Promising effects on ameliorating mitochondrial function and enhancing Akt signaling in SH-SY5Y cells by (M)-bicelaphanol A, a novel dimeric podocarpane type trinorditerpene isolated from Celastrus orbiculatus.
Oxidative stress plays an important role in the pathological processes of various neurodegenerative diseases. In this study, we investigated the neuroprotective effects of (M)-bicelaphanol A, which has been the first dimeric podocarpane type trinorditerpene isolated from Celastrus orbiculatus, against hydrogen peroxide ([H.sub.2][O.sub.2])-induced injury in human SH-SY5Y neuroblastoma cells. Our study showed that cells pretreated with (M)-bicelaphanol A significantly attenuated [H.sub.2][O.sub.2]-induced cell viability reduction and cell apoptosis. These neuroprotective effects of (M)-bicelaphanol A were associated with a reduction of reactive oxygen species and an increase in the level of adenosine triphosphate. In addition, (M)-bicelaphanol A pretreatment markedly increased the phosphorylation level of Akt in SH-SY5Y cells. In conclusion, our results for the first time demonstrate that the protection of (M)-bicelaphanol A on SH-SY5Y cells against [H.sub.2][O.sub.2]-induced oxidative stress may attribute, at least partially, to its attenuation of mitochondrial dysfunction and activation of Akt signaling pathway. Above results shed more light on the molecular mechanisms involved in the neuroprotective effects of (M)-bicelaphanol A, which could be a potential drug candidate for the treatment of oxidative stress-associated neurodegenerative diseases.
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Oxidative stress has long been implicated in neuronal cell death of variety neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and ischemic injury (Barnham et al. 2004; Chong et al. 2005; Gilgun-Sherki et al. 2001; Lin and Beal 2006). Oxidative damage is resulted from excessive generation of reactive oxygen species (ROS) following cell lysis, oxidative bursts or an excess of free transition metals. ROS can attack proteins, deoxynucleic acids and lipid membranes, which may eventually induce apoptosis (Martindale and Holbrook 2002; Valko et al. 2007). In the brain, several antioxidant molecules such as superoxide dismutase, glutathione peroxidase and carotenoid can scavenge ROS and protect against oxidative stress. With the aggravated oxidative insults and weakened antioxidant defense system in the brain of neurodegenerative diseases, pharmacological approach to resist oxidative stress seems to be a promising therapeutic strategy. In fact, great efforts have been put on finding novel chemicals with remarkable antioxidative activity, and some compounds, such as catecholamines and flavonoids, have been proved to possess promising neuroprotective effects on oxidative stress (Choi et al. 2011; Esposito et al.2002; Vauzour et al. 2008).
Hydrogen peroxide ([H.sub.2][O.sub.2]), a major ROS, is produced during normal metabolism. When the balance between oxidation and antioxidation is disturbed, [H.sub.2][O.sub.2] is overproduced and then induced oxidative stress. [H.sub.2][O.sub.2] itself can directly react with cellular macromolecules to damage mitochondria and result in cellular apoptosis. [H.sub.2][O.sub.2] has been used extensively as an oxidative stress inducer in several experimental situations for the development of antioxidant drug candidates to treat neurodegenerative disorders (Datta et al. 2002; Rojkinda et al. 2002; Zhang et al. 2007).
Celastrus orbiculatus is a twining liana that is traditionally used in China as an anti-inflammatory agent to treat rheumatism arthritis, fever, edema and bacterial infection (Tong and Moudgil 2007; Wu et al. 2004). Celastrus orbiculatus and its extracts have been reported to possess various pharmacological properties including anti-inflammatory and antioxidant effects (Allison et al. 2001; Jin et al. 2002). In our effort to further elucidate the antioxidant components of Celastus orbiculatus, phytochemical isolation, structural elucidation and biological activity screening was undertaken, and (M)-bicelaphanol A (Fig. 1), the first example of dimeric podocarpane type trinorditerpene isolated from nature was found to exhibit promising protective effect against [H.sub.2][O.sub.2]-induced neurotoxicity (Wang et al. 2013).
In this study, we further evaluated the neuroprotective effects of (M)-bicelaphanol A against [H.sub.2][O.sub.2]-induced cell damage in SH-SY5Y neuroblastoma cells. We also evaluated mitochondrial function and Akt expression to further elucidate the possible molecular pharmacological mechanisms of (M)-bicelaphanol A.
