International Journal of Nutrition, Pharmacology, Neurological Diseases

ORIGINAL ARTICLE
Year
: 2013  |  Volume : 3  |  Issue : 3  |  Page : 294--302

Antioxidant and anti-inflammatory potential of hesperidin against 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine-induced experimental Parkinson's disease in mice


Kuppusamy Tamilselvam1, Jagadhesan Nataraj1, Udaiyappan Janakiraman1, Thamilarasan Manivasagam1, Mustafa Mohammed Essa2,  
1 Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Tamil Nadu, India
2 Department of Food Science and Nutrition, CAMS, Sultan Qaboos University, Muscat, Oman

Correspondence Address:
Thamilarasan Manivasagam
Department of Biochemistry and Biotechnology, Annamalai University, Annamalainagar 608 002, Tamil Nadu
India

Abstract

Background : There is mounting evidence that flavonoid consumption is potentially beneficial to those suffering from neurodegenerative diseases, cardiovascular disease, and cancer. These beneficial properties are largely attributed to their medicinal values. Objectives: In this study, we evaluated the neuro-protective effect of black hesperidin against 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) induced behavioral deficits, oxidative stress, and inflammation. Results: Behavioral analyses showed that hesperidin ameliorates MPTP-induced motor dysfunction. Elevated level of thiobarbituric acid reactive substances and enhanced activities of superoxide dismutase and catalase with deprived levels of reduced glutathione and activities of glutathione peroxidase in MPTP group was attenuated significantly in hesperidin-treated group. Administration of MPTP-induced glial activation observed by primary marker Glial Fibrillary Acidic Protein increased the release of pro-oxidant Cyclooxygenase - 2 and inflammatory cytokines such as Interleukin-1β, Tumor necrosis factor-α, IL-6, IL-4, and IL-10 in striatum and substania nigra. Treatment of hesperidin significantly protects microglia activation and reduces the release of inflammatory cytokines proving the anti-inflammatory effect of hesperidin. Conclusion: These findings suggest that hesperidin partially attenuates MPTP-induced neurotoxicity through its antioxidant and anti-inflammatory properties.



How to cite this article:
Tamilselvam K, Nataraj J, Janakiraman U, Manivasagam T, Essa MM. Antioxidant and anti-inflammatory potential of hesperidin against 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine-induced experimental Parkinson's disease in mice.Int J Nutr Pharmacol Neurol Dis 2013;3:294-302


How to cite this URL:
Tamilselvam K, Nataraj J, Janakiraman U, Manivasagam T, Essa MM. Antioxidant and anti-inflammatory potential of hesperidin against 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine-induced experimental Parkinson's disease in mice. Int J Nutr Pharmacol Neurol Dis [serial online] 2013 [cited 2021 Oct 16 ];3:294-302
Available from: https://www.ijnpnd.com/text.asp?2013/3/3/294/114875


Full Text

 Introduction



Parkinson's disease (PD), first described by James Parkinson [1] as paralysis agitans, or the "shaking palsy," is an age-related second neurodegenerative disorder after Alzheimer's disease (AD). [2] The neuronal loss is slow and progressive, and the motor symptoms of the disease appear only after a given threshold of dopamine depletion (70-80%) in the corpus striatum (ST) and/or dopaminergic neuronal loss (50-60%) in substantia nigra has been reached. [3] The cause of PD is still unknown but aging, environmental factors, oxidative stress, neuroinflammation, and genetic factors may be involved in neurodegeneration. [4] Although the etiology of PD remains enigmatic, a better understanding of the pathophysiology of PD has come from the studies utilizing the neurotoxin, 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP), which produces a Parkinsonian syndrome in humans, primates, and rodents. The MPTP animal model is a useful model for the study of neurodegeneration in PD because it produces clinical, biochemical, and neuropathological changes similar to those observed in human PD. [5]

Considerable interest has been grown in the development of anti-parkinsonic drugs from natural origin. [6] Citrus fruits and their products are reported to have various medicinal properties and are widely consumed around the world. [7] Hesperidin is a phytoflavanone that exists abundance in citrus plants and can be isolated in large amounts from the peels of Citrus aurantium (bitter orange), Citrus sinensis (sweet orange) and Citrus unshiu (satsuma mandarin). [8] Hesperidin exerts numerous pharmacological functions such as antioxidant, anti-inflammatory, anti-hypercholesterolemic, and anti-carcinogenic actions. [9] It is also demonstrated that hesperidin implies its potential role in protecting neurons against various types of insults associated with many neurodegenerative diseases. [10] Therefore, the present study was aimed to evaluate the effect of hesperidin against subacute MPTP-induced behavioral, biochemical, and molecular changes in a mouse model of PD.

