Effect of Buspirone , Fluoxetine and 8-OH-DPAT on Striatal Expression of Bax , Caspase-3 and Bcl-2 Proteins in 6-Hydroxydopamine-Induced Hemi-Parkinsonian Rats

© 2015 The Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, as long as the original authors and source are cited. No permission is required from the authors or the publishers. Adv Pharm Bull, 2015, 5(4), 491-495 doi: 10.15171/apb.2015.067 http://apb.tbzmed.ac.ir Advanced Pharmaceutical Bulletin


Introduction
The pathogenesis of sporadic Parkinson disease (PD) is still enigmatic; many factors are speculated to operate in the mechanism of cell death of the nigrostriatal dopaminergic neurons in PD.Recent studies have been focused on the factors of the pathways of programmed cell death, i.e., apoptosis, that might be involved in the neurodegeneration in PD. 1 By end-stage disease in PD there is 80-95% loss of neurons in the substantia nigra. 2 Increased levels of proteins that signal for apoptosis have been demonstrated not only in neurons of postmortem PD but also in experimental models of PD strongly suggested involvement of apoptosis in the death of nigrostriatal neurons. 3The major anti-apoptotic family members, Bcl-2 and Bcl-xL, which are thought to exert their effect at the mitochondrial outer membrane contribute to maintenance of membrane integrity.In contrast, one of the major pro-apoptotic family members, Bax, exerts its effects by compromising the membrane integrity leading to leakage of apoptogenic factors such as cytochrome c into the cytosol, resulting in caspase-3 activation and demise of the cell. 4Altered expressional levels of Bcl-2, Bax and increased caspase 1 and 3 activity have been reported in dopaminergic neurons of PD patients 5,6 and in 6-OHDA-lesioned rats 7 but the effect of serotonergic drugs on these proteins in PD have not studied.The stimulation of 5HT 1A receptors induces a variable level of neuroprotection in different animal models of CNS injury. 8In vitro evidence indicates that 5HT 1A agonists are able to protect neurons from apoptosis. 9Thus in this study we attempted to investigate the effect of chronic administration of serotonergic drugs (8-OH-DPAT, buspirone and fluoxetine) on 6-OHDA-induced PD in

Chemicals
All chemicals were obtained from Sigma Chemical Co.except for antibodies used for western blotting technique which were purchased from Abcam Co. Drugs and 6-OHDA were dissolved in physiological saline (0.9% NaCl) and 0.9% saline containing 0.2% (w/v) ascorbic acid respectively.

Animals and Treatment Protocol
The experiments were carried out on male Wistar rats weighing 270-300 g.The animals were given food and water ad libitum and were housed in standard polypropylene cages, four per cage at an ambient temperature of 25±2 °C under a 12-h light/12-h dark cycle.Animals were habituated to the testing conditions including being transferred to the experimental environment, handled, weighed, and restrained on the test platform for 10 min; 2 days before the investigations were conducted.The present study was carried out in accordance with the ethical guidelines for the Care and Use of Laboratory Animals of Tabriz University of Medical Sciences, Tabriz, Iran (National Institutes of Health Publication No. 85-23, revised 1985).The parkinsonian rats were randomly allocated to equal groups (six rats per group) and were treated with 8-OH-DPAT (1mg/kg, i.p), fluoxetine (1mg/kg, i.p) and buspirone (1 mg/kg, i.p) for ten days.According to our previous study [10][11][12] 1mg/kg dose of these drugs had more profound anti-cataleptic effect in 6-OHDA-induced hemiparkinsonian rats.Then their striatal apoptotic proteins (Bcl-2, Bax and Caspase 3) were assessed by western blotting technique.

6-OHDA-Induced SNc Lesion
Animals were anesthetized with an intraperitoneal (i.p) injection of ketamine (50 mg/kg) and xylazine (5 mg/kg) and were mounted in a stereotaxic frame in the flat skull position.A central incision made to expose the skull.6-OHDA was injected thorough a guide cannula (23 gauge stainless steel) implanted in the SNc.The coordinates for this site were based on the rat brain atlas 13 anteroposterior (AP): -5.0 mm from the bregma; mediolateral (ML): -2.1 mm from the midline and dorsoventral (DV): -7.7 from the skull.Desipramine (25 mg/kg, i.p) was injected 30 min before the intra-SNc injection of 6-OHDA to avoid the destruction of noradrenergic neurons.Then, 6-OHDA (8 μg/per rat in 2 μl saline with 0.2% ascorbic acid) was infused with an infusion pump at a constant flow rate of 0.2 μl/min into the left SNc.