Materials and methods
Preparation of (M)-bicelaphanol A
(M)-bicelaphanol A (powder, purity > 99%; provided by Prof. Wei-min Zhao of Shanghai Institute of Materia Medica, Chinese Academy of Sciences) was isolated from the root bark of Celastrus orbiculatus. The botanical determination of the plant Celastrus orbiculatus has been described in detail previously (Wang et al. 2013). The root bark of Celastrus orbiculatus were percolated with 95% EtOH to give a crude extract, which was suspended in water and partitioned with EtOAc. (M)-bicelaphanol A was isolated from the EtOAc extract by repeated column chromatography. The purity of (M)-bicelaphanol A was determined using a Shimadzu LCMS-2020 system, and its structure was determined by spectroscopic and single-crystal X-ray diffraction analyses (Wang et al. 2013). (M)-bicelaphanol A was dissolved in dimethylsulfoxide (DMSO) to 10 mM as a stock solution stored at -20 [degrees]C and diluted with culture medium before usage. The chemical structure of (M)-bicelaphanol A is shown in Fig. 1.
SH-SY5Y cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained at 37 [degrees]C in a humidified atmosphere containing 5% C[O.sub.2]. Cells were seeded into 96-well plates and 6-well plates (Corning, Corning, NY, USA) at a density of 1 x [10.sup.5] cells/ml in Dulbecco's modified Eagle's medium (DMEM; Gibco. Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY). 100U/ml penicillin, 100 [micro]g/ml streptomycin, and 2mM L-glutamine. Twenty-four hours after cells were seeded, medium was replaced with DMEM and cells were pre-incubated with different concentrations of (M)-bicelaphanol A for 2h. To produce oxidative stress, [H.sub.2][O.sub.2] was freshly prepared from 8.8 M stock solution prior to each experiment, and the final concentration in the culture medium was 100 [micro]M in the entire study. Assays were carried out at different time after [H.sub.2][O.sub.2] treated, as specified in the following methods.
Cell viability test
Cell viability was evaluated by morphological observation under microscope (Nikon TE2000, Melville, NY. USA) with amplification of lOOx and 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT, Sigma, St Louis, MO, USA) assay. After cells were treated with 100 [micro]M [H.sub.2][O.sub.2] for 24 h. MTT was added to culture medium at a final concentration of 0.5mg/ml and incubated at 37 [degrees]C for 4h. When obtained by the live cell. MTT is transformed to formazan. The formazan product was dissolved by adding 100 [micro]l DMSO each well. Cell plates were shaken for 5 min and the absorbance of each well was recorded on a DTX 800 Multimode Detector (Beckman Coulter, Fullerton, CA) at 490 nm.
Flow cytometric determination of apoptosis
Double staining for Annexin V-FITC binding to membrane phosphatidylserine (PS) and for cellular DNA using propidium iodide (PI) was performed according to the protocol provided by the manufacturer (KeyGen Biotech, Nanjing, China). In brief, after exposure to 100 [micro]M [H.sub.2][O.sub.2] for 3 h, cells plated in 6-well plates were harvested by centrifugation (1000 x g, 10 min), washed twice with PBS, and resuspended in 500 [micro]l binding buffer containing 5 [micro]l Annexin V-FITC and 5 [micro]I PI, then incubated for 15 min in the dark at room temperature. Samples were kept on ice and acquired immediately on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA) using the CellQuest software (BD Biosciences, San Jose, CA). Annexin V-FITC and PI emissions were detected in the channel FL 1 and FL 3, respectively. For each analysis we recorded one hundred thousand counts. Data analysis was performed using FCS Express V3.00 (DeNovo Software. Thornhill, Canada). In each plot, the lower left quadrant represents viable cells, the upper left quadrant necrotic cells, the lower right (LR) quadrant early apoptotic cells, and the upper right quadrant (UR) late apoptotic cells.
