Myricitrin attenuates 6-hydroxydopamine-induced mitochondrial damage and apoptosis in PC12 cells via inhibition of mitochondrial oxidation

JOURNAL OF FUNCTIONAL FOODS

5 (2 0 1 3) 3 3 7–34 5

Available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/jff

Myricitrin attenuates 6-hydroxydopamine-induced mitochondrial damage and apoptosis in PC12 cells via inhibition of mitochondrial oxidation Yue-Hua Wanga,b, Zhao-Hong Xuanc, Shuo Tiana, Guo-Rong Hea, Guan-Hua Dua,b,* a Beijing Key Laboratory of Drug Target Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, PR China b State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, PR China c Shenyang Pharmaceutical University, Shenyang 110016, PR China

A R T I C L E I N F O

A B S T R A C T

Article history:

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by progressive loss of

Received 1 June 2012

dopaminergic (DA) neurons at the substantia nigra. 6-Hydroxydopamine (6-OHDA) is a

Received in revised form

dopamine analog, which specifically to damage dopaminergic neurons. Myricitrin, a flava-

1 November 2012

noid isolated from the root bark of Myrica cerifera, has antinociceptive activity, anti-inflam-

Accepted 7 November 2012

matory, antioxidant, and immunomodulatory properties. In the present study, the potential

Available online 10 January 2013

protection and mechanism of myricitrin against 6-OHDA-induced damage and apoptosis in PC12 cells was studied. The results showed that myricitrin attenuated 6-OHDA-induced cell

Keywords:

damage and mitochondrial dysfunction in a dose-dependent manner, which was correlated

Myricitrin

with decreased intracellular ATP content and mitochondrial membrane potential. Further-

Lipid peroxidation

more, it was found that myricitrin inhibited the apoptosis of PC12 cells induced by 6-OHDA

Mitochondria Apoptosis

in relation to reduction of cytochrome C release from mitochondria and inhibition of the activity of caspase-3. Finally, the antioxidation of myricitrin in PC12 cells and brain mitochondria was investigated. The results showed that myricitrin decreased the production of reactive oxygen species in PC12 cells and inhibited lipid peroxidation in rat brain mitochondria (IC50 = 3.19 ± 0.34 lM). Thus, myricitrin has the neuroprotective capacity to antagonize 6-OHDA-induced neurotoxicity in PC12 cells and may be useful in treating PD.  2012 Elsevier Ltd. All rights reserved.

1.

Introduction

Metabolic or neurotoxic insults often cause oxidative stressmediated neuronal apoptosis and thereby contribute to the pathogenesis and progression of neurodegenerative diseases, including Parkinson’s disease (PD) and Alzheimer’s disease (AD) (Stadtman, 2006). Oxidative stress often causes extensive damage to lipids, proteins, and DNA, resulting in cell death by

a variety of different mechanisms including activation or inactivation of various apoptotic cell signaling molecules. Typically, upregulated reactive oxygen species (ROS) generation results in calcium dysregulation that leads to mitochondrial dysfunction, resulting in activation of the apoptotic caspase cascade (Cannon & Greenamyre, 2010; Henchcliffe & Beal, 2008; Kanthasamy et al., 2010).

* Corresponding author at: Beijing Key Laboratory of Drug Target Identification and Drug Screening, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, PR China. Tel./fax: +86 10 63165184. E-mail address: [email protected] (G.-H. Du). 1756-4646/$ - see front matter  2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jff.2012.11.004

338

JOURNAL OF FUNCTIONAL FOODS

Parkinson’s disease (PD) is a typical neurodegenerative disorder, characterized by symptoms including rest tremors, postural instability, gait abnormality, bradykinesia and rigidity (Alonso, Otero, D’Regules, & Figueroa, 1986). PD is mainly characterized by the selective loss of the dopaminergic neurons in the substantia nigra (Moghal, Rajput, D’Arcy, & Rajput, 1994). The presence of apoptotic nigral neurons was demonstrated in post-mortem brains of PD patients (Anglade et al., 1997). Although the cause of the cellular destruction in PD remains unclear, it is now often attributed to the action of neurotoxins which act on the mitochondrial respiratory chain by depleting the production of ATP (Soto-Otero, Me´ndez-Alvarez, Hermida-Ameijeiras, Lo´pez-Real, & Labandeira-Garcı´a, 2002). 6-Hydroxydopamine (6-OHDA) is a dopamine analog, which specifically to damage dopaminergic neurons either via uncoupling mitochondrial oxidative phosphorylation resulting in energy deprivation or alternatively, is associated with its ability to produce hydrogen peroxide, hydroxyl and superoxide radicals under physiological pH conditions (SotoOtero et al., 2008). Evidence demonstrates that 6-OHDA generates ROS and induces apoptosis in dopaminergic cells of rat substantia nigra (Soto-Otero et al., 2002). It has also been reported that 6-OHDA inhibits complexes I and IV of the mitochondrial respiratory chain (Glinka, Tipton, & Youdim, 1996; Glinka & Youdim, 1995). So 6-OHDA is used to investigate the cellular and molecular mechanisms underlying selective degeneration of dopaminergic neurons in PD (Lee et al., 2005). Myricitrin, one of the principal components of the root bark of Myrica cerifera, is known to possess antinociceptive activity (Meotti et al., 2006a), anti-inflammatory (Wang et al., 2010), antioxidant (Moser, 2008), and antiallodynia activity (Meotti et al., 2006b). However, the effects of myricitrin on mitochondria are not studied. In this study, we studied the myricitrin against the lipid peroxidation in brain mitochondria and also observed the protective effects of myricitrin on mitochondrial damage and apoptosis induced by 6OHDA in PC12 cells.