Western Blot Analysis
Animals were sacrificed after ten days administration of drugs and their striatum was dissected and homogenized in lysis buffer (Cocktail Protease Inhibitor 10 µl/ml and Phenyl Methane Sulfonyl Fluoride 10 µl/ml).Lysates (30 µg protein) were resolved by SDS-PAGE for Bax, Caspase3 and Bcl-2 assay; then were transferred to polyvinylidene difluoride filters (PVDF) which were blocked with 5% milk and were incubated overnight at 4°C with the following primary antibodies: Bax, Bcl-2 and GAPDH, 1:1000 , Caspase-3, 1:500.Subsequently, PVDF membranes were incubated for 1 hr at room temperature with an HRP-conjugated secondary antibody (1:3000); incubated with the chemiluminescent substrate for 90 s; and exposed for 30 s to 5 min, to Hyperfilm.Finally the developed films were scanned, and density of immonoreactive bands was measured using "Image J" software and was normalized to the internal control bands (GAPDH was the loading control).

Statistical Analysis
Statistical analysis of each data set was performed by use of SPSS software (version 16.0).Data were expressed as the mean±SEM, and were analyzed by one-way ANOVA in behavioral and biochemical experiments.In the case of significant variation (p<0.05), the values were compared by Tukey test.

The Effects of Buspirone, Fluoxetine and 8-OH-DPAT on Bax Expression
The Expression of striatal Bax protein in 6-OHDAlesioned rats increased significantly (p<0.001) when compared to non-lesioned (control) rats.The amount of this protein in buspirone, fluoxetine and 8-OH-DPATtreated parkinsonian rats was almost same as the control level.On the other hand, these drugs decreased expression of Bax protein in 6-OHDA-lesioned rats (Figure 1).

The Effects of Buspirone, Fluoxetine and 8-OH-DPAT on Caspase-3 Expression
The striatum of aforementioned groups was investigated for evaluation of pro-apoptotic Caspase-3 protein activity.Results showed that in 6-OHDA-lesioned rats expression of this protein was more than control group (p<0.001),while in 6-OHDA-lesioned rats which were treated with buspirone, fluoxetine and 8-OH-DPAT expression of Caspase-3 protein was reduced to control level (Figure 2).

The Effects of Buspirone, Fluoxetine and 8-OH-DPAT on Bcl-2 Expression
As it has been shown in Figure 3, the expression of antiapoptotic Bcl-2 protein in the striatum of 6-OHDAlesioned rats was less than (p<0.001)control group.In 6-OHDA-lesioned rats which were treated with buspirone, fluoxetine and 8-OH-DPAT, expression of Bcl-2 protein was greater (p<0.001)than 6-OHDA-lesioned rats.