 Materials and Methods



Experimental animals and diet

Adult male C57BL/6 mice (25-30 g) were 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. The experimental protocols met with the National Guidelines on the Proper Care and Use of Animals in Laboratory Research (Indian National Science Academy, New Delhi, 2000) and were approved by the Animal Ethics Committee of the Institute (Reg. No. 160/1999/CPCSEA, Proposal No. 715/04-2010).

Chemicals

MPTP, hesperidin, thiobarbituric acid (TBA), reduced GSH, and 5, 5-Dithiobis [2-nitrobenzoic acid (DTNB)] were procured from Sigma Chemical Co. (St. Louis, MO, USA). Anti-GFAP and COX-2 antibodies were obtained from Cell Signaling (USA). IL-1 β, IL-6 and TNF-α, anti-inducible nitricoxide synthase, anti-IL-4, anti-IL-10, and β-actin antibodies were obtained from Santa Cruz Biotechnology (USA). Enhanced chemiluminescence (ECL)-kit was purchased from GenScript ECL kit, USA. All other chemicals were of analytical grade.

Experimental design

After a period of 1 week acclimatization, mice were randomly divided into six groups of six animals each. Group I mice treated with Dimethyl Sulphoxide (0.05%) served as control. Group II received intra-peritoneal injection of MPTP (30 mg/kg b. w once in a day) [11] for five consecutive days. In addition, group III mice received hesperidin (50/kg b. w) respectively for fourteen days consecutively 24 h after the last dose of MPTP. Group VI mice received hesperidin (100 mg/kg. b.w) dissolved in DMSO (0.05%) and administered for 14 days by oral gavage. At the end of the experiment (19 th day), the following behavioral tests were performed.

Behavioral assessment

Akinesia

Akinesia was measured by noting the latency in seconds (s) of the animals to move all the four limbs, and the test was terminated if the latency time exceeded 180 s. Each animal was initially acclimatized for 5 min, on an elevated wooden platform (40 × 40 × 30 cm). The time taken by the animals to move all four limbs were noticed. [12] The exercise was repeated six times for each animal.

Catalepsy

Catalepsy is the inability of an animal to correct an externally imposed posture. The catalepsy test was evaluated by placing both forepaws of the mouse on a horizontal wooden bar, placed 5 cm above from the floor. [12] The time during which the animals maintained this position before lifting their hind paws onto the bar was recorded. This experiment was repeated 6 times for each animal, and the mean value was taken.

Tissue preparation

On the 19 th day, following the behavioral assessment, mice was sacrificed by terminal anesthesia, and then perfused via intracardial infusion with saline (0.9%). After intracardial perfusion, ST and SN were collected quickly. For biochemical analysis, the ST were quickly removed and homogenized in phosphate buffer (10% w/v, pH 7.0) and centrifuged at 10,500 rpm for 20 min at 4°C to get post-mitochondrial supernatant (PMS) which was used in the assay of antioxidants and lipid peroxidation. For western blot studies, ST and SN were dissected, immediately frozen on dry ice, and stored at −80°C.

Biochemical analysis

Thiobarbituric acid reactive substances

The activity of Thiobarbituric acid reactive substances (TBARS) was determined as described previously Bhattacharya et al. [13] Briefly, the tissue extracts were incubated with 0.2 ml phenyl methosulfate at 37°C in metabolic water bath shaker. After 1 h of incubation, 0.4 ml of 5% tricarboxylic acid and 0.4 ml of 0.67% TBA was added. The reaction mixture was centrifuged at 4000 rpm for 15 min, and the supernatant was boiled for 10 min. After cooling, the samples were read at 535 nm. The rate of lipid peroxidation was expressed as nmol of TBARS formed/h/g tissue.