2.

Materials and methods

2.1.

Reagents

Myricitrin was obtained from National Institutes for Food and Drug Control (Beijing, China). RPMI-1640 and fetal bovine serum were purchased from Gibco products (USA). 6-OHDA, MTT, Ac-DEVD-AFC, Propidium iodide and 2,7-dichlorodihydrofluorescein diacetate were purchased from Sigma–Aldrich (St. Louis, MO, USA). The ATP Bioluminescent Assay Kit was purchased from Promega company (Madison, WI, USA). Cytochrome C assay kit was purchased from Friendship Biotechnology Co., Ltd (Beijing, China). The JC-1 assay kit was purchased from Beyotime Institute of Biotechnology (Haimen city, Jiangsu, China).

2.2.

Cell culture and treatment

PC12 cells have been widely employed as a neuronal cell model and a large number of studies were generated. The cells were grown in RPMI-1640 supplemented with 10% horse

5 ( 2 0 1 3 ) 3 3 7 –3 4 5

serum, 5% fetal bovine serum. Conditions were maintained at 37 C in a humidified atmosphere containing 5% CO2. The medium was changed every 2–3 days (Wang & Du, 2009). 6OHDA was dissolved in 0.2% ascorbic acid and used at a final concentration of 100 lM. The cells were plated into 96-well plates at a density of 2 · 105 cells/ml. After 80% confluence, the cells were pre-incubated with different concentration of myricitrin in a serum-free RPMI-1640 medium for 1 h. Then, 6-OHDA was added to the wells at a final concentration of 100 lM and incubated for another 24 h at 37 C.

2.3. cells

Morphological changes and viability assay of PC12

After treatment, cell viability was evaluated by MTT assay (Kang et al., 2012; Wang & Du, 2009). This method measures mitochondrial activity based on the reductive cleavage of yellow tetrazolium salt to a purple formazan compound by the dehydrogenase activity of intact mitochondria. Briefly, after treatment, MTT 0.5 mg/ml was added to each well and incubated for 4 h at 37 C. Then, the supernatant was removed and the formazan product obtained was dissolved in 100 ll of dimethylsulphoxide (DMSO) with stirring for 15 min on a microtiter plate shaker and the absorbance was read at 540 nm. The percentage of viable cells in each treatment group was determined by comparing their respective absorbance with that of untreated group.

2.4.

Intracellular ATP level assay

Cellular ATP level was measured with an ATP Bioluminescent Assay Kit according to the manufacturer s instructions. After treatment, culture medium was removed and cells were lysed by adding 100 ll of ATP releasing reagent. Aliquots of 100 ll were transferred to white 96 well assay plates. Luminescence was monitored on a Spectramax M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA) in the luminescence mode following the addition 100 ll of luciferin and luciferase (Tirmenstein et al., 2005).

2.5.

Measurement of mitochondrial membrane potential

The fluorescent probe JC-1 (5,5 0 ,6,6 0 -tetrachloro-1,1 0 ,3,3 0 -tetraethylbenzimidazolylcarbocyanine iodide) was used to estimate mitochondrial membrane potential (Mashimo & Ohno, 2006). JC-1 localizes to the inner mitochondrial membrane where it forms either monomers or aggregates based on mitochondrial membrane potential. At high membrane potential, JC-1 accumulates sufficiently in the mitochondria to form aggregates that exhibit red fluorescence. On the other hand, at lower membrane potential less dye enters mitochondria resulting in monomers that show green fluorescence. JC-1 is sensitive to mitochondrial membrane potential, and the changes in the ratio between aggregate (red) and monomer (green) fluorescence can provide information regarding the mitochondrial membrane potential. JC-1 was dissolved in DMSO and further diluted in PBS the final concentration of 1 mg/ml. After treatment, the culture medium were removed and loaded with JC-1 for 15 min at 37 C in the dark. After two more rinses with Hank’s solution, fluorescence intensity of

JOURNAL OF FUNCTIONAL FOODS

the red/green ratio was determined on a Spectramax M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA) at an excitation of 490 nm and emission of 530 nm (green fluorescent monomers) and 590 nm (red fluorescent aggregates) respectively.

