|Year : 2011 | Volume
| Issue : 2 | Page : 192-196
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
|Date of Submission||20-Mar-2010|
|Date of Decision||22-Nov-2010|
|Date of Acceptance||03-Jan-2011|
|Date of Web Publication||6-Mar-2011|
Sangeeta Pilkhwal Sah
Department of Pharmaceutical Sciences, Kumaun University, Nainital, Uttarakhand
Source of Support: None, Conflict of Interest: None
Objectives : Gram-negative infections and control infusion of recombinant cytokines in human have been shown to induce sickness behavior characterized by fever, prolong sleep, decreased food and water intake, reduced mobility, depression, and anxiety. Therefore, the present study was undertaken to investigate the effect of bioflavonoid quercetin in lipopolysaccharide (LPS)-induced sickness behavior.
Materials and Methods : Wistar albino rats were divided into six groups (n=6). Three groups received vehicle and two doses of quercetin (2 and 25 mg/kg, i.p.) respectively for 2 weeks before being challenged with LPS (1 mg/kg, i.p). One group received vehicle for 2 weeks and was challenged with saline on day 15. The per se effect of quercetin (2 and 25 mg/kg, i.p.) was also seen after 2 weeks of dosing. LPS-induced sickness behavior in rats was quantified by measuring time in social exploration, anxiety, food and water consumption, and weight loss. Levels of cytokines (TNF-α, IL-1β, and IL-6) and oxidative stress in rat brain were also analyzed.
Results : Quercetin (2 and 25 mg/kg) administration significantly (P<0.05) attenuated LPS-induced sickness behavior by modulating cytokines production as well inhibiting LPS-induced oxidative stress.
Conclusions : Adequate intake of dietary flavonoids (like quercetin) may help promote recovery from sickness behavior.
Keywords: Lipopolysaccharide, oxidative stress, polyphenols, Quercetin, sickness behavior
|How to cite this article:|
Sah SP, Tirkey N, Kuhad A, Chopra K. Effect of quercetin on lipopolysaccharide induced-sickness behavior and oxidative stress in rats. Indian J Pharmacol 2011;43:192-6
|How to cite this URL:|
Sah SP, Tirkey N, Kuhad A, Chopra K. Effect of quercetin on lipopolysaccharide induced-sickness behavior and oxidative stress in rats. Indian J Pharmacol [serial online] 2011 [cited 2019 Nov 22];43:192-6. Available from: http://www.ijp-online.com/text.asp?2011/43/2/192/77365
| » Introduction|| |
An endotoxin or lipopolysaccharide (LPS) challenge results in highly differentiated changes in brain neurotransmission probably subserving the coordinate processing of immune information.  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.  In the late phase of inflammatory response in sickness behavior (also seen during gram negative bacterial infections),  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.  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.  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.  Another study reports that quercetin significantly inhibited TNF-α production and gene expression in a dose-dependent manner via modulation of the NFkβ system. 
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|| |
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.
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. 
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. 
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.  Quercetin can cross the blood--brain barrier and once daily dosing was chosen for the study.  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.  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.  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.  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.  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.
The protein content was measured according to the method of Lowry et al. using bovine serum albumin as standard. 
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.
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].
|Table 1: Effect of chronic administration of quercetin on body weight, water, and food intake in rats|
Click here to view
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].
|Table 2: Effect of chronic administration of quercetin on ambulations and rearing in rats|
Click here to view
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].
|Table 3: Effect of chronic administration of quercetin in plus maze in rats|
Click here to view
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].
|Table 4: Effect of chronic administration of quercetin on brain IL-1β, IL-6 and TNF- α levels in rats after LPS administration|
Click here to view
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].
|Figure 1: Effect of chronic QT administration on MDA levels after LPS treatment.|
Click here to view
|Figure 2: Effect of chronic QT administration on glutathione levels after LPS treatment.|
Click here to view
| » 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. 