Superoxide dismutase (SOD)

SOD activity was assayed using an indirect inhibition assay, in which xanthine and xanthine oxidase serve as a superoxide generator, and nitro blue tetrazolium (NBT) is used as a superoxide indicator. The assay mixture consisted of 960 μl of 50 mM sodium carbonate buffer (pH 10.2), containing 0.1 mM xanthine, 0.025 mM NBT, and 0.1 mM Ethylenediaminetetraacetic acid, 20 μl of xanthine oxidase, and 20 μl of the brain supernatant. Changes in absorbance were observed spectrophotometrically at 560 nm. The activity was expressed as units/min/mg protein. [14]

Catalase

Catalase activity was assayed by measuring the rate of decomposition of hydrogen peroxide at 240 nm. The assay mixture consisted of 50 μl of 1 M Tris-HCl buffer (pH 8.0) containing 5 mM EDTA, 900 μl of 10 mM H 2 O 2 , 30 μl of MQ water, and 20 μl of the brain tissue supernatant. The rate of decomposition of hydrogen was observed spectrophotometrically at 240 nm. The enzyme activity was expressed as nmol of hydrogen peroxide decomposed/min/mg protein. [15]

Glutathione peroxidase (GPx)

The GPx assay mixture consisted of 100 μl of 1 M Tris-HCl (pH 8.0) containing 5 mM EDTA, 20 μl of 0.1 M GSH, 100 μl of GSH reductase solution (10 units/ml), 100 μl of 2 mM Nicotinamide adenine dinucleotide phosphate, 650 μl of distilled water, 10 μl of 7 mM tert-butyl hydroperoxide, and 10 μl of the brain 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 μmol of NADPH per min. [16]

Reduced glutathione

The level of GSH in the brain homogenate was measured by the method described by Jollow et al. [17] The brain 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 5, 5-DTNB in 1 M phosphate buffer (pH 8).The optical density was read at 412 nm in a spectrophotometer.

Western blot analysis of inflammatory markers

Striatal and nigral tissues were homogenized in an ice-cold Radioimmunoprecipitation assay buffer [1% Triton, 0.1% sodium dodecyl sulphate 0.5% deoxycholate, 1 mmol/L EDTA, 20 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 10 mmol/L NaF, and 0.1 mmol/L phenylmethylsulfonyl fluoride (PMSF)]. The homogenate was centrifuged at 12,000 rpm/min for 15 min at 4°C to remove debris. Protein concentration was measured by the method of Lowry et al. [18] (1951). Samples containing 50 μg of total cellular protein were loaded and separated on 10% SDS polyacrylamide gel electrophoresis. The gel was then transferred on to a polyvinyliene sulfhide membrane (Millipore). The membranes were incubated with the blocking buffer containing 5% non-fat dry milk powder or bovine serum albumin for 2 h to reduce non-specific binding sites, and then incubated in β-actin (rabbit polyclonal; 1:500 dilution in 5% BSA in Tris-buffered saline and 0.05% Tween-20 (TBST), IL-1β, IL-6, IL-4 and IL-10 (rabbit polyclonal; 1:500), TNF-α (mouse polyclonal 1:700), and anti-GFAP (mouse monoclonal 1:500) with gentle shaking overnight at 4°C. After this, membranes were incubated with their corresponding secondary antibodies (anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase) for 2 h at room temperature. The membrane was washed thrice with TBST for 30 min. Immunoreactive protein was visualized by the chemiluminescence protocol (GenScript ECL kit, USA). Densitometric analysis was performed with a computer using a gel image analysis program.

Statistical analysis

Values were expressed as mean ± SD. One-way analysis of variance (ANOVA) followed by suitable post-hoc Duncan's multiple range test (DMRT) test was applied to calculate the statistical significance between various groups. A value of P < 0.05 was considered to be statistically significant.

