Elsevier

Neurochemistry International

Volume 79, December 2014, Pages 1-11
Neurochemistry International

Mechanism of the beneficial effect of melatonin in experimental Parkinson's disease

https://doi.org/10.1016/j.neuint.2014.09.005Get rights and content

Highlights

  • Melatonin treatment decreased COX activity which was increased with 6-OHDA.

  • Melatonin treatment decreased PGE2 level which was increased with 6-OHDA.

  • Melatonin treatment decreased caspase-3 activity which was increased with 6-OHDA.

  • Melatonin treatment decreased neuron death which was increased with 6-OHDA.

  • Melatonin treatment increased Bcl-2 amount which was decreased with 6-OHDA.

Abstract

This study aimed to elucidate locomotor activity changes in 6-hydroxydopamine (6-OHDA) induced Parkinson's disease (PD) and investigate the possible beneficial effects of melatonin on altered levels of locomotor activity, cyclooxygenase (COX), prostaglandin E2 (PGE2), nuclear factor kappa-B (NF-κB), nitrate/nitrite and apoptosis. Male Wistar rats were divided into five groups: vehicle (V), melatonin-treated (M), 6-OHDA-injected (6-OHDA), 6-OHDA-injected + melatonin-treated (6-OHDA-Mel) and melatonin treated + 6-OHDA-injected (Mel-6-OHDA). Melatonin was administered intraperitoneally at a dose of 10 mg/kg/day for 30 days in M and Mel-6-OHDA groups, for 7 days in 6-OHDA-Mel group. Experimental PD was created stereotactically via unilateral infusion of 6-OHDA into the medial forebrain bundle (MFB). The 6-OHDA-Mel group started receiving melatonin when experimental PD was created and treatment was continued for 7 days (post-treatment). In the Mel-6-OHDA group, experimental PD was created on the 23rd day of melatonin treatment and continued for the remaining 7 days (pre- and post-treatment). Locomotor activity performance decreased in 6-OHDA group compared with vehicle; however melatonin treatment did not improve this impairment. Nuclear factor kappa Bp65 and Bcl-2 levels were significantly decreased while COX, PGE2 and caspase-3 activity were significantly increased in 6-OHDA group. Melatonin treatment significantly decreased COX, PGE2 and caspase-3 activity, increased Bcl-2 and had no effect on NF-κB levels in experimental PD. 6-Hydroxydopamine injection caused an obvious reduction in TH positive dopaminergic neuron viability as determined by immunohistochemistry. Melatonin supplementation decreased dopaminergic neuron death in 6-OHDA-Mel and Mel-6-OHDA groups compared with 6-OHDA group. Melatonin also protected against 6-OHDA-induced apoptosis, as identified by increment in Bcl-2 levels in dopaminergic neurons. The protective effect of melatonin was more prominent for most parameter following 30 days treatment (pre- and post-) than 7 days post-treatment. In summary, melatonin treatment decreased dopaminergic neuron death in experimental PD model by increasing Bcl-2 protein level and decreasing caspase-3 activity.

Introduction

Melatonin is a natural hormone synthesized and released by the pineal gland (Niranjan et al., 2010). Melatonin is an extremely potent free-radical scavenger and antioxidant. Its protective and anti-inflammatory effect in numerous neurological disorders including Parkinson's disease (PD) have been previously demonstrated (Lin, 2013, Niranjan et al, 2010, Patki, Lau, 2011).

Parkinson's disease is the second most common neurodegenerative disease that affects about 1.8% of old people over 65 years of age. Parkinson's disease is characterized by progressive loss of dopaminergic neurons in the substantia nigra (SN) and depletion of dopamine (DA) in the corpus striatum (Hornykiewicz and Kish, 1987). The etiology of PD is not completely clear yet, however several hypotheses to explain neuronal death has been put forward, such as; mitochondrial dysfunction, oxidative stress, exitotoxicity and inflammation (Dauer, Przedborski, 2003, Moore, 2005). In line with these hypotheses, there are several studies indicating oxidative stress in the pathology of PD (Adams et al, 2001, Przedborski, 2005). Since reactive oxygen species (ROS) are accumulated more in dopaminergic neurons, it makes these neurons more vulnerable to neural degeneration (Graham, 1978). As a result of enzymatic metabolism of DA, hydrogen peroxide (H2O2) and superoxide radicals (O2) are formed, also when the auto-oxidation product of DA combines with iron (Fe2+), it leads to the formation of DA-quinones that causes the formation of hydroxyl radicals (.OH) (Ben-Shachar, 1991, Graham, 1978). Furthermore, the synthesis of DA by tyrosine hydroxylase (TH) and catabolism by monoamine oxidase (MAO) can result in the formation of H2O2 (Andersen, 1994, Graham, 1984). Reactive oxygen species have been detected in the SN of Parkinsonian patients and experimental animal models (Przedborski, 2005). Mitochondria which is the main source of ROS, contributes to the pathogenesis of PD (Przedborski, 2005).

