Evaluation of Neuroprotective Effect of Flax Lignan Against Parkinson’s Disease in Animal Model

Abstract

Parkinson’s disease (PD) is likely to increase in the years to come as are related to motor defects such as resting tremor often referred as “pill-rolling”, bradykinesia and rigidity of skeletal muscle, postural instability, stooped posture, and freezing of gait. The objective of the present study was to evaluate the neuroprotective activity of natural polyphenols (Flax Lignan) against Parkinson’s disease. Evaluation of anti-Parkinson’s activity of polyphenols (Flax Lignan) using in-vitro, viz; metal chelation assay, nitric oxide scavenging assays and in-vivo models viz;1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model and rotenone model using zebra fish was performed. Various behavioural parameters like, muscle grip strength, locomotor activity, muscle rigidity, complete immobility, extent of anxiety, number and duration of freezing, total distance moved and swimming velocity and biochemical parameters were evaluated viz; dopamine, catalase, glutathione, super oxide dismutase, nitrite (NO) concentration, gamma-aminobutyric acid (GABA) levels, lipid peroxidation (TBARs), catalase activity, myeloperoxidase activity (MPO). Statistical analysis was done using GraphPad prism software using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. A significant (* p?0.05, ** p?0.01) reduction in test drug Flax Lignan in muscle rigidity and increased locomotor activity was observed, when compared with disease group. Brain neurotransmitter levels of antioxidant (p?0.01) were increased and oxidative stress (p?0.01) was reduced to that of the standard drug Selegiline. From above studies it can be concluded that ethanoic extract of flax lignan (EEFL) exhibits significant anti-parkinsonism activity in MPTP and rotenone model in mouse and zebrafish respectively.

Keywords

Parkinson’s Disease, Excitatory Amino Acids (EAA’s), Neurotoxicity, Neuroprotective, Flax Lignan,1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), Rotenone.

Introduction

 

 

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Values were expressed as Mean ± SEM for 6 rats in each group. Significance was determined by One- way ANOVA followed by Tukey’s multiple comparison test’s * p?0.05, ** p?0.01, when compared with disease group. Actophotometer score was found to be increased in groups administered with EEFL then Standard Seligeline when compared with disease group. Hence it can be concluded that may help in improving locomotor activity in Parkinson’s disease.

 

 

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Values were expressed as Mean ± SEM for 6 rats in each group. Significance was determined by One- way ANOVA followed by Tukey’s multiple comparison test’s ** p?0.01, *** p?0.001, when compared with disease group. Antioxidant Glutathione was found to be more in groups administered with EEFL then Standard Selegiline when compared with disease group. Hence it can be concluded that increase in Glutathione may help in improving symptoms of Parkinson’s disease.

       
           
       
Figure no.1.e:  Dopamine Level

Values were expressed as Mean ± SEM for 6 rats in each group. Significance was determined by One way ANOVA followed by Tukey’s multiple comparison test’s ** p?0.01, when compared with disease group. Dopamine was found to be more in EEFL when compared with disease group. Hence it can be concluded that increase in dopamine may help in improving symptoms of Parkinson’s disease.

       
           
       
Figure no.1.f:  Catalase Level

Values were expressed as Mean ± SEM for 6 rats in each group. Significance was determined by One- way ANOVA followed by Tukey’s multiple comparison test’s * p?0.001, ** p?0.01, when compared with disease group. Antioxidant Catalase was found to be more in groups administered with EEFL when compared with disease group. Hence it can be concluded that increase in catalase may help in improving symptoms of Parkinson’s disease.

       
           
       
Figure 2.a. Examination Tank^{[ ]}

Experimental Work:

 

 

 

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Values were expressed as Mean ± SEM for 8 zebrafish in each group. Significance determined was ** p?0.01, when compared with disease group. Number of freezing episodes were absent in group administered with EEFL and Selegiline as compared to disease   control. Hence it can be concluded that Dyskinesia –a symptom of PD was improved by significantly by EEFL as then Selegiline as compared to Rotenone treated group.

 

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Values were expressed as Mean ± SEM for 8 zebrafish in each group. Significance determined was *** p?0.001, when compared with disease group. Duration of freezing episodes were absent in group administered with EEFL and Seligiline as compared to disease   control. Hence it can be concluded that Dyskinesia –a symptom of PD was improved by the EEFL.

 

 

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Values were expressed as Mean ± SEM for 8 zebrafish in each group. Significance determined was, ** p?0.01, when compared with disease group. Complete cataleptic time was absent in group administered with EECC as compared to disease control. Hence it can be concluded that complete immobility –a symptom of PD was improved by the EEFL.

 

 

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Values were expressed as Mean ± SEM for 8 zebrafish in each group. Significance was determined by One way ANOVA followed by Tukey’s multiple comparison test’s* p?0.05, ** p?0.01, when compared with disease group. Time spent near the bottom of the tank was more in group administered with EEFL as compared to disease control i.e. midline crossing was more in test group than disease. Hence it can be concluded that anxiety –a symptom of PD was improved by the EEFL.