ROS produced by LPS modulates production of inflammatory cytokines which are responsible for causing depression of social exploration, anorexia, and body weight loss.  TNF-α and IL-6 are known to depress food intake by a centrally mediated effect leading to loss of body weight.  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  as well as increased concentration of corticotrophin-releasing hormone (CRH), known to promote anxiety.  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.  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.  QT interferes with the transcription of the TNF-α and NO genes  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. , 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|| |
|1.||Linthorst AC, Reul JM. Brain neurotransmission during peripheral inflammation. Ann N Y Acad Sci 1998;840:139-52. |
|2.||Turnbull AV, Rivier C. Inhibition of gonadotropin-induced testosterone secretion by the intracerebroventricular injection of interleukin-1 beta in the male rat. Endocrinology 1997;138:1008-13. |
|3.||Kelley KW, Bluthé RM, Dantzer R, Zhou JH, Shen WH, Johnson RW, et al. Cytokine-induced sickness behavior. Brain Behav Immun 2003;17:S112-S8. |
|4.||Asehnoune K, Strassheim D, Mitra S, Kim JY, Abraham E. Involvement of reactive oxygen species in Toll-like receptor 4-dependent activation of NF-kappa B. J Immunol 2004;172:2522-9. |
|5.||Berg BM, Godbout JP, Kelley KW, Johnson RW. Alpha-tocopherol attenuates lipopolysaccharide-induced sickness behavior in mice. Brain Behav Immun 2004;18:149-57. |
|6.||Wadsworth TL, Koop DR. Effects of the wine polyphenolics quercetin and resveratrol on pro-inflammatory cytokine expression in RAW 264.7 macrophages. Biochem Pharmacol 1999;15:941-9. |
|7.||Nair MP, Mahajan S, Reynolds JL, Aalinkeel R, Nair H, Schwartz SA, et al. The flavonoid quercetin inhibits proinflammatory cytokine (tumor necrosis factor alpha) gene expression in normal peripheral blood mononuclear cells via modulation of the NF-kappa beta system. Clin Vaccine Immunol 2006;13:319-28. |
|8.||Kulkarni SK. Handbook of Experimental Pharmacology, New Delhi: Vallabh Prakashan; 1999. p. 36. |
|9.||Naidu PS, Singh A, Kulkarni SK. Quercetin, a bioflavonoid, attenuates haloperidol-induced orofacial dyskinesia. Neuropharmacology 2003;44:1100-6. |
|10.||Youdim KA, Qaiser MZ, Begley DJ, Rice-Evans CA, Abbott NJ. Flavonoid permeability across an in situ model of the blood-brain barrier. Free Radic Biol Med 2004;36:592-604. |
|11.||Castanon N, Bluthé RM, Dantzer R. Chronic treatment with the atypical antidepressant tianeptine attenuates sickness behavior induced by peripheral but not central lipopolysaccharide and interleukin-1beta in the rat. Psychopharmacology (Berl) 2001;154:50-60. |
|12.||Wills ED. Mechanisms of lipid peroxide formation in animal tissues. Biochem J 1999;99:667-76. |
|13.||Jollow DJ, Mitchell JR, Zampaglione N, Gillette JR. Bromobenzene-induced liver necrosis. Protective role of glutathione and evidence for 3,4-bromobenzene oxide as the hepatotoxic metabolite. Pharmacology 1974;11:151-69. |
|14.||Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin-Phenol reagent. J Biol Chem 1951;193:265-75. |
|15.||Dantzer R. Cytokine-induced sickness behavior: Mechanisms and implications. Ann N Y Acad Sci 2001;933:222-34. |
|16.||Layé S, Gheusi G, Cremona S, Combe C, Kelley K, Dantzer R, et al. Endogenous brain IL-1 mediates LPS-induced anorexia and hypothalamic cytokine expression. Am J Physiol Regul Integr Comp Physiol 2000;279:R93-8. |
|17.||Reichenberg A, Kraus T, Haack M, Schuld A, Pollmächer T, Yirmiya R. Endotoxin-induced changes in food consumption in healthy volunteers are associated with TNF-alpha and IL-6 secretion. Psychoneuroendocrinology 2002;27:945-56. |
|18.||Schöbitz B, De Kloet ER, Holsboer F. Gene expression and function of interleukin 1, interleukin 6 and tumor necrosis factor in the brain. Prog Neurobiol 1994;44:397-432. |
|19.||Heilig M, Koob GF, Ekman R, Britton KT. Corticotropin-releasing factor and neuropeptide Y: Role in emotional integration. Trends Neurosci 1994;17:80-5. |
|20.||Manjeet KR, Ghosh B. Quercetin inhibits LPS-induced nitric oxide and tumor necrosis factor-alpha production in murine macrophages. Int J Immunopharmacol 1999;21:435-43. |
|21.||Bureau G, Longpre F, Martinoli MG. Resveratrol and quercetin, two natural polyphenols, reduce apoptotic neuronal cell death induced by neuroinflammation. J Neurosci Res 2008;86:403-10. |
|22.||Xie QW, Kashiwabara Y, Nathan C. Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase. J Biol Chem 1994;269:4705-8. |
|23.||Dias AS, Porawski M, Alonso M, Marroni N, Collado PS, González-Gallego J. Quercetin decreases oxidative stress, NF-kappaB activation, and iNOS overexpression in liver of streptozotocin-induced diabetic rats. J Nutr 2005;135:2299-04. |
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]
|This article has been cited by|
||Beneficial effect of honokiol on lipopolysaccharide induced anxiety-like behavior and liver damage in mice
| ||Kunjbihari Sulakhiya,Parveen Kumar,Satendra S. Gurjar,Chandana C. Barua,Naba K. Hazarika |
| ||Pharmacology Biochemistry and Behavior. 2015; 132: 79 |
|[Pubmed] | [DOI]|
||Effect of Ginkgo Biloba Extract on Lipopolysaccharide-induced Anhedonic Depressive-like Behavior in Male Rats
| ||Kuei-Ying Yeh,Sing-Siang Shou,Yi-Xuan Lin,Chao-Cin Chen,Chen-Yen Chiang,Chien-Yu Yeh |
| ||Phytotherapy Research. 2014; : n/a |
|[Pubmed] | [DOI]|
||Effects of long-term etanercept treatment on anxiety- and depression-like neurobehaviors in rats
| ||Dilek Bayramgürler,Ayse Karson,Cüneyt Özer,Tijen Utkan |
| ||Physiology & Behavior. 2013; 119: 145 |
|[Pubmed] | [DOI]|
||In depression, bacterial translocation may drive inflammatory responses, oxidative and nitrosative stress (O&NS), and autoimmune responses directed against O&NS-damaged neoepitopes
| ||M. Maes,M. Kubera,J.-C. Leunis,M. Berk,M. Geffard,E. Bosmans |
| ||Acta Psychiatrica Scandinavica. 2013; 127(5): 344 |
|[Pubmed] | [DOI]|