Discussion
14][15][16][17] Furthermore, 8-OH-DPAT (1 mg/kg, i.p), 10 fluoxetine (1 mg/kg, i.p) 11 and buspirone (1 mg/kg, i.p) 12 attenuated 6-OHDA-induced catalepsy in hemiparkinsonian rats.Thus, in this study we attempted to assess the effect of these drugs on expression of proapoptotic proteins, Bax and Caspase-3 as well as antiapoptotic Bcl-2 protein in 6-OHDA-lesioned rats.Our results showed that expression of Bax protein was increased significantly in 6-OHDA-lesioned rats.0] In those groups of parkinsonian rats which were treated sub-chronically by buspirone, fluoxetine and 8-OH-DPAT, striatal expression of Bax protein was dropped markedly to the level of control group.The attenuating effect of buspirone and 8-OH-DPAT on expression of Bax Protein was greater than fluoxetine.Caspase family has 14 members that many of these proteases have been implicated in neuronal apoptosis.The first evidence that caspase activity is required for neuronal apoptosis came from experiments using pharmacological inhibitors of caspases.Moreover caspases may play pivotal roles in a number of acute and chronic neurologic disorders including stroke, Amyotrophic lateral sclerosis (ALS), PD and Huntington's disease. 21For example, mice transgenic for a dominant negative mutant of caspase-1 exhibit reduced sensitivity to ischemia as well as trophic factor deprivation.Accordingly, inhibition of caspase-3 expression plays a crucial neuroprotective role in a number of animal and human models of neurodegenerative disease such as Alzheimer disease (AD) and PD. 22Our data demonstrate that in 6-OHDAlesioned rats there was a significant increase in striatal Caspase-3 level when compared to control group.This is in accordance with previous studies reporting an increase of in 6-OHDA, Methyl-4-phenyl-1,2,3,6tetrahydropyridine ( MPTP) and lipopolysaccharide (LPS)-treated rats. 3,23Additionally we assessed Caspase-3 activity in striatum of buspirone, fluoxetine and 8-OH-DPAT-treated parkinsonian rats.In parkinsonian rats which were treated with these drugs Caspase-3 expression diminished to the level of non-lesioned (control) animals.The effect of 8-OH-DPAT on Caspase-3 expression was more profound than buspirone and fluoxetine.In a previous study it has been shown that 8-OH-DPAT can reduce morphine-induced apoptosis in hippocampus of rats. 24Since 8-OH-DPAT is a pure agonist of 5-HT 1A receptors, therefore we may suggest possible involvement of these receptors in apoptosis process of PD.Bcl-2 is specifically considered as an important antiapoptotic protein and is thus classified as an oncogene.Many members of the Bcl-2 family have been implicated in the regulation of neuronal cell death.The exact mechanism by which the Bcl-2 proteins control apoptosis is still not entirely clear but studies have shown that it's over expression in cell culture and in transgenic mice increases resistance of neurons to death during development and protects from apoptosis induced by excitotoxic, metabolic and oxidative insults relevant to AD, stroke and other disorder. 22,25In this study, Bcl-2 level was decreased in 6-OHDA-lesioned rats. 26ollowing treatment with buspirone, fluoxetine and 8-OH-DPAT, striatal level of this protein was elevated significantly in comparison to 6-OHDA-lesioned rats.The effect of 8-OH-DPAT and buspirone (as a partial agonist of 5-HT 1A receptors) was greater than fluoxetine.1][12] The stimulation of 5HT 1A receptors induces a variable level of neuroprotection in different animal models of CNS injury such as ischemia, N-methyl-d-aspartate (NMDA) excitotoxicity, acute subdural hematoma, and traumatic brain injury.Furthermore, in vitro evidence indicates that 5HT 1A agonists are able to protect neurons from apoptosis. 24There are different hypotheses on the mechanisms involved in 5HT 1A -mediated neuroprotection, including neuronal membrane hyperpolarization that reduces excitability, reduced glutamate release, and blockade of voltage-sensitive Na+ channels. 24Other neuroprotective mechanisms have also been proposed for 5HT 1A agonists such as stimulation of the BCL-2 expression through the MAPK/ERK signaling pathway and suppression of the pro-apoptotic protein caspase-3 in a MAPK-and protein kinase C-α dependent manner. 31,32Thus, we may suggest that the observed antiapoptotic effects of serotonergic drugs i.e. buspirone, fluoxetine and 8-OH-DPAT is exerted through 5-HT 1A receptors.According to the obtained results buspirone, fluoxetine and 8-OH-DPAT diminished striatal expression of Bax and Caspase-3 proteines in parkinsonian rats, whereas they increased Bcl-2 level.On the other hand, these drugs could attenuate apoptotic process in 6-OHDAinduced experimental model of PD.This correlates well with the results of our previous behavioral studies [10][11][12] reporting an anti-cataleptic effect for buspirone, fluoxetine and 8-OH-DPAT in 6-OHDA-lesioned rats.

Conclusion
In conclusion, we suggest that serotonergic agents may have beneficial effect in preventing the progression of PD through attenuating apoptotic events.However, further clinical investigations should be carried out to prove this.