 

 

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Values were expressed as Mean ± SEM for 8 zebrafish in each group.Significance was determined by One way ANOVA followed by Tukey’s multiple comparison test’s ** p?0.01, *** p?0.001, when compared with disease group.Latency to travel from one point to another decreased in group administered with EEFL as compared to disease control. Hence it can be concluded that muscle rigidity –a symptom of PD was improved by the EEFL.

 

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Values were expressed as Mean ± SEM for 8 zebrafish in each group. Significance was determined by One way ANOVA followed by Tukey’s multiple comparison test’s * p?0.05, *** p?0.001, when compared with disease group. Total distance moved by zebrafish was increased in group administered with EEFL as compared to disease control. Hence it can be concluded that bradykinesia –a symptom of PD was improved by the EEFL.

 

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Values were expressed as Mean ± SEM for 8 zebrafish in each group.Significance was determined by One way ANOVA followed by Tukey’s multiple comparison test’s * p?0.05, *** p?0.001, when compared with disease group.Swimming velocity of zebrafish was increased in group administered with EEFL as compared to disease control. Hence it can be concluded that bradykinesia –a symptom of PD was improved by the EEFL.

CONCLUSION:

In the above research animal models were used which are pivotal for understanding the mechanism of neuroprotection and anti-Parkinsonian activity and development of effective therapy for its optimal management. Oxidative stress is a major player in the pathology of neurodegenerative disorders. The relationship between oxidative stress and neuronal death has been extensively investigated. Oxidative stress generated as a result of mitochondrial dysfunction and oxidative metabolism of DA plays an important role in the PD pathogenesis ^{[ 15].} Excitotoxicity defined as cell death resulting from the toxic actions of excitatory amino acids. Excessive activation of glutamate receptors by excitatory amino acids leads to a number of deleterious consequences, including impairment of calcium buffering, generation of free radicals, activation of the mitochondrial permeability transition and secondary excitotoxicity^[16].. The evaluation of neuroprotection in humans is complex because most of the stimuli required to induce neuronal damage produce irreversible damage. Drug development intended for humans require the demonstration of safety and efficacy in animal models. To reach at a conclusive result and before using it on human beings, the animal study is necessary for dose interpolation for human beings and safety data. Neuroprotection refers to the strategies and relative mechanisms able to defend the central nervous system (CNS) against neuronal injury due to both acute (e.g. stroke or trauma) and chronic neurodegenerative disorders (e.g. Alzheimer's disease, AD, and Parkinson's disease, PD). Neuroprotection is a broad term to cover any therapeutic strategy to prevent nerve cells called neurons from dying, and it usually involves an intervention, either a drug or treatment. The goal of neuroprotection is to limit neuronal dysfunction after injury and attempt to maintain the possible integrity of cellular interactions in the brain resulting in undisturbed neural function. These products may be of various kinds and can be classified as free radical scavengers, anti-excitotoxic agents, apoptosis inhibitors, neurotrophic factors etc ^[17]. Kovacsova M. et.al.  2014 reported Natural polyphenols have been reported to exert beneficial effects in preventing cardiovascular diseases but their neuroprotective mechanisms were studied much less. They focused on biochemical pathways and molecular neuroprotective mechanisms of natural polyphenols in the brain particularly; the evidence that antioxidant activity, mainly inhibition of the NADPH oxidase and subsequent reactive oxygen species generation; a balance in NO production from different NO synthase isoforms; reduction of neuroinflammation via attenuation of the release of cytokines and downregulation of the pro-inflammatory transcription factors; and the potential to modulate signalling pathways such as mitogen-activated protein kinase cascade and cAMP response element-binding protein are responsible for the neuroprotective actions of different natural polyphenols^[18]. Yacoubian T. et.al.  2009 reported that Neuroprotection in PD remains an important but elusive goal. A successful neuroprotective treatment could transform PD from a relentlessly progressive and disabling disease to a problem that can be managed with only a modest effect on quality of life. Currently, there are no treatments that are clearly established as neuroprotective in PD, and the discovery of such treatments is an important goal of much work in the field ^[19]. Natural polyphenols have been reported to exert beneficial effects in preventing cardiovascular diseases but their neuroprotective mechanisms were studied much less. The polyphenols a chemical widespread within the plant kingdoms, has antioxidant, antiinfectious, and antitumor activities. The neuroprotective actions of polyphenols against N-methyl-d-aspartate (NMDA) investigated in primary cultured cortical neurons by MTT assay are reported in invitro studies^{[ 20]} . The neuroprotective actions of FLL against N?methyl?d?aspartate (NMDA) are investigated in primary cultured cortical neurons by MTT assay. The expression levels of proteins related to apoptosis and GluN2?containing receptor were detected by Western blot analysis. Intracellular Ca²⁺ was measured under a confocal laser scanning microscope ^[21].Test compound Ethanolic Extract of Flax Lignan (EEFL) was selected for favorable physical and chemical properties and its ability to cross the blood brain barrier make it a suitable candidate as a potential treatment approach of neuroprotection in Parkinson’s disease.   