Protective effect of mangiferin against lipopolysaccharide-induced depressive and anxiety-like behaviour in mice

European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Behavioural pharmacology

Protective effect of mangiferin against lipopolysaccharide-induced depressive and anxiety-like behaviour in mice Ashok Jangra a, Manish M. Lukhi a, Kunjbihari Sulakhiya a, Chandana C. Baruah b, Mangala Lahkar c,n a Laboratory of Neuroscience, Department of Pharmacology & Toxicology, National Institute of Pharmaceutical Education & Research (NIPER), Guwahati, Assam 781032, India b Department of Pharmacology and Toxicology, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, Assam 781022, India c Department of Pharmacology, Guwahati Medical College, Guwahati, Assam 781032, India

art ic l e i nf o

a b s t r a c t

Article history: Received 18 November 2013 Received in revised form 12 May 2014 Accepted 10 July 2014

Numerous studies have demonstrated that inflammation, oxidative stress and altered level of neurotrophins are involved in the pathogenesis of depressive illness. Mangiferin, a C-glucosylxanthone is abundant in the stem and bark of Mangifera indica L. The compound has been shown to possess antioxidant, anti-inflammatory and immunomodulatory activities. The present study was performed to investigate the effect of mangiferin pretreatment on lipopolysaccharide-induced increased proinflammatory cytokines, oxidative stress and neurobehavioural abnormalities. Mice were challenged with lipopolysaccharide (0.83 mg/kg, i.p.) after 14 days of mangiferin (20 and 40 mg/kg, p.o.) pretreatment. Mangiferin pretreatment significantly ameliorated the anxiety-like behaviour as evident from the results of an elevated plus maze, light-dark box and open field test. Mangiferin pretreatment also improved the anhedonic behaviour as revealed by sucrose preference test and increased social interaction time. It also prevented the lipopolysaccharide-evoked depressive-like effect by reducing the immobility time in forced swim and tail suspension test. Lipopolysaccharide-induced elevated oxidative stress was decreased with mangiferin pretreatment due to its potential to increase reduced glutathione concentration, Superoxide dismutase and catalase activity and decrease lipid peroxidation and nitrite level in the hippocampus as well as in the prefrontal cortex. Mangiferin pretreatment also attenuated neuroinflammation by reducing the interleukin-1 beta (IL-1β) level in hippocampus and prefrontal cortex. In conclusion, our results demonstrated that mangiferin possessed antidepressant and anti-anxiety properties due to its ability to attenuate IL-1β level and oxidative stress evoked by intraperitoneal administration of lipopolysaccharide. Mangiferin may be a potential therapeutic agent for the treatment of depressive and anxiety illness. & 2014 Published by Elsevier B.V.

Keywords: Anxiety Depression Lipopolysaccharide Mangiferin Oxidative stress

1. Introduction Depression is a common, recurring and sometimes lethal psychiatric disorder resulting in personal suffering and suicides, in addition to social and economic burden. World health organization estimated 350 million people suffering from depression worldwide, and reported depression as a major contributor to the global burden of disease (World Health Organization, 2012). Core symptoms of major depressive disorder include loss of interest, loss of energy, dysfunctional thoughts, self guilt, suicidal ideation, disturbed sleep and appetite, and sexual dysfunction. Currently, various drug

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Corresponding author. Tel.: þ 91 9706806533. E-mail address: [email protected] (M. Lahkar).

therapies are available for the treatment of depression. However, lower efficacy, delayed action and more side effects of the current medications warrant a requirement of a novel antidepressant, which is more efficacious and shows a promising approach for the treatment of depression. Several experimental studies have shown that oxido-nitrosative stress is involved in the pathophysiology of depression and anxiety. The observed effects include lipid peroxidation, reduced glutathione level, DNA damage and reduction in the level of antioxidant enzymes (De Oliveira et al., 2007; Gibson et al., 2012; Maes et al., 2008, 2011; Salim et al., 2010; Suzuki et al., 2001). Therefore, targeting oxidative and nitrosative stress with strong antioxidants can be a beneficial approach to provide protection against depression. Patients with major depressive disorder show marked rise in inflammatory markers, including pro-inflammatory cytokines (IL-1,

http://dx.doi.org/10.1016/j.ejphar.2014.07.031 0014-2999/& 2014 Published by Elsevier B.V.

Please cite this article as: Jangra, A., et al., Protective effect of mangiferin against lipopolysaccharide-induced depressive and anxietylike behaviour in mice. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.07.031i

