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Myricetin attenuated MPP+-induced cytotoxicity by anti-oxidation and inhibition of MKK4 and JNK activation in MES23.5 cells
Neuropharmacology 61 (2011) 329e335
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Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm
Myricetin attenuated MPPþ-induced cytotoxicity by anti-oxidation and inhibition of MKK4 and JNK activation in MES23.5 cells Kai Zhang, ZeGang Ma, Jun Wang, AnMu Xie, JunXia Xie* Department of Physiology, Shandong Provincial Key Laboratory of Pathogenesis and Prevention of Neurological Disorders and State Key Disciplines: Physiology, Medical College of Qingdao University, Qingdao 266071, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 December 2010 Received in revised form 6 April 2011 Accepted 12 April 2011
Increasing evidence suggests that oxidative stress may be implicated in the degeneration of dopaminergic neurons in Parkinson’s disease (PD), and anti-oxidation have been shown to be effective to PD treatment. Myricetin has been reported to have the biological functions of anti-oxidation, anti-apoptosis, anti-inflammation and iron-chelation. The aim of the present study is to investigate the neuroprotective effect of myricetin on 1-methyl-4-phenylpyridinium (MPPþ)-treated MES23.5 cells and the underlying mechanisms. The results showed that myricetin treatment significantly attenuated MPPþ-induced cell loss and nuclear condensation. Further experiments demonstrated that myricetin could suppress the production of intracellular reactive oxygen species (ROS), restore the mitochondrial transmembrane potential (6Jm), increase Bcl-2/Bax ratio and decrease caspase-3 activation that induced by MPPþ. Futhermore, we also showed myricetin decreased the phosphorylation of mitogen-activated protein kinase (MAPK) kinase 4 (MKK4) and c-Jun N-terminal kinase (JNK) caused by MPPþ. These results suggest that myricetin protected the MPPþ-treated MES23.5 cells by anti-oxidation and inhibition of MKK4 and JNK activation. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Myricetin MPPþ Oxidative stress MKK4
1. Introduction Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by selectively loss of dopaminergic neurons in the substantia nigra (SN), giving rise to dopamine (DA) depletion in the striatum. Although the precise pathogenic mechanism leading to neurodegeneration in PD is not known, a number of factors have been implicated in the pathogenesis of DA neuron loss. The generation of reactive oxygen species (ROS), mitochondrial dysfunction (Anantharam et al., 2007; Gardoni and Di Luca, 2006; Linazasoro, 2002; Mandel et al., 2000; McNaught et al., 2002), excitotoxicity (Gardoni and Di Luca, 2006; Moldzio et al., 2006) and inflammation (Koprich et al., 2008; Thomas et al., 2008) are all considered important mediators of neuronal death in PD. It is more
Abbreviations: MPTP, 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine; MPPþ, 1-methyl-4-phenylpyridinium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide; JNK, c-Jun N-terminal kinase; DA, dopamine; DMEM/F12, Eagle’s medium Nutrient Mixture-F12; 6J, mitochondrial transmembrane potential; MKK4, mitogen-activated protein kinase kinase 4; PD, Parkinson’s disease; ROS, reactive oxygen species; RT-PCR, Reverse transcription-polymerase chain reaction; SN, substantia nigra. * Corresponding author. Tel.: þ86 532 85955891; fax: þ86 532 83780136. E-mail address: [email protected] (JunXia Xie). 0028-3908/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2011.04.021
likely that multiple factors converge to give rise to PD than any single reason. The approaches to prevent dopamine neuron degeneration reflect the current notion on the pathogenesis of PD. Drugs directed against a single target will be ineffective and a single drug with multiple pharmacological properties maybe more suitable (Grunblatt et al., 2004; Van der Schyf et al., 2006; Youdim and Buccafusco, 2005), in which flavonoids are considered to be the most promising candidate because of their well-reported biological properties. Myricetin is one of such flavonoids. It has many biological functions, including anti-oxidation (Kang et al., 2010), anti-apoptosis (Shimmyo et al., 2008), free radical scavenging and antiinflammation (Lee and Choi, 2008; Hladovec, 1977). Recently, several studies demonstrated the neuroprotective effects of myricetin. It has been shown that myricetin protect against rotenoneinduced cytotoxicity in SHSY5Y cells (Molina-Jimenez et al., 2003), Myricetin also inhibits the formation and extension of amyloidb protein for the prevention of Alzheimer’s disease (Ono et al., 2003). Myricetin also inhibits 6-hydroxydopamine-induced neurotoxicity in rats (Ma et al., 2007). However, the underlying mechanisms of myricetin-mediated neuroprotection are uncertain. 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) is a common neurotoxin for inducing PD models. Chronic administration of MPTP induced apoptosis was found in the SN of mice.
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Activation of the mitogen-activated protein kinase (MAPK) kinase 4 (MKK4) and c-Jun N-terminal kinase (JNK) pathway has been shown to play a critical role in MPTP or 1-methyl-4-phenylpyridinium (MPPþ) induced PD models (Xia et al., 2001). In the present study, we aim to elucidate whether myricetin could protect MPPþ-induced neurotoxicity and the underlying mechanisms in MES23.5 cells. 2. Materials and methods
2.7. Measurement of caspase-3 activation The active caspase-3 was measured by PE-conjugated monoclonal active caspase-3 antibody apoptosis kit. Briefly, cells were seeded on 6-well plates and treated as described above. After washing twice with cold PBS, cells were resuspended in Citofix/CytopermTM solution at a concentration of 1 106 cells/ 0.5 ml and incubated for 20 min. The cells were then washed with Perm/Washing buffer twice and incubated in Perm/Washing buffer with antibody (1:5) for 30 min. After washing once with Perm/Washing buffer, the cells were re-suspended with 0.5 mL Perm/Washing buffer and analyzed by flow cytometry. The extent of apoptosis was determined by counting the number of active caspase-3 immunoreactive cells as a percentage of total MES23.5 cells using Cellquest Software.