2.6.

Analysis of DNA fragmentation

After the treatment, the DNA extract was performed as described previously. The DNA was resuspended in 30 ll TE (10 mM Tris–HCl, 1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0) and 4 ll RNase A for 1.5 h at 37 C and was electrophoresed in a 1.2% agarose gel in TAE buffer (40 mM Tris–HCl, 20 mM acetic acid, and 1 mM Na2EDTA, pH 8.0). The gel was stained with 0.5 mg/ml EB, visualized by UV transillumination and photographed.

2.7.

PI uptake

This method relies upon the fact that propidium iodide (PI) is impermeable to cells with an intact plasma membrane. However, when cell membranes disintegrate, PI gains access to the nucleus where it complexes with DNA rendering the nucleus highly fluorescent. After treatment, PI was added to the cultures at the final concentration of 10 lM in colorless DMEM and the cultures were incubated at 37 C for 15 min in the dark. After washing with colourless medium, the DNA content was measured by using a Flow Cytometer (Hou et al., 2008; Zunino, Storms, Zhang, & Seeram, 2009).

2.8.

Quantification of cytochrome C release

Cytochrome C was shown to redistribute from mitochondria to cytosol during apoptosis in intact cells. The cytosolic cytochrome C levels were measured in cytosolic fractions obtained from untreated and 6-OHDA-treated PC12 cells using an ELISA Kit as described previously (Eliseev et al., 2009). Briefly, after treatment, PC12 cells were washed once with ice-cold PBS and resuspended in 1 ml of ice-cold homogenization buffer (10 mM Tris–HCl, pH 7.5, 0.3 M sucrose, 1 mM phenylmethylsulphonyl fluoride, 25 g/ml aprotinin, and 10 g/ml leupeptin) and homogenized on ice. Cells were then centrifuged for 10,000g for 60 min at 4 C. The supernatants were collected as cytoplasmic fraction and used for cytochrome C release measurements. The optical density of each well is then measured at 450 nm using a Spectramax microplate reader. The concentration of cytochrome C is calibrated from a standard curve based on reference standards.

2.9.

Determination of caspase-3 activities

The activities of caspase-3 were measured using caspase-3 specific fluorescence substrate Ac-DEVD-AFC as described previously (Kaul, Kanthasamy, Kitazawa, Anantharam, & Kanthasamy, 2003). Briefly, cells (2 · 105 cells/well) were subcultured in 24-well tissue culture plates and treated with 100 lM 6-OHDA for 24 h. Formation of 7-amino-4-methylcoumarine (AMC), resulting from caspase substrate cleavage, was

5 ( 20 1 3) 3 3 7–34 5

339

measured using a Spectramax M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA) with excitation at 400 nm and emission at 505 nm. Enzyme activities are expressed as fluorescence units/mg protein/h.

2.10. level

Determination of intracellular reactive oxygen species

Intracellular reactive oxygen species level was measured using 2,7-dichlorofluorescein-diacetate (H2DCFH-DA) staining method. H2DCF-DA is a nonpolar compound that is converted into a nonfluorescent polar derivative (H2DCF) by cellular esterases after incorporation into cells. H2DCF is membraneimpermeable and rapidly oxidized to the highly fluorescent 2,7-dichlorofluorescein (DCF) in the presence of intracellular reactive oxygen species (Ham et al., 2012; Sauer, Klimm, Hescheler, & Wartenberg, 2001). After incubation with 6OHDA, cells were loaded with 10 lM H2DCFH-DA for 30 min at 37 C in the dark. Cells were then analyzed on a Spectramax M5 microplate reader (Molecular Devices, Sunnyvale, CA, USA) with excitation at 488 nm and emission at 525 nm.

