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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
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Neuroprotective effect of lycopene against MPTP induced experimental Parkinson’s disease in mice
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Asokan Prema, Udaiyappan Janakiraman, Thamilarasan Manivasagam ∗ , Arokiasamy Justin Thenmozhi
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Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar 608002, Tamilnadu, India
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Lycopene ameliorates MPTP induced behavioral deficts. Lycopene reverses MPTP mediated neurochemical depletion. Lycopene attenuates MPTP induced oxidative stress. Lycopene reduces apoptosis in PD mice.
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Article history: Received 27 March 2015 Received in revised form 10 May 2015 Accepted 13 May 2015 Available online xxx
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Keywords: Experimental Parkinson’s disease Lycopene Oxidative stress Apoptosis Behavior
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1. Introduction
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Parkinson’s disease (PD) is the second most common neurodegenerative disorder that mainly affects the movement of the aged populations. Lycopene is a carotenoid with unique pharmacological properties and its efficacy on experimental Hunginton’s disease and brain ischemia has shown intense neuroprotective effects. The present study was aimed to explore the neuroprotective effect of lycopene against 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced PD mice. Administration of lycopene (5, 10 and 20 mg/kg/day orally) protected MPTP induced depletion of striatal dopamine (DA) and its metabolites in a dose dependent manner. It also attenuated MPTP-induced oxidative stress and motor abnormalities seen in PD mice. Our western blot studies showed that treatment with lycopene reversed MPTP induced apoptosis may be due to its antioxidant and antiapoptotic properties. As to conclude, lycopene reverses neurochemical deficts, oxidative stress, apoptosis and physiological abnormalities in PD mice and offer promise strategy in the treatment of this neurodegenerative disease. © 2015 Published by Elsevier Ireland Ltd.
Parkinson’s disease (PD) is a devastating neurodegenerative disorder characterized by degeneration of dopaminergic neurons in the substantia nigra (SN) and diminution of dopamine (DA) concentration in striatum (ST) that leads to severe motor disabilities [21]. MPTP is considered as the gold standard for toxin based PD models because, it can easily cross the blood brain barrier and where it is taken by glial cells and metabolized to form 1-methyl-4-phenyl pyridinium (MPP+ ) by monoamine oxidase-B [10]. Released MPP+ is selectively taken up into dopaminergic neurons by dopamine transporters (DAT) and causing mitochondrial dysfunction and oxidative stress [25] which finally induces apoptosis [7].
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∗ Corresponding author. Tel.: +91 8940505861. E-mail address: mani [email protected] (T. Manivasagam).
Now interest has been focused on lycopene, an aliphatic hydrocarbon carotenoid, present in the ripened tomatoes as a potential useful agent in the management of neurodegenerative disorders because of its ability to cross the blood–brain barrier [14] and strong antioxidant property [22]. About 10–30% of dietary lycopene is absorbed by humans and is distributed in various tissues including adrenal gland, liver, prostate gland, brain and testes. Though lycopene undergoes oxidative metabolism and forms several oxidized forms and polar metabolites, their biological significance is not yet elucidated [24]. Lycopene protects cultured hippocampal neurons against Aˇ and glutamate toxicity [18], microglial activation in focal cerebral ischemia in rats [11] and attenuated cognition impairment in elderly population [1]. In addition, lycopene has been shown to mediate its neuroprotective effect in 3-nitropropionic acid induced Huntington’s disease [15] and rotenone induced PD [16]. In the present study, we investigated the neuroprotective effect of lycopene on MPTP-induced mouse model of PD by analysing the
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Please cite this article in press as: A. Prema, et al., Neuroprotective effect of lycopene against MPTP induced experimental Parkinson’s disease in mice, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.05.024
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protein expressions of pro-apoptotic (Bax cytochrome-c, caspases 3, 8 and 9) and anti-apoptotic (Bcl-2) markers.
supernatant was boiled for 10 min. After cooling, the samples were read at 535 nm [23].
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2. Materials and methods
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2.1. Animals
2.3.6. Assay of SOD The assay mixture consisted of 960 l of sodium carbonate buffer containing 0.1 mM xanthine, 0.025 mM NBT, and 0.1 mM EDTA, 20 l of xanthine oxidase and 20 l of the brain supernatant were taken. Changes in absorbance were observed spectrophotometrically at 560 nm [23].
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Male C57bL/6 mice (25–30 g) were purchased from the National Institute of Nutrition, Hyderabad and acclimatized to laboratory conditions prior to experimentation. The animals were kept under standard conditions with food and water ad libitum. The protocol was approved by the Institutional Animal Ethics Committee (approval no: 890/2012) and carried out in accordance with the Indian National Science Academy guidelines for the use and care of animals.
