MPP+-treated model group increased (59.89 ± 5.70 vs.

1376 Lu F, et al. / Neural Regeneration Research. 2011;6(18):1375-1379.

146.98 ± 11.79, P < 0.01). When incubated with plays an important role in the PD process[19-20]. 10 μg/mL Eleutheroside B, fluorescent intensity was Acanthopanax senticosus Harms has been shown to markedly reduced compared with the model group have a protective effect against PD, however, the (102.69 ± 7.91 vs. 146.98 ± 11.79, P < 0.01; Figure 3, mechanism of its action remains unclear. The current supplementary Table 3 online). study demonstrates for the first time that Eleutheroside B, extracted from the dry root and rhizome of Acanthopanax A B C senticosus Harm, exerts significant neuroprotective effect against MPP+-induced cytotoxicity in PC12 cells. Our previous work has indicated that an MPP+ concentration of 300 μmol/L with a treatment duration of 48 hours is a stable cellular model of PD. According to our experimental results on survival and apoptosis rate, we determined that 10 μg/mL Eleutheroside B is an optimal neuroprotective dose. In order to further Figure 2 Eleutheroside B attenuates mitochondrial transmembrane potential loss in PC12 cells exposed to investigate the protective effects of Eleutheroside B 300 μmol/L 1-methyl-4-phenylpyridinium ion, as assayed (10 μg/mL), the release of LDH was monitored. This by flow cytometry. Cells were resuspended with experiment revealed that Eleutheroside B could Rhodamine123. Fluorescence was read using an excitation wavelength at 480 nm and an emission wavelength at attenuate the release of LDH. This study indicated that at 530 nm with a fluorescence plate reader. (A) Control group; lower concentrations Eleutheroside B induces a (B) Model group; (C): Eleutheroside B group. neuroprotective effect, but at higher doses it can be toxic (40 and 80 μg/mL).

A B C

35 kDa Caspase 3

β-actin 42 kDa 1 2 3 Figure 4 Western blot analysis of the inhibitory effect of 2+ 10 μg/mL Eleutheroside B on caspase-3 activity in PC12 Figure 3 Eleutheroside B reduces intracellular Ca cells exposed to 300 μmol/L 1-methyl-4- phenylpyridinium concentration in PC12 cells exposed to 300 μmol/L ion for 48 hours. 1: Control group; 2: model group; 3: 1-methyl-4-phenylpyridinium ion, as assayed by flow Eleutheroside B group. cytometry. Cells were collected and resuspended with Fluo-3/AM. Fluorescence was measured using an excitation wavelength of 480 nm and an emission wavelength of To determine whether Eleutheroside B was effective in 530 nm with a fluorescence plate reader. (A) Control group; preventing apoptosis through an antioxidant capacity, (B): model group; (C): Eleutheroside B group. SOD activity and MDA levels were monitored. The present study confirmed that SOD activity increased and Eleutheroside B suppressed caspase-3 expression that MDA content decreased after Eleutheroside B in PC12 cells treatment following MPP+-induced apoptosis in PC12 Active caspase-3 plays an important role in programmed cells. According to the above results, the cytotoxic effects cell death[17]. In this study, activation of caspase-3 was of MPP+ may be mediated by oxidative stress in PC12 monitored using western blot. After PC12 cells were cells, and the protective effect of Eleutheroside B may be treated with MPP+ for 48 hours, levels of active via an antioxidant effect. caspase-3 significantly increased compared with the Mitochondria are organelles that play a primary role in control group (1.98 ± 0.12 vs. 1.65 ± 0.05, P < 0.01). the regulation of cell death. Disruption of MMP causes However, activity significantly decreased following the release of cytochrome C from mitochondria to the Eleutheroside B treatment compared with the model cytosol[21], which leads to the activation of caspases that group (1.90 ± 0.07 vs. 1.98 ± 0.12, P < 0.05; Figure 4, can trigger apoptosis[22-23]. This study showed that supplementary Table 4 online). The present study compared with the model group, Eleutheroside B indicates that Eleutheroside B treatment can inhibit prevents MPP+-induced collapse of MMP and MPP+-induced activation of caspase-3. significantly suppresses MPP+-induced activation of caspase-3, which is involved in the impairment of DISCUSSION dopaminergic neurons in PD[24]. In addition, it may relieve intracellular calcium overload. Overall, these data Previous data has demonstrated that oxidative damage suggest that Eleutheroside B may have intracellular may occur in the Parkinsonian brain[18], which leads to anti-apoptotic therapeutic potential. neuronal apoptosis. In addition, necrosis is a significant In summary, we hypothesized that Eleutheroside B cause of nervous system degenerative diseases and exerts its neuroprotective effects by its anti-oxidative and