2.1. Materials Unless otherwise stated, all chemicals including myricetin were purchased from Sigma Chemical Co. The purity of myricetin used is 95%. Dulbecco’s modified Eagle’s medium Nutrient Mixture-F12 (DMEM/F12) was from Invitrogen.
2.2. Cell culture MES23.5 cells were cultured in DMEM/F12 containing Sato’s components growth medium supplemented with 5% fetal bovine serum, 100 U/mL of penicillin and 100 mg/mL of streptomycin at 37 C, in a humid 5% CO2, 95% air environment. For experiments, cells were seeded at a density of 1 105/cm2 in the plastic flasks.
2.3. Cell viability assay MES23.5 cells were seeded in a 96-well plate at a density of 2 104 cells per well. Cell viability was measured by the conventional 3-(4,5-dimethylthiazol-2-yl)2,5- diphenyltetrazolium bromide (MTT) assay. The cells were divided into vehicle group, MPPþ treatment group, MPPþ and myricetin co-treatment group. After incubation with the chemicals for 24 h respectively, the cells were incubated in MTT (5 mg/mL) for 4 h and cell viability was measured at 570 nm by colorimetric assay (Rayto RT-2100C, Shenzhen, China).
2.4. Hoechst 33258 staining Nuclear morphology was detected using the method previously described in our lab (Xu et al., 2008; Zhang et al., 2009). MES23.5 cells were seeded on sterile cover glasses in 24-well plates and treated with vehicle, MPPþ, MPPþ and myricetin for 24 h respectively. The cells were fixed in 4% paraformaldehyde for 30 min, washed in phosphate-buffered saline, and stained with Hoechst 33258 dye for 30 min at room temperature. After washing 3 times to remove the excessive dye, the cells were examined and photographed under a fluorescence microscope (Olympus, Japan) with an excitation wavelength of 330e380 nm. Apoptotic cells were defined on the basis of nuclear morphological changes, such as chromatin condensation and fragmentation. The total number of condensed cells was counted manually by researchers blinded to the treatment schedule using unbiased stereology. For each well, we delineated a 400 mm2 frame and counted all condensed and normal nuclei in at least 10 different fields. Average sum of condensed and normal nuclei was calculated per well. The data were presented as the percentage of condensed nuclear number to the total number.
2.8. Western blots analysis MES23.5 cells were incubated with vehicle, MPPþ, myricetin, MPPþ and myricetin for 5 min respectively, then, cells were lysed in a buffer containing 50 mM TriseHCl (pH 7.4), 1 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and 10 mg/mL aprotinin. Cell lysates were separated by 8% sodium dodecyl sulfate polyacrylamide gel and then transferred to polyvinylidene difluoride membrane. Blots were probed with anti-pMKK4 and anti-pJNK monoclonal antibody (Cell Signaling, 1:1000). Blots were also probed with antiebeactin monoclonal antibody (Sigma, 1:10,000) as a loading control. Results were analyzed through scanning densitometry by Tanon Image System.
2.9. Reverse transcription-polymerase chain reaction (RT-PCR) MES23.5 cells were incubated with vehicle, MPPþ, myricetin, MPPþ and myricetin for 24 h respectively, Total RNA was isolated by Trizol Reagent (Invitrogen). 5 mg of total RNA was reversed transcribed in a 20 ml reaction using the AMV reverse transcription system (Promega Corporation, Madison, WI, USA). We amplified Bcl-2 cDNA fragment (amplified products were 409 bp length) with the primers (forward: 50 - GTC CCG CCT CTT CAC CTT -30 ; reverse: 50 - CCC ACT CGT AGC CCC TCT -30 ), Bax cDNA fragment (amplified products were 307 bp length) with the primers (forward: 50 - GGC GAA TTG GAG ATG AAC -30 ; reverse: 50 - CCG AAG TAG GAG AGG AGG -30 ) and GAPDH cDNA fragment (amplified products were 236 bp length) with the primers (forward: 50 - TTC ACC ACC ATG GAG AAG GC -30 ; reverse 50 - GGC ATG GAC TGT GGT CAT GA -30 ). The expression of house-keeping gene, GAPDH mRNA, was used as an internal standard. PCRs were run for 36 cycles in an Eppendorf Mastercycler. Denaturing, annealing, and extension reactions were performed at 94 C for 30 s, 51 C for 30 s, and 72 C for 45 s. The PCR products were electrophoresed on 1% agarose gel, stained with ethidium bromide, and detected by UV irradiation. The levels of Bcl-2 and Bax mRNA were expressed as their respective ratios to GAPDH mRNA.
2.10. Statistical analysis Data were expressed as mean S.E.M. and analyzed using the SPSS11.5 software. One-way analysis of variance (ANOVA) followed by Tukey test was used to compare the differences between means. A probability value of less than 0.05 was considered to be statistically significant.
2.5. Reactive oxygen species (ROS) assay Intracellular ROS in MES23.5 cells were measured by carboxy-H2DCFDA as described before (Sanelli et al., 2007; Zhang et al., 2009; Zhu and Liu, 2004). The fluorescence coming from carboxy-H2DCFDA reflects the intracellular ROS generation. The cells treated as described above were incubated in HEPES-buffered saline (HBS) containing carboxy-H2DCFDA in a final concentration of 5 mmol/L for 30 min at 37 C and followed by washing twice with HBS. Fluorescent intensity was recorded at 488 nm excitation and 525 nm emission wavelengths. Results were demonstrated as FL1-H (Fluorescence 1-Histogram), setting of the gated region M1 and M2 as a marker to observe the changing levels of fluorescence intensity by Cellquest Software. 2.6. Detection of mitochondrial transmembrane potential (6Jm) The changes of mitochondrial membrane potential were measured by rhodamine123 using flow cytometry (Becton Dickinson, USA) as described before (Sanelli et al., 2007; Zhang et al., 2009; Zhu and Liu, 2004). The uptake of rhodamine123 into mitochondria is an indicator of the 6Jm. After 24 h treatment, the cells were incubated with rhodamine123 in a final concentration of 5 mmol/L for 30 min at 37 C. The fluorescent intensity was recorded at 488 nm excitation and 525 nm emission wavelengths. Results were demonstrated as described in ROS assay.
