Indian Journal of Pharmacology Medknow Publications on behalf of Indian Pharmacological Society ISSN: 0253-7613 EISSN: 1998-3751 Vol. 43, Num. 2, 2011, pp. 192-196

Indian Journal of Pharmacology, Vol. 43, No. 2, March-April, 2011, pp. 192-196

Research Article

Effect of quercetin on lipopolysaccharide induced-sickness behavior and oxidative stress in rats

Sangeeta Pilkhwal Sah¹, Naveen Tirkey², Anurag Kuhad², Kanwaljit Chopra²

¹ Department of Pharmaceutical Sciences, Kumaun University, Nainital, Uttarakhand, India
² Pharmacology Division, University Institute of Pharmaceutical Sciences, Punjab University, Chandigarh, India

Correspondence Address: Sangeeta Pilkhwal Sah, Department of Pharmaceutical Sciences, Kumaun University, Nainital, Uttarakhand, India, spilkhwal@yahoo.com

Date of Submission: 20-Mar-2010
Date of Decision: 22-Nov-2010
Date of Acceptance: 03-Jan-2011

Code Number: ph11050

DOI: 10.4103/0253-7613.77365

Abstract

Keywords: Lipopolysaccharide, oxidative stress, polyphenols, Quercetin, sickness behavior

Introduction

An endotoxin or lipopolysaccharide (LPS) challenge results in highly differentiated changes in brain neurotransmission probably subserving the coordinate processing of immune information. ^[1] LPS-induced activation of peripheral innate immune cells elicits secretion of inflammatory cytokines, including IL-1, IL-6, and TNF-α (tumor necrotic factor) which then relay signal to the CNS macrophages and microglia to produce the same cytokines, targeting neuronal substrates and eliciting sickness behavior. Sickness behavior is characterized by non-specific symptoms like fever, prolong sleep, decrease in food and water intake, reduced mobility, depression, and anxiety. ^[2] In the late phase of inflammatory response in sickness behavior (also seen during gram negative bacterial infections), ^[3] LPS creates an abundance of reactive oxygen species (ROS) primarily from macrophages and infiltrating neutrophils. ROS serve as an intracellular messenger to induce signal transduction and activates transcription factors such as nuclear factor kappa B (NFkB); therefore, production of ROS is important for host defense and may influence sickness behavior via NFkB-dependent cytokine production. ^[4] However, the levels of ROS should be optimum for clearing infections and if the levels overcome endogenous antioxidant defenses it results in a state of oxidative stress-mediated pathology. Moreover, an amplified inflammatory cytokine response in the brain is also associated with a myriad of complications like cognitive dysfunction and depression like behavior. Antioxidants like N-acetyl-l-cysteine, β-carotene, and α-tocopherol have been shown to completely or partially block LPS-induced ROS and NFkB activation in cell cultures and animal models. ^[5] Quercetin (QT), a bioflavonoid, is the most potent scavenger of ROS and it is suggested to substantially empower the endogenous antioxidant system. LPS activates NFkβ in macrophages of rat lung tissue, and mouse leukemic monocyte macrophage cell line (RAW 264.7 macrophage) is used in vitro to study the effect of LPS on macrophages. A study demonstrates that QT inhibits LPS-stimulated NFkβ activation in RAW 264.7 macrophage. ^[6] Another study reports that quercetin significantly inhibited TNF-α production and gene expression in a dose-dependent manner via modulation of the NFkβ system. ^[7]

However, little research has been done discerning the mechanisms and interactions of antioxidant treatment on cytokine regulated behaviors of sick animals. Hence, the present study was designed to evaluate the effect of QT on LPS-induced sickness behavior and oxidative stress in rats.

Materials and Methods

Animals

Wistar albino rats (150--200 g), bred in central animal house of Punjab University (India), were used. The animals were housed under standard conditions of light and dark cycle with free access to food and water. The experimental protocols were approved by the Punjab University Animal Ethics Committee, Chandigarh.

Drugs

Quercetin (QT) (Sigma, St. Louis, MO, USA) was prepared in a dimethyl sulfoxide (4% DMSO) solution and LPS (serotype E. Coli 0111:B4) (Fluka) was prepared in pyrogen-free saline. The drug solutions were prepared freshly at the beginning of each experiment.