2.11. Lipid peroxidation in brain mitochondria induced by FeSO4–cystine Brain mitochondria were obtained by differential centrifugation with minor modifications to a previously published method (Katyare, Bangur, & Howland, 1994). Male Sprague– Dawley rats weighing 200–250 g were used. All animal experiments were approved by the Laboratories Institutional Animal Care and Use Committee of Chinese Academy of Medical Sciences. Briefly, animals were killed by decapitation and the brain was immediately removed and washed in icecold isolation medium (mannitol 210 mM, sucrose 70 mM, EDTA 0.5 mM, Tris–HCl 10 mM and 0.2% bovine serum albumin. pH 7.4). After removing blood vessels and pial membranes, the brains were manually homogenized with 10% (w/v) of the isolation medium. Then, the homogenate was centrifuged at 1000 g for 10 min at 4 C. The supernatant was subjected to a further centrifugation at 12,000g for 10 min. The mitochondrial pellet was then washed once with isolation medium and recentrifuged at 12,000g for 10 min. Finally, the mitochondrial pellet was reconstituted in a Locke’s buffer (in mM: NaCl 154 mM, KCl 5.6 mM, CaCl2 2.3 mM, MgCl2 1.0 mM, NaHCO3 3.6 mM, glucose 5.0 mM, Hepes 5.0 mM. pH 7.4). All procedures were performed at 4 C. Mitochondrial protein quantification was determined by the Bradford method (Bradford, 1976) using BSA as standard. Lipid peroxidation was determined by the formation of thiobarbituric acid reactive substances (TBARS) as described and modification (Gassen, Glinka, Pinchasi, & Youdim, 1996). Briefly, rat brain mitochondria 100 lg/well in 0.2 M histidine buffer including FeSO4 50 lM and cystine 500 lM was added into 96-well plate. Then, different concentration of myricitrins were added and incubated at 37 C for 30 min. The incubation was stopped by the addition of 1.0% thiobarbituric acid (TBA) solution and incubated for 30 min at 60 C. The absorption was measured at 532 nm.

340

2.12.

JOURNAL OF FUNCTIONAL FOODS

Statistical analysis

The data were expressed as the means ± SD. Significance of differences between group means was determined by Oneway analysis of variance (ANOVA) followed by t-test. A P-value < 0.05 was considered statistically significant.

3.

Results

3.1. Effect of myricitrin on cell viability in PC12 cells damaged by 6-OHDA Within 24 h of treatment with 6-OHDA alone, the majority of PC12 cells had undergone morphological changes such as membrane blebbing and cell shrinkage. Co-treatment with myricitrin protected the cells from 6-OHDA damage (Fig. 1A). Treatment of PC12 cells with 6-OHDA alone resulted in an approximately 25% reduction in cell survival within 24 h, whereas co-treated with 1 and 10 lM myricitrin showed a reduction of 6-OHDA-mediated cytotoxicity (Fig. 1B).

5 ( 2 0 1 3 ) 3 3 7 –3 4 5

3.2. Myricitrin increases the intracellular ATP level induced by 6-OHDA ATP is the primary energy currency of living systems. Virtually all energy requiring processes utilize the chemical energy stored in the phosphate bond of ATP. ATP is formed exclusively in the mitochondria and a variety of genetic diseases can affect ATP formation in the mitochondria. Cellular ATP concentrations were significantly decreased following 24 h incubations with 100 lM 6-OHDA. Whereas pre-treated with 0.1, 1 and 10 lM myricitrin showed a reduction of 6-OHDA-mediated ATP decrease (Fig. 2).

3.3. Myricitrin blocked reduction of mitochondria membrane potential induced by 6-OHDA in PC12 cells Changes in mitochondrial membrane potential (MMP) were measured by determining the red/green fluorescence ratio of JC-1. The ratio was normalized by comparison with the control red/green ratio. As shown in Fig. 3, treatment of

Fig. 1 – Effect of myricitrin on the morphological changes and viability of PC12 cells induced by 6-OHDA. PC12 cells were preincubated for 1 h with different concentration of myricitrin before 100 lM 6-OHDA treatments and co-cultured for 24 h. (A) Morphological changes were observed by light microscopy (40·); (B) Cell viability was estimated by MTT assay. Data were expressed as percent cell viability of untreated cells. Data are the mean ± SD, n = 4, ##P < 0.01 vs untreated group; *P < 0.05 vs vehicle group.

JOURNAL OF FUNCTIONAL FOODS

Fig. 2 – Effect of myricitrin on the generation of ATP induced by 6-OHDA. PC12 cells were pre-incubated for 1 h with different concentration of myricitrin before 100 lM 6-OHDA treatments and co-cultured for 24 h. Data are the mean ± SD, n = 4, ##P < 0.01 vs untreated group; **P < 0.01 vs vehicle group.

5 ( 20 1 3) 3 3 7–34 5

341

Fig. 3 – Effect of myricitrin on mitochondria membrane potential assayed by JC-1. PC12 cells were pre-incubated for 1 h with different concentration of myricitrin before 100 lM 6-OHDA treatments and co-cultured for 24 h. Data are the mean ± SD, n = 4, ##P < 0.01 vs untreated group; *P < 0.05, **P < 0.01 vs vehicle group.

cultures with 100 lM 6-OHDA for 24 h resulted in significant decrease of MMP compared with untreated group (P < 0.01). However, the decrease of MMP induced by 6-OHDA were significantly attenuated by treatment with 0.1, 1 and 10 lM myricitrin (P < 0.01) (Fig. 3).

3.4.

