Pharmacological, Biochemical and Molecular Investigations in 6-OHDA Induced Parkinson’s Disease Rats on the Protection Benefits of Mangiferin in Behavioral, Inflammatory and Oxidative Biomarkers

Background: Persistent up regulation of NF-κB leads to chronic inflammation and subsequent microglial activation and takes neurons towards death by activating death receptor domains and the p53 pathway. Thus, inhibition of NF-κB may lead to more effective treatment for Parkinson’s disease. Therefore, we have used mangiferin, specific inhibitor of NF-κB in this study. Method: The study utilized male Wistar rats weighing 200-250 gm (n=8 in each group). Stereotactic surgery of rats was done to induce 6-OHDA lesioning in rats. On day 42, rats were subjected to behavioural studies to evaluate effect of mangiferin and their brains were taken out after euthanasia to perform biochemical and molecular studies. Results: Mangiferin significantly increases locomotor parameters in 6-OHDA lesioned rats. It also decreases activity of Cyclooxygenase enzyme which then leads to decrease concentration of inflammatory cytokines. Microglial inflammation was also substantially reduced by reducing MPO concentration. Oxidative stress burden was also reduced after treatment with mangiferin as indicated by increase in Total Antioxidant Capacity, SOD and Catalase and reduction in concentration of MDA. Treatment with mangiferin also reduces burden of oxidative stress by increasing the activity of NRF2/ARE pathway. Activity of Caspase 3 and 9 was also significantly reduced after treatment with mangiferin. Significant decrease in activity of both Cox1 and Cox 2 was also observed. Maximum improvement in all parameters was observed in rats treated with grouping of mangiferin 45mg.kg-1 and levodopa 10mg.kg-1. Treatment with levodopa alone has no significant effect on biochemical and molecular parameters though it significantly improves behavioural parameters. Conclusion and Implications: Results of this study suggest that mangiferin has protective effect in hemi-parkinsonian rats by inhibiting NF-κB. Current treatment of Parkinson’s disease does not target the underlying problem of the disease. Therefore, combination therapy of mangiferin and levodopa can be helpful in better management of Parkison’s.


Introduction
Parkinson's disease (PD) is a progressive locomotor disorder characterized by death of neurons in the nigrostriatal area of basal ganglia. Bradykinesia, muscular rigidity, rolling tremors in the pill, postural abnormalities and gait issues are some of the clinical features of PD [1], [2], [3], [4].
Though PD is an idiopathic disorder with its etiopathology not fully known, after decades of research, researchers believe that dysregulation of transcription factors controlling inflammation is the key reason behind the advancement of PD and PD like symptoms.
Various studies have suggested that the expression of transcription factor NF-κB increases during inflammation. Increased expression of transcription factor NF-κB then results in downstream activation of Toll like receptors (TLRs) and Interleukin 1 Receptors (IL1R), which then triggers Myd88 gene to recruit Interleukin1 Receptor Activated Kinase 1 (IRAK1) to this receptor signaling complex for phosphorylation. After phosphorylation, IRAK1 form a complex with Tumor necrosis factor Receptor Associated Factor 6 (TRAF6).
All these factors then further increase the expression of NF-κB, which then regulates apoptosis through the nuclear buildup of RelA. Nuclear translocation of RelA then increases the membrane permeability of mitochondria by chaperon mediated activation of Bax onto the outer membrane of mitochondria and inhibiting the anti-apoptotic protein Bcl-XL [18].
In recent years, research interest in natural compounds has been revivified owning to their slighter toxic effect as compared to chemical compounds. In our study, we have used polyphenolic compound Mangiferin obtained from plants belonging to Anacardiaceae and Gentianaceae family.
Thus, owing to its anti-inflammatory and anti-oxidant effect, mangiferin could be useful in pharmacotherapy of PD to rescue the neurons from cell death pathways.