1377 Lu F, et al. / Neural Regeneration Research. 2011;6(18):1375-1379. anti-apoptotic properties. This neuroprotective effect may cells (1 × 106) were washed twice with PBS and then offer a practical strategy for PD treatment. Therefore, resuspended with 500 μL buffer. Cells were incubated in further research on the neuroprotective effect of dark with 5 μL Annexin V-FITC (Nanjing Keygen Biotech. Eleutheroside B is necessary to determine its Co., Ltd., Nanjing, Jiangsu Province, China) and 10 μL PI mechanism of action before definite conclusions can be (Nanjing Keygen Biotech. Co., Ltd.) at room temperature drawn. for 5 minutes. Apoptosis was determined by flow cytometry (BD Company, San Jose, CA, USA). The MATERIALS AND METHODS obtained data was analyzed by Cell Quest software. LDH release assay Design Based on cell viability studies, 10 μg/mL was chosen as A randomized, controlled experiment pertaining to the final working concentration of Eleutheroside B. Cells cellular biology. (0.5 × 105 cells/mL) were seeded in 6-well plates for 24 Time and setting hours, with 1 mL in each well. Cells were treated with The experiment was performed at the laboratory, 300 μmol/L MPP+ and 10 μg/mL Eleutheroside B. At the Heilongjiang University of Traditional Chinese Medicine, end of treatment, the culture medium was collected and China from May 2009 to August 2010. LDH levels were determined using a lactate Materials dehydrogenase reagent kit (Nanjing Jiancheng Eleutheroside B (> 98% purity) was purchased from the Bioengineering Institute, Nanjing, Jiangsu Province, National Institute for the Control of Pharmaceutical and China) according to the manufacturer’s protocol. The Biological Products (Beijing, China; license No: absorbance of the samples was read at 756 nm using a 111574-200502). ultraviolet-visible spectrophotometer (Hangzhou Astra Methods Precision Instruments Co., Ltd., Hangzhou, Zhejiang PC12 cell culture and treatment Province, China). PC12 cells (Shanghai Institute of Cell Biology, Chinese SOD activity and MDA levels Academy of Sciences, China) were cultured in Following the above cell treatment protocol with MPP+ Dulbecco's Modified Eagle Medium (DMEM; Gibco, and/or the Eleutheroside B for 48 hours, cells were Carlsbad, CA, USA) supplemented with 10% (v/v) harvested, washed with PBS and degraded with normal blood serum (Hangzhou Sijiqing Company, ultrasonic cells in 1 mL PBS containing 0.05 mmol/L Hangzhou, China), 1% (v/v) mycillin mixed liquor and 1% EDTA. The cell suspension was centrifuged and the

(v/v) L-glutamine. Cells were maintained in a humidified supernatant fluid was used to assay SOD and MDA incubator at 37°C in 5% CO2 and 95% air. Culture levels according to the manufacturer’s specifications solution was renewed every 2 or 3 days. Cells were (Nanjing Jiancheng Bioengineering Institute). The passaged at 80% confluence with 0.25% (w/v) trypsin. All absorbance of the samples was read at 756 nm using a the cells used in experiments were in the logarithmic ultraviolet-visible spectrophotometer. phase of growth. PC12 cells treated with 300 μmol/L MMP measurement MPP+ (Sigma, St. Louis, MO, USA) for 48 hours were Following the above cell treatment protocol with MPP+ selected as the model group[13]. Cells simultaneously and/or the Eleutheroside B, cells (1 × 106) were collected treated with MPP+ and various concentrations of and rinsed twice with PBS. The cells were resuspended Eleutheroside B (2.5, 5, 10, 20, 40, 80 μg/mL) served as with Rhodamine123 (final concentration, 5 mg/L; Sigma) the Eleutheroside B groups. The control group was and incubated for 45 minutes in the dark at 37°C. Cells administered with the same amount of DMEM were washed twice with PBS and re-suspended in

