Mochamad Rasjad Indra 1*, Satuman Karyono 1, Retty Ratnawati 1 and Safarina G Malik 2

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1 Indr et l. BMC Reserch Notes 2013, 6:275 SHORT REPORT Open Access Quercetin suppresses inflmmtion y reducing ERK1/2 phosphoryltion nd NF kpp B ctivtion in Leptin-induced Humn Umilicl Vein Endothelil Cells (HUVECs) Mochmd Rsjd Indr 1*, Stumn Kryono 1, Retty Rtnwti 1 nd Sfrin G Mlik 2 Astrct Bckground: High concentrtions of plsm leptin nd the relese of pro-inflmmtory cytokines in leptin-resistnce in oesity hve een reported to trigger endothelil dysfunction. The ojective of this study ws to elucidte the role of quercetin in modulting leptin-induced inflmmtion s ssessed y the levels of O-R expression, ERK1/2 phosphoryltion, NF-kpp B ctivtion nd TNF-lph secretion in umilicl vein endothelil cells (HUVECs) in vitro. Findings: HUVECs were exposed to either control levels (0 ng/ml) or 500 ng/ml leptin (L) for 48 hours, followed y control or 125 um quercetin (Q) for nother 6 h. The experimentl groups were s follows: L0Q0, L0Q125, L500Q0, L500Q125. The presence of the short chin leptin receptor isoform O-R in HUVECs ws determined y Western lot nd immunocytochemistry nlyses. O-R expression, ERK1/2 phosphoryltion, NF-kpp B ctivtion nd TNF-lph secretion were quntified y ELISA, nd NF-kpp B ctivtiony immunofluorescence stining. Our results showed tht O-R expression, ERK1/2 phosphoryltion nd NF-kpp B ctivtion incresed significntly fter 500 ng/ml leptin exposure (1.8x, 1.5x, 6.2x for O-R, ERK1/2 nd NF-kpp B, respectively), ut were reduced y ddition of 125 um quercetin (0.7x, 0.3x nd 0.4x for O-R, ERK1/2 nd NF-kpp B, respectively), nd tht quercetin could lso prtilly suppress leptin-induced TNF-lph secretion (3.8x) y 0.8x. Conclusion: Exposure of HUVECs to leptin up-regulted O-R expression nd elevted ERK1/2 phosphoryltion nd NFkB ctivtion, nd incresed TNF-lph secretion. These effects strongly suppressed y quercetin, with the exception of TNF-lph which ws prtilly suppressed. The findings might e of clinicl significnce, s endothelil dysfunction tht could led to crdiovsculr disese is preventle, nd quercetin is nturl compound found in vrious plnts nd fruits. Keywords: Leptin, Quercetin, O-R, ERK1/2, NFκB, TNFα, HUVECs Findings Oesity hs ecome glol helth prolem, with the prevlence of overweight nd oesity reching criticl levels throughout the world, including Indonesi. A ntionl survey in 2007 in 12 Indonesin provinces showed tht 18.8% of the popultion older thn 15 yers old re oese [1]. Oesity is mjor risk fctor for crdiovsculr * Correspondence: rsjdindr@yhoo.com 1 Deprtment of Physiology, Fculty of Medicine, Brwijy University, Jl. Vetern, Mlng 65145, Est Jv, Indonesi Full list of uthor informtion is ville t the end of the rticle disese, hypertension, dyslipidemi nd dietes mellitus, ll of which reduce oth the qulity of life nd life expectncy. Oesity is ssocited with excessive dipose tissue ccumultion due to excessive energy intke nd insufficient energy expenditure [2], nd is chrcterized y the ltertion of leptin levels, cytokine produced y dipocytes nd one of the regultors of energy metolism. Studies hve shown tht most oese ptients re leptin resistnt, nd high leptin levels were oserved in these individuls [3]. An ssocition etween leptin nd incresed crdiovsculr risk hs een reported [4], nd is ssocited with incresed levels of inflmmtory fctors exhiiting 2013 Indr et l.; licensee BioMed Centrl Ltd. This is n Open Access rticle distriuted under the terms of the Cretive Commons Attriution License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited.