Fig. 1. MTT analysis of cell viability in MES23.5 cells. 200 mmol/L MPPþ decreased the cell viability compared with that of control group, this effect was partly inhibited by treated with myricetin. (##P < 0.01, compared with the control; *P < 0.05, compared with MPPþ treatment group; n ¼ 4).
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Fig. 2. Morphological changes in MES23.5 cells. (A). Representative photographs of Hoechst 33258 staining from vehicle, MPPþ, MPPþ and myricetin co-treatment groups. MPPþ treatment resulted in nuclear condensation; however, 109 mol/L myricetin treatment significantly attenuated MPPþ-induced nuclear condensation (Magnification 400). (B). Statistical analysis. Data were presented as means SEM of 6 independent experiments. (###P < 0.001, compared with the control; *P < 0.05, compared with MPPþ treatment group).
3. Results 3.1. Myricetin attenuated MPPþ-induced cytotoxicity in MES23.5 cells The cell viability was unchanged when treated with myricetin up to 50 mmol/L. However, a significant reduction of cell viability was observed when cells when treated with above 50 mmol/L myricetin (data not shown). Therefore, a concentration of lower than 50 mmol/L of myricetin was chosen to do the experiment. As shown in Fig. 1, the cell viability reduced significantly by 64% when treated with 200 mmol/L MPPþ. Co-treated with 109 mol/L and 108 mol/L myriceitn could significantly increase the cell viability compared to MPPþ treatment.
3.2. Myricetin attenuated MPPþ-induced cell apoptosis indicated by Hoechst 33258 Morphological changes of the cells were further observed by Hoechst 33258 staining. As shown in Fig. 2, 200 mmol/L MPPþ treatment caused nuclear condensation in MES23.5 cells, 109 mol/ L myricetin treatment significantly attenuated this effect. 3.3. Myricetin prevented the 6Jm reduction in the MPPþ-treated MES23.5 cells Mitochondrial membrane potential is the marker of mitochondria function, which is involved in a variety of key events in oxidative stress and apoptosis. When treated with MPPþ, MES23.5
Fig. 3. Measurement of mitochondrial transmembrane potential (6Jm) by flow cytometry in MES23.5 cells. 200 mmol/L MPPþ treatment decreased the 6Jm compared with that of control, 109 mol/L myricetin treatment prevented against the 6Jm reduction induced by MPPþ. (A) Representatives of the fluorometric assay on 6Jm in different groups. (B) Statistical analysis. Data were presented as mean SEM of 6 independent experiments. Fluorescence values of the control were set to 100%. (#P < 0.05, compared with the control, *P < 0.05, compared with MPPþ treatment group).
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Fig. 4. Measurement of ROS production by flow cytometry in MES23.5 cells. 200 mmol/L MPPþ treatment increased the production of ROS compared with that of control, 109 mol/L myricetin treatment protected against MPPþ-induced increase of ROS production. (A) Representatives of the fluorometric assay on ROS production in different groups. (B) Statistical analysis. Data were presented as mean SEM of 5 independent experiments. Fluorescence values of the control were set to 100%. (##P < 0.01, compared with the control; *P < 0.05, compared with MPPþ treatment group).
cells showed a markedly decrease of 6Jm, this effect was partially reversed by myricetin (Fig. 3). This suggested that myricetin protect against MPPþ-induced cytotoxicity by restoring the mitochondria function. 3.4. Myricetin inhibited ROS production in the MPPþ-treated MES23.5 cells ROS played an important role in cell apoptosis, the changes of
6Jm were considered to be involved in ROS production. We
further measured ROS production using fluorescent dye H2DCFDA. As shown in Fig. 4, 200 mmol/L MPPþ treatment increased the production of ROS compared with that of control. Myricetin could markedly inhibit ROS production in the MPPþ-treated MES23.5 cells. 3.5. Myricetin inhibited caspase-3 activity in the MPPþ-treated MES23.5 cells Caspase-3 is a key protein in the process of apoptosis. We further observed the caspase-3 activity in different treatment groups. Consistent with the above observation, increased caspase-3
activity was observed after treated with MPPþ. This effect was inhibited by treated with 109 mol/L myricetin (Fig. 5). 3.6. Myricetin inhibited the phosphorylation of MKK4 and JNK in the MPPþ-treated MES23.5 cells The mechanisms of MPPþ-induced cell damage were also tested by measuring the MKK4 and JNK activity using anti-pMKK4 and anti-pJNK monoclonal antibody. As shown in Fig. 6, 200 mmol/L MPPþ treatment significantly increased the phosphorylated MKK4 and JNK protein expressions. These effects were partially blocked by 109 mol/L myricetin. Treated by 109 mol/L myricetin alone has no effect on the activity of MKK4 and JNK. 3.7. Myricetin increased the ratio of Bcl-2/Bax in the MPPþ-treated MES23.5 cells The Bcl-2 family is associated with mitochondrial function during apoptosis. Bcl-2, as an anti-apoptotic member of the Bcl-2 family, can bind Bax to form Bcl-2/Bax heterodimers, thereby attenuating the pro-apoptotic effect of Bax (Oltvai et al., 1993) In the present study, MPPþ resulted in a markedly down-regulation of
Fig. 5. Measurement of caspase-3 activity by flow cytometry in MES23.5 cells. 200 mmol/L MPPþ increased the activity of caspase-3, this effect was inhibited by treated with 10s9 mol/L myricetin. (A) Representatives of the fluorometric assay on the activity of caspase-3 in different groups. (B) Statistical analysis. Data were presented as mean SEM of 4 independent experiments. Fluorescence values of the control were set to 100%. (##P < 0.01, compared with the control; *P < 0.05, compared with MPPþ treatment group).