Myricitrin inhibits the apoptosis induced by 6-OHDA

One of the important hallmarks of apoptosis is DNA fragmentation into multiples of 180–200 bp, which appears as a typical ‘‘DNA laddering’’ pattern on DNA electrophoresis gel. The obvious DNA ladder was observed in the cells treated with 100 lM of 6-OHDA for 24 h. 6-OHDA-induced DNA fragmentation was rescued by myricitrin at the experimental concentrations (Fig. 4). PI is membrane impermeant and generally excluded from viable cells. PI is commonly used for identifying dead cells in a population. The apoptosis rate was significantly increased following 24 h incubations with 100 lM 6-OHDA. Whereas pre-treated with 1 and 10 lM myricitrin showed a significant reduction of 6-OHDA-mediated apoptosis (Fig. 5).

3.5. Myricitrin reduces the accumulation of cytochrome C in the cytosol induced by 6-OHDA Cytochrome C plays an important role in apoptosis. The protein is located in the space between the inner and outer mitochondrial membranes. An apoptotic stimulus triggers the release of cytochrome C from the mitochondria into cytosol where it then activates caspase-3 and other downstream caspases. The results (Fig. 6) showed that 6-OHDA exposure resulted in an increase in cytosolic cytochrome C by 167%

Fig. 4 – Effect of myricitrin on DNA fragmentation induced by 6-OHDA. PC12 cells were pre-incubated for 1 h with different concentration of myricitrin before 100 lM 6-OHDA treatments and co-cultured for 24 h. DNA mark (1), untreated cells (2), cells damaged with 100 lM 6-OHDA only (3), or cells pretreated with 0.1, 1 and 10 lM myricitrin (4–6).

over the untreated group (P < 0.01). Myricitrin at the dose of 0.1, 1 and 10 lM could significantly reduce the cytochrome C release into cytosol induced by 6-OHDA, respectively (P < 0.05, P < 0.01, and P < 0.01).

342

JOURNAL OF FUNCTIONAL FOODS

5 ( 2 0 1 3 ) 3 3 7 –3 4 5

ZFig. 5 – Flow cytometric analysis of effect of myricitrin on apoptosis induced by 6-OHDA. PC12 cells were pre-incubated for 1 h with different concentration of myricitrin before 100 lM 6-OHDA treatments and co-cultured for 24 h. After treatment with 6-OHDA for 24 h, the cells were harvested and stained with PI. Percentage (%) represented the rate of apoptotic PC12 cell nuclei with hypodiploid DNA content (Sub-G1 peak). Data are the mean ± SD, n = 4, ##P < 0.01 vs untreated group; *P < 0.05 vs vehicle group.

3.6. Myricitrin inhibits the activation of caspase-3 induced by 6-OHDA

3.8. Inhibitory effect of myricitrin on lipid peroxidation in brain mitochondria

The results (Fig. 7) showed that caspase-3 activity increased 3.3-fold compared with the untreated group after 24 h of incubation with 6-OHDA (P < 0.01). Myricitrin at 1 and 10 lM could inhibit the activity of caspase-3, respectively (P < 0.05, P < 0.05).

The results are presented in Fig. 9. Myricitrin inhibited the lipid peroxidation in brain mitochondria in a concentrationdependent manner. IC50 value is 3.19 ± 0.34 lM.

3.7. Myricitrin reduces the production of ROS induced by 6-OHDA

Flavonoid glycosides are the main form in which flavonoids occur in nature. Importantly, glycosylation greatly influences chemical and physical properties altering in vivo bioavailability and possible pharmacological actions. Indeed, flavonoid glycosides have been shown to exert CNS-mediated activities (Fernandez et al., 2009). For example, myricitrin were shown to possess antinociceptive effects after systemic administration (Meotti et al., 2006a, 2006b). However, the action of myricitrin on PD has not been explored. This study was the first to investigate neuroprotective effects of myricitrin on brain mitochondria and neurotoxin 6-OHDA-induced apoptosis in PC12 cells. The results showed that myricitrin strongly inhibited iron-induced lipid peroxidation in brain mitochondria

In order to determine the degree to which ROS are involved in 6-OHDA-induced apoptosis, PC12 cells were treated for 24 h with 100 lM 6-OHDA and determined the intracellular ROS levels by DCF fluorescence. Cells treated with 6-OHDA showed a significant increase (about 1.4-fold) of intracellular ROS compared with untreated cells. This increase was significantly attenuated by co-incubation with 0.1, 1 and 10 lM myricitrin, respectively. This result indicates that the co-incubation of PC12 cells with myricitrin effectively prevents 6OHDA-induced the production of ROS (Fig. 8).

4.

Discussion

JOURNAL OF FUNCTIONAL FOODS

Fig. 6 – Effect of myricitrin on the release of cytochrome C into cytoplasm by ELISA assay. PC12 cells were preincubated for 1 h with different concentration of myricitrin before 100 lM 6-OHDA treatments and co-cultured for 24 h. Data are the mean ± SD, n = 4, ##P < 0.01 vs untreated group; *P < 0.05, **P < 0.01 vs vehicle group.