Quantification of intracellular ROS
Intracellular ROS was determined following the oxidation of 2', 7'-dichlorodihydrofluorescein diacetate ([H.sub.2]DCFDA; Molecular Probes) (Sigma-Aldrich Corporation, St Louis, MO, USA) to fluorescent DCF. Cells seeded into 6-well plates were incubated with 100 [micro]M [H.sub.2][O.sub.2] for 3h, then loaded in darkness with 10 [micro]M [H.sub.2]DCF-cliacetate for 45 min at 37 [degrees]C in a saline medium containing(in mM): 132.0 NaCl, 4.0 KCl, 1.0 Ca[Cl.sub.2], 1.4 Mg[Cl.sub.2],1.2 Na[H.sub.2]P[O.sub.4], 6.0 glucose, 10.0 Hepes (PH 7.4). Next, cells were washed twice with the same medium, harvested by centrifugation (3000 x g, 5 min) and resuspended in 500 [micro]l PBS. ROS was measured on the FACSCalibur flow cytometer with 488 nm excitation/520 nm emission filters. Data acquisition and analysis were performed using CellQuest and FCS Express V3.00. The mean fluorescence intensity was used to quantify the responses. A minimum of ten thousand cells were acquired for each sample.
Detection of ATP content
The intracellular adenosine triphosphate (ATP) content was measured using the CellTiter-Glo[R] luminescent assay (Promega, Madison. WI. USA) based on ATP bioluminescence. After cells in 96-well plates were treated with 100 [micro]M [H.sub.2][O.sub.2] for 24 h. 100 [micro]l of CellTiter-Glo reagent was added to each well and contents mixed for 2 min on a shaker. The plates were then incubated at room temperature for 10 min following the procedure recommended by the manufacturer. The luminescence produced by the luciferase-catalyzed reaction of luciferin with ATP was detected on a Wallac EnVision 2104 multilabel reader (Perkin Elmer. Waltham. MA. USA). The ATP levels were normalized by control group and expressed as percentage of control.
Western blot analysis
After exposure to 100 [micro]M [H.sub.2][O.sub.2] for 1 h. cells in 6-well plates were collected using a scraper and washed with PBS. After centrifugation (1000 x g. 10 min), cells were lysed in 100 [micro]l RIPA buffer [Tris 50 mM. NaCl 150mM, sodium-deoxycholate 0.25% (v/v), NP-40 1% (v/v), NaF 1 mM. [Na.sub.3]V[O.sub.4] 1 mM, pH 7.4] on ice for 30 min before centrifuging at 12,000 x g for 10 min at 4 [degrees]C. The supernatant was collected and followed by protein concentration determination using BCA Assay kit (Pierce. Rockford, IL, USA). After addition of loading buffer, protein samples were electrophoresed on a 12% SDSPAGE and subsequently transferred to nitrocellulose membrane (Millipore, Bedford. MA, USA). The membrane was blocked with 5% skimmed milk in T-TBS [Tween-20 0.005% (v/v). Tris 50 mM, NaCl 150 mM, pH 7.51 at room temperature for 1 h and then probed with primary anti-Akt (1:1000), anti-p-Akt (1:1000) (Cell Signaling, Beverly, MA, USA) at 4 [degrees]C overnight. The membrane was washed three times for 10 min each with T-TBS buffer. After that it was incubated with horseradish peroxidase-conjugated secondary antibody (1:5000; Kangcheng, Shanghai. China) for 1 h at room temperature and washed three times in T-TBS buffer, then the membrane was developed using the ECL reagent (Millipore, Billerica, MA, USA) according to the instructions of manufacture and visualized with autoradiography film. The intensity of each band was quantified with Image J software (NIH, Bethesda, MD. USA).
The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay was carried out followed by the previous report (Blois 1958). Briefly, 1 ml of (M)-bicelaphanol A in water at different concentrations (1-10 [micro]M) was added to 5ml of DPPH solution in 95% ethanol. The mixture was shaken vigorously and kept standing at room temperature for 15 min. Then the absorbance of the mixture was measured at 517 nm on a 752-C Spectrophotometer (The 3rd Analytical Instruments Factory, Shanghai, China). Vitamin C, a well-known anti-oxidant, was used as a positive control. The decrease in the absorbance indicates an increase in DPPH-radical scavenging activity. The percentage inhibition was calculated by the following equation, where Ac is the absorbance of control and As is absorbance of sample:
DPPH radical scavenging(%) = [1 - ([A.sub.s]/[A.sub.c]] x 100
All data were shown only if at least three independent experiments showed consistent results. Data were expressed as mean [+ or -] SD. Multiple comparisons were analyzed by one-way ANOVA followed by Turkey's test using a computerized statistical package. Statistical significance was established at p value < 0.05.