 Results



Effects of hesperidin on behavioral paradigm

[Figure 1] and [Figure 2] revealed that movement coordination impairment of control and experimental mice. Systemic administration of MPTP caused impaired ability to initiate movement (akinesia) as well as inability to correct an externally imposed posture (catalepsy) as compared to control mice ( P < 0.05). Post-treatment of hesperidin significantly attenuate MPTP-induced akinesia [Figure 1] and catalepsy [Figure 2]. Moreover, no significant changes were observed between control and hesperidin alone treated mice.{Figure 1}{Figure 2}

Effects of hesperidin on the levels tyhiobarbituric acid reactive substances and antioxidants in the striatum

We measured the levels of TBARS, activities of enzymatic and non-enzymatic antioxidants in the ST of control and experimental mice. The levels of TBARS and activities of SOD and catalase were increased in the ST of MPTP-treated mice as compared with control mice [Table 1]. However, increase in TBARS and SOD and catalase activities were significantly ameliorated when mice were orally treated with hesperidin (50 mg/kg. b.w) ( P < 0.05). In addition, the levels of GSH and activities of GPx were drastically decreased in MPTP-treated mice as compared with control mice ( P < 0.05), and hesperidin treatment largely attenuated this decrease significantly ( P < 0.05). There is no significant difference between control and hesperidin administrated mice.{Table 1}

Effects of hesperidin on 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine-induced pro-inflammatory cytokines

Sub-acute MPTP treatment significantly increases the expression of IL-1β, TNF-α and IL-6 in ST and SN [Figure 3]a and b as compared to control. Administration of hesperidin to MPTP lessioned mice significantly reduces the expression of proinflammatory cytokines ( P < 0.05) as compared to MPTP alone treated mice.{Figure 3}

Effects of hesperidin on 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine-induced anti-inflammatory cytokines

The anti-inflammatory response was assessed by western blot analysis of IL-4 and IL-10 expression in ST and SN. MPTP treatment significantly increases the release of IL-4 and IL-10 in ST and SN [Figure 4]a and b. Oral administration of hesperidin significantly reduces the release of anti-inflammatory cytokines ( P < 0.05) as compared to MPTP mice.{Figure 4}

Effects of hesperidin on 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine-induced astroglial activation

The astrocytic reaction was assessed by western blot analysis of GFAP and COX-2 expression in ST and SN. iNOS, GFAP, and COX-2 expression reveals robust increase induced by MPTP treatment [Figure 5]a and b. However, prolonged administration of hesperidin to MPTP-injected mice significantly reduces the iNOS, GFAP, and COX-2 expressions when compared with MPTP alone induced mice ( P < 0.05). However, there was no difference in cytokine expression between ST and SN of control and hesperidin-treated animals.{Figure 5}

 Discussion



This study aims to investigate the neuroprotective effect of hesperidin on MPTP intoxicated mouse model of PD by analyzing behavior patterns and biochemical variables. The key symptom of PD involves delayed motor initiative, slow performance of voluntary movements, rapid fatigue with repetitive movements, and disorders in postural fixation, performance of associated muscles etc. [19] Motor functions in experimental PD are normally measured by performing behavioral assessments. One of those behavioral effects is catalepsy, which manifests as inability to correct an externally imposed posture. These cataleptic responses in rodents are thought to represent a correlate of the acute extrapyramidal motor side-effects (e.g., dystonia, parkinsonism) seen in humans following antipsychotic drug administration. [20] Akinesia in animal models of PD by means of the stepping test performance is thought to resemble limb akinesia and gait problems seen in PD patients. [21],[22] Akinesia appears to be a highly useful assay for MPTP-induced changes subsequent to down regulation of dopaminergic neurons in the SN. [23] Systemic administration of MPTP caused impaired ability to initiate movement (akinesia) as well as inability to correct an externally imposed posture (catalepsy). Thus, a reduction in nigral dopaminergic neurons results in a decrease in stratial dopamine that is believed to cause PD symptoms. [24] There is increasing evidence indicating that oxidative stress may contribute to several central nervous system pathologies, including PD, aging, and AD. [25] In the present study, the levels of lipid peroxidation and the activities of SOD and catalase were increased in the SN of MPTP-induced mice, which are an indication of the release of more free radicals. Results from other studies clearly shows that animals treated with MPTP [26],[27],[28] have increased levels of lipid peroxidation products compared to controls [29] , showed that treatment with hesperidin amoliterates oxidative stress by reducing enhanced lipid peroxidation.