5 ( 20 1 3) 3 3 7–34 5

343

Fig. 8 – Effect of myricitrin on generation of ROS induced by 6-OHDA. PC12 cells were pre-incubated for 1 h with different concentration of myricitrin before 100 lM 6-OHDA treatments and co-cultured for 24 h. ROS was measured by incubation with H2DCF-DA. Data were expressed as percent cell viability of untreated cells. Data are the mean ± SD, n = 4, ## P < 0.01 vs untreated group; **P < 0.01 vs vehicle group.

Fig. 7 – Effect of myricitrin on caspase-3 activity. PC12 cells were pre-incubated for 1 h with different concentration of myricitrin before 100 lM 6-OHDA treatments and cocultured for 24 h. The activity of caspsae-3 was assessed by fluorogenic substrate Ac-DEVD-AMC. Data are the mean ± SD, n = 4, ##P < 0.01 vs untreated group; *P < 0.05, **P < 0.01 vs vehicle group.

Fig. 9 – Inhibitory effect of myricitrin on lipid peroxidation in brain mitochondrial. Results are expressed as mean ± SD of triplicate assays.

and inhibited the production of ROS in PC12 cells, and also significantly enhances the PC12 cells viability and rescue PC12 cells from apoptosis induced by 6-OHDA through

protecting mitochondria. Our results suggest that myricitrin may serve as a neuroprotective candidate for the treatment of PD.

344

JOURNAL OF FUNCTIONAL FOODS

It has been known that 6-OHDA selectively could cause degeneration of nigrostriatal dopaminergic neuronal pathway in several animals (Glinka & Youdim, 1995) and could cause the PC12 cells apoptotic death (Xu et al., 2001), so the 6OHDA-damaged PC12 cells was used as a vitro PD model in our studies to investigate the possible protective effect of myricitrin. 6-OHDA is a highly reactive substance, which is readily auto-oxidized and oxidatively deaminated by monoamine oxidase, to give rise to H2O2 and ROS (Levites, Youdim, Maor, & Mandel, 2002). ROS, in turn, can cause DNA strand breaks, damage protein residues and initiate lipid peroxidation reactions (Halliwell, 1992). These results are supported by our findings that myricitrin exhibited potent neuroprotection against 6-OHDA-induced mitochondrial damage and apoptosis in PC12 cells. This is the first time that the protective effect of myricitrin has been described under PD-like conditions. Our results indicated that the initial blockage of ROS by myricitrin might be a very important factor for the protection of PC12 cells. Mitochondria have an integral role in the apoptotic cell pathway (Henchcliffe & Beal, 2008). A reduction in mitochondrial transmembrane potential has been reported to accompany early apoptosis. Mitochondrial cytochrome C was identified as a component required for the crucial steps in apoptosis (Skulachev, 1998). Cytochrome C is a water soluble protein of 15 kDa with a net positive charge, residing loosely attached in the mitochondrial intermembrane space. Cytochrome C was shown to redistribute from mitochondria to cytosol during apoptosis in intact cells. The release of cytochrome C into the cytosol leads to an activation of an apoptotic program via activation of a caspase dependent pathway (Slee et al., 1999). Measurement of cytochrome C release from the mitochondria is a tool to detect the first early steps for initiating apoptosis in cells. Caspase-3 is believed to be the final executor of apoptotic DNA damage, as a marker of apoptosis, studies indicated neuronal death in PD has been associated with activation of caspase-3. 6-OHDA has been shown to cause apoptotic actions of dopaminergic cells via activation of caspase-3-like proteases in experimental models. Our results showed 6-OHDA caused the reduction of mitochondria membrane potential, and subsequently cytochrome C release from the damaged mitochondria and constituting a significant factor for the activation of caspase-3, eventually leading to cell apoptosis. Whereas, the co-incubation with myricitrin attenuated the 6-OHDA neurotoxicity. The results suggested that one possible mechanism underlying the effect of myricitrin against 6-OHDA neurotoxicity may involve its anti-apoptosis through protecting mitochondria. In summary, 6-OHDA-induced apoptosis of PC12 cells is caused principally by generation of ROS, reduced mitochondria membrane potential, increased the release cytochrome C, elevated the activity of caspase-3 and induced caspases activation. These factors are regarded as pathological features in PD, and these features are all significantly attenuated by co-incubation with myricitrin. In other words, our results provide evidence that myricitrin may have the neuroprotective effect against 6-OHDA neurotoxicity in vitro. Next, further investigation into the pharmacodynamic effect in vivo conditions will be underway.

5 ( 2 0 1 3 ) 3 3 7 –3 4 5

Acknowledgements This work was supported by Grants from the Young Foundation of 2011 in CAMS & PUMC, the National Science and Technology Major Project (2012ZX09103101-078), and the research special fund for public welfare industry of health (200902008).