Protective effects of (M)-bicelaphanol A on [H.sub.2][O.sub.2]-induced cytotoxicity in SH-SY5Y cells SH-SY5Y cells
SH-SY5Y cells exposed to 100 [micro]M [H.sub.2][O.sub.2] for 24 h resulted in cell body swelling, dendrites fragmentation and cell number decrease (Fig. 2A) with simultaneous decrease in cell viability (53.95 [+ or -] 2.17%) as compared with control (p < 0.01, Fig. 2B). However, pretreatment with (M)-bicelaphanol A markedly attenuated [H.sub.2][O.sub.2]-induced cytotoxicity in a dose dependent manner with the concentration of 10 [micro]M showed a maximal protection restored cell viability (99.92 [+ or -] 6.32%; p < 0.01 vs. [H.sub.2][O.sub.2] group) (Fig. 2B). Consistent with the result from MTT assay, the cell morphological damages induced by [H.sub.2][O.sub.2] were also significantly attenuated by 10 [micro]M (M)-bicelaphanol A pretreatment (Fig. 2A). Additionally, no notable changes in cell viability were observed by incubation with 1-10 [micro]M (M)-bicelaphanol A for 24 h in [H.sub.2][O.sub.2] free condition (Fig. 2C).
(M)-bicelaphanol A pretreatment protected SH-SY5Y cells against [H.sub.2][O.sub.2]-induced apoptosis
Annexin V-FITC binding analysis and PI staining (Fig. 3A) were performed to identify cells undergoing early and late apoptosis, respectively, while intact cells can not be stained with Annexin V-FITC or PI. In control group (Fig. 3B), only a small number of cells (9.18 [+ or -] 2.44%) were positive to Annexin V-FITC and PI staining. After exposure to 100 [micro]M [H.sub.2][O.sub.2] for 3h, the percentage of apoptosis significantly increased (17.89 [+ or -] 3.50%; p < 0.05 vs. control) (Fig. 3B). Preincubation with (M)-bicelaphanol A(1, 3 and 10 [micro]M) for 2h markedly reduced the number of apoptotic cells, and the rate of apoptosis were dose-dependently decreased (18.23 [+ or -] 2.10%, 16.88 [+ or -] 1.20% and 7.64 [+ or -] 1.58%; respectively) (Fig. 3B).
(M)-bicelaphanol A pretreatment decreased [H.sub.2][O.sub.2]-induced intracellular ROS accumulation
After being loaded with [H.sub.2]DCFDA, normal SH-SY5Y cells displayed substantial fluorescence signal (Fig.4A). However, exposing these cells to 100 [micro]M [H.sub.2][O.sub.2] for 3h caused a 4-fold increase of fluorescent intensity, which led to a right shift of the emission maximum (Fig. 4A) and statistical significance (Fig. 4B; p < 0.01 vs. control). Pretreatment of the cells with (M)-bicelaphanol A (1, 3, 10 [micro]M) before [H.sub.2][O.sub.2] exposure robustly reduced ROS production in a dose-dependent manner (Fig. 4B). Moreover, (M)-bicelaphanol A at the concentration of 3 and 10 [micro]M significantly inhibited the increase of DCF fluorescence (p < 0.01 vs. [H.sub.2][O.sub.2] group. Fig. 4A), which was almost reduced to control level (Fig. 4B).
(M)-bicelaphanol A pretreatment ameliorated [H.sub.2][O.sub.2]-induced ATP reduction
To evaluate whether the antioxidant effect of (M)-bicelaphanol A was related to the improvement of mitochondrial function, the ATP content was evaluated. Exposure of the SH-SY5Y cells to 100 [micro]M [H.sub.2][O.sub.2] for 24 h significantly reduced ATP content (41.31 [+ or -] 10.45%. p < 0.01 vs. control) (Fig. 5). In contrast, 2 h of pretreatment with (M)-bicelaphanol A (1, 3 and 10 [micro]M) prevented the [H.sub.2][O.sub.2]-induced loss of ATP content in a dose-dependent manner (54.10 [+ or -] 14.83%. 70.20 [+ or -] 12.01%, 82.79 [+ or -] 12.30%, respectively) (Fig. 5).
