|Year : 2018 | Volume
| Issue : 2 | Page : 53-58
Isolongifolene Attenuates Oxidative Stress and Behavioral Impairment in Rotenone-Induced Rat Model of Parkinson’s Disease
Rengasamy Balakrishnan, Kuppusamy Tamilselvam, Ahmedsha Sulthana, Thangavel Mohankumar, Dharmar Manimaran, Namasivayam Elangovan
Department of Biotechnology, School of Biosciences, Periyar University, Salem, Tamil Nadu, India
|Date of Web Publication||26-Apr-2018|
Department of Biotechnology, School of Biosciences, Periyar University, Periyar Palkalai Nagar, Salem - 636 011, Tamil Nadu
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Introduction: Parkinson’s disease (PD) is a progressive age-related disease, in which dopaminergic neurons in the nigrostriatal pathway are destroyed, resulting in movement and behavioral impairment. Oxidative stress and the generation of reactive oxygen species play a key role in the progression and pathology of neurodegenerative diseases such as PD. Rotenone is a common pesticide that induces PD through the generation of oxidative stress. Isolongifolene (ILF), a tricyclic sesqueterpene of Murraya koenigii, has antioxidant and neuroprotective effects. The current study was aimed to investigate the effect of ILF against oxidative stress and movement impairment on rotenone-induced rat model of PD. Materials and Methods: Biochemical measures, including the activities of catalase, glutathione peroxidase (GPx), superoxide dismutase (SOD) and the levels of reduced glutathione (GSH) and lipid peroxidation products [thiobarbituric acid reactive substances (TBARS) and behavioral analysis (hang and catalepsy test) were performed. Results: The muscle strength and cataleptic score of the ILF co-treated groups were significantly improved. Treatment with ILF prevented the increases in the levels of TBARS, significantly improved the SOD, catalase, GPx activities, and GSH levels. Conclusion: These findings suggested that ILF has neuroprotective properties through its potent antioxidant activities.
Keywords: Behavioral impairments, isolongifolene, oxidative stress, Parkinson’s disease, rotenone
|How to cite this article:|
Balakrishnan R, Tamilselvam K, Sulthana A, Mohankumar T, Manimaran D, Elangovan N. Isolongifolene Attenuates Oxidative Stress and Behavioral Impairment in Rotenone-Induced Rat Model of Parkinson’s Disease. Int J Nutr Pharmacol Neurol Dis 2018;8:53-8
|How to cite this URL:|
Balakrishnan R, Tamilselvam K, Sulthana A, Mohankumar T, Manimaran D, Elangovan N. Isolongifolene Attenuates Oxidative Stress and Behavioral Impairment in Rotenone-Induced Rat Model of Parkinson’s Disease. Int J Nutr Pharmacol Neurol Dis [serial online] 2018 [cited 2018 Oct 20];8:53-8. Available from: http://www.ijnpnd.com/text.asp?2018/8/2/53/231270
| Introduction|| |
Parkinson’s disease (PD), a second most common neurodegenerative disease, is characterized by the slow and irreversible degeneration of nigrostriatal neurons that project from the substantia nigra (SN) to the striatum (ST). Following the progressive destruction of the dopaminergic neurons, the levels of dopamine in the ST are reduced, which results in motor and coordination impairments in patients with PD. Oxidative stress is thought to play critical roles in the pathogenesis and progression of PD. SN neurons are prone to oxidative stress because it has high levels of iron. Decreased levels of glutathione (GSH) and high amount of reactive oxygen species (ROS) production as a result of dopamine metabolism. However, the neuroinflammation and mitochondrial dysfunction that are observed in patients with PD increases the ROS levels in the SN, which results in apoptosis.
Rotenone is a pesticide and insecticide that is derived from the root and bark of the Derris and Lonchorcarpus species. Recently, rotenone has been used to induce in vitro and rodent model of PD. Rotenone conveniently crosses the blood brain barrier and inactivates complex I of the mitochondrial electron transport chain, which results in increased ROS production and causes neurodegeneration. Rats exposed to rotenone exhibit a number of pathological signs, such as the loss of dopaminergic neurons in the SNpc, striatal dopamine depletion, α-synuclein aggregation, and impaired balance and coordination. These pathological features are similar to the symptoms of human patients with PD.