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IL-6, TNF-α) and their soluble receptors, both peripherally as well as centrally (Raison et al., 2006). Inflammatory challenge by peripheral administration of lipopolysacchride (LPS) exhibits both depressivelike and anxiety-like behaviour in animal model by causing a systemic inflammation through increase in the production of proinflammatory mediators such as tumour necrosis factor-alpha (TNFα), interferon-gamma (IF-γ), interleukin-6 (IL-6) and interleukin-1 beta (IL-1β). Furthermore, these pro-inflammatory cytokines produce sickness behaviour syndrome such as hyperthermia, anorexia, sleepiness, reduction of locomotor activity, exploration, libido, loss of body weight and anhedonia (Godbout et al., 2005; Huang et al., 2008; Kelley et al., 2003; Qin et al., 2007; Swiergiel et al., 1997). Moreover, LPS-induced inflammation leads to significant reduction in the neurotrophic factors like brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin-3 (NT-3) levels in different regions of the brain (Guan and Fang, 2006). Mangiferin (2-C-β-D-glucopyranosyl-1,3,6,7-tetrahydroxyxanthone), a natural C-glucosylxanthone is an active phytochemical present in the leaves, bark and root of Mangifera indica (Singh et al., 2009). It has been reported to exhibit antioxidant (Rao et al., 2012), analgesic, anti-inflammatory (Garrido et al., 2001), antitumor, immunomodulatory (Guha et al., 1996), antidiabetic (Aderibigbe et al., 2001), cardioprotective (Hou et al., 2013), hepatoprotective (Das et al., 2012) and monoamine oxidase inhibition properties (Bhattacharya et al., 1972). Mangiferin inhibits LPS-induced chronic inflammation by regulating MAPK (Mitogen-Activated Protein Kinase) signalling pathway through alteration in the expressions of ERK (Extracellular signal-regulated kinase) and JNK (c-Jun Nterminal kinase) (Wei et al., 2011). It also shows neuroprotective action by preventing neuroinflammation and oxidative damage in brain induced by restraint stress exposure (Márquez et al., 2012). The anti-depressant and anti-anxiety activities of mangiferin in LPS induced depressive-like behaviour model has not been studied so far. Thus, in the present study, we investigated the possible anti-depressant and anxiolytic effects of mangiferin through its effect on oxidative stress and inflammation. Furthermore, we assessed the effects of mangiferin pre-treatment on oxidative stress, pro-inflammatory cytokines and BDNF level in the brain following an immune challenge with LPS in mice.

Lipopolysaccharide and mangiferin were prepared freshly for the study. All other chemicals used were of analytical grade. 2.2. Animals The experiments were performed in male Swiss mice (weight: 22–30 g) from 8.00 to 14.00 h in accordance with the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) Government of India guidelines. The study was approved by the Institutional Animal Ethics Committee (IAEC) (Approval no. MC/32/2012/40), Gauhati Medical College & Hospital (CPCSEA Registration no. 351, 3/1/2001). The animals were kept at room temperature (2471 1C), with 65 710% humidity, 12 h light and dark cycles. Standard laboratory animal feed (Pranav Agro Industries Ltd. Pune, India) and water were provided ad libitum. Animals were acclimatized under the experimental conditions for a period of 1 week prior to the commencement of the experiment. 2.3. Preparation of doses Different doses of mangiferin (20 and 40 mg/kg) were selected based on the previous experimental study (Biradar et al., 2012). Mangiferin was dissolved in 30% dimethyl sulfoxide (DMSO) and administered daily by oral route at the dose volume of 10 ml/kg. Lipopolysaccharide (0.83 mg/kg) serotype 0127:B8 was dissolved in endotoxin free normal saline and administered intraperitoneally (i.p.) at the dose volume of 10 ml/kg. 2.4. Experimental design At the beginning of the experiment, mice were randomly divided into six experimental groups, each group consisting of 8 mice (Fig. 1):

 Group I was treated with vehicle (30% DMSO) of mangiferin for  

2. Materials and methods



2.1. Chemicals Lipopolysaccharide from Escherichia coli, serotype 0127:B8 and mangiferin were purchased from Sigma-Aldrich, St. Louis, MO, USA.



14 days and then challenged with saline on the 15th day. This group served as the control group. Group II was treated with vehicle (30% DMSO) of mangiferin for 14 days and then challenged with LPS (0.83 mg/kg, i.p.) on the 15th day. This group served as the LPS control group. Group III was treated with mangiferin (20 mg/kg, p.o.) for 14 days and then challenged with LPS (0.83 mg/kg, i.p.) on the 15th day. Group IV was treated with mangiferin (40 mg/kg, p.o.) for 14 days and then challenged with LPS (0.83 mg/kg, i.p.) on the 15th day. Group V was treated with mangiferin (20 mg/kg, p.o.) for 14 days.

Fig. 1. Illustration of experimental timeline and study plan.

Please cite this article as: Jangra, A., et al., Protective effect of mangiferin against lipopolysaccharide-induced depressive and anxietylike behaviour in mice. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.07.031i

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 Group VI was treated with mangiferin (40 mg/kg, p.o.) for 14 days. Two days before the initiation of the experiment, baseline food and water consumption were determined by keeping the animals in separate cages. After 2 weeks of dosing, i.e., on the 15th day, mice were challenged with either saline or LPS (0.83 mg/kg, i.p.). Anxiety-like behaviour was assessed by elevated plus maze test, light–dark box test, social interaction test and open field test 3 h after the LPS or saline challenge. Depressive-like behaviour was assessed by forced swim test (FST) and tail suspension test (TST) after 24 and 28 h respectively after the LPS or saline challenge. Anxiety and depressive-like behaviour were assessed in different animal groups. All the behavioural parameters were evaluated in a dimly illuminated, quiet and isolated room. Food and water consumption were measured at 6 and 24 h after saline or LPS administration. Neurochemical and neurobehavioral assessments were performed on different animal groups to avoid the possible consequences of behavioural assessment on biochemical parameters. The animals used for the neurochemical estimation were killed by cervical dislocation after 24 h of saline or LPS challenge. Brain regions such as hippocampus (HC) and prefrontal cortex (PFC) were isolated quickly and stored at  80 1C.