(supplementary Figure 1 online). 400 μL of PBS, containing KH2PO4, K2HPO4, NaCl and Analysis of cell viability KCl. Fluorescence was read using an excitation PC12 cells (5 × 103 cells/mL) were seeded in a 96-well wavelength at 480 nm and an emission wavelength at plate for 24 hours. Culture medium was changed and 530 nm with a fluorescence plate reader (BD Company). cells were incubated for a further 48 hours. MTT (5 g/L; Measurement of intracellular Ca2+ concentration Sigma) was added into each well and cells were Cells (1 × 106) from each group were rinsed twice with incubated at 37°C for an additional 4 hours. At the end of PBS, collected, trypsinized and re-suspended with the incubation period, MTT was removed and 150 μL Fluo-3/AM (final concentration, 5 μmol/L; Sigma) before DMSO was added into each well to dissolve the being incubated for 45 minutes in the dark at 37°C, with formazan crystal product. Absorbance was measured at gentle agitation. Cells were rinsed twice with PBS and 570 nm with a Microplate Reader (Thermo, Beijing, left for 30 minutes. Fluorescence was read using an China). Cell viability was obtained by the mean value of excitation wavelength of 480 nm and an emission absorbance in 12 wells of each group. Cell survival = wavelength of 530 nm with a fluorescence plate reader. (Absorbance value of the treatment group / Absorbance Measurement of caspase-3 activity by western blot value of the control group) × 100%. Using the traditional method of extracting cellular Analysis of apoptosis by flow cytometry proteins[25], 20 μL of protein was electrophoresed on a Following the above cell treatment protocol, the PC12 10% (w/v) SDS-polyacrylamide gel. Protein in the gel

1378 Lu F, et al. / Neural Regeneration Research. 2011;6(18):1375-1379. was transferred onto a nitrocellulose membrane, [3] Halliday GM, McCann H. The progression of pathology in followed by incubation overnight at 4°C with a rabbit Parkinson's disease. Ann N Y Acad Sci. 2010;1184:188-195. [4] Kadota T, Yamaai T, Saito Y, et al. Expression of dopamine polyclonal caspase-3 antibody (1: 1 000; Cell Signaling transporter at the tips of growing neurites of PC12 cells. J Technology Inc., Beverly, MA, USA) and mouse Histochem Cytochem. 1996;44(9):989-996. monoclonal anti-β-actin antibody (1: 1 000; Cell Signaling [5] Shafer TJ, Atchison WD. Transmitter, ion channel and receptor Technology Inc.). The membranes were extensively properties of pheochromocytoma (PC12) cells: a model for washed three times with Tris-buffered saline Tween-20 neuiotoxicological studies. Neurotoxicology. 1991;12(3):473-492. [6] Chinese Pharmacopocia Commission. Pharmacopoeia of the for 5 minutes each. Samples were incubated for 1 hour People's Republic of China (2010). Beijing: China Medicine with goat anti-mouse IgG-horseradish peroxidase Science and Technology Press. 2010:192. (1: 1 000; Cell Signaling Technology Inc.) at room [7] Fujikawa T, Yamaguchi A, Morita I, et al. Protective effects of temperature. Tyrosine hydroxylase (Cell Signaling Acanthopanax senticosus Harms from Hokkaido and its components on gastric ulcer in restrained cold water stressed rats. Technology Inc.) was visualized using an ECL detection Biol Pharm Bull. 1996;19(9):1227-1230. reagent. The absorbance value analysis of protein bands [8] Jung HJ, Park HJ, Kim RG, et al. In vivo anti-inflammatory and was performed using the Quantity one analysis system antinociceptive effects of liriodendrin isolated from the stem bark (Bio-Rad, Hercules, CA, USA). The relative amount of of Acanthopanax senticosus. Planta Med. 2003;69(7):610-616. [9] Hibasami H, Fujikawa T, Takeda H, et al. Induction of apoptosis by caspase-3 protein expression = (absorbance of Acanthopanax senticosus HARMS and its component, sesamin in caspase-3/absorbance of β-actin) × 100%. human stomach cancer KATO III cells. Oncol Rep. 2000;7(6): Statistical analysis 1213-1216. Unless otherwise stated, experiments were carried out [10] Fujikawa T, Miguchi S, Kanada N, et al. Acanthopanax senticosus with n = 9. Data are shown as the mean ± SEM and were Harms as a prophylactic for MPTP-induced Parkinson's disease in rats. J Ethnopharmacol. 2005;97(2):375-381. analyzed with SPSS 13.0 software (SPSS, Chicago, IL, [11] Kuang SX, Xie M, Yang D, et al. Clinical studies of Acanthopanax USA). Statistical analysis was performed using one-way senticosus in treatment of Parkinson’s disease. Jilin Zhongyiyao. analysis of variance or the Student t-test. A P < 0.05 was 2004;25(01):14-15. considered statistically significant. [12] Zhang L. Studies on effectives parts of Acanthopanax senticosus for treating Parkinson’s disease. Harbin: Heilongjiang Zhongyiyao