Animals:
Male wistar rats (200-250 gm) were used in the study. Rats (n=8/group) were kept in the

Induction of Parkinson's disease in Rats
6-OHDA lesions were performed in rats using stereotactic frame. Rats were anesthetized with ketamine-xylazine (ketamine 60mg.kg -1 and xylazine 7.5mg.kg -1 ) cocktail before placing them in to stereotactic chamber. Top of rat's head was shaved and cleaned through 70% ethanol. Stereotactic coordinates for substantia nigra region was then determined using Paxinos and Watson, The Rat Brain in Coordinates [39]. For substantia nigra pars compacta (SNpc) (dorsal part) lesion coordinates in reference to bregma were anteroposterior (A/P) -2 mm; mediolateral (M/L) -5 mm and dorsoventral 8.2 mm. A midline incision was done and lambda and bregma were identified at the intersection of coronal and saggital sutures. A burr hole was then drilled in to rat brain using these coordinates. After hole was drilled, rats were cannulated with a 20 gauge cannula. Length of the cannula was kept at 8 mm. After implanting the cannula, it was then fixed using denture material.
Proposed treatment from mangiferin (15μg, 30μg and 45μg) was done for 28 days, from day 14 to day 42. After treatment with mangiferin, rats were subjected to behavioural studies to evaluate effect of mangiferin on locomotor changes in 6-OHDA lesioned rats. After completion of study rats were sacrificed with high dose of anaesthesia (pentobarbital 100mg.kg -1 ) and their brain were taken out to perform biochemical and molecular studies.

Animal Activity Meter: Opto-Varimex-5 Auto-Track
Opto-Varimex5 (Columbus Instruments, Columbus, Ohio, USA) is modern software that helps in quantification of locomotive parameters such as total distance travelled (cm), average speed (cm.s -1 ), total ambulatory time (s), resting time (s) and stereotypic time

(s).Any-maze Video Tracking System
Rat's behavioural and locomotive activity was evaluated utilizing Any-Maze video tracking system of Stoelting, USA. Rat was placed for a short time in an open field corner and the required parameters, including the distance travelled; average speed; freezing duration and episodes of freezing were chosen through Any-maze software and were recorded with the aid of over-mounted camera movement of rats.

Cylinder Test
Cylinder test assesses lop-sidedness in locomotor activity in rodents. This test measures the forelimb activity of rodents inside an open-top transparent plastic cylinder. Cylinder test assesses lop-sidedness in locomotor activity in rodents. This test measures the forelimb activity of rodents inside an open-top transparent plastic cylinder. The magnitude of forelimb activity of rats was measured when the rat places its entire paw on the cylinder wall for body support while rearing. A total of 20 such forelimb contacts were, measured for each rat. The numbers of impaired and non-impaired forelimb contacts were calculated as a percentage of total contacts [40].

Grip Strength Meter
Forelimb grip strength was measured using Grip Strength Meter (Columbus Instruments, USA).

Cook's Pole Climbing Test
This test was used to determine the latency to climb the pole by 6-OHDA lesioned rats. In pole climbing test, rats are accustomed to climb onto the pole to steer clear of the shock. A tone of 50 Hz and current of 1mili ampere was passed onto wooden floor to condition the rats which is then succeeded by current of zero amperes as unconditioned stimuli. Time taken by the rats to climb the wooden pole (shock free zone) in the middle of instrument was then recorded [41].

Stepping Test
The stepping test was used to assess the contralateral forepaw's initiation deficiency. In this test, the number of adjustment steps taken by rats was recorded while rats travel sideways on a 60 cm wide flat surface with one of his forelimb being restrained [42].

MDA levels in brain tissue homogenate
After completion of the study animals were euthanized (pentobarbitone sodium 100mg.kg -1 i.p) and their brain was isolated and homogenized. The homogenate thus obtained was assayed for the MDA concentration by the method of Ohkawa et al [43] and was expressed as nmol.mg-1 protein. Protein estimation was done by using method of Lowry et al [44].