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Fig. 6. The phosphorylated MKK4 and JNK protein levels in MES23.5 cells. (A) Myricetin inhibited the phosphorylation of MKK4 in the MPPþ-treated MES23.5 cells. Increased protein expression of phospharylated MKK4 was observed in MPPþ-treated MES23.5 cells. The up-regulation of phospharylated MKK4 protein was inhibited by myricetin. Data were presented as mean SEM of 3 independent experiments. (B) Myricetin inhibits the phosphorylation of JNK in the MPPþ-treated MES23.5 cells. Increased protein expression of phospharylated JNK was observed in MPPþ-treated MES23.5 cells. Myricetin treatment prevents the up-regulation of phospharylated JNK protein. Data were presented as mean SEM of 3 independent experiments. b-actin was used as a loading control. (##P < 0.01, compared with the control; **P < 0.01,*P < 0.05, compared with MPPþ treatment group).
Bcl-2 mRNA and up-regulation of Bax mRNA in MES23.5 cells. Cotreatment with 109 mol/L myricetin could inhibit the decrease of Bcl-2 and increase of Bax caused by MPPþ. Therefore, the Bcl-2/Bax ratio in the MPPþ-treated cells significantly increased by myricetin (Fig. 7). 4. Discussion In the present study, we demonstrated that myricetin effectively protected MES23.5 cells from MPPþ-induced cell death. The MES23.5 cells were chosen because it exhibits several properties similar to the primary neurons originated in the SN (Crawford et al., 1992). Therefore, the results from this cell line will give direct evident correlation to the degenerated dopamine neurons in PD. MPPþ is a common neurotoxin used as a chemical for inducing PD models. In this study, MPPþ injured the mitochondria function and increased ROS generation. When treated with myricetin, a fall in 6Jm was blocked and increased ROS level was decreased. These may attribute to the anti-oxidation and free radical scavenging properties of myricetin (Hanasaki et al., 1994). It is well known that MPPþ could enter the cell through the dopamine reuptake system, and then inhibit complex I of the mitochondrial respiratory chain, and may induce oxidative stress (Cassarino et al., 1999; Desai et al.,
1996). Oxidative stress plays an important role in PD (Andersen, 2004; Jenner, 2003; Kidd, 2000). The dopamine-rich areas of the brain are particularly vulnerable to oxidative stress, because metabolism of dopamine itself leads to the generation of ROS, including hydrogen peroxide and hydroxyl radicals (Lotharius and Brundin, 2002). Mitochondrion is not only the main target of ROS, but also the major site of ROS production as well as primary target of oxidative molecular damage. The damaged mitochondrial membrane is implicated as key events in the pathogenic cascades; it breaks the balance of Bcl/Bax system and leads to cells death. Our study also showed that myricetin could partially reverse the MPPþinduced dysfunction of Bcl/Bax system and then inhibit the activation of Caspase-3. These observations also highlighted that myricetin could protect the cells by anti-oxidation and mitochondria protection. We further observed that the phosphorylated MKK4 and JNK protein levels markedly decreased after myricetin treatment compared with the MPPþ treatment group. It has been reported that activation of the JNK pathway plays a critical role in many models of neuronal degeneration. Inhibition of MKK4 and JNK activation could protect dopamine neurons in the MPTP or MPPþ models of PD (Herdegen and Waetzig, 2001; Xia et al., 2001). Although the exact protective mechanisms of myricetin on
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Fig. 7. Changes of Bcl-2 and Bax mRNA level in MES23.5 cells. Myricetin treatment inhibited MPPþ-induced Bcl-2 mRNA level down-regulation (A), Bax mRNA level up-regulation (B) and down-regulation of Bcl-2/Bax ratio (C). Bcl-2 and Bax band intensities were normalized with GAPDH band intensity. Each value represented the means SEM. (##P < 0.01, # P < 0.05, compared with the control; *P < 0.05, compared with MPPþ treatment group; n ¼ 3).
dopamine neuron degeneration are still unclear, we hypothesize that the inhibitory effect of myricetin on MKK4 and JNK activation may play an important role. It has been shown that myricetin may bind MKK4 directly by competing with ATP and then trigger chemopreventive effects against TNF-a-related disease by inhibiting the MKK4 and downstream JNK activation (Kim et al., 2009). It seems likely that the protective action of myricetin on the toxicity
myricetin
MPP+
ROS
of MPPþ may partly be attributed to the myricetin-suppressed MKK4 activity. The simplified depiction of effects of myricetin on MPPþinduced cytotoxitity was summarized by Fig. 8. The in vitro neuroprotective action of myricetin may be mediated by a reduction of oxidative stress and an inhibition of MKK4 activation. However, in an in vivo situation, where myricetin is injested orally, it can suffer biotransformation altering its structure. Further studies should be carried out to test the in vivo protective action and try to find the promising metabolite of myricetin to treat PD. These studies may lead to new development of promising drugs for prevention and treatment of PD. Acknowledgements
myricetin
MKK4 promotion effect
JNK
Mitochondrial dysfunction
inhibition effect
Bcl/Bax system
We thank Dr. Wei-dong Le for giving us the MES23.5 cell line. This work was supported by grants from National Basic Research Program of China (2011CB504102), Natural Science Foundation of China, (30800353) and the Bureau of Science and Technology of Qingdao (09-1-1-29-nsh).
c-Jun
References
Cytc Caspase-9 Caspase-3
Cells death
Fig. 8. Simplified depiction of the effects of myricetin on MPPþ-induced cytotoxicity.