Elevated Plus maze

The elevated plus maze apparatus for rats consisted of two open (50 × 10 cm) and two closed arms (50 × 10 × 40 cm), and an open roof with the entire maze elevated 50 cm from the floor. Each rat was placed in the center of elevated plus maze with head facing toward the open arm. During the 5 min test, the number of entries into the open arms and the time spent in open arm of the maze were recorded. ^[8]

Open field test

An open field apparatus consisted of a circular arena (wall height 27 cm; diameter 84 cm) with 25 houses equipped with light and sound sources. Each rat was placed in the circular arena of apparatus. One ambulation was recorded when the animal moves from one segment to another. Similarly, one rearing score was recorded when animal stand on its hind limbs with or without support of the wall. Ambulation and rearing scores of rats were recorded over a period of 5 min. ^[8]

Experimental protocol and procedure

At the beginning of the experiment, rats were divided into six groups of six animals each and treated for 2 weeks:

• Group I was treated with vehicle (4% DMSO) of QT and then challenged with saline on 15 ^th day. This group served as control.
• Group II was treated with equivalent volume of vehicle of QT and then challenged with LPS (1 mg/kg) on 15th day.
• Group III was treated with QT (2 mg/kg, i.p.) and challenged with LPS (1 mg/kg) on 15 ^th day.
• Group IV was treated with QT (25 mg/kg, i.p.) and challenged with LPS (1 mg/kg) on 15th day.
• Group V was treated with QT (2 mg/kg, i.p.).
• Group VI was treated with QT (25 mg/kg, i.p.).

The concentration of drug solutions was so prepared that not more than 0.3-0.4 ml of drug was injected to the animal. The doses were selected based on previous studies done at our lab. ^[9] Quercetin can cross the blood--brain barrier and once daily dosing was chosen for the study. ^[10] Two days before the experiment, baseline food consumption, water consumption, and body weight were measured by keeping them in individual cages. After 2 weeks of dosing i.e. on 15 ^th day, they were challenged with injection of either saline or LPS (1 mg/kg, i.p.) as per the protocol and immediately after injection, 50 g of food pellets and 100 ml of water were supplied to each animal for measurement of food and water consumption. Anxiety was assessed in the elevated plus maze 3 h after the LPS or saline challenge and activity in the open field test was assessed 4 h after the LPS or saline challenge. ^[11] All behavioral parameters were assessed in a dimly illuminated, quiet room. Body weight, food, and water consumption was measured 6 h after LPS or saline injection because a study depicts that LPS induces body weight loss with peak effect at 6 h post-injection. ^[11] Immediately after this, all the animals were sacrificed, brains were removed, frozen in liquid nitrogen, stored at -80°C, and homogenates were later used for the study.

Assessment of oxidative stress

Brain homogenate preparation

Rat brain was rinsed with normal saline and the homogenate prepared in 10% (w/v) cold phosphate buffered saline (0.1 mol/l, pH 7.4) with the help of a homogenizer was used to estimate cytokine levels, thiobarbituric acid-reacting substances (TBARS), and reduced glutathione (GSH) in rat brain.

Estimation of lipid peroxidation

Lipid peroxidation was assayed in the form of thiobarbituric acid reacting substances (TBARS), in brain homogenate according to the method of Wills. ^[12] The amount of TBARS, formed was measured at 532 nm using Erba Chem 5 Plus (Transasia, India). The results were calculated as nmol of TBARS/mg protein using the molar extinction coefficient of chromophore (1.56 x 10 ^-1 cm ^-1 ) and then expressed as percentage of control.

Estimation of reduced glutathione

Reduced glutathione in the brain homogenate was estimated by the method of Jollow et al. ^[13] 1.0 ml of tissue homogenate (10%) was precipitated with 1.0 ml of sulfosalicylic acid (4%). The samples were kept at 4ºC for at least 1 h and then subjected to centrifugation at 1200g for 15 min at 4ºC. The assay mixture contained 0.1 ml aliquot and 2.7 ml phosphate buffer (0.1M pH 7.4) to which is added 0.05 ml of DTNB (5-5′-dithio bis-2 nitrobenzoic acid). The yellow color developed was read immediately at 412 nm using an Erba Chem 5 Plus (Transasia, India) semiautoanalyzer. The results were calculated as nmol of GSH per mg protein and expressed as percentage of control.

Protein estimation

The protein content was measured according to the method of Lowry et al. using bovine serum albumin as standard. ^[14]

Enzyme-linked immunosorbent assay

Cytokines were measured from tissue samples (brain homogenate) using commercially available ELISAs for rat IL-1β, TNF-α, and IL-6 (R & D Systems, Minneapolis, MN). The ELISAs were run according to the manufacturer′s instructions. The intensity of the color measured is in proportion to the amount of rat cytokine bound in the initial steps. The sample values were then read off from the standard curve.

Statistical analysis

Results were expressed as mean±SEM. The intergroup variation was measured by one-way analysis of variance (ANOVA) followed by Tukey′s test using the Jandel Sigma Stat Statistical Software version 2.0. P<0.05 was considered statistical significant.

Results

Assessment of food and water consumption

LPS challenge led to a marked decrease in food and water consumption in rats. This decrease was significantly (P<0.05) prevented by QT (2 and 25 mg/kg) treatment. However, chronic treatment with QT for 14 days did not alter food and water consumption [Table - 1].

Assessment of body weight

LPS produced a significant (P<0.05) reduction in body weight in rats. Although LPS reduced body weight in both the groups, the reduction in vehicle treated rats was significantly more as compared to the QT treated group [Table - 1].