Myeloperoxidase assay in brain tissue homogenate
Myeloperoxidase (MPO) activity was evaluated in to assess microglial activity as described by Barone et al. 1992 [45]. Rats were sacrificed by high dose of pentobarbitone sodium 100mg.kg -1 i.p and brain was taken out and homogenized. Supernatant thus obtained was then assayed for MPO at 460 nm and was represented in mU.gm -1 weight of wet tissue.

SOD activity in brain tissue homogenate
Analysis of SOD activity in brain tissue homogenates was done by the method of Marklund and Marklund [46] and expressed in U.gm -1 of protein. Protein estimation was done by Lowry's method [44].

Catalase assay in brain tissue homogenate
Catalase activity in rat brain tissue homogenate was determined as per method described by Sinha et al. 1972 andAebi et al. 1974 and was expressed as U.mg -1 of protein [47], [48].

Total antioxidant capacity assay
Evaluation of Total antioxidant capacity in striatum was done by ferric reduction antioxidant power (FRAP) assay [49].

Th1/Th2 Cytokine assay
Solid phase sandwich ELISA kits obtained from Diaclone, France were used in Th1 (IFN-γ) and Th2 (IL-4) cytokine assay. In this assay, monoclonal antibody specific for rat IL-4 and IFN-γ were coated on to the wells of the micro titer strips. Antigen and antibodies were then incubated simultaneously at 37 ºC for 1 hr. Streptavidin horseradish peroxidase and chromogen TMB (3, 3', 5, 5;-tetramethylbenzidine) were used in the revelation step. Rest of the protocol was followed as described in the assay kit. The plates were read on Microscan-5405A (ECIL) and results were expressed in pg/ml.

Pro-inflammatory (TNF-α, IL-1β, IL-4 and IL-6) cytokines estimation
At the end of study all animals were sacrificed using high dose of anesthesia and their brain was isolated and homogenized. Tissue levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-4 and IL-6was determined using commercially available enzyme linked immunosorbent assay (ELISA) kits from Cloud Clone Corp and was represented as pg.μg -1 tissue.

NF-κB estimation
At the end of study, rats were sacrificed using high dose of anesthesia and their brain was isolated and homogenized in 10% PBS. NF-κB was then determined in brain tissue homogenates using commercially available ELISA kit from Cloud Clone Corp and results were expressed in pg.μg -1 tissue.

Caspase-3 activity
Caspase-3 activity was assayed using fluorometric assay system (Biovision, USA) brain tissue homogenates as per the manufacturer's protocol. Final reading was taken at 360/460 nm and was expressed in μmol.mg -1 of protein.

Caspase 9 activity
Caspase-9 activity was calculated using the ready to use fluorometric assay system (BioVisionInc, USA). Aliquots of the brain tissue homogenate were re-suspended in lysis buffer and subjected to further homogenization and were assayed for Caspase 9 activity as per the manufacturer's protocol.Final reading was taken at 400/505nm respectively and expressed in μmol.mg -1 of protein.

Cyclooxygenase Activity
Cyclooxygenase (Cox1 and Cox 2) activity was measured in rat brain tissue homogenates using Cox-1 and Cox-2 ELISA kit from CUSABIO Houston, Texas, USA.

Statistics
The data obtained was analyzed by two-way ANOVA followed by Newman Keul posthoc test for multiple group analysis by using Graph Pad Prism 6.0. The p value <0.05 was considered as significant in all parameters. The data was analyzed and represented as Mean ± SEM.

Effect of mangiferin on ambulatory, stereotypic and resting time in 6-OHDA lesioned rats
Ambulatory, stereotypic and resting times were evaluated in rats after 42 days of 6-OHDA

Effect of Mangiferin on 6-OHDA induced changes in track plot of rats
Heat Map of hemi-parkinsonian rats also showed that locomotor activity was markedly suppressed after 6-OHDA lesioning. Daily treatment with mangiferin (15-45μg) for 28 days extensively improves locomotor activity which was conspicuous through their heat map.
Treatment with levodopa 10 mg.kg -1 alone and in combination with mangiferin 45μg also significantly improves locomotor activity as evident through their heat map (Fig. 3a-3g).