(M)-bicelaphanol A pretreatment increased Akt phosphorylation level in SH-SY5Y cells
To reveal potential molecular mechanism involved in the anti-apoptotic function of (M)-bicelaphanol A, the phosphorylation level of Akt was examined. Exposure of SH-SY5Y cells with [H.sub.2][O.sub.2] for 1 h increased the ratio of phorspho-Akt (Serine 473)/total Akt (131.6 [+ or -] 19.27%; p < 0.05 vs. control). After (M)-bicelaphanol A (10 [micro]M) preincubation for 2h, then exposed to [H.sub.2][O.sub.2] for another 1 h. Akt signaling in SH-SY5Y cells was markedly enhanced, with a significantly increased phosphor-Akt/total Akt ratio (175.3 [+ or -] 23.70%; p < 0.05 vs. [H.sub.2][O.sub.2] group. Fig. 6).
The ability of (M)-bicelaphanol A scavenging DPPH radicals
To obtain information about the mechanisms of the antioxidative effects of (M)-bicelaphanol A, we examined its radical scavenging capacity by using DPPH radicals, and vitamin C was used as a standard. DPPH is a stable radical that loses its purple color when it accepts an electron from an antioxidant molecule (Zou et al. 2004). In our experiment, (M)-bicelaphanol A at concentrations of 1, 3 and 10 [micro]M inhibited 1.34 [+ or -] 0.61 %. 3.32 [+ or -] 0.43% and 11.49 [+ or -] 0.30% of DPPH absorbance, respectively, indicating a low ability to scavenge DPPH radicals. Data were from three independent experimental results and presented as mean [+ or -] SD.
Considerable efforts have been attempted to search for potent antioxidant drugs (Swalwell et al. 2012), especially natural compounds with antioxidant properties and neuroprotective effects. In this study, we for the first time reported the possible molecular mechanisms involved in the strong antioxidative effects of (M)-bicelaphanol A, a novel dimeric podocarpane type trinorditerpene isolated from Celastrus orbiculatus. These results indicate that, with the potent effects on mitochondrial function and Akt signaling, (M)-bicelaphanol A could be a promising drug candidate for the treatment of neurodegenerative diseases.
[H.sub.2][O.sub.2], a major ROS, has been extensively used as an inducer of oxidative stress to develop antioxidant for neurodegenerative diseases therapy (Finkel 2003). Exogenous addition of [H.sub.2][O.sub.2] into cultured cells induced cells undergoing a series of process such as mitochondrial dysfunction, protein misfolding, genetic mutation and finally cell death. In the present study, we found that pretreatment of SH-SY5Y cells with different concentrations of (M)-bicelaphanol A decreased [H.sub.2][O.sub.2]-induced morphological damages (Fig. 2A) in a dose-dependent manner and the result was further confirmed by cell viability assay in SH-SY5Y cells (Fig. 2B). These results indicate that (M)-bicelaphanol A is able to protect SH-SY5Y cells from [H.sub.2][O.sub.2]-induced neurotoxicity.
It has been demonstrated that [H.sub.2][O.sub.2] can damage proteins and induce apoptosis (Chen et al. 2010; Klamt et al. 2009). Consistent with these previous reports, when double stained with fluorescent Annexin V-FITC and PI, [H.sub.2][O.sub.2] exposure markedly increased the apoptotic cells (Fig. 3). Similar as the aforementioned results in cell survival test, (M)-bicelaphanol A pretreatment significantly attenuated this phenomenon, indicating that the neuroprotective effect of (M)-bicelaphanol A may partially attribute to its anti-apoptosis effect.
Mitochondria are extremely sensitive to oxidative stress (Halliwell 2012) and also play a vital role in cell apoptosis cascade (Eckert et al. 2003; Kroemer et al. 1997). Apoptosis triggered by oxidative stress is associated with the increased production of intracellular ROS, most of which are generated from mitochondrial respiratory chain and subsequently produce toxic metabolic byproducts (Niatsetskaya et al. 2012). In our study, SH-SY5Y cells exposed to [H.sub.2][O.sub.2] resulted in a significant increase of ROS. However, the effect was inhibited by pretreatment with (M)-bicelaphanol A (Fig. 4). We proposed that the ability of (M)-bicelaphanol A to reduce the free-radical production produced by an oxidative insult is not mainly attribute to its ability to scavenge radical entities, since (M)-bicelaphanol A treatment almost reversed the overproduction of ROS both at 3 and 10 [micro]M (Fig. 4), while 3 [micro]M (M)-bicelaphanol A only had a very low DPPH radical-scavenging capacity (the inhibition rate is 3.32%).