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Myricetin attenuated MPPþ-induced cytotoxicity by anti-oxidation and inhibition of MKK4 and JNK activation in MES23.5 cells Kai Zhang, ZeGang Ma, Jun Wang, AnMu Xie, JunXia Xie* Department of Physiology, Shandong Provincial Key Laboratory of Pathogenesis and Prevention of Neurological Disorders and State Key Disciplines: Physiology, Medical College of Qingdao University, Qingdao 266071, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 December 2010 Received in revised form 6 April 2011 Accepted 12 April 2011
Increasing evidence suggests that oxidative stress may be implicated in the degeneration of dopaminergic neurons in Parkinson’s disease (PD), and anti-oxidation have been shown to be effective to PD treatment. Myricetin has been reported to have the biological functions of anti-oxidation, anti-apoptosis, anti-inflammation and iron-chelation. The aim of the present study is to investigate the neuroprotective effect of myricetin on 1-methyl-4-phenylpyridinium (MPPþ)-treated MES23.5 cells and the underlying mechanisms. The results showed that myricetin treatment significantly attenuated MPPþ-induced cell loss and nuclear condensation. Further experiments demonstrated that myricetin could suppress the production of intracellular reactive oxygen species (ROS), restore the mitochondrial transmembrane potential (6Jm), increase Bcl-2/Bax ratio and decrease caspase-3 activation that induced by MPPþ. Futhermore, we also showed myricetin decreased the phosphorylation of mitogen-activated protein kinase (MAPK) kinase 4 (MKK4) and c-Jun N-terminal kinase (JNK) caused by MPPþ. These results suggest that myricetin protected the MPPþ-treated MES23.5 cells by anti-oxidation and inhibition of MKK4 and JNK activation. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Myricetin MPPþ Oxidative stress MKK4
1. Introduction Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by selectively loss of dopaminergic neurons in the substantia nigra (SN), giving rise to dopamine (DA) depletion in the striatum. Although the precise pathogenic mechanism leading to neurodegeneration in PD is not known, a number of factors have been implicated in the pathogenesis of DA neuron loss. The generation of reactive oxygen species (ROS), mitochondrial dysfunction (Anantharam et al., 2007; Gardoni and Di Luca, 2006; Linazasoro, 2002; Mandel et al., 2000; McNaught et al., 2002), excitotoxicity (Gardoni and Di Luca, 2006; Moldzio et al., 2006) and inflammation (Koprich et al., 2008; Thomas et al., 2008) are all considered important mediators of neuronal death in PD. It is more
Abbreviations: MPTP, 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine; MPPþ, 1-methyl-4-phenylpyridinium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide; JNK, c-Jun N-terminal kinase; DA, dopamine; DMEM/F12, Eagle’s medium Nutrient Mixture-F12; 6J, mitochondrial transmembrane potential; MKK4, mitogen-activated protein kinase kinase 4; PD, Parkinson’s disease; ROS, reactive oxygen species; RT-PCR, Reverse transcription-polymerase chain reaction; SN, substantia nigra. * Corresponding author. Tel.: þ86 532 85955891; fax: þ86 532 83780136. E-mail address: [email protected] (JunXia Xie). 0028-3908/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2011.04.021
likely that multiple factors converge to give rise to PD than any single reason. The approaches to prevent dopamine neuron degeneration reflect the current notion on the pathogenesis of PD. Drugs directed against a single target will be ineffective and a single drug with multiple pharmacological properties maybe more suitable (Grunblatt et al., 2004; Van der Schyf et al., 2006; Youdim and Buccafusco, 2005), in which flavonoids are considered to be the most promising candidate because of their well-reported biological properties. Myricetin is one of such flavonoids. It has many biological functions, including anti-oxidation (Kang et al., 2010), anti-apoptosis (Shimmyo et al., 2008), free radical scavenging and antiinflammation (Lee and Choi, 2008; Hladovec, 1977). Recently, several studies demonstrated the neuroprotective effects of myricetin. It has been shown that myricetin protect against rotenoneinduced cytotoxicity in SHSY5Y cells (Molina-Jimenez et al., 2003), Myricetin also inhibits the formation and extension of amyloidb protein for the prevention of Alzheimer’s disease (Ono et al., 2003). Myricetin also inhibits 6-hydroxydopamine-induced neurotoxicity in rats (Ma et al., 2007). However, the underlying mechanisms of myricetin-mediated neuroprotection are uncertain. 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) is a common neurotoxin for inducing PD models. Chronic administration of MPTP induced apoptosis was found in the SN of mice.
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Activation of the mitogen-activated protein kinase (MAPK) kinase 4 (MKK4) and c-Jun N-terminal kinase (JNK) pathway has been shown to play a critical role in MPTP or 1-methyl-4-phenylpyridinium (MPPþ) induced PD models (Xia et al., 2001). In the present study, we aim to elucidate whether myricetin could protect MPPþ-induced neurotoxicity and the underlying mechanisms in MES23.5 cells. 2. Materials and methods
2.7. Measurement of caspase-3 activation The active caspase-3 was measured by PE-conjugated monoclonal active caspase-3 antibody apoptosis kit. Briefly, cells were seeded on 6-well plates and treated as described above. After washing twice with cold PBS, cells were resuspended in Citofix/CytopermTM solution at a concentration of 1 106 cells/ 0.5 ml and incubated for 20 min. The cells were then washed with Perm/Washing buffer twice and incubated in Perm/Washing buffer with antibody (1:5) for 30 min. After washing once with Perm/Washing buffer, the cells were re-suspended with 0.5 mL Perm/Washing buffer and analyzed by flow cytometry. The extent of apoptosis was determined by counting the number of active caspase-3 immunoreactive cells as a percentage of total MES23.5 cells using Cellquest Software.