2.5. Assessment of food and water consumption Two days before the experiment, baseline food and water consumption were measured by keeping the animals in individual cages. After saline or LPS (0.83 mg/kg, i.p.) administration, 50 g of food pellets and 100 ml of water were supplied to each animal cage for the measurement of food and water consumption at 6 and 24 h after saline or LPS administration.

2.6. Assessment of behavioural parameters 2.6.1. Sucrose preference test This test was performed to assess the anhedonic response after LPS injection. We used two-bottle paradigm in which mice could choose between two bottles, one having water and other one 2% sucrose solution. One week before the initiation of the experiment, all mice included in this test were given daily 2% sucrose solution and drinking water for 24 h period to find out the baseline consumption and to reduce the response to novelty. Food and water were given to the animals ad libitum before and during the test. Sucrose preference test was performed after 24 h of saline or LPS administration by keeping the bottles containing drinking water and sucrose solution for the next 24 h. Sucrose consumption was measured by weighing the bottles and calculated by using an equation: % Sucrose consumption ¼sucrose intake/ total fluid intake (waterþsucrose intake)  100.

2.6.2. Light-dark box test This test was used to assess the anxiety behaviour in the rodents. The test involved the use of two different compartments: a light side (42  30  20 cm3; white walls and brightly illuminated with 40 W bulb) and a dark side (42  30  20 cm3; opaque black walls and dark), with an opening (6  6 cm2) between the two compartments and a video camera located 50 cm above the box. Individual mouse was placed on a dark side with head facing towards the light side and allowed to explore for 10 min. Time spent in the light compartment, number of light-dark transitions and time of risk assessments were recorded, evaluated and presented (Lacosta et al., 1999).

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2.6.3. Elevated plus maze test Elevated plus maze (EPM) was used to evaluate the influence of LPS on the anxiety behaviour in mice. It was made of two open arms (35  5 cm2) perpendicular to two closed arms of the same size with a small central square (5  5 cm2) between arms. The maze was elevated 50 cm from the floor in a dim room. Each mouse was placed at the centre of elevated plus maze with head facing toward the open arm and 5 min free exploration of mice was recorded by a video camera. The total number of entries into the open arm and closed arm, open arm time and end-arm exploration during the test was evaluated and presented (Espejo, 1997). 2.6.4. Open field test Open field test is a useful tool to assess the effect of LPS on motor and behavioural changes in the mice. Acrylic transparent box (72  72  36 cm3) with its floor divided into 16 equal sized squares (18  18 cm2) was used. Four squares were considered as the centre, and the 12 squares along the walls were considered as the periphery. Each mouse was put in the centre of the box, and number of central and peripheral crossings, rearing movements and the immobility time of mice were observed for 10 min by a video camera (Bassi et al., 2012). 2.6.5. Social interaction test Social interaction behaviour of mice was assessed (Mizunoya et al., 2013). Individual mouse was habituated to the transparent acrylic test box (45  45  34 cm3) for 10 min on the testing day. Then, the unfamiliar mouse of the same species having the same weight, in a triangular prism like plastic box having holes to allow the social interaction between them under the dimly lit condition was introduced. Social interaction behaviour consisting of social sniffing, anogenital sniffing, aggressive behaviour and social grooming was recorded for 10 min under the dimly lit condition. 2.6.6. Forced swim test This test was performed by using the method described by Porsolt et al. (1977). A vertical glass cylinder (25 cm high, 10 cm in diameter) containing tap water to a depth of 20 cm at 25 1C was used. For testing, mouse was placed in the cylinder for 6 min duration and the duration of immobility was scored during the last 5 min after 1 min of acclimatization. Each animal was considered as being immobile when the mouse was either static or made only little movements necessary to keep their head above the water. At the end of experiment, mice were placed under a warming lamp before returning to their home cage. 2.6.7. Tail suspension test This test was performed by using the method described by Steru et al. (1985). Mice were suspended 40 cm above the floor by small adhesive tape placed 1 cm from the tip of the tail. The immobility time of each mouse was observed for 6 min, but the data scored during the last 5 min were analyzed and presented. 2.7. Estimation of biochemical and molecular parameters 2.7.1. Superoxide dismutase (SOD) activity The SOD assay kit (Sigma-Aldrich, St. Louis, MO, USA) was used for the quantitative determination of Superoxide dismutase (SOD) in prefrontal cortex (PFC) and hippocampus (HC) of mouse brain. Protein level was measured by the method of Lowry et al. (1951). Hippocampus and prefrontal cortex were isolated from mice killed 24 h after LPS challenge and homogenized by sonication in phosphate buffer saline (pH 7.4, 8% w/v). The homogenates were centrifuged at 15,000 g for 20 min at 4 1C. The supernatants were collected, and SOD activity was estimated using a kit according to

Please cite this article as: Jangra, A., et al., Protective effect of mangiferin against lipopolysaccharide-induced depressive and anxietylike behaviour in mice. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.07.031i