R E F E R E N C E S

Alonso, M. E., Otero, E., D’Regules, R., & Figueroa, H. H. (1986). Parkinson’s disease: A genetic study. Canadian Journal Neurological Science, 13, 248–251. Anglade, P., Vyas, S., Javoy-Agid, F., Herrero, M. T., Michel, P. P., Marquez, J., Mouatt-Prigent, A., Ruberg, M., Hirsch, E. C., & Agid, Y. (1997). Apoptosis and autophagy in nigral neurons of patients with Parkinson’s disease. Histology and Histopathology, 12(1), 25–31. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry, 72, 248–254. Cannon, J. R., & Greenamyre, J. T. (2010). Neurotoxic in vivo models of Parkinson’s disease recent advances. Progress in Brain Research, 184, 17–33. Eliseev, R. A., Malecki, J., Lester, T., Zhang, Y., Humphrey, J., & Gunter, T. E. (2009). Cyclophilin D interacts with Bcl2 and exerts an anti-apoptotic effect. The Journal of Biological Chemistry, 284(15), 9692–9699. Fernandez, S. P., Nguyen, M., Yow, T. T., Chu, C., Johnston, G. A., Hanrahan, J. R., & Chebib, M. (2009). The flavonoid glycosides, myricitrin, gossypin and naringin exert anxiolytic action in mice. Neurochemical Research, 34(10), 1867–1875. Gassen, M., Glinka, Y., Pinchasi, B., & Youdim, M. B. (1996). Apomorphine is a highly potent free radical scavenger in rat brain mitochondrial fraction. European Journal of Pharmacology, 308(2), 219–225. Glinka, Y., Tipton, K. F., & Youdim, M. B. (1996). Nature of inhibition of mitochondrial respiratory complex I by 6hydroxydopamine. Journal of Neurochemistry, 66, 2004–2010. Glinka, Y., & Youdim, M. B. (1995). Inhibition of mitochondrial complexes I and IV by 6-hydroxydopamine. European Journal of Pharmacology, 292(3–4), 329–332. Halliwell, B. (1992). Reactive oxygen species, and the central nervous system. Journal of Neurochemistry, 59, 1609–1623. Ham, Y. M., Yoon, W. J., Park, S. Y., Song, G. P., Jung, Y. H., Jeon, Y. J., Kang, S. M., & Kim, K. N. (2012). Quercitrin protects against oxidative stress-induced injury in lung fibroblast cells via upregulation of Bcl-xL. Journal of Functional Foods, 4, 253–262. Henchcliffe, C., & Beal, M. F. (2008). Mitochondrial biology and oxidative stress in Parkinson disease pathogenesis. Nature Clinical Practice Neurology, 4(11), 600–609. Hou, L., Ju, C., Zhang, J., Song, J., Ge, Y., & Yue, W. (2008). Antitumor effects of Isatin on human neuroblastoma cell line (SH-SY5Y) and the related mechanism. European Journal of Pharmacology, 589(1–3), 27–31. Kang, S. M., Kim, A. D., Heo, S. J., Kim, K. N., Lee, S. H., Ko, S. C., & Jeon, Y. J. (2012). Induction of apoptosis by diphlorethohydroxycarmalol isolated from brown alga, Ishige okamurae. Journal of Functional Foods, 4, 433–439. Kanthasamy, A., Jin, H., Mehrotra, S., Mishra, R., Kanthasamy, A., & Rana, A. (2010). Novel cell death signaling pathways in neurotoxicity models of dopaminergic degeneration: Relevance to oxidative stress and neuroinflammation in Parkinson’s disease. Neurotoxicology, 31(5), 555–561.