2.1. Materials Unless otherwise stated, all chemicals including myricetin were purchased from Sigma Chemical Co. The purity of myricetin used is 95%. Dulbecco’s modified Eagle’s medium Nutrient Mixture-F12 (DMEM/F12) was from Invitrogen.
2.2. Cell culture MES23.5 cells were cultured in DMEM/F12 containing Sato’s components growth medium supplemented with 5% fetal bovine serum, 100 U/mL of penicillin and 100 mg/mL of streptomycin at 37 C, in a humid 5% CO2, 95% air environment. For experiments, cells were seeded at a density of 1 105/cm2 in the plastic flasks.
2.3. Cell viability assay MES23.5 cells were seeded in a 96-well plate at a density of 2 104 cells per well. Cell viability was measured by the conventional 3-(4,5-dimethylthiazol-2-yl)2,5- diphenyltetrazolium bromide (MTT) assay. The cells were divided into vehicle group, MPPþ treatment group, MPPþ and myricetin co-treatment group. After incubation with the chemicals for 24 h respectively, the cells were incubated in MTT (5 mg/mL) for 4 h and cell viability was measured at 570 nm by colorimetric assay (Rayto RT-2100C, Shenzhen, China).
2.4. Hoechst 33258 staining Nuclear morphology was detected using the method previously described in our lab (Xu et al., 2008; Zhang et al., 2009). MES23.5 cells were seeded on sterile cover glasses in 24-well plates and treated with vehicle, MPPþ, MPPþ and myricetin for 24 h respectively. The cells were fixed in 4% paraformaldehyde for 30 min, washed in phosphate-buffered saline, and stained with Hoechst 33258 dye for 30 min at room temperature. After washing 3 times to remove the excessive dye, the cells were examined and photographed under a fluorescence microscope (Olympus, Japan) with an excitation wavelength of 330e380 nm. Apoptotic cells were defined on the basis of nuclear morphological changes, such as chromatin condensation and fragmentation. The total number of condensed cells was counted manually by researchers blinded to the treatment schedule using unbiased stereology. For each well, we delineated a 400 mm2 frame and counted all condensed and normal nuclei in at least 10 different fields. Average sum of condensed and normal nuclei was calculated per well. The data were presented as the percentage of condensed nuclear number to the total number.
2.8. Western blots analysis MES23.5 cells were incubated with vehicle, MPPþ, myricetin, MPPþ and myricetin for 5 min respectively, then, cells were lysed in a buffer containing 50 mM TriseHCl (pH 7.4), 1 mM EDTA, 150 mM NaCl, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, and 10 mg/mL aprotinin. Cell lysates were separated by 8% sodium dodecyl sulfate polyacrylamide gel and then transferred to polyvinylidene difluoride membrane. Blots were probed with anti-pMKK4 and anti-pJNK monoclonal antibody (Cell Signaling, 1:1000). Blots were also probed with antiebeactin monoclonal antibody (Sigma, 1:10,000) as a loading control. Results were analyzed through scanning densitometry by Tanon Image System.
2.9. Reverse transcription-polymerase chain reaction (RT-PCR) MES23.5 cells were incubated with vehicle, MPPþ, myricetin, MPPþ and myricetin for 24 h respectively, Total RNA was isolated by Trizol Reagent (Invitrogen). 5 mg of total RNA was reversed transcribed in a 20 ml reaction using the AMV reverse transcription system (Promega Corporation, Madison, WI, USA). We amplified Bcl-2 cDNA fragment (amplified products were 409 bp length) with the primers (forward: 50 - GTC CCG CCT CTT CAC CTT -30 ; reverse: 50 - CCC ACT CGT AGC CCC TCT -30 ), Bax cDNA fragment (amplified products were 307 bp length) with the primers (forward: 50 - GGC GAA TTG GAG ATG AAC -30 ; reverse: 50 - CCG AAG TAG GAG AGG AGG -30 ) and GAPDH cDNA fragment (amplified products were 236 bp length) with the primers (forward: 50 - TTC ACC ACC ATG GAG AAG GC -30 ; reverse 50 - GGC ATG GAC TGT GGT CAT GA -30 ). The expression of house-keeping gene, GAPDH mRNA, was used as an internal standard. PCRs were run for 36 cycles in an Eppendorf Mastercycler. Denaturing, annealing, and extension reactions were performed at 94 C for 30 s, 51 C for 30 s, and 72 C for 45 s. The PCR products were electrophoresed on 1% agarose gel, stained with ethidium bromide, and detected by UV irradiation. The levels of Bcl-2 and Bax mRNA were expressed as their respective ratios to GAPDH mRNA.
2.10. Statistical analysis Data were expressed as mean S.E.M. and analyzed using the SPSS11.5 software. One-way analysis of variance (ANOVA) followed by Tukey test was used to compare the differences between means. A probability value of less than 0.05 was considered to be statistically significant.
2.5. Reactive oxygen species (ROS) assay Intracellular ROS in MES23.5 cells were measured by carboxy-H2DCFDA as described before (Sanelli et al., 2007; Zhang et al., 2009; Zhu and Liu, 2004). The fluorescence coming from carboxy-H2DCFDA reflects the intracellular ROS generation. The cells treated as described above were incubated in HEPES-buffered saline (HBS) containing carboxy-H2DCFDA in a final concentration of 5 mmol/L for 30 min at 37 C and followed by washing twice with HBS. Fluorescent intensity was recorded at 488 nm excitation and 525 nm emission wavelengths. Results were demonstrated as FL1-H (Fluorescence 1-Histogram), setting of the gated region M1 and M2 as a marker to observe the changing levels of fluorescence intensity by Cellquest Software. 2.6. Detection of mitochondrial transmembrane potential (6Jm) The changes of mitochondrial membrane potential were measured by rhodamine123 using flow cytometry (Becton Dickinson, USA) as described before (Sanelli et al., 2007; Zhang et al., 2009; Zhu and Liu, 2004). The uptake of rhodamine123 into mitochondria is an indicator of the 6Jm. After 24 h treatment, the cells were incubated with rhodamine123 in a final concentration of 5 mmol/L for 30 min at 37 C. The fluorescent intensity was recorded at 488 nm excitation and 525 nm emission wavelengths. Results were demonstrated as described in ROS assay.