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the manufacturer's instructions. Absorbance was recorded at 440 nm on a microplate reader, and the results were expressed as U/mg of protein. 2.7.2. Catalase activity The catalase assay kit (Sigma-Aldrich, St. Louis, MO, USA) was used to investigate the enzymatic activity in the peroxidation function of catalase in PFC and HC of mouse brain. Hippocampus and prefrontal cortex were homogenized by sonication in phosphate buffer saline (pH 7.4, 8% w/v). The supernatants were collected and catalase activity was estimated using a kit according to the manufacturer's instructions. The resulting red quinoneimine dye formed by oxidative coupling reaction of 4-aminophenazone with 3,5-dichloro-2-hydroxybenzenesulfonic acid catalyzed by horseradish peroxidase was recorded at 520 nm on microplate reader. Results were expressed as mMol/min/mg of protein. 2.7.3. Malondialdehyde (MDA) level Malondialdehyde (MDA) level in PFC and HC of mice was determined according to the method of Ohkawa et al. (1979). After 24 h of LPS or saline exposure, brain was homogenized in 1.15% KCl buffer (pH 7.0) using homogenizer. Homogenate was added to a mixture of 0.2 ml of 8.1% sodium dodecyl sulphate (SDS), 1.5 ml of 20% acetic acid (pH 3.4) and 1.5 ml of 0.8% thiobarbituric acid. The final reaction mixture was heated to 95 1C for 60 min in a water bath and then centrifuged at 10,000 rpm for 10 min. Absorbance of the supernatant was recorded at 532 nm. Protein estimation was performed according to the method described by Lowry et al. (1951). Results were expressed as mmole MDA/g of tissue weight. 2.7.4. Reduced glutathione (GSH) levels The reduced glutathione content in PFC and HC of mice was determined by using the method of Beutler et al. (1963). The supernatant of the homogenate was mixed with trichloroacetic acid (10% w/v) in 1:1 ratio and centrifuged at 1000 g for 10 min at 4 1C. The supernatant obtained (0.5 ml) was mixed with 2 ml of 0.3 M disodium hydrogen phosphate. Then 0.25 ml of 0.001 M freshly prepared DTNB [5, 50 -dithiobis (2-nitro benzoic acid) dissolved in 1% w/v sodium citrate] was added and absorbance was recorded spectrophotometrically at 412 nm. The results were expressed as nmol of GSH per mg protein. 2.7.5. Nitrite assay Nitrite content was determined by the method described by Green et al. (1982). The assay was based on Griess reaction to determine the production of nitric oxide. 100 ml of the supernatant was incubated with 100 ml of the Griess reagent, which contained equal parts (1:1:1:1) of 1% sulphanilamide dissolved in 1% H3PO4, 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride and distilled water at room temperature for 10 min. Nitrite content was determined from a standard curve generated by using sodium nitrite as a standard. The absorbance was recorded at 560 nm in a microplate reader and the results were expressed as mM/mg tissue.

2.7.6. IL1-β, TNF-α and BDNF immunoassays Quantitative determination of Interleukin1-β, Tumour Nuclear Factor (TNF)-α and Brain Derived Neurotrophic Factor (BDNF) was performed by using immunoassay kits purchased from Invitrogen co., Carlsbad, CA, USA (IL-1β and TNF-α assay kit) and Promega, Madison, WI, USA (BDNF assay kit). Prefrontal cortex and hippocampus were homogenized in 8 volumes of PBS buffer and centrifuged (10,000 rpm for 5 min). The concentration of the cytokines in 100 mL samples was determined according to the manufacturer's protocol, and the sample values were then read off from the standard curve. IL-1β, and TNF-α concentrations were measured in HC and PFC and expressed as pg/ml, whereas BDNF concentration was measured in HC and expressed as ng/mg of protein. 2.8. Statistical analysis Results were expressed as Mean 7S.E.M. Jandel Sigma Stat Version 3.5 software was used for the statistical analysis. The data were analyzed statistically by using one way analysis of variance (ANOVA). In case ANOVA showed significant difference, post-hoc analysis was performed with Tukey's test. Pr0.05 was considered to be statistically significant.

3. Results 3.1. Effect of mangiferin on LPS-induced changes in food and water consumption LPS administration significantly reduced the food intake at 6 h (P o0.001), at 24 h (Po 0.001), and water intake at 6 h (P o0.01), at 24 h (Po0.001) as compared to the vehicle treated control group (Table 1). Two weeks of pretreatment with mangiferin (20 and 40 mg/kg, p.o.) did not show any significant effect on the LPSinduced reduction in food and water consumption. 3.2. Effect of mangiferin on LPS-induced changes in behavioural parameters LPS administration produced significant effect on time spent in the light compartment (P o0.001), light-dark transitions (Po0.01) and on risk assessment time (P o0.001) in the light-dark box test (Table 2). No significant effect was observed on time spent in the light compartment and light-dark transitions after 14 days of pretreatment with mangiferin (20 mg/kg, p.o.) group. However, mangiferin (20 mg/kg, p.o.) pretreatment significantly (P o0.05) enhanced the risk assessment time. Mangiferin (40 mg/kg, p.o.) pretreatment showed significant improvement on all the parameters of light-dark box apparatus viz. time spent in the light compartment (P o0.05), light-dark transitions (P o0.05) and on risk assessment time (Po 0.001) as compared to LPS-treated control group. LPS administration induced an anxiogenic effect as evident from the reduction in the number of entries in open arm (Po0.01)

Table 1 Effect of mangiferin pretreatment on LPS induced changes in water and food intake in mice. Values are expressed as the mean 7 SEM (n¼ 8 mice/group). Sl. no.