Oxidative stress is central to the development of mitochondrial dysfunction, as mitochondria are the primary source of cellular oxidants. Mitochondrial oxidative phosphorylation depends on an intact membrane. Overproduction of ROS induced the opening of the mitochondrial permeability pore and caused the decline in membrane potential (Wei and Lee 2002). Moreover, a decrease in the integrity of the mitochondria would destruct transmembrane proton gradient and decrease ATP production. In agreement with these reports, it was observed that [H.sub.2][O.sub.2] exposure caused diminished ATP production in SH-SY5Y cells (Fig. 5), while pretreatment of (M)-bicelaphanol A maximally reduced the decrease of ATP, which may be due to the ability of (M)-bicelaphanol A to inhibit intracellular ROS accumulation.
Akt is an important serine threonine kinase in cell proliferation and survival (Downward 2004). Acute activation of the Akt can inhibit necrosis and apoptosis of cells induced by deleterious stimuli (Brunet et al. 2001; Hetman et al. 1999). We wondered whether the neuroprotective effects of (M)-bicelaphanol A are a result of the activation of the Akt pathway. Consistent with other studies (Sadidi et al. 2009). Akt phosphorylation at serine473 is increased by [H.sub.2][O.sub.2] exposure in SH-SY5Y cells in our study. This phenomenon indicates that the cells may generate a compensative mechanism to against the attack of [H.sub.2][O.sub.2]. Unfortunately, these compensations are not strong enough to prevent or block the oxidative damage. Compared with those treated by [H.sub.2][O.sub.2] alone, much higher phosphorylation levels of Akt were observed in (M)-bicelaphanol A pre-incubated cells. Indicating that the further activation of Akt signaling might mediate the protection of (M)-bicelaphanol A against [H.sub.2][O.sub.2]-induced cell apoptosis. Above pharmacological mechanism is similar as that of other nature compounds (Cheng et al. 2013; Dal-Cim et al. 2012; Zhang et al. 2005), however, the precise regulatory effects of (M)-bicelaphanol A on Akt signaling pathway deserve further investigation.
In summary, the present results for the first time elucidated the molecular targets of (M)-bicelaphanol A on mitochondrial function and Akt signaling in [H.sub.2][O.sub.2]-exposcd SH-SY5Y cells. The antioxidative and neuroprotective effects indicate the potential of (M)-bicelaphanol A as a new therapeutic candidate for neurodegenerative diseases.
The authors are grateful to Prof. Lu Qi Huang providing leaf picture of Celastrus orbiculatus. This work is supported by the Ministry of Science and Technology of China (No. 2011CB510004); the National Natural Science Foundation of China (Nos. 81173034, 81072646); National Science & Technology Major Project "Key New Drug Creation and Manufacturing Program" of China (Nos. 2011ZX09307-002-03. 2012ZX09301001-001. 2012ZX09301001-004), the State Key Laboratory of Drug Research (No. SIMM1203KF-02).
Abbreviations: AD. Alzheimer's disease; ATP, adenosine triphosphate; [H.sub.2]DCFDA. 2',7'-dichlorodihydrofluorescein diacetate; DMEM. Dulbecco's modified Eagle's medium; DMSO. dimethylsulfoxide: DPPH. 1.1-diphenyl-2-picrylhydrazyl; FBS, fetal bovine serum; [H.sub.2][O.sub.2], hydrogen peroxide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide; Pl, propidium iodide; PS, phosphatidylserine; ROS, reactive oxygen species.
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Xu Jie Wang (a), Luo Yi Wang (a), Yan Fu (a), Jian Wu (a), Xi Can Tang (a), Wei Min Zhao (a), *, Hai Yan Zhang (a), (b), *
(a) State Key Laboratory of Drug Research Shanghai Institute of Materia Medica. Chinese Academy of Sciences. People's Republic of China
(b) CAS Key Laboratory of Receptor Research. Shanghai Institute of Materia Medica. Chinese Academy of Sciences. People's Republic of China
* Corresponding authors at: Shanghai Institute of Materia Medica, 555 Zu Chong Zhi Road. Zhangjiang Hi-Tech Park. Shanghai 201203. People's Republic of China. Tel.: +86 2150806710: fax: +86 2150806710.
E-mail addresses: email@example.com (W.M. Zhao). firstname.lastname@example.org, email@example.com (H.Y. Zhang).
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|Author:||Wang, Xu Jie; Wang, Luo Yi; Fu, Yan; Wu, Jian; Tang, Xi Can; Zhao, Wei Min; Zhang, Hai Yan|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Sep 15, 2013|
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