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MPTP, lycopene, thiobarbituric acid (TBA), reduced glutathione and 3,5-dithio-bisnitrobenzoic acid (DTNB) were purchased from Sigma–Aldrich, Bangalore, India. Anti caspase-3,-8, -9, cytochromec (cyto-c), bax and bcl-2 (Santa Cruz Biotechnology Inc.), anti-actin (cell signalling) primary antibodies produced from rabbits and anti rabbit HRP conjugated secondary antibody (Sigma chemical, USA) were used in the present study.
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2.3.1. Phase I Mice were randomized and divided into six groups (n = 6). Group I animals were treated with saline; group II mice were injected intraperitoneally with MPTP (30 mg/kg body weight) [23] once daily in saline for last four consecutive days (from 4th to 7th day); group III, IV and V animals were injected with MPTP as group II and administered with lycopene (5, 10 and 20 mg/kg/day orally for 7 days, respectively); group VI mice were treated with lycopene (20 mg/kg/day orally for 7 days) alone. Seven days after the last injection of MPTP, behavior tests such as narrow beam walking test and hang test were performed. Then, animals were killed by cervical dislocation and striatal and nigral tissues were procured rapidly and utilized for the estimation of DA and oxidative stress related indices.
2.3.7. Determination of activity of catalase The assay mixture consisted of 50 l of 1 M Tris–HCl buffer (pH 8.0) containing 5 mM EDTA, 900 l of 10 mM H2 O2 , 30 l of MQ water and 20 l of the brain tissue supernatant. The rate of decomposition of hydrogen peroxide was observed spectrophotometrically at 240 nm [26]. 2.3.8. Assay of GPx The GPx assay mixture consisted of 100 l of 1 M Tris–HCl (pH 8.0) containing 5 mM EDTA, 20 l of 0.1 M GSH, 100 l of GSH reductase solution (10 U/ml), 100 l of 2 mM NADPH, 650 l of distilled water, 10 l of 7 mM tert-butyl hydroperoxide and 10 l of the brain supernatant. Oxidation of NADPH was determined spectrophotometrically at 340 nm [23]. 2.3.9. Estimation of GSH Brain tissue homogenate was centrifuged at 16,000 g for 15 min at 4 ◦ C. The supernatant (0.5 ml) was added to 4 ml of ice-cold 0.1 mM solution of 5,5-dithiobis[2-nitrobenzoic acid] in 1 M phosphate buffer (pH 8). The optical density was read at 412 nm in a spectrophotometer [26]. 2.4. Phase II Based on DA levels, effective dose of lycopene was found to be 10 mg/kg bodyweight and used for further study involving the following groups – control, MPTP treated, MPTP and lycopene (10 mg/kg bodyweight) treated and lycopene alone treated (10 mg/kg bodyweight). Seven days after the last injection of MPTP, open field test (with effective dose) was performed. After performing behavioral test, animals were killed by cervical dislocation and striatal and nigral
2.3.2. Narrow beam test Pre-trained animals were allowed to walk on a narrow flat stationary wooden beam (length –100 cm × width – 1 cm) placed at a height of 100 cm from the floor. The time taken to cross the beam from one end to the other along with foot slip errors were measured [12]. 2.3.3. Hang test Mice were allowed to hold on a horizontal grid. Then, the grid was inverted so that the mice were allowed to hang upside down and hanging time was measured [20]. 2.3.4. Estimation of DA and its metabolites Immediately after the procurement, ST was sonicated in ice-cold 0.1 M HClO4 containing 0.01% EDTA and centrifuged at 10,000 g for 5 min. Supernatant was collected and injected (10 l) into the HPLC system and results were expressed in ng/mg wet weight [20]. 2.3.5. Estimation of TBARS Briefly, the nigral extracts were incubated with 0.2 ml of phenyl methosulfate at 37 ◦ C for 1 h of incubation and 0.4 ml of tricarboxylic acid and 0.4 ml of thiobarbituric acid were added. The reaction mixture was centrifuged at 4000 rpm for 15 min, and the
Fig. 1. Measurement of balance and muscular co-ordination by narrow beam walking test. MPTP treated mice took more time to cross the narrow beam with high number of foot slip errors, whereas preadministration of lycopene significantly attenuated motor deficts induced by MPTP. Results given are mean ± SD, (n = 6), values not sharing common superscript are significant with each other p < 0.05, one way ANOVA followed by DMRT.