Parameters

1.

Water intake

2.

Food intake

a b

Vehicle control (ml/6 h) (ml/24 h) (g/6 h) (g/24 h)

1.377 0.15 47 0.37 1.65 7 0.12 3.5 7 0.32

LPS control a

0.687 0.10 1.5 7 0.25b 0.377 0.15b 1.62 7 0.18b

MF-20 mg/kg þ LPS

MF-40 mg/kg þ LPS

MF-20 mg/kg

MF-40 mg/kg

0.80 7 0.07 1.577 0.22 0.87 7 0.18 1.85 7 0.22

0.95 7 0.14 1.25 7 0.31 0.93 7 0.11 1.92 7 0.36

1.46 7 0.17 3.75 7 0.36 1.62 7 0.20 3.127 0.36

1.34 7 0.23 4.25 7 0.31 1.687 0.16 3.56 7 0.34

P o0.01 compared with the vehicle treated control group. Po 0.001 compared with the vehicle treated control group.

Please cite this article as: Jangra, A., et al., Protective effect of mangiferin against lipopolysaccharide-induced depressive and anxietylike behaviour in mice. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.07.031i

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Table 2 Effect of mangiferin pretreatment on LPS induced changes on the exploratory behaviour of mice in the light-dark transition test. Values are expressed as the mean 7 SEM (n¼ 8 mice/group). Sl. no.

Light-dark transition test (Parameters)

Vehicle control

LPS control

MF-20 mg/kg þ LPS

MF-40 mg/kg þLPS

MF-20 mg/kg

MF-40 mg/kg

1. 2. 3.

Time spent in light compartment (s) Light-dark transitions Time of risk assessment (s)

192.88 7 29.30 71.87 7 3.18 25.127 3.61

587 13.45a 43.377 7.01c 69.87 7 6.83a

104.38 719.49 53.87 74 50.5 72.69b

148.887 25.17b 63.62 7 5.25b 397 2.40d

199.50 7 36.26 72.62 7 4.04 27.75 7 3.37

200.25 723.7 70.6273.06 26.5 73.62

a

P o0.001 compared with the vehicle treated control group. Po 0.05 compared with the vehicle treated control group. c Po 0.01 compared with the LPS control group. d Po 0.001 compared with the LPS control group. b

Table 3 Effect of mangiferin pretreatment on LPS induced changes on the anxiety-like behaviour of mice in the elevated plus-maze test. Values are expressed as the mean 7 SEM (n¼ 8 mice/group). Sl. no.

Elevated plus maze test (Parameters)

Vehicle control

LPS control

MF-20 mg/kg þ LPS

MF-40 mg/kg þ LPS

MF-20 mg/kg

MF-40 mg/kg

1. 2. 3. 4.

No. of entries in open arm (Frequency) No. of entries in closed arm (Frequency) Open arm time (in s) End-arm explorations (Frequency)

6.5 7 0.65 9.75 7 0.73 100.75 7 8.59 37 0.37

2.87 7 0.61a 4.377 0.60c 33.127 4.40c 0.87 7 0.22c

3.87 70.66 5.62 70.60 59 74.50 1.25 70.36

5.62 7 0.41b 7.62 7 0.56b 75.127 13.13b 2.25 7 0.41d

6.62 7 0.63 9.25 7 0.88 977 7.95 2.87 7 0.29

77 0.50 10.127 0.91 105.25 7 5.89 3.127 0.22

a

P o0.01 compared with the vehicle treated control group. Po 0.01 compared with the vehicle treated control group. c Po 0.001 compared with the LPS control group. d Po 0.05 compared with the LPS control group. b

Table 4 Effect of mangiferin pretreatment on LPS induced changes on the exploratory behaviour of mice in the open-field test. Values are expressed as the mean 7 SEM (n¼ 8 mice/ group). Sl. no. 1. 2. 3. 4.

Open field exploration test (Parameters) Centre crossings (Frequency) Peripheral crossings (Frequency) Rearings (Frequency) Immobility (Frequency)

Vehicle control 20.25 7 2.02 62.137 4.68 35.887 2.15 77 0.59

MF-20 mg/kg þ LPS

LPS control a

9.25 7 1.06 37.62 7 4.14 c 107 1.70 a 26.62 7 1.87 a

12.50 7 1.31 44.5 7 4.66 18.137 1.46 20.377 2.04

MF-40 mg/kg þLPS b

15.50 7 1.71 56.38 7 4.87b 23.63 7 3.56b 17.127 1.65d

MF-20 mg/kg

MF-40 mg/kg

19.63 7 1.63 64.38 7 3.57 34.007 2.01 7.5 7 0.68

19.137 1.26 637 3.19 36.38 7 3.20 8.5 7 1.45

a

P o0.001 compared with the vehicle treated control group. Po 0.05 compared with the vehicle treated control group. Po 0.01 compared with the LPS control group. d Po 0.01 compared with the LPS control group. b c