Interest in the use of natural products in complementary medicine has recently increased. Medicinal plants contain a variety of bioactive molecules, such as flavonoids and alkaloids, which have potent antioxidant and anti-inflammatory properties that have been reported to reduce oxidative stress and inflammation by scavenging ROS. Isolongifolene (ILF) is tricyclic sesqueterpene that is found in essential oil from plant such as Murraya koenigii. The leaves of M. koenigii mainly contain carbazole alkaloids that are known to possess antioxidant and anti-inflammatory activities., Previous experiments reported that M. koenigii leaf extract ameliorates the cognitive deficit and protects against oxidative stress in the aged mice by reducing of the cholesterol levels and brain cholinesterase activity, possibly through the action of carbazole alkaloids. Therefore, the main objective of the present study was to investigate the effect of ILF on rotenone-induced oxidative stress and behavior impairments in the rat model of PD.
| Materials and Methods|| |
Adult male Wistar rats (225–250 g) purchased from the National Institute of Nutrition, Hyderabad, were used in the present study. The animals were kept under 12-h light/dark cycles, at 22°C and 60% humidity with food and water ad libitum. All the experimental procedures used in the present study were approved by the Committee of National Guidelines on the Proper Care and Use of Animals in Laboratory Research (Indian National Science Academy, New Delhi, 2000) and approved by Albino Research & Training Institute (Reg. No. 1722/RO/Ere/S/13/CPCSEA, Proposal No. ARTI/CBCSEA/2015/ARTI 20).
Rotenone, ILF, Thiobarbituric acid (TBA), GSH, 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) were purchased from Sigma Chemical Company, Bangalore, India. All other chemicals were of analytical grade.
In the present study, safety guidelines for handling of rotenone as reported in the literature were followed. The rats were randomized and divided into four groups of six animals each.
Group I: Rats were injected with 0.5 ml sunflower oil i.p. served as control.
Group II: Rats were injected with rotenone intraperitoneally at 2.5 mg/kg b.w. (it was first dissolved dimethyl sulfoxide and diluted in sunflower oil to obtain a final concentration 2.5 mg/ml) for 35 days.
Group III: Rats were administered orally with ILF (10 mg/kg) dissolved in sunflower oil (1 h prior to each rotenone injection) for 35 days.
Group IV: Rats were administered orally with ILF (10 mg/kg) was dissolved in sunflower oil for 35 days.
After the end of the experimental period (36th day), hang test was performed and SN from control and experimental rats were dissected for the estimation of biochemical analysis.
Motor functional analysis
Hang test: The effect of ILF on neuromuscular strength was analyzed by grid hang test. Briefly, animals were placed on a horizontal grid and supported until they held the grid firmly with all their four paws. The grid was then kept in an inverted position allowing the animals to hang upside down and the maximum hanging time was noted. Proper care was taken to prevent injury/damage to animals in case of falling. Maximum latency time was fixed as 300 s.
Catalepsy test: To test catalepsy, using bar test, rats were positioned such that their hind limbs were on the base and their forelimbs rested on a wooden bar 9 cm above from the base. The rats were placed with both the front paws on the bar in half rearing position; and they were timed with stopwatch. When the animals removed one paw from the bar the stopwatch was stopped and the time noted. The maximum cutoff for bar test was fixed at 180 s.
Lipid peroxidation: The thiobarbituric acid reactive substances (TBARS) level was determined as previously described Selvakumar et al. SN tissue extracts were incubated with 1:1:1 ratio of 0.37% TBA, 15% trichloroacetic acid and 0.25 N hydrochloric acid. The reaction mixture was centrifuged at 4000 rpm for 15 min, and supernatant was collected and boiled in water bath for 15 min. After cooling, the clear supernatant was measured at 535 nm spectrophotometrically and unit was reported in μmol of TBARS formed/mg protein.
Superoxide dismutase: Superoxide dismutase (SOD) activity was assayed based on the method of Selvakumar et al. In brief, the assay mixture contained of 960 L of 50 mM sodium carbonate buffer (pH 10.2) containing 0.1 mM xanthine, 0.025 mM NBT, and 0.1 mM EDTA, 20 L of xanthine oxidase and 20 L of the supernatant. Changes in absorbance were observed spectrophotometrically at 560 nm. The activity of SOD is expressed as units/min/mg protein.
Catalase: Catalase activity was measured based on the ability of the enzyme to break down hydrogen peroxide. The reaction mixture consisted of 50 L of 1 M Tris–HCl buffer (pH 8.0) containing 5 mM EDTA, 900 L of 10 mM H2O2, 30 L of MQ water, and 20 L of the tissue supernatant. The rate of decomposition of H2O2 was observed spectrophotometrically at 240 nm. The enzyme activity was calculated as nmol of hydrogen peroxide decomposed/min/mg protein.