Fig. 1. MTT analysis of cell viability in MES23.5 cells. 200 mmol/L MPPþ decreased the cell viability compared with that of control group, this effect was partly inhibited by treated with myricetin. (##P < 0.01, compared with the control; *P < 0.05, compared with MPPþ treatment group; n ¼ 4).
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Fig. 2. Morphological changes in MES23.5 cells. (A). Representative photographs of Hoechst 33258 staining from vehicle, MPPþ, MPPþ and myricetin co-treatment groups. MPPþ treatment resulted in nuclear condensation; however, 109 mol/L myricetin treatment significantly attenuated MPPþ-induced nuclear condensation (Magnification 400). (B). Statistical analysis. Data were presented as means SEM of 6 independent experiments. (###P < 0.001, compared with the control; *P < 0.05, compared with MPPþ treatment group).
3. Results 3.1. Myricetin attenuated MPPþ-induced cytotoxicity in MES23.5 cells The cell viability was unchanged when treated with myricetin up to 50 mmol/L. However, a significant reduction of cell viability was observed when cells when treated with above 50 mmol/L myricetin (data not shown). Therefore, a concentration of lower than 50 mmol/L of myricetin was chosen to do the experiment. As shown in Fig. 1, the cell viability reduced significantly by 64% when treated with 200 mmol/L MPPþ. Co-treated with 109 mol/L and 108 mol/L myriceitn could significantly increase the cell viability compared to MPPþ treatment.
3.2. Myricetin attenuated MPPþ-induced cell apoptosis indicated by Hoechst 33258 Morphological changes of the cells were further observed by Hoechst 33258 staining. As shown in Fig. 2, 200 mmol/L MPPþ treatment caused nuclear condensation in MES23.5 cells, 109 mol/ L myricetin treatment significantly attenuated this effect. 3.3. Myricetin prevented the 6Jm reduction in the MPPþ-treated MES23.5 cells Mitochondrial membrane potential is the marker of mitochondria function, which is involved in a variety of key events in oxidative stress and apoptosis. When treated with MPPþ, MES23.5
Fig. 3. Measurement of mitochondrial transmembrane potential (6Jm) by flow cytometry in MES23.5 cells. 200 mmol/L MPPþ treatment decreased the 6Jm compared with that of control, 109 mol/L myricetin treatment prevented against the 6Jm reduction induced by MPPþ. (A) Representatives of the fluorometric assay on 6Jm in different groups. (B) Statistical analysis. Data were presented as mean SEM of 6 independent experiments. Fluorescence values of the control were set to 100%. (#P < 0.05, compared with the control, *P < 0.05, compared with MPPþ treatment group).
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Fig. 4. Measurement of ROS production by flow cytometry in MES23.5 cells. 200 mmol/L MPPþ treatment increased the production of ROS compared with that of control, 109 mol/L myricetin treatment protected against MPPþ-induced increase of ROS production. (A) Representatives of the fluorometric assay on ROS production in different groups. (B) Statistical analysis. Data were presented as mean SEM of 5 independent experiments. Fluorescence values of the control were set to 100%. (##P < 0.01, compared with the control; *P < 0.05, compared with MPPþ treatment group).
cells showed a markedly decrease of 6Jm, this effect was partially reversed by myricetin (Fig. 3). This suggested that myricetin protect against MPPþ-induced cytotoxicity by restoring the mitochondria function. 3.4. Myricetin inhibited ROS production in the MPPþ-treated MES23.5 cells ROS played an important role in cell apoptosis, the changes of
6Jm were considered to be involved in ROS production. We
further measured ROS production using fluorescent dye H2DCFDA. As shown in Fig. 4, 200 mmol/L MPPþ treatment increased the production of ROS compared with that of control. Myricetin could markedly inhibit ROS production in the MPPþ-treated MES23.5 cells. 3.5. Myricetin inhibited caspase-3 activity in the MPPþ-treated MES23.5 cells Caspase-3 is a key protein in the process of apoptosis. We further observed the caspase-3 activity in different treatment groups. Consistent with the above observation, increased caspase-3
activity was observed after treated with MPPþ. This effect was inhibited by treated with 109 mol/L myricetin (Fig. 5). 3.6. Myricetin inhibited the phosphorylation of MKK4 and JNK in the MPPþ-treated MES23.5 cells The mechanisms of MPPþ-induced cell damage were also tested by measuring the MKK4 and JNK activity using anti-pMKK4 and anti-pJNK monoclonal antibody. As shown in Fig. 6, 200 mmol/L MPPþ treatment significantly increased the phosphorylated MKK4 and JNK protein expressions. These effects were partially blocked by 109 mol/L myricetin. Treated by 109 mol/L myricetin alone has no effect on the activity of MKK4 and JNK. 3.7. Myricetin increased the ratio of Bcl-2/Bax in the MPPþ-treated MES23.5 cells The Bcl-2 family is associated with mitochondrial function during apoptosis. Bcl-2, as an anti-apoptotic member of the Bcl-2 family, can bind Bax to form Bcl-2/Bax heterodimers, thereby attenuating the pro-apoptotic effect of Bax (Oltvai et al., 1993) In the present study, MPPþ resulted in a markedly down-regulation of
Fig. 5. Measurement of caspase-3 activity by flow cytometry in MES23.5 cells. 200 mmol/L MPPþ increased the activity of caspase-3, this effect was inhibited by treated with 10s9 mol/L myricetin. (A) Representatives of the fluorometric assay on the activity of caspase-3 in different groups. (B) Statistical analysis. Data were presented as mean SEM of 4 independent experiments. Fluorescence values of the control were set to 100%. (##P < 0.01, compared with the control; *P < 0.05, compared with MPPþ treatment group).