and closed arm (Po 0.001), duration in open arm (P o0.001) and end arm exploration (P o0.001) as compared to vehicle treated group (Table 3). Mangiferin (20 mg/kg, p.o.) pretreatment failed to show any significant improvement on all the EPM parameters. However, mangiferin (40 mg/kg, p.o.) pretreatment was able to reverse the effect of LPS on all the EPM parameters significantly. It showed significant effect on the number of entries in open arm (P o0.01) and closed arm (P o0.01), open arm duration (P o0.01), and end arm exploration (Po0.05) as compared to LPS treated control group. LPS administration produced a significant difference between the number of central (P o0.001) and peripheral crossings (P o0.01), rearing (P o0.001), and immobility time (P o0.001). No significant effect was observed on all the OFT parameters by the mangiferin (20 mg/kg, p.o.) pretreatment. However, mangiferin (40 mg/kg, p.o.) pretreatment produced a significant increase in the frequency of the central (Po 0.05) and peripheral crossings (P o0.05), rearings (Po0.05) and a significant reduction in the immobility time (P o0.01) (Table 4). We found that immobility time in FST and TST was significantly (P o0.001) increased after the LPS administration as shown in Figs. 2 and 3. Mangiferin (40 mg/kg, p.o.) pretreatment significantly reversed the effect of LPS on the immobility time in FST (P o0.001) and TST (P o0.01). LPS-treated mice spent significantly (P o0.001) lesser time in social interactions as compared to the

vehicle treated control group (Fig. 4). Mangiferin (20 mg/kg, p.o., Po 0.05) and mangiferin (40 mg/kg, p.o., Po 0.01) significantly improved the social interaction time as compared to LPS-treated group. We found that LPS-treated group exhibited a significant decrease in sucrose preference (P o0.001) that was increased by mangiferin (40 mg/kg, p.o.) significantly (P o0.01) as shown in Fig. 5. 3.3. Effect of mangiferin on LPS-induced changes in oxidative stress markers SOD level was significantly reduced both in HC (P o0.001) and PFC (P o0.001) of mice after 24 h of LPS administration when compared with vehicle control group (Fig. 6). Mangiferin (40 mg/ kg, p.o.) pretreatment prevented and reversed LPS induced decrease in SOD level both in HC (Po0.01) and PFC (P o0.01). On the other hand, no significant alterations were observed in SOD level by mangiferin (20 mg/kg, p.o.) group. We found that LPS after 24 h significantly reduced the catalase activity both in HC (P o0.001) and PFC (P o0.001) of mice as compared to the vehicle control group as shown in Fig. 7. Mangiferin (40 mg/kg, p.o.) pretreatment restored the LPSinduced decrease in catalase level both in HC (P o0.01) and PFC (P o0.01). But mangiferin (20 mg/kg, p.o.) pretreatment failed to produce any significant effect on catalase level.

Please cite this article as: Jangra, A., et al., Protective effect of mangiferin against lipopolysaccharide-induced depressive and anxietylike behaviour in mice. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.07.031i

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Fig. 2. Effect of mangiferin pretreatment on LPS induced changes in forced swim test. Values are expressed as the mean 7 SEM (n¼ 8 mice/group). ###P o0.001 compared with the normal control group. ***Po 0.001 compared with the LPS control group.

Fig. 3. Effect of mangiferin pretreatment on LPS induced changes in tail suspension test. Values are expressed as the mean 7 SEM (n¼ 8 mice/group). ###P o0.001 compared with the normal control group. **Po 0.01 compared with the LPS control group.

Fig. 4. Effect of mangiferin pretreatment on LPS induced changes in social interaction test. Values are expressed as the mean 7 SEM (n ¼8 mice/group). ### Po 0.001 compared with the normal control group. *Po 0.05 and **Po 0.01 compared with the LPS control group.

Fig. 5. Effect of mangiferin pretreatment on LPS induced changes sucrose preference test. Values are expressed as the mean7 SEM (n¼8 mice/group). ### Po 0.001 compared with the normal control group. *Po 0.05 and **Po 0.01 compared with the LPS control group.

Fig. 6. Effect of mangiferin on Superoxide dismutase (SOD) activity in hippocampus and prefrontal cortex of mice brain. Values are expressed as the mean 7 SEM (n ¼8 mice/group). ###Po 0.001 compared with the normal control group. **Po 0.01 compared with the LPS control group.

Fig. 7. Effect of mangiferin on catalase activity in hippocampus and prefrontal cortex of mice brain. Values are expressed as the mean 7SEM (n¼8 mice/group). ### Po 0.001 compared with the normal control group. **P o0.01 compared with the LPS control group.

Fig. 8. Effect of mangiferin on thiobarbituric acid reactive substances (TBARS) level in hippocampus and prefrontal cortex of mice brain. Values are expressed as the mean7 SEM (n¼ 8 mice/group). ###Po 0.001 compared with the normal control group. **Po 0.01 compared with the LPS control group.

LPS after 24 h significantly enhanced the TBARS level both in HC (P o0.001) and PFC (Po 0.001) of mice when compared with vehicle control group (Fig. 8). Mangiferin (40 mg/kg, p.o.) pretreatment resulted in significant reduction in TBARS level both in HC (Po 0.01) and PFC (Po0.01). However, mangiferin (20 mg/kg, p.o.) pretreatment failed to show any significant effect on TBARS level. GSH level was found to be decreased both in HC (P o0.001) and PFC (P o0.001) of mice after 24 h of LPS exposure as compared to the vehicle control group. Mangiferin (40 mg/kg, p.o.) pretreatment showed a significant increase in GSH level in HC (P o0.01) and PFC (P o0.05), whereas mangiferin (20 mg/kg, p.o.)