Glutathione peroxidase: Glutathione peroxidase (GPx) activity was assayed by using the method of Anandhan et al. 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 supernatant. Oxidation of NADPH was determined spectrophotometrically at 340 nm. One unit of activity was defined as the amount of GPx required to oxidize 1 mol of NADPH per min.
Reduced glutathione: The level of reduced GSH in the tissue homogenate was measured by the method described by Anandhan et al. Briefly, tissue homogenate was centrifuged at 16,000×g for 15 min at 40°C. The supernatant (0.5 ml) was added to 4 ml of ice-cold 0.1 mM solution of DTNB in 1 M phosphate buffer (pH 8). The optical density was read at 412 nm in a spectrophotometer.
Statistical significance was evaluated by one-way analysis of variance using the Statistical Package for the Social Sciences version 16.0 software (SPSS Inc., Chicago, IL, United States), and individual comparisons were obtained using Duncan’s multiple range test (DMRT). All data are expressed as mean ± SD for six rats in each group. Values were considered statistically significant if P < 0.05.
| Results|| |
Hang test: The neuromuscular strength was observed by hang test as shown in [Figure 1]; the average hanging time of rotenone was administered, which was evidenced by the inability of rat to hold the grid when compared with the control rat. Moreover, pretreatment with ILF to rotenone significantly improved the hanging ability in group III rat when compared with rotenone alone-treated rat (P < 0.05). No significant changes observed between ILF alone treated animal and control animal (P < 0.05).
|Figure 1: Shows the effect of ILF on rotenone-induced reduction in neuromuscular strength in hang test. Animals were allowed to hang on the grid and the bestfall values were recorded. Values are given as mean ± SD for six animals in each group. P < 0.05, compared with the control group; P < 0.05, compared with the rotenone group|
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Catalepsy test: As shown in [Figure 2] the cataleptic behavior of rotenone treated rats was found to significantly increase latency in when compared to control animals. Co-treatment with ILF significantly reduced cataleptic score when compared to rotenone-treated group (P < 0.05). No significant changes were observed between ILF-alone-treated animal and control animal (P < 0.05).
|Figure 2: The assessment of ILF on rotenone-induced catalepsy test control and experimental groups. Animals were allowed to latency of movement or maintain imposed posture in bar values were noted. Values are given as mean ± SD for six animals in each group. Values not sharing a common superscript letter differ significantly at P < 0.05 (DMRT)|
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The effect of ILF on the levels of lipid peroxidation products and antioxidant status
To explore the antioxidant effect of ILF on rotenone-induced oxidative stress, the levels of TBARS and GSH and activities of SOD, catalase, and GPx in the SN of control and experimental rats were measured. On the one hand, [Figure 3] showed the rat-administered with rotenone caused a significant increase in the TBARS levels and decrease in the levels of GSH and the activities SOD, catalase, and GPx as compared to control group (P < 0.05), but on the other hand, ILF co-treatment significantly and dose dependently attenuated the rotenone-induced oxidative stress by decreasing the level of TBARS and restoring the levels and activities of antioxidants (P < 0.05). Interestingly, rats treated with ILF alone did not show significant alterations in the levels and activities of oxidative and antioxidative indices as compared to that of control rats.
|Figure 3: Deficits the effect of ILF on rotenone-induced oxidative stress in control and experimental group. Values are given as mean ± SD for six animals in each group. Values not sharing a common superscript letter differ significantly at P < 0.05 (DMRT). A = enzyme required for 50% inhibition of NBT reduction. B = μmol of H2O2 utilized /min. C = μmol of glutathione utilized/min|
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| Discussion|| |
This model mimics many of the neuropathological features of human PD, such as motor dysfunction that is directly related to dopaminergic cell lesions. In this current study, we used the four-limb hang test and latency in cataleptic test, which is a reliable test to determine the effects of potential therapeutic compounds on muscle strength to evaluate the motor skills of the animals. The result of the current study, the impaired motor coordination of muscle strength and cataleptic behavior below the baseline value in the rotenone model group which confirmed that the loss of dopaminergic cells in the SN as has been shown previously. Motor dysfunctions in rat PD models are attenuated by antioxidant supplements. The results of our study showed that ILF improved motor dysfunction in muscle weakness and catalepsy that were induced by rotenone may be due to its potent antioxidative properties.