In many studies, when compared with the control groups of the same age, lipid peroxidation was proven to be higher in the SN of patients with PD (Dexter, 1989, Yoritaka, 1996). Because of these reasons, systems that produce oxidative stress are very important in the pathology of PD.

In addition to oxidative stress, the amount of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) are increased in PD (Aras, 2014, Levecque, 2003). Together with this increase, peroxynitrite (ONOO) is also formed (Chabrier et al., 1999). It is known that nitric oxide (NO) is a free radical and forms nitrogen derivatives such as nitrites and nitrates in the presence of oxygen. It has been detected that oxygen radicals formed as a result of auto-oxidation react with NO to form ONOO (Beckman and Koppenol, 1996). It has also been demonstrated that ONOO is a strong oxidant that can destroy several biologic molecules and independent of metal catalysis dissociates to form .OH (Beckman and Koppenol, 1996). Nitric oxide also affects nuclear factor kappa-B (NF-κB) activation to cause apoptosis (Pannu and Singh, 2006).

It has been demonstrated that 6-OHDA causes the activation COX-2 and increases the level of mRNA (Jin, 2008, Pyo, 2013). It is also known that the amount of PGE2 metabolite is increased in tissues as an indicator of high level of COX-2 (Esposito, 2007, Jin, 2008, Lee, 2010, Ozsoy, 2011, Pyo, 2013). Lipid peroxidation is known to increase as a result of all these events.

Conducted studies demonstrated that melatonin decreases 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced lipid peroxidation in-vivo (Thomas and Mohanakumar, 2004) and caspase-3 enzyme activity in-vitro (Sharma et al., 2005). Attempts have been made to explain the mechanism of this process using Fe2+, the Haber–Weiss reaction and glial cell line-derived neurotrophic factor (GDNF) (Ortiz et al., 2013). However the effect of melatonin on 6-OHDA induced lipid peroxidation and caspase-3 enzyme activity is not known. It has been demonstrated in separate studies that melatonin inhibits iNOS and COX-2 activity (Absi, 2000, Dabbeni-Sala, 2001, Vilar, 2014). Its protective effects could be explained by the oxidative enzymes in mitochondria (Dastgheib, Moezi, 2014, Raghavendra, Kulkarni, 1999). It has been shown in microglia cell line studies that melatonin inhibits iNOS (Tocharus et al., 2008). Additionally, it has been detected that it protects neurons by inhibiting iNOS (Cardinali et al., 2013). Furthermore, it has been detected that melatonin increases the amount of anti-apoptotic Bcl-2 and decreases the amount of pro-apoptotic Bax protein (Ma et al., 2009). It is also known that death of dopaminergic neurons containing TH decreased by the administration of melatonin in experimental PD (Grealish, 2008, Sharma et al, 2007). However, there is no study in the literature concerning the effect of melatonin on COX, PGE2, NF-κB, iNOS and Bcl-2 in 6-OHDA lesion model of PD.

This study was performed to clarify the anti-apoptotic mechanism of melatonin in 6-OHDA-induced hemiparkinsonian rat model. The role of COX, PGE2, NF-κB, iNOS, caspase-3 and Bcl-2 on this effect was evaluated. Our study is important in terms of demonstrating the protective effect of melatonin in the progression of 6-OHDA model of PD.

Section snippets

Animals

Male Wistar rats (3 month old, weighing 250–300 g) were obtained from Akdeniz University Animal Care Unit. The animals were housed in stainless steel cages (5–6 per cage) in an air-conditioned room (22 ± 2 °C with a 12:12 h light:dark cycle). All experimental protocols conducted on rats were performed in accordance with the standards established by the Institutional Animal Care and Use Committee at Akdeniz University Medical School.

Experimental design

Rats were randomly divided into five experimental groups as

Locomotor activity test

Changes in locomotor activity performances are shown in Fig. 1. 6-Hydroxydopamine-injected rats exhibited significantly reduced ambulatory, vertical, horizontal and total locomotor activity compared with vehicle rats. In addition, total distance moved by rats in 6-OHDA-injected group was significantly decreased compared with vehicle. There was an increase in locomotor activity in 6-OHDA-lesioned rats after melatonin treatment but it did not reach significance. There was no significant

Discussion

We examined the effects of melatonin on motor activity and dopaminergic cell death in rats injected with 6-OHDA, an experimental model of PD. To explore the mechanism of melatonin effect, wedetermined nigral PGE2, NF-κB, nitrate/nitrite levels, also the activities of COX, caspase-3 enzymes, and the immunostaining of Bcl-2 was evaluated. Our findings indicate that the beneficial effects of melatonin treatment on experimental PD was associated with decreased dopaminergic neuron death in the SN, a

Acknowledgments

This study was supported by the Akdeniz University Research Projects Unit (Project number: 2011.01.0103.021).

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