Please cite this article in press as: A. Prema, et al., Neuroprotective effect of lycopene against MPTP induced experimental Parkinson’s disease in mice, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.05.024
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tissues were procured rapidly and stored in −80 ◦ C for further analysis. 2.4.1. Open field test Mice were placed in one corner of open field chamber, its movement and activities were observed for 5 min. When the animal enters a square with both of its forelimb, one count is made. The number of center and peripheral squares explored by the animal was noted. Activities of mice including grooming and rearing were also counted [2]. 2.4.2. Western blotting Nigral tissues were homogenized in an ice-cold RIPA buffer and were centrifuged at 12,000 rpm/min for 15 min at 4 ◦ C. Protein concentration was measured by the method of Lowry et al. [17]. Samples containing 50 g of total cellular protein were loaded and separated on 10% SDS–polyacrylamide gel electrophoresis. The gel was then transferred onto a PVDF membrane. The membranes were incubated with the blocking buffer containing BSA for 2 h and then incubated with primary antibodies of -actin (rabbit monoclonal; 1:500 dilution), cyto-c, caspases-3,-8, -9 (1:500), bcl2 and bax (1:1000) with gentle shaking overnight at 4 ◦ C. After this, membranes were incubated with their corresponding secondary antibodies for 2 h at room temperature and washed thrice. Protein bands were visualized by an enhanced chemiluminescence’s method and scanned bands were quantitated by Total lab Quant, the control was set to 100.
Fig. 2. Determination of neuromuscular strength by hang test. Hanging time of MPTP induced mice were drastically decreased, meanwhile, treatment with lycopene significantly increased hanging time of MPTP induced mice. Results given are mean ± SD, (n = 6), values not sharing common superscript are significant with each other p < 0.05, one way ANOVA followed by DMRT.
2.5. Statistical analysis All values were expressed as mean ± SD of the number of experiments (n = 6). Statistical analysis was carried out using one-way analysis of variance (ANOVA) using SPSS version 15.0 software
Fig. 3. Determination of acclimatization ability and motor performance in open field test. Administration of MPTP led to significant reduction in movement and activities; however, these reductions were prevented by administration of lycopene. Results given are mean ± SD, (n = 6), values not sharing common superscript are significant with each other p < 0.05, one way ANOVA followed by DMRT.
Please cite this article in press as: A. Prema, et al., Neuroprotective effect of lycopene against MPTP induced experimental Parkinson’s disease in mice, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.05.024
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Fig. 4. MPTP-induced depletion of dopamine and its metabolites can be conserved by lycopene treatment. Administration of MPTP depleted the levels of dopamine and its metabolites, which are attenuated dose-dependently by treatment of lycopene. Results given are mean ± SD, (n = 6), values not sharing common superscript are significant with each other p < 0.05, one way ANOVA followed by DMRT.
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and individual comparisons were obtained using Duncan’s multiple range test (DMRT).
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in MPTP induced mice. However, it was observed that both the 10 and 20 mg/kg b.w showed similar induction, but more significant than 5 mg/kg b.w. So we have chosen the 10 mg/kg b.w as the optimum dose for further studies.
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3.1. Behavioral assessment
3.3. Oxidative and antioxidative indices Oxidative stress is the main culprit that can induce neurodegeneration in PD. The levels of TBARS and activities of SOD and catalase were increased in the SN of MPTP-treated mice, whereas pre-treatment of lycopene significantly lowered their levels and activities as compared with MPTP injected mice. In addition, the levels of GSH and activities of GPx were drastically decreased MPTP-treated mice, whereas lycopene pretreatment significantly attenuated the levels of GSH and activities of GPx significantly (p < 0.05) (Fig. 5).
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Behavioral studies such as narrow beam walking test (to examine the balance and motor coordination skills), open field test (to measure the locomotion and activity) and hang test (to accesses the neuromuscular strength and coordination) were performed in control and experimental animals. MPTP treated mice exhibited more time to cross the wooden beam with increased foot slip errors in narrow beam walking test, more hanging time in hang test, reduced movement (peripheral and centre) and activities (grooming and rearing) in open field test as compared with the control animals (p < 0.05), whereas the lycopene pre-treatement attenuated the behavioral abnormalities seen in MPTP animals (Figs. 1–3).
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Apoptosis is reported mode of cell death in various neurodegenerative diseases including PD. The expression patterns of bcl-2-associated X, caspases-3, 8 and 9 were significantly increased in MPTP treated mice, whereas lycopene pretreatment significantly decreased the expression pattens of these proapoptotic markers as compared with MPTP alone treated animals. The expressions of bcl-
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Fig. 4 revealed a significant decline in the levels of striatal DA and its metabolites in the MPTP alone treated mice as compared with control mice, whereas pre-administration of lycopene dose dependently enhanced the levels of dopamine and its metabolites
3.4. Western blot analysis
Please cite this article in press as: A. Prema, et al., Neuroprotective effect of lycopene against MPTP induced experimental Parkinson’s disease in mice, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.05.024
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Fig. 5. MPTP induced oxidative stress can be attenuated by lycopene. The levels of TBARS and activities of SOD and catalase were increased along with a decreased levels of GSH and activities of GPx in the SN of MPTP-treated mice, meanwhile, treatment with lycopene significantly ameliorated oxidative stress. Results given are mean ± SD, (n = 6), values not sharing common superscript are significant with each other p < 0.05, one way ANOVA followed by DMRT. A Amount of enzyme required to inhibit 50% of NBT reduction. B nmol H2 O2 consumed/minute/mg protein. C nmol NADPH oxidized/minute/mg protein.