Daxue. 2005:8-37. Author contributors: Fang Lu and Yang Dong had full access [13] An LF, Liu SM, Dong Y, et al. Protective effect of effective part of to all data and participated in data integrity and data analysis Acanthopanacis senticosus on damage of PC12 cells induced by accuracy. Shumin Liu, Fang Lu and Lifeng An were responsible MPP+. Zhongguo Zhongyao Zazhi. 2010;35(15):2021-2025. [14] Ding AS, Wang FZ, Zhao GL. Effects of hypoxia on natal for study design. Fang Lu, Yang Dong, Shihui Zhou and Bo hippocampal neuronal cells. Biochim Biophys Acta. 1993;15: Tang participated in data collection. Lifeng An, Bo Tang and 167-169. Laijun Deng were responsible for data analysis and [15] Ilhan N, Halifeoglu I, Qzercan HI, et al. Tissue malondialdehyde interpretation. Shumin Liu and Fang Lu participated in and adenosine triphosphatase level after experimental liver manuscript revision and study supervision. ischemia reperfusion damage. Cell Biochem Funct. 2001;19:207. [16] Bernardi P. The permeability transition pore. Control points of a Conflicts of interest: None declared. cyclosporin A-sensitive mitochondrial channel involved in cell Funding: This study was financially sponsored by Major death. Biochim Biophys Acta. 1996;1275(1-2):5-9. Projects of National Science and Technology, No. [17] Chandra J, Samali A, Orrenius S. Triggering and modulation of 2009ZX09103-329; the National Natural Science Foundation for apoptosis by oxidative stress. Free Radic Biol Med. 2000;29(3-4): 323-333. Distinguished Young Scholars of China, No. 30901974; [18] Mattson MP. Apoptosis in neurodegenerative disorders. Nat Rev Outstanding Youth Science Fund Program of Heilongjiang Mol Cell Biol. 2000;1(2):120-129. Province, No. JC200705; "Spring Sunshine" Plan of Ministry of [19] Przedborski S, Ischiropoulos H. Reactive oxygen and nitrogen Education, No. 2006. species: Weapons of neuronal destruction in models of Acknowledgments: We would like to express our sincere Parkinson's disease. Antioxid Redox Signal. 2005;7(5-6):685-693. [20] Hald A, Lotharius J. Oxidative stress and inflammation in thanks to Qiaoyun Chen from the Institute of Jiamusi, Parkinson’s disease: Is there a causal link? Exp Neurol. Heilongjiang University of Traditional Chinese Medicine, China, 2005;193(2):279-290. for technical support. [21] Nicholls DG, Budd SL. Mitochondria and neuronal survival. Supplementary information: Supplementary data associated Physiol Rev. 2000;80(1):315-360. [22] Green DR, Reed JC. Mitochondria and apoptosis. Science. with this article can be found in the online version, by visiting 1998;281(5381):1309-1312. www.nrronline.org, and entering Vol. 6, No. 18, 2011 after [23] Gogvadze V, Orrenius S, Zhivotovsky B. Multiple pathways of selecting the “NRR Current Issue” button on the page. cytochrome c release from mitochondria in apoptosis. Biochim Biophys Acta. 2006;1757(5-6):639-647. REFERENCES [24] Hartmann A, Hunot S, Michel PP, et al. Caspase-3: a vulnerability factor and final effector in apoptotic death of dopaminergic

neurons in Parkinson’s disease. Proc Natl Acad Sci U S A. [1] Braak H, Del Tredici K, Rub U, et al. Staging of brain pathology 2000;97(6):2875-2880. related to sporadic Parkinson’s disease. Neurobiol Aging. [25] Xu SS, Yan CL, Liu LM, et al. Effects of different cell lysis buffers 2003;24(2):197-211. on protein quantification. Zhejiang Univ Medical Sci. 2008;37(1): [2] Béné R, Antić S, Budisić M, et al. Parkinson's disease. Acta Clin 45-50. Croat. 2009;48(3):377-380. (Edited by Zhao JG, Liang DH/Yang Y/Wang L)

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