Effect of mangiferin on sensorimotor forelimb function in 6-OHDA lesioned rats
Sensorimotor forelimb function was measured in 6-OHDA induced hemi-parkinsonian rats to reckon the asymmetry of forelimb function. Significant diminution in spontaneous use of contralateral front paw was observed in rats after lesioning with 6-OHDA. Upswing in the contralateral forelimb use was observed in lesioned rats after treatment with mangiferin (15-45μg) on daily basis for 28 days. Treatment with levodopa 10 mg.kg -1 also spurts contralateral forelimb use in parkinsonian rats. Significant upturn in the use of contralateral forelimb was also observed in 6-OHDA lesioned rats treated with combination of levodopa 10 mg.kg -1 and mangiferin 45μg) (F (8, 18) 20.70; = p<0.0001) (Fig. 4a).  (Fig. 4b).

Effect of mangiferin on cook's pole climbing test in 6-OHDA lesioned rats
6-OHDA lesioning significantly increases the time to climb the pole as compared to rats in which sham surgery was performed. Daily treatment with mangiferin (15-45μg) for 28 days significantly decreases the time to climb the pole in hemi-parkinsonian rats. Significant decrease in time to climb the pole was observed in rats treated with levodopa 10 mg.kg -1 .

Effect of mangiferin on forelimb akinesia in 6-OHDA lesioned rats
Forelimb akinesia in 6-OHDA lesioned rats was assessed through stepping test. 6-OHDA lesioning significantly decreases the number of adjusting steps of contralateral forelimb which is suggestive of forelimb akinesia in rats. Treatment with mangiferin (15-45μg) significantly decreases forelimb akinesia by increasing number of adjusting steps taken by contralateral forelimb in lesioned rats. Substantial decrease in forelimb akinesia was also observed in rats treated with levodopa 10 mg.kg -1 alone and in combination with mangiferin 45μg (F (8, 18) = 2.556; p<0.0001) (Fig. 4d).

Effect of mangiferin on myeloperoxidase activity in 6-OHDA lesioned rats
6-OHDA lesioning in rats leads to neuro-inflammation and microglial activation.

Effect of mangiferin on SOD and Catalase activity in 6-OHDA lesioned rats
SOD activity was measured to estimate oxidative stress in 6-OHDA lesioned rats. SOD activity was observed to be significantly reduced in hemi-parkinsonian rats. Mangiferin (15-45μg) treatment for 28 days significantly increases SOD activity in hemi-parkinsonian rats.
Significant decrease in catalase activity was observed in rats after 6-OHDA lesioning.

Effect of mangiferin on 6-OHDA induced changes in Th1 and Th2 cytokine assay
It was observed that 6-OHDA lesioning significantly increases Th1 and Th2 cytokines levels.