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2 and cyto c were significantly diminished in MPTP treated mice as compared with controls, whereas lycopene pre-treatment significantly enhanced the expressions of bcl-2 and cyto-c as compared with MPTP alone treated animals (Fig. 6). 4. Discussion DA is an important neurotransmitter responsible for balance and control of movements including walking. MPTP administration caused a selective destruction of dopaminergic neurons in nigrostriatal regions and leads to depletion in the levels of striatal DA.
Administration of tomato powder enriched in lycopene or tomato diet ameliorated MPTP [27] and 6-OHDA [6] induced DA depletion, which is concordant with the results of our study Fig. 7. Q4 Open field test, narrow beam walking test and hang test are used as indices to measure the motor impairments in MPTP-induced behavioral abnormalities in animal models [20]. MPTP induced behavior deficits are mainly due to pathological loss of dopaminergic neurons in midbrain with degeneration of their striatal nerve terminals. However, lycopene treatment caused a recovery of behavioral deficits induced by MPTP, by enhancing the levels of DA.
Please cite this article in press as: A. Prema, et al., Neuroprotective effect of lycopene against MPTP induced experimental Parkinson’s disease in mice, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.05.024
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Fig. 6. MPTP persuaded apoptosis can be amolierated by lycopene administration. Expressions of bax, cyto-c, caspase-3, -8 and -9, bcl-2 in the SN were analyzed by Western blot (A and B), with -actin used as the loading control of total proteins; lane 1: control, saline control, lane 2: MPTP, MPTP-treated only, lane 3: MPTP-lycopene, lycopene with MPTP, lane 4: lycopene, lycopene alone administrated. The band density was quantified by scanning densitometry (C and D). Results given are mean ± SD, (n = 3), values not sharing common superscript are significant with each other p < 0.05, one way ANOVA followed by DMRT.
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Inhibition of electron transport chain I by MPP+ , a toxic metabolite of MPTP, leads to the leaking of electrons, which may generate the production of ROS (5–7 times more) by reacting with excessively available O2 . Previous studies from our lab, clearly indicated that the administration of MPTP has been reported to enhance the levels of TBARS, a marker of lipid peroxidation processes and activities of SOD and catalase and diminished the levels of GSH and activity of GPx [26], which may reflect an adaptive response to oxidative stress. Lycopene is described as the most effective antioxidants in the carotenoid family and exhibits 10-fold, 47fold and 100-fold more effective in quenching singlet oxygen than ␣-tocopherol, -carotene and vitamin E, respectively [5,19]. Moreover its administration modulates positively the activities and levels of SOD, catalase, GPx and GSH in the striatum of 3nitropropionic acid treated HD rats [25] and regulating antioxidant
response element genes [3]. In the present study, pre-treatment of lycopene significantly ameliorated oxidative stress induced by MPTP due to its free radical scavenging and antioxidant activities. In the present study, MPTP treatment decreased the expression of bcl-2 and increased cyto-c release with increased expressions of bax, caspases-3, 8 and 9 [20], thereby favoring apoptosis. Fujita et al. [9] reported that the administration of lycopene inhibits ischemia/reperfusion-induced neuronal apoptosis in gerbil hippocampal tissue by enhancing bcl-2 and diminishing caspase 3 levels. The dosage of 10 mg/kg of the lycopene, which markedly exceeds the estimated daily intake of humans (0.5–27 mg/person/day), even no toxic effects were observed in rats that were orally administered with 2000 mg/kg/day [13]. Large numbers of studies indicated that the neuroprotective effect of lycopene attributed through amelioration of mitochon-
Please cite this article in press as: A. Prema, et al., Neuroprotective effect of lycopene against MPTP induced experimental Parkinson’s disease in mice, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.05.024
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MPTP Monaoamine oxidase B in astrocytes
MPP+, a toxic metabolite of MPTP
Released from astrocytes and enters specifically into dopaminergic neurons by dopamine transporter MPP+ inhibits mitochondrial complex I activity and causes mitochondrial dysfunction
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Fig. 7. Shows the mechanism of action of lycopene.
drial impairment [25] and inflammation [4], that contributes directly or indirectly to the pathogenesis of Alzheimer’s disease, PD, amyotrophic lateral sclerosis, stroke and trauma. Further research is needed to elucidate the mitochondrial protective and anti-inflammatory effect of lycopene against MPTP induced neurotoxicity to confirm its neuroprotective effect. Conflict of interest The authors declare that there are no conflicts of interest. Uncited reference [8]. Acknowledgment
Financial assistance in the form of a major research project from Q6 the Indian Council of Medical Research, New Delhi, is gratefully 283 acknowledged. 284 282
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
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Neuroprotective effect of lycopene against MPTP induced experimental Parkinson’s disease in mice
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Asokan Prema, Udaiyappan Janakiraman, Thamilarasan Manivasagam ∗ , Arokiasamy Justin Thenmozhi
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Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar 608002, Tamilnadu, India
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Lycopene ameliorates MPTP induced behavioral deficts. Lycopene reverses MPTP mediated neurochemical depletion. Lycopene attenuates MPTP induced oxidative stress. Lycopene reduces apoptosis in PD mice.