Please cite this article as: Jangra, A., et al., Protective effect of mangiferin against lipopolysaccharide-induced depressive and anxietylike behaviour in mice. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.07.031i

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Fig. 9. Effect of mangiferin on reduced glutathione level in hippocampus and prefrontal cortex of mice brain. Values are expressed as the mean 7 SEM (n ¼8 mice/group). ###P o0.001and ##Po 0.01 compared with the normal control group. **P o 0.01and *Po 0.05 compared with the LPS control group.

Fig. 10. Effect of mangiferin on nitrite level in hippocampus and prefrontal cortex of mice brain. Values are expressed as the mean 7 SEM (n¼ 8 mice/group). ### P o0.001 compared with the normal control group. **Po 0.01 and *P o 0.05 compared with the LPS control group.

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Fig. 11. Effect of mangiferin on Interlekin-1β level in hippocampus and prefrontal cortex of mice brain. Values are expressed as the mean 7 SEM (n¼ 8 mice/group). ### Po 0.001 compared with the normal control group. ***Po 0.001, **Po 0.01 and *P o 0.05 compared with the LPS control group.

Fig. 12. Effect of mangiferin on TNF-α level in hippocampus and prefrontal cortex of mice brain. Values are expressed as the mean 7 SEM (n ¼8 mice/group). #Po 0.05 compared with the normal control group.

pretreatment showed a significant increase GSH level only in HC (P o0.05) when compared with LPS-treated group (Fig. 9). Nitrite level was increased both in HC (Po0.001) and PFC (Po0.001) of mice after 24 h of LPS exposure when compared to the vehicle control group (Fig. 10). Mangiferin (40 mg/kg, p.o.) pretreatment prevented the LPS-induced increase in nitrite level both in HC (Po0.01) and PFC (Po0.05). Mangiferin (20 mg/kg, p.o.) pretreatment did not produce any significant effect on nitrite level. 3.4. Effect of mangiferin on LPS-induced changes in proinflammatory cytokines and BDNF level LPS after 24 h resulted in a significant increase in IL-1β level both in HC (P o0.001) and PFC (Po0.001) of mice that was significantly decreased by mangiferin (40 mg/kg, p.o.) both in HC (Po0.001) and PFC (Po0.05), whereas mangiferin (20 mg/kg, p.o.) pretreatment showed its effect only in HC (Po0.01) (Fig. 11). TNF-α level was significantly increased in HC (Po0.05) and PFC (Po0.05) of mice after 24 h of LPS exposure. Both doses of mangiferin did not show any effect on the TNF-α level in HC and PFC (Fig. 12). We found that LPS after 24 h significantly reduced the BDNF level (P o0.001) and only mangiferin (40 mg/kg, p.o.) significantly prevented the reduction of BDNF level in HC (P o0.05) as shown in Fig. 13.

4. Discussion There are many perpetrator interacting pathways that are involved in the pathogenesis of depression due to its complexity and heterogeneity. Depressive illness is closely associated with chronic inflammatory pathway, which is manifested by elevated levels of proinflammatory cytokines, chemokines, and adhesion

Fig. 13. Effect of mangiferin on BDNF level in hippocampus and prefrontal cortex of mice brain. Values are expressed as the mean 7 SEM (n ¼8 mice/group). ### Po 0.001 compared with the normal control group. *Po 0.05 compared with the LPS control group.

molecules in the periphery and central nervous system (Dunbar et al., 1992; Hestad et al., 2003; Maes, 1995, 1999; Raison et al., 2006). There are ample evidences that central or peripheral injection of LPS (a potent immune activator) induces oxidative stress, neuroinflammation, depressive and anxiety-like behaviour Q3 in rodents (Bassi et al., 2012; Biesmans et al., 2013; Lawson et al., 2013; Qin et al., 2007; Swarnkar et al., 2009; Swiergiel and Dunn, 2007). Experimental studies have demonstrated that mangiferin exhibits neuroprotective activity via inhibition of inflammation and oxidative stress in rat brain exposed to stress and also inhibits chronic inflammation induced by lipopolysacchride (Biradar et al., 2012; Márquez et al., 2012). Based on these facts, the present study was designed to evaluate the protective effect of mangiferin (20 and 40 mg/kg, orally) against LPS-induced behavioural and biochemical alterations in mice. We evaluated different behavioural and biochemical parameters in our study, which demonstrated that 14 days pretreatment with mangiferin prevented the

Please cite this article as: Jangra, A., et al., Protective effect of mangiferin against lipopolysaccharide-induced depressive and anxietylike behaviour in mice. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.07.031i