JOURNAL OF FUNCTIONAL FOODS

Katyare, S. S., Bangur, C. S., & Howland, J. L. (1994). Is respiratory activity in the brain mitochondria responsive to thyroid hormone action: A critical re-evaluation. Biochemical Journal, 302, 857–860. Kaul, S., Kanthasamy, A., Kitazawa, M., Anantharam, V., & Kanthasamy, A. G. (2003). Caspase-3 dependent proteolytic activation of protein kinase C delta mediates and regulates 1methyl-4-phenylpyridinium (MPP+)-induced apoptotic cell death in dopaminergic cells: Relevance to oxidative stress in dopaminergic degeneration. European Journal of Neuroscience, 18(6), 1387–1401. Lee, H. J., Noh, Y. H., Lee, D. Y., Kim, Y. S., Kim, K. Y., Chung, Y. H., Lee, W. B., & Kim, S. S. (2005). Baicalein attenuates 6hydroxydopamine-induced neurotoxicity in SH-SY5Y cells. European Journal of Cell Biology, 84(11), 897–905. Levites, Y., Youdim, M. B., Maor, G., & Mandel, S. (2002). Attenuation of 6-hydroxydopamine (6-OHDA)-induced nuclear factor-kappaB (NF-kappaB) activation and cell death by tea extracts in neuronal cultures. Biochemistry Pharmacology, 63(1), 21–29. Mashimo, K., & Ohno, Y. (2006). Ethanol hyperpolarizes mitochondrial membrane potential and increases mitochondrial fraction in cultured mouse myocardial cells. Archives of Toxicology, 80(7), 421–428. Meotti, F. C., Luiz, A. P., Pizzolatti, M. G., Kassuya, C. A., Calixto, J. B., & Santos, A. R. (2006a). Analysis of the antinociceptive effect of the flavonoid myricitrin: Evidence for a role of the larginine-nitric oxide and protein kinase C pathways. Journal Pharmacology and Experimental Therapeutics, 316(2), 789–796. Meotti, F. C., Missau, F. C., Ferreira, J., Pizzolatti, M. G., Mizuzaki, C., Nogueira, C. W., & Santos, A. R. (2006b). Anti-allodynic property of flavonoid myricitrin in models of persistent inflammatory and neuropathic pain in mice. Biochemical Pharmacology, 72, 1707–1713. Moghal, S., Rajput, A. H., D’Arcy, C., & Rajput, R. (1994). Prevalence of movement disorders in elderly community residents. Neuroepidemiology, 13(4), 175–178. Moser, B. R. (2008). Efficacy of myricetin as an antioxidant in methyl esters of soybean oil. European Journal of Lipid Science Technology, 110, 1167–1174. Sauer, H., Klimm, B., Hescheler, J., & Wartenberg, M. (2001). Activation of p90RSK and growth stimulation of multicellular tumor spheroids are dependent on reactive oxygen species

5 ( 20 1 3) 3 3 7–34 5

345

generated after purinergic receptor stimulation by ATP. The FASEB Journal, 15(13), 2539–2541. Skulachev, V. P. (1998). Cytochrome c in the apoptotic and antioxidant cascades. FEBS Letters, 423(3), 275–280. Slee, E. A., Harte, M. T., Kluck, R. M., Wolf, B. B., Casiano, C. A., Newmeyer, D. D., Wang, H. G., Reed, J. C., Nicholson, D. W., Alnemri, E. S., Green, D. R., & Martin, S. J. (1999). Ordering the cytochrome c-initiated caspase cascade: Hierarchical activation of caspases-2,-3,-6,-7,-8, and -10 in a caspase-9dependent manner. The Journal of Cell Biology, 144(2), 281–292. Soto-Otero, R., Me´ndez-Alvarez, E., Hermida-Ameijeiras, A., Lo´pez-Real, A. M., & Labandeira-Garcı´a, J. L. (2002). Effects of ()-nicotine and ()-cotinine on 6-hydroxydopamine-induced oxidative stress and neurotoxicity: Relevance for Parkinson’s disease. Biochemical Pharmacology, 64, 125–135. Soto-Otero, R., Me´ndez-Alvarez, E., Sa´nchez-Iglesias, S., Zubkov, F. I., Voskressensky, L. G., Varlamov, A. V., de Candia, M., & Altomare, C. (2008). Inhibition of 6-hydroxydopamine-induced oxidative damage by 4,5-dihydro-3H-2-benzazepine N-oxides. Biochemical Pharmacology, 75(7), 1526–1537. Stadtman, E. R. (2006). Protein oxidation and aging. Free Radical Research, 40, 1250–1258. Tirmenstein, M. A., Hu, C. X., Scicchitano, M. S., Narayanan, P. K., McFarland, D. C., Thomas, H. C., & Schwartz, L. W. (2005). Effects of 6-hydroxydopamine on mitochondrial function and glutathione status in SH-SY5Y human neuroblastoma cells. Toxicology In Vitro, 19(4), 471–479. Wang, Y. H., & Du, G. H. (2009). Ginsenoside Rg1 inhibits betasecretase activity in vitro and protects against Abeta-induced cytotoxicity in PC12 cells. Journal of Asian Natural Products Research, 11(7), 604–612. Wang, S. J., Tong, Y., Lu, S., Yang, R., Liao, X., Xu, Y. F., & Li, X. (2010). Anti-inflammatory activity of myricetin isolated from Myrica rubra Sieb. et Zucc. leaves. Planta Medica, 76(14), 1492–1496. Xu, R., Liu, J., Chen, X., Xu, F., Xie, Q., Yu, H., Guo, Q., Zhou, X., & Jin, Y. (2001). Ribozyme-mediated inhibition of caspase-3 activity reduces apoptosis induced by 6-hydroxydopamine in PC12 cells. Brain Research, 899(1–2), 10–19. Zunino, S. J., Storms, D. H., Zhang, Y., & Seeram, N. P. (2009). Growth arrest and induction of apoptosis in high-risk leukemia cells by strawberry components in vitro. Journal of Functional Foods, 1, 153–160.