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Article history: Received 27 March 2015 Received in revised form 10 May 2015 Accepted 13 May 2015 Available online xxx
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Keywords: Experimental Parkinson’s disease Lycopene Oxidative stress Apoptosis Behavior
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1. Introduction
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Parkinson’s disease (PD) is the second most common neurodegenerative disorder that mainly affects the movement of the aged populations. Lycopene is a carotenoid with unique pharmacological properties and its efficacy on experimental Hunginton’s disease and brain ischemia has shown intense neuroprotective effects. The present study was aimed to explore the neuroprotective effect of lycopene against 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced PD mice. Administration of lycopene (5, 10 and 20 mg/kg/day orally) protected MPTP induced depletion of striatal dopamine (DA) and its metabolites in a dose dependent manner. It also attenuated MPTP-induced oxidative stress and motor abnormalities seen in PD mice. Our western blot studies showed that treatment with lycopene reversed MPTP induced apoptosis may be due to its antioxidant and antiapoptotic properties. As to conclude, lycopene reverses neurochemical deficts, oxidative stress, apoptosis and physiological abnormalities in PD mice and offer promise strategy in the treatment of this neurodegenerative disease. © 2015 Published by Elsevier Ireland Ltd.
Parkinson’s disease (PD) is a devastating neurodegenerative disorder characterized by degeneration of dopaminergic neurons in the substantia nigra (SN) and diminution of dopamine (DA) concentration in striatum (ST) that leads to severe motor disabilities [21]. MPTP is considered as the gold standard for toxin based PD models because, it can easily cross the blood brain barrier and where it is taken by glial cells and metabolized to form 1-methyl-4-phenyl pyridinium (MPP+ ) by monoamine oxidase-B [10]. Released MPP+ is selectively taken up into dopaminergic neurons by dopamine transporters (DAT) and causing mitochondrial dysfunction and oxidative stress [25] which finally induces apoptosis [7].
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∗ Corresponding author. Tel.: +91 8940505861. E-mail address: mani [email protected] (T. Manivasagam).
Now interest has been focused on lycopene, an aliphatic hydrocarbon carotenoid, present in the ripened tomatoes as a potential useful agent in the management of neurodegenerative disorders because of its ability to cross the blood–brain barrier [14] and strong antioxidant property [22]. About 10–30% of dietary lycopene is absorbed by humans and is distributed in various tissues including adrenal gland, liver, prostate gland, brain and testes. Though lycopene undergoes oxidative metabolism and forms several oxidized forms and polar metabolites, their biological significance is not yet elucidated [24]. Lycopene protects cultured hippocampal neurons against Aˇ and glutamate toxicity [18], microglial activation in focal cerebral ischemia in rats [11] and attenuated cognition impairment in elderly population [1]. In addition, lycopene has been shown to mediate its neuroprotective effect in 3-nitropropionic acid induced Huntington’s disease [15] and rotenone induced PD [16]. In the present study, we investigated the neuroprotective effect of lycopene on MPTP-induced mouse model of PD by analysing the
http://dx.doi.org/10.1016/j.neulet.2015.05.024 0304-3940/© 2015 Published by Elsevier Ireland Ltd.
Please cite this article in press as: A. Prema, et al., Neuroprotective effect of lycopene against MPTP induced experimental Parkinson’s disease in mice, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.05.024
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protein expressions of pro-apoptotic (Bax cytochrome-c, caspases 3, 8 and 9) and anti-apoptotic (Bcl-2) markers.
supernatant was boiled for 10 min. After cooling, the samples were read at 535 nm [23].
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2. Materials and methods
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2.1. Animals
2.3.6. Assay of SOD The assay mixture consisted of 960 l of sodium carbonate buffer containing 0.1 mM xanthine, 0.025 mM NBT, and 0.1 mM EDTA, 20 l of xanthine oxidase and 20 l of the brain supernatant were taken. Changes in absorbance were observed spectrophotometrically at 560 nm [23].
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Male C57bL/6 mice (25–30 g) were purchased from the National Institute of Nutrition, Hyderabad and acclimatized to laboratory conditions prior to experimentation. The animals were kept under standard conditions with food and water ad libitum. The protocol was approved by the Institutional Animal Ethics Committee (approval no: 890/2012) and carried out in accordance with the Indian National Science Academy guidelines for the use and care of animals.