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Fig. 6. The phosphorylated MKK4 and JNK protein levels in MES23.5 cells. (A) Myricetin inhibited the phosphorylation of MKK4 in the MPPþ-treated MES23.5 cells. Increased protein expression of phospharylated MKK4 was observed in MPPþ-treated MES23.5 cells. The up-regulation of phospharylated MKK4 protein was inhibited by myricetin. Data were presented as mean SEM of 3 independent experiments. (B) Myricetin inhibits the phosphorylation of JNK in the MPPþ-treated MES23.5 cells. Increased protein expression of phospharylated JNK was observed in MPPþ-treated MES23.5 cells. Myricetin treatment prevents the up-regulation of phospharylated JNK protein. Data were presented as mean SEM of 3 independent experiments. b-actin was used as a loading control. (##P < 0.01, compared with the control; **P < 0.01,*P < 0.05, compared with MPPþ treatment group).
Bcl-2 mRNA and up-regulation of Bax mRNA in MES23.5 cells. Cotreatment with 109 mol/L myricetin could inhibit the decrease of Bcl-2 and increase of Bax caused by MPPþ. Therefore, the Bcl-2/Bax ratio in the MPPþ-treated cells significantly increased by myricetin (Fig. 7). 4. Discussion In the present study, we demonstrated that myricetin effectively protected MES23.5 cells from MPPþ-induced cell death. The MES23.5 cells were chosen because it exhibits several properties similar to the primary neurons originated in the SN (Crawford et al., 1992). Therefore, the results from this cell line will give direct evident correlation to the degenerated dopamine neurons in PD. MPPþ is a common neurotoxin used as a chemical for inducing PD models. In this study, MPPþ injured the mitochondria function and increased ROS generation. When treated with myricetin, a fall in 6Jm was blocked and increased ROS level was decreased. These may attribute to the anti-oxidation and free radical scavenging properties of myricetin (Hanasaki et al., 1994). It is well known that MPPþ could enter the cell through the dopamine reuptake system, and then inhibit complex I of the mitochondrial respiratory chain, and may induce oxidative stress (Cassarino et al., 1999; Desai et al.,
1996). Oxidative stress plays an important role in PD (Andersen, 2004; Jenner, 2003; Kidd, 2000). The dopamine-rich areas of the brain are particularly vulnerable to oxidative stress, because metabolism of dopamine itself leads to the generation of ROS, including hydrogen peroxide and hydroxyl radicals (Lotharius and Brundin, 2002). Mitochondrion is not only the main target of ROS, but also the major site of ROS production as well as primary target of oxidative molecular damage. The damaged mitochondrial membrane is implicated as key events in the pathogenic cascades; it breaks the balance of Bcl/Bax system and leads to cells death. Our study also showed that myricetin could partially reverse the MPPþinduced dysfunction of Bcl/Bax system and then inhibit the activation of Caspase-3. These observations also highlighted that myricetin could protect the cells by anti-oxidation and mitochondria protection. We further observed that the phosphorylated MKK4 and JNK protein levels markedly decreased after myricetin treatment compared with the MPPþ treatment group. It has been reported that activation of the JNK pathway plays a critical role in many models of neuronal degeneration. Inhibition of MKK4 and JNK activation could protect dopamine neurons in the MPTP or MPPþ models of PD (Herdegen and Waetzig, 2001; Xia et al., 2001). Although the exact protective mechanisms of myricetin on
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Fig. 7. Changes of Bcl-2 and Bax mRNA level in MES23.5 cells. Myricetin treatment inhibited MPPþ-induced Bcl-2 mRNA level down-regulation (A), Bax mRNA level up-regulation (B) and down-regulation of Bcl-2/Bax ratio (C). Bcl-2 and Bax band intensities were normalized with GAPDH band intensity. Each value represented the means SEM. (##P < 0.01, # P < 0.05, compared with the control; *P < 0.05, compared with MPPþ treatment group; n ¼ 3).
dopamine neuron degeneration are still unclear, we hypothesize that the inhibitory effect of myricetin on MKK4 and JNK activation may play an important role. It has been shown that myricetin may bind MKK4 directly by competing with ATP and then trigger chemopreventive effects against TNF-a-related disease by inhibiting the MKK4 and downstream JNK activation (Kim et al., 2009). It seems likely that the protective action of myricetin on the toxicity
myricetin
MPP+
ROS
of MPPþ may partly be attributed to the myricetin-suppressed MKK4 activity. The simplified depiction of effects of myricetin on MPPþinduced cytotoxitity was summarized by Fig. 8. The in vitro neuroprotective action of myricetin may be mediated by a reduction of oxidative stress and an inhibition of MKK4 activation. However, in an in vivo situation, where myricetin is injested orally, it can suffer biotransformation altering its structure. Further studies should be carried out to test the in vivo protective action and try to find the promising metabolite of myricetin to treat PD. These studies may lead to new development of promising drugs for prevention and treatment of PD. Acknowledgements
myricetin
MKK4 promotion effect
JNK
Mitochondrial dysfunction
inhibition effect
Bcl/Bax system
We thank Dr. Wei-dong Le for giving us the MES23.5 cell line. This work was supported by grants from National Basic Research Program of China (2011CB504102), Natural Science Foundation of China, (30800353) and the Bureau of Science and Technology of Qingdao (09-1-1-29-nsh).
c-Jun
References
Cytc Caspase-9 Caspase-3
Cells death
Fig. 8. Simplified depiction of the effects of myricetin on MPPþ-induced cytotoxicity.
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