Brain is more vulnerable to oxidative stress as compared to other tissues, because (i) high metabolic activity, (ii) utility of more oxygen, (iii) auto-oxidation of DA, its metabolites and its precursors to form semiquinones and quinones able of adducting sulfhydryl groups of protein, including GSH, and (iv) presence of enhanced iron levels in the SN leading to the reduction of H2O2 to form the highly reactive hydroxyl radical (.OH) (Fenton chemistry). Lipid peroxidation is the process of oxidative destruction of PUFA and its occurrence in membranes causes impaired structural integrity and membrane function, inactivation of numerous membrane bound enzymes and decreased fluidity. Oxidative stress generated by free radicals and subsequent lipid peroxidation plays an important role in PD pathogenesis. During oxidative injury, accumulation of oxidants makes the cell membrane more susceptible to injury and results in formation of the lipid peroxidation product, TBARS. The brain has less efficient antioxidant defense mechanisms and is rich in polyunsaturated fatty acids, so it is more sensitive to oxidative damage than other tissue. The extent of lipid peroxidation processes was measured by quantifying the levels of TBARS, collective products of lipid peroxidation. Bashkatova et al. showed increased NO and TBARS levels in the ST and cortex following the chronic rotenone treatment, which corroborates with our study. Increased production of ROS during NDDs is an indication of the oxidative stress and leads to a rapid consumption of endogenous scavenging antioxidants.
Endogenous antioxidant defense networks consist of enzymatic (SOD, catalase, and GPx) and non-enzymatic (GSH) molecules that neutralize the oxygen-free radicals that lead to oxidative stress, if the antioxidant system is compromised.,, Perturbation of antioxidant defense system components such as GSH, SOD, catalase, and GPx has been well documented in the PD brain.,, The decrease in GSH content following rotenone administration in the current study likely occurred to minimize the deleterious consequence of oxidative stress. A fault in individual or more endogenous antioxidant molecules particularly GSH is an important factor in the etiology of PD. Sian et al. described about 40% reduction in the levels of GSH in SN of the patients with PD as compared to control subjects. This may impair H2O2 clearance and enhance hydroxyl radical formation leading to the generation of pro-oxidant conditions. However, following ILF treatment to rotenone-administered rats, significant recovery or restoration of GSH levels clearly demonstrates the antioxidant and free radical scavenging activity of ILF.
Rotenone treatment reduced the activities of enzymatic antioxidants, which are in consistent with previous studies., SOD is a ubiquitous enzymatic antioxidant, present in most tissues and being one of the key antioxidants in the Central Nervous System (CNS) together with catalase and GPx. Diminished activity of SOD in the rotenone treated rats indicated inactivation of SOD by ROS and leads to the scenario of increased superoxide radical production. An increase in oxidative damage is often correlated with a simultaneous decline in the activity of the intracellular antioxidant enzymes, SOD, catalase, and GPx. Following rotenone administration, a significant reduction in the activities of nigral SOD, catalase, and GPx was observed in our study. Low activity of SOD, catalase, and GPx in rotenone-treated rats may result from the inactivation of the enzymes by H2O2. Enhanced superoxide radicals spontaneously dismutated to form H2O2. Elevated levels of H2O2 could lead to depletion of their metabolising enzymatic antioxidants such as catalase and GPx. ILF offered neuroprotective effect by reducing the levels of lipid peroxidation and augmenting the activities of antioxidant enzymes,, which is consistent with our study.
Our results showed that lipid peroxidation resulting from oxidative stress in the brain was clearly increased after the rotenone challenge, in agreement with previous studies., The decrease in TBARS levels following treatment with ILF can be ascribed to the enhanced activity of antioxidant defense mechanisms, as evidenced by increased activity of antioxidant enzymes and increased GSH availability. However, after administration of ILF and rotenone, the significant improvement in the activity of SOD, catalase, and GPx demonstrates the antioxidant activity of ILF. Consistent with previous observations, our present findings suggest that the neuroprotective action of ILF can be attributed to its direct free radical quenching properties or augmentation of antioxidant enzymes. ILF has been shown to protect dopaminergic neurons in vitro, an effect that was attributed to its free radical scavenging activity.
| Conclusion|| |
Based on our previous and present study findings, we conclude that ILF impedes rotenone-induced dopaminergic neurodegeneration by restoration of the inhibition of lipid peroxidation, antioxidant system, mitochondrial dysfunction, and apoptosis.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]