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augmentation of pro-inflammatory cytokines, oxidative stress, depressive and anxiety-like behaviour. LPS challenged animals exhibited marked reduction in the food and water intake as compared to the vehicle treated group. Decreased food and water intake by LPS treated animals might be due to the augmentation of IL-1, IL-6 and TNF-α that have effect on the hypothalamic region (Layé et al., 2000; Reichenberg et al., 2002). Mangiferin was unable to produce any significant effect on the food and water consumption in LPS challenged group. Anhedonic behaviour is a basic characteristic feature of depression; hence the anhedonic behaviour was assessed by measuring the consumption of sucrose solution (Lemke et al., 1999). It was found that LPS challenged animals showed significant decrease in sucrose solution consumption during test period i.e. 24 h. In contrast, mangiferin (40 mg/kg) pretreatment group exhibited more preference for the sucrose solution as compared to LPS treated group. Our findings showed that LPS administration leads to anxietylike behaviour after 3 h in mice. We found significant reduction in the time spent in the light compartment, reduction in light-dark transitions, and increase in the time of risk assessment in the light-dark test, which evidently confirmed the anxiety-like behaviour of LPS-treated group. In the elevated plus maze test, LPStreated group showed anxiety as revealed by significant reduction in the number of entries in open and closed arms, time duration in open arm, and end-arm explorations as compared to the vehicletreated group. Similarly, LPS-treated group showed significant reduction in the central as well as peripheral crossings, rearing movements and increased immobility time in the open field exploration test. Social interaction time of LPS-exposed mice was significantly less as compared to vehicle treated. Our behavioural experimental data reveals that LPS administration evokes anxiogenic effect, which is corroborated by previous studies (Bassi et al., 2012; Lacosta et al., 1999; Salazar et al., 2012; Swiergiel and Dunn, 2007). The anxiogenic effect of LPS-treated mice observed in various behavioural test paradigms may be due to increased proinflammatory cytokines, oxidative stress or by increase in corticotrophin-releasing hormone (CRH) that is evoked by peripheral as well as central administration of LPS (Agelaki et al., 2002; Koo and Duman, 2009; Risbrough and Stein, 2006; Simen et al., 2006). Treatment with mangiferin (40 mg/kg) decreased the anxiety-like behaviour by significantly improving all the anxietyrelated behavioural parameters probably via reducing the level of proinflammatory cytokines, oxidative stress in HC as well as in PFC and by preventing the depletion of BDNF in HC. Many experimental studies illustrate the role of oxidative stress and neuroinflammation in the pathogenesis of depression (Leonard and Maes, 2012; Maes, 2008; Sluzewska et al., 1996). Proinflammatory cytokines and activated microglia induced by LPS cause a drastic increase in the production of reactive oxygen species and peroxides, which may further lead to inflammation, lower antioxidant status, and consequently cause neurobehavioural alterations (Chung et al., 2010; Dikalov et al., 2002). Earlier several antioxidants like N-acetylcysteine, quercetin, α-tocopherol, curcumin, liquiritin showed the antidepressant-like effect via inhibition of oxidative stress in various animal models (Berg et al., 2004; Berk et al., 2008; Sah et al., 2011; Zhao et al., 2008). In our study, significant reduction in antioxidant status in HC and PFC of LPS-treated mice after 24 h was significantly ameliorated by two weeks mangiferin pretreatment at the dose of 40 mg/kg. LPS-treated mice showed a significant increase in IL-1β and TNF-α in the HC and PFC, which might be responsible for the neuroinflammation, oxidative stress and neurobehavioural changes. Mangiferin (40 mg/kg) pretreatment significantly attenuated the increased level of IL-1β but failed to restrict LPS for augmentation of TNF-α level both in hippocampus and prefrontal cortex. Neurotrophins like brain-derived

neurotrophic factor (BDNF) signalling mechanisms play a vital role in brain neuroplasticity and depression pathogenesis. Disrupted neuroplasticity and low level of BDNF had been observed in major depressive disorders (MDD) patients in various previous studies (Duman and Monteggia, 2006; Karege et al., 2002; Lee and Kim, 2010; Sen et al., 2008; Varambally et al., 2013). Peripheral immune activation by LPS significantly reduced the amount of BDNF in the rat brain (Guan and Fang, 2006). Our study demonstrated that BDNF level was declined in HC by LPS administration after 24 h, which was prevented by mangiferin pretreatment at the dose of 40 mg/kg. Preventive effect of mangiferin on hippocampal BDNF level may be responsible for the improved neurobehavioural effect. The results of the present study shows that i.p. injection of LPS causes increased immobility time in the TST and FST, which was significantly improved by mangiferin (40 mg/kg) pretreatment probably via inhibition of oxidative stress and proinflammatory cytokines. In conclusion, our experimental results demonstrated the protective effect of mangiferin in LPS-evoked depressive and anxiety-like behaviour in mice. This protective effect is likely due to inhibition of neuroinflammation, oxidative stress and prevention of the depletion of BDNF level in the mouse brain. Hence, our study suggests that mangiferin could be an intriguing therapeutic approach for the treatment of neuropsychiatric disorders associated with oxidative stress and neuroinflammation caused by proinflammatory cytokines.

Acknowledgement We would like to thank the Department of Pharmaceuticals, Q4 Ministry of Chemicals and Fertilizers, Government of India for financial support. The authors are immensely thankful to Priyanka Trivedi, Department of Pharmacology & Toxicology, NIPER-Mohali, for proof-reading this article.

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Please cite this article as: Jangra, A., et al., Protective effect of mangiferin against lipopolysaccharide-induced depressive and anxietylike behaviour in mice. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.07.031i