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2.2. Chemicals
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MPTP, lycopene, thiobarbituric acid (TBA), reduced glutathione and 3,5-dithio-bisnitrobenzoic acid (DTNB) were purchased from Sigma–Aldrich, Bangalore, India. Anti caspase-3,-8, -9, cytochromec (cyto-c), bax and bcl-2 (Santa Cruz Biotechnology Inc.), anti-actin (cell signalling) primary antibodies produced from rabbits and anti rabbit HRP conjugated secondary antibody (Sigma chemical, USA) were used in the present study.
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2.3.1. Phase I Mice were randomized and divided into six groups (n = 6). Group I animals were treated with saline; group II mice were injected intraperitoneally with MPTP (30 mg/kg body weight) [23] once daily in saline for last four consecutive days (from 4th to 7th day); group III, IV and V animals were injected with MPTP as group II and administered with lycopene (5, 10 and 20 mg/kg/day orally for 7 days, respectively); group VI mice were treated with lycopene (20 mg/kg/day orally for 7 days) alone. Seven days after the last injection of MPTP, behavior tests such as narrow beam walking test and hang test were performed. Then, animals were killed by cervical dislocation and striatal and nigral tissues were procured rapidly and utilized for the estimation of DA and oxidative stress related indices.
2.3.7. Determination of activity of catalase The assay mixture consisted of 50 l of 1 M Tris–HCl buffer (pH 8.0) containing 5 mM EDTA, 900 l of 10 mM H2 O2 , 30 l of MQ water and 20 l of the brain tissue supernatant. The rate of decomposition of hydrogen peroxide was observed spectrophotometrically at 240 nm [26]. 2.3.8. Assay of GPx The GPx assay mixture consisted of 100 l of 1 M Tris–HCl (pH 8.0) containing 5 mM EDTA, 20 l of 0.1 M GSH, 100 l of GSH reductase solution (10 U/ml), 100 l of 2 mM NADPH, 650 l of distilled water, 10 l of 7 mM tert-butyl hydroperoxide and 10 l of the brain supernatant. Oxidation of NADPH was determined spectrophotometrically at 340 nm [23]. 2.3.9. Estimation of GSH Brain tissue homogenate was centrifuged at 16,000 g for 15 min at 4 ◦ C. The supernatant (0.5 ml) was added to 4 ml of ice-cold 0.1 mM solution of 5,5-dithiobis[2-nitrobenzoic acid] in 1 M phosphate buffer (pH 8). The optical density was read at 412 nm in a spectrophotometer [26]. 2.4. Phase II Based on DA levels, effective dose of lycopene was found to be 10 mg/kg bodyweight and used for further study involving the following groups – control, MPTP treated, MPTP and lycopene (10 mg/kg bodyweight) treated and lycopene alone treated (10 mg/kg bodyweight). Seven days after the last injection of MPTP, open field test (with effective dose) was performed. After performing behavioral test, animals were killed by cervical dislocation and striatal and nigral
2.3.2. Narrow beam test Pre-trained animals were allowed to walk on a narrow flat stationary wooden beam (length –100 cm × width – 1 cm) placed at a height of 100 cm from the floor. The time taken to cross the beam from one end to the other along with foot slip errors were measured [12]. 2.3.3. Hang test Mice were allowed to hold on a horizontal grid. Then, the grid was inverted so that the mice were allowed to hang upside down and hanging time was measured [20]. 2.3.4. Estimation of DA and its metabolites Immediately after the procurement, ST was sonicated in ice-cold 0.1 M HClO4 containing 0.01% EDTA and centrifuged at 10,000 g for 5 min. Supernatant was collected and injected (10 l) into the HPLC system and results were expressed in ng/mg wet weight [20]. 2.3.5. Estimation of TBARS Briefly, the nigral extracts were incubated with 0.2 ml of phenyl methosulfate at 37 ◦ C for 1 h of incubation and 0.4 ml of tricarboxylic acid and 0.4 ml of thiobarbituric acid were added. The reaction mixture was centrifuged at 4000 rpm for 15 min, and the
Fig. 1. Measurement of balance and muscular co-ordination by narrow beam walking test. MPTP treated mice took more time to cross the narrow beam with high number of foot slip errors, whereas preadministration of lycopene significantly attenuated motor deficts induced by MPTP. Results given are mean ± SD, (n = 6), values not sharing common superscript are significant with each other p < 0.05, one way ANOVA followed by DMRT.