Post-mortem analysis has revealed increased lipid peroxidation and SOD activities along with elevated free iron levels in the substantia nigra of Parkinsonian patients. [30] This increase may reflect an adaptive response due to a leakage of superoxide anion resulting from mitochondrial respiration impairment, as suggested by Thiffault et al. [30] In the present study, MPTP increased both SOD and catalase activities in SN, a finding that supports previous studies. [31],[32] Alternatively, increased enzyme activity due to MPTP-induced gliosis could not be ruled out. However, oral treatment of hesperidin significantly prevented catalase induction and SOD levels partially, due to its free radical scavenging activity. [33]

In the present study, MPTP-treated animals showed marked depletion of GSH and GPx in the SN, which is in support of previous findings. [12],[32],[34] The decreased levels of GSH and GPx in MPTP-lesioned mice in our study also confirm that MPTP induces a state of oxidative stress in the brain. Interestingly, treatment of PD mice with hesperidin produces a notable improvement in the levels of GSH and GPx. [35] Interestingly, treatment of hesperidin to MPTP-induced mice produces a notable improvement in the levels of GSH and GPx. The antioxidant/neuroprotective properties of flavanoids including hesperidin, involves chelation of metal ions such as iron and copper resulted in inhibition of transition metal-catalyzed free radical formation. [36] Taken together, these results suggested that the neuroprotective effect of hesperidin as demonstrated by protecting dopamine depletion may partly owe to its antioxidant properties.

Apart from dopaminergic degeneration, oxidative stress, and mitochondrial dysfunction, other hall marks of PD includes accumulation of activated microglia and astrocytes in the SN and ST. Activated microglia may damage the CNS by releasing various interleukines and pro-inflammatory molecules, [37],[38] or on the other hand play neuroprotective roles by producing neurotrophic factors such as brain- derived neurotrophic factor and glial cell line derived neurotrophic factor. [39] Astrocytes in CNS either possess neurotrophic properties through producing large amount of molecules which may benefit the injured nervous system or play toxic effect by releasing cytokines. [40]

Several lines of evidence indicate the presence of activated glial cells expressing the pro-inflammatory cytokines IL-1 β and TNF-α in the SN of PD patients, [41] and MPTP-treated mice. [42],[43] Astrogliosis induced by cytokines such as IL-1 β and IL-6 could accelerate GFAP release, which favored neuroinflammatory response and neuronal loss. [44] On the other hand, it is observed that treatment of hesperidin significantly diminished GFAP expression, which indicates that astrogliosis activation has been mitigated. Alternatively, the levels of anti-inflammatory cytokines (IL-4 and IL-10) were also less elevated in MPTP-treated mice because of compensatory mechanism, [45] against neuronal inflammation. Furthermore, oral treatment of hesperidin effectively decreased MPTP-induced over-expression of IL-1 β, TNF-α and IL-6, which consequently alleviate inflammatory stress and in turn secured the formation of anti-inflammatory cytokines IL-4 and IL-10. [46] In addition, hesperidin possesses anti-inflammatory properties and has been shown to protect experimental animals against chemically induced neurotoxicity. [47] Furthermore, hesperidin reduced inflammation and the production of inflammatory cytokines induced by acetaminophen and streptozotocin. [48]

Cyclooxygenase activity is the rate-limiting activity in the biosynthesis of prostaglandins. COX-2 is mainly responsible for the production of prostanoids linked to pathologic events. [49],[50] Previous studies indicate that the neurodegenerative process in PD is associated with increased expression of COX-2 and elevated levels of prostoglandin E- 2. [51],[52] Inhibition of COX-2 activity improves behavioral impairment, and protects dopaminergic neurons against degeneration caused by Parkinsonism-inducing neurotoxins. [53],[54] In the present study, elevated expression of COX-2 in brain of MPTP-treated mice indicates that COX-2 was responsible for increased production of inflammatory cytokines which were corroborated with others. [55] Hesperidin protected against inflammation in mice by inhibiting arachidonic acid metabolism via both 5-lipoxygenase and cyclooxygenase pathways. [49]

 Conclusion



In conclusion, the present study confirmed protective effects of hesperidin in MPTP-induced neurotoxicity by normalizing oxidative stress and inflammation. Our present study speculates that hesperidin might be a therapeutic agent for the treatment of PD, but further clinical studies are warranted.

 Acknowledgments



The financial assistance by the Indian Council of Medical Research (ICMR), Government of India, New Delhi, in the form of Senior Research Fellowship to Mr. K. Tamilselvam, to carry out the above study is gratefully acknowledged.

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