MPTP is considered as the gold standard for toxin based Parkinson Disease 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. Released MPP+ is selectively taken up into dopaminergic neurons by dopamine transporters (DAT) and causing mitochondrial dysfunction, Excitotoxicity, oxidative stress which finally induces apoptosis leads to damage to dopaminergic neurons ^[22].  Selegiline (Standard) pretreatment can protect neurons against a variety of neurotoxins, such as 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP), 6-hydroxydopamine, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4), methyl-beta-acetoxyethyl-2-chloroethylamine (AF64A), and 5,6-dihydroxyserotonin, which damage dopaminergic, adrenergic, cholinergic, and sertoninergic neurons, respectively. Selegiline produces an amphetamine-like effect, enhances the release of dopamine, and blocks the reuptake of dopamine. Selegiline reduces the production of oxidative radicals, up-regulates superoxide dismutase and catalase, and suppresses nonenzymatic and iron-catalyzed autooxidation of dopamine. Also causes over activation of NMDA receptor exerts excitotoxicity. Over activation of NMDA along with other glutamate / glycine receptors disturb the calcium homeostasis, which is the key mediator if glutamate – induced excitotoxicity neuronal damage. Selegiline, diminishes potentiation of the NMDA receptors by the polyamine binding site^[23] hence Selegiline was used as standard.  Rotenone generates an experimental animal model of Parkinson disease that Mimics and elicits Parkinson like symptoms such as muscular rigidity, bradykinesia ,postural instability ,unsteady gait and sleep disturbances . Due to its high lipophillicity, rotenone cab readily cross Blood brain barrier and enter all cells without being dependent on specific transporter . Application of low doses of rotenone in vitro and in vivo have been shown to affect many of the mechanisms involves in Pathogenesis of Parkinson Disease, like altered calcium signalling , induction of oxidative stress and apoptosis , loss of tyrosine hydroxylase, proteasomal dysfunction,nigral iron accumulation. Since rotenone, a complex I inhibitor, can cause many of the pathological features of PD in rats and complex I dysfunction has been associated with PD in humans. Rotenone used as agent for PD model due to inhibit mitochondrial complex I (c-I), decreasing endogenous antioxidants and generating oxidative stress from complexes I and III (c I, c III, respectively) which leads to oxidation of macromolcules. Additionally, cytochrome c (Cyt c) is released from the intermitochondrial space, activating caspase signaling and subsequent apoptotic cell death^[24] .  Zebrafish is becoming an increasingly attractive model organism for understanding biology and developing therapeutics, because as a vertebrate, it shares considerable similarity with mammals in both genetic compositions and tissue/organ structures, and yet remains accessible to high throughput phenotype-based genetic and small molecule compound screening. Zebrafish models have significantly contributed to our understanding of vertebrate development and, more recently, human disease ^{[24 ]} In the present study, results obtained from MPTP induced Parkinson’s disease in mice showed improvement in locomotor activity, decrease in muscle rigidity, neuromuscular strength was improved in Parkinson’s disease when treated with Ethanolic extract of flax lignan (EEFL) as compared to standard drug Selegiline and disease group induced with Parkinson’s disease using MPTP. Biochemical estimations done on 8^th day of in-vivo study sacrificed animals brains, it was observed increase in glutathione activity, also prominent restoring activity of dopamine levels and catalase levels were observed. Rotenone is a pesticide, insecticide, etc. Application of low doses of rotenone in vivo have been shown to affect many of the mechanisms involved in pathogenesis of pd ,such as altered calcium signalling , induction of oxidative stress and apoptosis, loss of tyrosine hydroxylase nigral iron accumulation formation of fibrillary cytoplasmic inclusions . Other studies have also shown that exposure to Rotenone induced behavioural and motor deficits , which are similar to human PD, including muscle rigidity , bradykinesia , postural instability, unsteady gaits and sleep disturbances. In Rotenone model of Zebra fish for Parkinson’s disease the study of behavioral parameters like latency to travel from one point to another, time spent near the bottom of tank, complete immobility time, erratic swimming were found to be increased significantly (p<0> Patients with PD usually present with features indicative of degeneration of nigrostriatal pathways. A useful clinical definition for PD is ‘‘asymmetric onset of an akinetic (bradykinetic) rigid syndrome with resting tremor and a good response to levodopa’’. When applied by neurologists with an interest in movement disorders, this definition has a pathological correlation exceeding 98%.4 The use of dopaminergic drugs improves motor function, significantly reduces both the morbidity and mortality of PD, and improves quality of life. Levodopa remains the drug most commonly used in PD. It is very effective in improving bradykinesia and rigidity, and in practice remains the gold standard against which other drugs are judged. Some studies, predominantly in vitro, have suggested that levodopa. Current therapy options for PD remain focussed on the symptomatic improvement of motor features related predominantly to loss of dopaminergic neurones in the substantia nigra. Such treatment is effective in improving morbidity and mortality^[24]. Thus, the above research is based on the basis where recent insights into the aetiology, pathology, and pathogenesis of PD are providing important opportunities to develop disease modifying therapies that will have an even greater impact on the disease than did the introduction of levodopa

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