Assessment of ambulation and rearing

Both the ambulatory and rearing scores decreased significantly (P <0.05) after LPS administration. However, both the doses of QT significantly attenuated LPS-induced reduction in ambulation and rearing [Table - 2].

Assessment of plus maze response

LPS induced an anxiogenic effect in vehicle-treated group and significantly (P<0.05) decreased the time spent in open arm in plus maze. Chronic QT (2 and 25 mg/kg) treatment per se did not produce any change in the time spent in open arm but significantly reversed the effect of LPS [Table - 3].

Assessment of cytokines levels

Six hours after endotoxin administration, brain TNF-α, IL-1β, and IL-6 levels decreased significantly (P <0.05) in rats pretreated with QT (25 mg/kg) as compared to rats pretreated with the vehicle. A 2 mg/kg dose of quercetin significantly decreased IL-6 and TNF-α levels in LPS-challenged rats but no significant decrease was seen in brain levels of IL-1β [Table - 4].

Assessment of oxidative stress in brain

LPS induced a significant oxidative stress characterized by increased MDA levels [Figure - 1] and decreased level of glutathione in vehicle-treated groups (P <0.05). Chronic administration of QT (2 and 25 mg/kg) significantly (P <0.05) attenuated the effect of LPS [Figure - 2].

Discussion

In the present study, administration of LPS reduced appetite, body weight, suppressed locomotor and exploratory activity, and induced an anxiogenic response in the rodents. LPS also elicited a marked oxidative stress in rat brain. These findings replicate the results of previous studies that demonstrated that activation of the immune system by LPS, as well as other immune challenges, induces reduction in appetite and body weight, suppresses exploratory and social activity, fatigue and malaise, impairment in cognitive abilities, reduced libido and sexual behavior and anhedonia. ^[15]

ROS produced by LPS modulates production of inflammatory cytokines which are responsible for causing depression of social exploration, anorexia, and body weight loss. ^[16] TNF-α and IL-6 are known to depress food intake by a centrally mediated effect leading to loss of body weight. ^[17] This might be due to the direct action of peripheral cytokines on glucose-sensitive neurons in hypothalamic nuclei such as the lateral hypothalamic arc thus driving the suppression of food intake. In our study, both the doses of QT significantly decreased brain cytokines levels after 6 h of LPS administration as compared to rats treated with saline. It has been established that IL-1, TNF-α, and other cytokines produced in response to LPS activates HPA axis is associated with stereotypical behavioral depression including reduced locomotor activity in animals ^[18] as well as increased concentration of corticotrophin-releasing hormone (CRH), known to promote anxiety. ^[19] The present study observed that both the doses of QT significantly attenuated the depressed locomotion in LPS-challenged rats and reversed the LPS-induced anxiogenic effect in animals.

LPS activates macrophages and neutrophils thereby generating a large amount of superoxide and nitric oxide, a mechanism by which foreign organisms are destroyed. However, excessive production of superoxide can lead to multiple organ damage. QT is a potent antioxidant and our study also showed that oxidative damage caused by LPS was significantly attenuated by it. The protective effect against lipoperoxidative damage may be due to hydrogen donating capacity of hydroxyl groups and inhibition of iNOS overexpression. Besides, it is a potent inhibitor of human neutrophil degranulation and superoxide anion production.

Quercetin inhibits LPS-induced TNF-α production in macrophages. ^[20] Moreover, it was found that in glial cells quercetin can inhibit LPS-induced mRNA levels of two cytokines, i.e. TNF- α and IL-1. ^[21] QT interferes with the transcription of the TNF-α and NO genes ^[22] and also inhibits ERK1/2 phosphorylation and p38 MAPK (mitogen activated protein kinase) activity, which are important in the post-transcriptional regulation of a TNF-α mRNA. A possible explanation for the ameliorating effect of quercetin in sickness behavior may be found in the interplay between oxidative stress and inflammatory cytokines. As already discussed, ROS are not only involved in the occurrence of oxidative stress, but are also involved in modulation of NF-kappa β transcription, and the NFkβ activated by LPS induces many inflammatory genes that encode for pro-inflammatory cytokines, chemokines, that selectively induce the inflammatory enzymes such as inducible NO synthase, COX-2, and adhesion molecules. QT decreases LPS-induced NF-kB activation. ^[6],[23] Consequently, scavenging ROS would not only prevent the occurrence of oxidative stress but also help mitigate the symptoms of sickness behavior.

Thus, chronic QT ameliorated reductions in food, water consumption, body weight, reversed anxiogenic effect, and depressed locomotion induced by LPS by its ROS scavenging, TNF-α, IL-6, and IL-1β inhibiting property. The study suggests that ensuring adequate intake of flavonoids like quercetin can improve the general health by reducing the time required to recover from infection.

References

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