Discussion
Levodopa is the mainstay of current pharmacotherapy of PD; however, owing to its autooxidant property, it could be a necessary evil. Thus, due to its auto-oxidant nature, levodopa fails to stop the disease progression. Due to its auto-oxidant nature levodopa promotes the disease progression by increasing oxidative stress and stimulating TNF-α secretion which then leads to neuro-inflammation and subsequent death of dopaminergic neurons [12,50], [51]. Therefore, in this study we have tried to evaluate neuro-protective effect of NF-κB On the expected lines, treatment with mangiferin significantly reduces the reaction time in cook's pole climbing test due to increased motor activity in hemi-parkinsonian rats.
Maximum increase in this motor activity was observed in rats treated with combination of mangiferin 45μg and levodopa 10mg.kg -1 .
Mangiferin also attenuates 6-OHDA induced sensorimotor forelimb function and forelimb akinesia in hemi-parkinsonian rats. Maximum increase in these gait analysis parameters was observed in rats treated combinative therapy of mangiferin 45μg and levodopa 10mg.kg -1 .
Treatment with mangiferin 15-45μg significantly reduces oxidative stress by decreasing ARE pathway. Mangiferin promotes the nuclear translocation of NRF2 which thus results in increased nuclear expression of NRF2. Furthermore, treatment with mangiferin also up regulates the expression of NQO1 and promotes the binding NRF2 with NQO1-ARE complex. Increased expression of NRF2 then stimulates the expression of anti-oxidant enzymes such as SOD and Catalase and increases the total anti-oxidant capacity of 6-OHDA lesioned rats [52].
Treatment with levodopa has no significant effect on these oxidative stress markers due to its auto-oxidant property. Metabolism of levodopa increases oxidative stress burden by generating free radicals and molecules like H2O2. It also reacts with iron present in brain to increase oxidative stress.
Myeloperoxidase activity which measured index of inflammation and microglial activation was significantly reduced by treatment with mangiferin 15-45μg due to its property of inhibiting pro-inflammatory cytokine TNF-α and COX pathway. Mangiferin substantially reduces the transcript activity of both Cox-1 and Cox-2, by inhibiting the nuclear translocation of NF-κB, which is crucial for expression of pro-inflammatory factors such as Cox-1, Cox-2 and TNF-α.
Levodopa has no significant effect on myeloperoxidase activity as it promotes inflammation by stimulating TNF-α and IL-4 secretion.
Significant decrease in concentration of NF-κB was observed in striatum after treatment with mangiferin as it is a specific inhibitor of NF-κB. This decrease in NF-κB activation significantly reduces neuro-inflammation as NF-κB is considered as 'master regulator' of inflammation. After activation, NF-κB promotes activation of Toll like receptors 4 (TLR4) which in turn promote NF-κB activation thus promoting nefarious cycle of inflammation.
Mangiferin stops this cycle of inflammation by inhibiting NF-κB activation.
Balance between Th1/Th2 cytokines plays crucial role in inflammation. GATA 3 and T-bet control the Th1/Th2 differentiation. GATA-3 is regulator of Th2 while T-BET regulates the Th1expression. Increased expression of NF-κB promoted the GATA-3 mRNA and thereby increases the protein expression of GATA-3, while inhibiting the T-bet expression. This causes an imbalance between GATA-3 and T-bet which then results in Th1/Th2 imbalance nad increased expression of Th2 cytokine. Treatment with Mangiferin restores the imbalance between Th1/Th2 cytokine [53], [54] Significant increase in activity of caspase-3 and caspase-9 was observed in 6-OHDA lesioned rats which indicate programmed death of dopaminergic neurons. Induction of p53 causes an activation of NF-κB that correlates with the ability of p53 to induce apoptosis. Inhibition or loss of NF-κB activity abrogated p53-induced apoptosis, indicating that NF-κB is essential in p53-mediated cell death. Activation of NF-κB by p53 was distinct from that mediated by tumour-necrosis factor-alpha and involved MEK1 and the activation of pp90rsk [55].
Treatment with mangiferin significantly reduces caspase-3 and caspase-9 activity due to its property of inhibiting pro-apoptotic transcription factor NF-κB and promotes TGF-β activity which has anti-inflammatory and proliferative activity [56]. Treatment with levodopa has no significant effect on caspase-3 and caspase-9 activity. Probably, this is the reason why disease progression goes unchecked after treatment with levodopa [25], [57] .

Conclusion
The findings of this research indicate that mangiferin has a dose-dependent protective effect in 6-OHDA-lesioned rats. Mangiferin efficacy is amplified when combined with levodopa 10 mg/kg, which is the cornerstone of anti-parkinsonism treatment. Thus, we may conclude that the combination of mangiferin with levodopa may have therapeutic benefit in Parkinson's disease therapy on our results.

Ethics Approval
Before starting the study, necessary approval from the Institutional Animal Ethics Committee Availability of Data and Materials