Please cite this article in press as: A. Prema, et al., Neuroprotective effect of lycopene against MPTP induced experimental Parkinson’s disease in mice, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.05.024
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tissues were procured rapidly and stored in −80 ◦ C for further analysis. 2.4.1. Open field test Mice were placed in one corner of open field chamber, its movement and activities were observed for 5 min. When the animal enters a square with both of its forelimb, one count is made. The number of center and peripheral squares explored by the animal was noted. Activities of mice including grooming and rearing were also counted [2]. 2.4.2. Western blotting Nigral tissues were homogenized in an ice-cold RIPA buffer and were centrifuged at 12,000 rpm/min for 15 min at 4 ◦ C. Protein concentration was measured by the method of Lowry et al. [17]. Samples containing 50 g of total cellular protein were loaded and separated on 10% SDS–polyacrylamide gel electrophoresis. The gel was then transferred onto a PVDF membrane. The membranes were incubated with the blocking buffer containing BSA for 2 h and then incubated with primary antibodies of -actin (rabbit monoclonal; 1:500 dilution), cyto-c, caspases-3,-8, -9 (1:500), bcl2 and bax (1:1000) with gentle shaking overnight at 4 ◦ C. After this, membranes were incubated with their corresponding secondary antibodies for 2 h at room temperature and washed thrice. Protein bands were visualized by an enhanced chemiluminescence’s method and scanned bands were quantitated by Total lab Quant, the control was set to 100.
Fig. 2. Determination of neuromuscular strength by hang test. Hanging time of MPTP induced mice were drastically decreased, meanwhile, treatment with lycopene significantly increased hanging time of MPTP induced mice. Results given are mean ± SD, (n = 6), values not sharing common superscript are significant with each other p < 0.05, one way ANOVA followed by DMRT.
2.5. Statistical analysis All values were expressed as mean ± SD of the number of experiments (n = 6). Statistical analysis was carried out using one-way analysis of variance (ANOVA) using SPSS version 15.0 software
Fig. 3. Determination of acclimatization ability and motor performance in open field test. Administration of MPTP led to significant reduction in movement and activities; however, these reductions were prevented by administration of lycopene. Results given are mean ± SD, (n = 6), values not sharing common superscript are significant with each other p < 0.05, one way ANOVA followed by DMRT.
Please cite this article in press as: A. Prema, et al., Neuroprotective effect of lycopene against MPTP induced experimental Parkinson’s disease in mice, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.05.024
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Fig. 4. MPTP-induced depletion of dopamine and its metabolites can be conserved by lycopene treatment. Administration of MPTP depleted the levels of dopamine and its metabolites, which are attenuated dose-dependently by treatment of lycopene. Results given are mean ± SD, (n = 6), values not sharing common superscript are significant with each other p < 0.05, one way ANOVA followed by DMRT.
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and individual comparisons were obtained using Duncan’s multiple range test (DMRT).
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in MPTP induced mice. However, it was observed that both the 10 and 20 mg/kg b.w showed similar induction, but more significant than 5 mg/kg b.w. So we have chosen the 10 mg/kg b.w as the optimum dose for further studies.
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3.3. Oxidative and antioxidative indices Oxidative stress is the main culprit that can induce neurodegeneration in PD. The levels of TBARS and activities of SOD and catalase were increased in the SN of MPTP-treated mice, whereas pre-treatment of lycopene significantly lowered their levels and activities as compared with MPTP injected mice. In addition, the levels of GSH and activities of GPx were drastically decreased MPTP-treated mice, whereas lycopene pretreatment significantly attenuated the levels of GSH and activities of GPx significantly (p < 0.05) (Fig. 5).
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Behavioral studies such as narrow beam walking test (to examine the balance and motor coordination skills), open field test (to measure the locomotion and activity) and hang test (to accesses the neuromuscular strength and coordination) were performed in control and experimental animals. MPTP treated mice exhibited more time to cross the wooden beam with increased foot slip errors in narrow beam walking test, more hanging time in hang test, reduced movement (peripheral and centre) and activities (grooming and rearing) in open field test as compared with the control animals (p < 0.05), whereas the lycopene pre-treatement attenuated the behavioral abnormalities seen in MPTP animals (Figs. 1–3).
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Apoptosis is reported mode of cell death in various neurodegenerative diseases including PD. The expression patterns of bcl-2-associated X, caspases-3, 8 and 9 were significantly increased in MPTP treated mice, whereas lycopene pretreatment significantly decreased the expression pattens of these proapoptotic markers as compared with MPTP alone treated animals. The expressions of bcl-
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Fig. 4 revealed a significant decline in the levels of striatal DA and its metabolites in the MPTP alone treated mice as compared with control mice, whereas pre-administration of lycopene dose dependently enhanced the levels of dopamine and its metabolites
3.4. Western blot analysis
Please cite this article in press as: A. Prema, et al., Neuroprotective effect of lycopene against MPTP induced experimental Parkinson’s disease in mice, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.05.024
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Fig. 5. MPTP induced oxidative stress can be attenuated by lycopene. The levels of TBARS and activities of SOD and catalase were increased along with a decreased levels of GSH and activities of GPx in the SN of MPTP-treated mice, meanwhile, treatment with lycopene significantly ameliorated oxidative stress. Results given are mean ± SD, (n = 6), values not sharing common superscript are significant with each other p < 0.05, one way ANOVA followed by DMRT. A Amount of enzyme required to inhibit 50% of NBT reduction. B nmol H2 O2 consumed/minute/mg protein. C nmol NADPH oxidized/minute/mg protein.
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2 and cyto c were significantly diminished in MPTP treated mice as compared with controls, whereas lycopene pre-treatment significantly enhanced the expressions of bcl-2 and cyto-c as compared with MPTP alone treated animals (Fig. 6). 4. Discussion DA is an important neurotransmitter responsible for balance and control of movements including walking. MPTP administration caused a selective destruction of dopaminergic neurons in nigrostriatal regions and leads to depletion in the levels of striatal DA.
Administration of tomato powder enriched in lycopene or tomato diet ameliorated MPTP [27] and 6-OHDA [6] induced DA depletion, which is concordant with the results of our study Fig. 7. Q4 Open field test, narrow beam walking test and hang test are used as indices to measure the motor impairments in MPTP-induced behavioral abnormalities in animal models [20]. MPTP induced behavior deficits are mainly due to pathological loss of dopaminergic neurons in midbrain with degeneration of their striatal nerve terminals. However, lycopene treatment caused a recovery of behavioral deficits induced by MPTP, by enhancing the levels of DA.
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Fig. 6. MPTP persuaded apoptosis can be amolierated by lycopene administration. Expressions of bax, cyto-c, caspase-3, -8 and -9, bcl-2 in the SN were analyzed by Western blot (A and B), with -actin used as the loading control of total proteins; lane 1: control, saline control, lane 2: MPTP, MPTP-treated only, lane 3: MPTP-lycopene, lycopene with MPTP, lane 4: lycopene, lycopene alone administrated. The band density was quantified by scanning densitometry (C and D). Results given are mean ± SD, (n = 3), values not sharing common superscript are significant with each other p < 0.05, one way ANOVA followed by DMRT.
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Inhibition of electron transport chain I by MPP+ , a toxic metabolite of MPTP, leads to the leaking of electrons, which may generate the production of ROS (5–7 times more) by reacting with excessively available O2 . Previous studies from our lab, clearly indicated that the administration of MPTP has been reported to enhance the levels of TBARS, a marker of lipid peroxidation processes and activities of SOD and catalase and diminished the levels of GSH and activity of GPx [26], which may reflect an adaptive response to oxidative stress. Lycopene is described as the most effective antioxidants in the carotenoid family and exhibits 10-fold, 47fold and 100-fold more effective in quenching singlet oxygen than ␣-tocopherol, -carotene and vitamin E, respectively [5,19]. Moreover its administration modulates positively the activities and levels of SOD, catalase, GPx and GSH in the striatum of 3nitropropionic acid treated HD rats [25] and regulating antioxidant
response element genes [3]. In the present study, pre-treatment of lycopene significantly ameliorated oxidative stress induced by MPTP due to its free radical scavenging and antioxidant activities. In the present study, MPTP treatment decreased the expression of bcl-2 and increased cyto-c release with increased expressions of bax, caspases-3, 8 and 9 [20], thereby favoring apoptosis. Fujita et al. [9] reported that the administration of lycopene inhibits ischemia/reperfusion-induced neuronal apoptosis in gerbil hippocampal tissue by enhancing bcl-2 and diminishing caspase 3 levels. The dosage of 10 mg/kg of the lycopene, which markedly exceeds the estimated daily intake of humans (0.5–27 mg/person/day), even no toxic effects were observed in rats that were orally administered with 2000 mg/kg/day [13]. Large numbers of studies indicated that the neuroprotective effect of lycopene attributed through amelioration of mitochon-
Please cite this article in press as: A. Prema, et al., Neuroprotective effect of lycopene against MPTP induced experimental Parkinson’s disease in mice, Neurosci. Lett. (2015), http://dx.doi.org/10.1016/j.neulet.2015.05.024
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MPTP Monaoamine oxidase B in astrocytes
MPP+, a toxic metabolite of MPTP
Released from astrocytes and enters specifically into dopaminergic neurons by dopamine transporter MPP+ inhibits mitochondrial complex I activity and causes mitochondrial dysfunction
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Fig. 7. Shows the mechanism of action of lycopene.
drial impairment [25] and inflammation [4], that contributes directly or indirectly to the pathogenesis of Alzheimer’s disease, PD, amyotrophic lateral sclerosis, stroke and trauma. Further research is needed to elucidate the mitochondrial protective and anti-inflammatory effect of lycopene against MPTP induced neurotoxicity to confirm its neuroprotective effect. Conflict of interest The authors declare that there are no conflicts of interest. Uncited reference [8]. Acknowledgment
Financial assistance in the form of a major research project from Q6 the Indian Council of Medical Research, New Delhi, is gratefully 283 acknowledged. 284 282
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