2 Indr et l. BMC Reserch Notes 2013, 6:275 Pge 2 of 8 pro-therogenic effects [5-7]. Oesity hs lso een considered s stte of low-grde inflmmtion [8]; previous reserch hs shown tht therosclerosis is the result of chronic inflmmtion, nd erly therosclerosis formtion is induced y pro-inflmmtory cytokines nd other proteins produced y inflmmtory cells [9,10]. In oesity-relted high plsm leptin conditions, inflmmtion occurs when signl trnsduction pthwys re ctivted, such s ctivtion of NFκΒ, y the inding of leptin to its receptor (O-R), nd susequent relese of the inflmmtion fctors, for instnce tumour necrosis fctor lph (TNFα) [11]. Our preliminry results reveled tht 500 ng/ml leptin decreses cell prolifertion nd increses TNFα, monocyte chemottrctnt protein- 1 (MCP-1), nd intrcellulr C 2+ levels in humn umilicl vein endothelil cells (HUVECs) [12]. Quercetin, flvonoid compound found in plnts nd fruits, hs een reported to hve nti-inflmmtory effects [13], which re medited through the inhiition of pro-inflmmtory cytokines [14]. The im of this study ws to investigte the effect of quercetin in modulting the expression of O-R, phosphoryltion of ERK1/2, ctivtion of NFκB nd secretion of TNFα in leptin-induced humn umilicl vein endothelil cells (HUVECs) in vitro. Methods Smples Humn umilicl vein endothelil cells (HUVECs) were otined from umilicl cords of ptients tht hve undergone cesren section in Dr. Syiful Anwr Hospitl, Mlng, fter otining informed consent. This reserch ws pproved y the institutionl reserch ethicl committee from the Fculty of Medicine, Brwijy University, Mlng. Cell culture nd tretment HUVECs were isolted nd cultured s descried previously [15,16]. Briefly, HUVECs were cultured in M- 199 medium (Sigm-Aldrich, USA) supplemented with 10% fetl clf serum (Biochrom, Germny), g/ ml L-glutmine (Gico, USA), 50 U/mL penicillin (Gico, USA), nd 50 mg/ml streptomycin (Gico, USA), t 37 C in 5% CO2 incutor. Humn recominnt leptin (500 ng/ml; Sigm-Aldrich, USA) dissolved in dimethyl sulfoxide/dmso (MPBio, USA) ws dded to HUVECs nd incuted for 48 hours. Quercetin (125 μm; Sigm-Aldrich, USA) dissolved in methylcellulose (MPBio, USA), ws dded to leptinexposed HUVECS for 6 hours. HUVECs tht were not exposed to leptin were treted with 0 μm nd 125 μm quercetin for 6 hours. Quercetin concentrtion of 125 μm ws reported to hve mrginl cell toxicity [17]. The experimentl groups were: L0Q0 without leptin nd quercetin (control, with DMSO nd methylcellulose only); L0Q125 without leptin ut with 125 μm quercetin; L500Q0 with 500 ng/ml leptin ut without quercetin; L500Q125 with 500 ng/ml leptin nd 125 μm quercetin. The experiments were repeted 5 times for ech group. Western lot nlysis Proteins were extrcted from HUVECs y using mmmlin protein extrction regent (Pierce, IL). Protein concentrtions were determined using Coomssie protein regent (Bio-Rd, CA). Thirty microgrms of totl protein ws loded per lne nd seprted y 7.5% sodium dodecyl sulfte-tris-glycine polycrylmide gel electrophoresis. Proteins were trnsferred to nitrocellulose memrnes nd locked overnight in Tris-uffered sline (TBS) contining 0.1% Tween nd 5% nonft dry milk. Memrnes were proed with rit polyclonl ntiody directed ginst either humn short leptin receptor (OR) or humn phosphorylted ERK1/2 or humn p65 NFκB ntiodies (1:1000; Cell Signling, MA), or mouse ntihumn β-ctin monoclonl ntiody (1:1.000; Snt Cruz Biotech, USA), then were detected using Biotinconjugted got nti-rit IgG (Snt Cruz Biotech, USA). Visuliztion ws done fter incution with streptvidin-lkline phosphtse conjugte (Invitrogen, USA), y employing colorimetric detection using nitrolue tetrzolium/5-romo-4-chloro-3-indolylphosphte regent (Roche, IN). O-R Immunocytochemistry HUVECs were fixed with methnol in glss slides, then gently rinsed with phosphte uffer solution (PBS). Norml humn serum (1:10 dilution) (MPBio, USA) ws pplied nd incuted for minutes t 37 C. Rit nti-humn O-R ntiody (1:100) (Snt Cruz Biotech, USA) ws pplied nd the specimens were incuted overnight t 4 C. Lelled nti rit IgG-SA-HRP (KPL, USA) ws pplied for 60 minutes, then sustrtechromogensolution3,3 -diminoenzidine ws dded, nd the specimens were incuted for 10 minutes. Hemtoxylin counterstining ws performed to stin the nucleus. The slides were then covered with cover slips. Ezyme linked immunosorent ssy Cell lyste were otined from hrvested HUVECs, then ELISA ws performed to quntify the level of O-R (BioSource Interntionl, USA), totl phosphorylted ERK1/2 (Assy Designs, USA), nd p50/ p65 NFκB (Imgenex,USA). ELISA for secreted TNFα (Bender MedSystem, Austri) ws conducted using the superntnt of HUVECs culture.

3 Indr et l. BMC Reserch Notes 2013, 6:275 Pge 3 of 8 NFκB Immunofluorescence Su-confluent HUVECs grown on cover slips were fixed with methnol t room temperture for 10 minutes, leled with 10 μg/ml ntihumn p50/p65 NFκB ntiody (Thermo Scientific, USA) for 1 hour, wshed 3 times with PBS, then incuted with 8 μg/ml FITC-leled got nti-rit IgG (Snt Cruz Biotechnology Inc, USA) for 1 hour. Bound ntiody ws detected using Fluo view 1000 confocl microscope (Olympus, Jpn) equipped for epiluminescence. Sttisticl nlyses Results were the verge of t lest five repets. Differences etween groups were determined y ANOVA, followed y Tukey s test (SPSS vs. 17). All dt were shown s mens ± SD, nd p < 0.05 ws considered sttisticlly significnt. Results O-R ws expressed in HUVECs The short isoform of leptin receptor (O-R) ws expressed in HUVECs, s demonstrted y Western lot nlysis shown in Figure 1A. After leptin induction the expression of O-R ws incresed lmost two folds s compred to control (nd intensities re 1488 ± vs ± 380.8, without nd with leptin exposure, p = 0.001). Immunocytochemistry stining confirmed the incresed expression of O-R in leptin-induced HUVECs (Figure 1B). Quercetin down-regulted leptin receptor expression The short isoform of leptin receptor (O-R) expression ws significntly incresed 1.8x fter 48 hours leptin exposure (500 ng/ml) (9966 ± vs ± 1823 ng/ml, L0Q0 vs. L500Q0, p =10-4 ). Quercetin (125 μm) down-regulted the leptin-induced O-R expression y 0.7x(13449 ± vs ± 1823 ng/ ml, L500Q125 vs. L500Q0, p = 0.028). Quercetin lone (L0Q125) incresed leptin receptor expression (13660 ± 1452 vs ± ng/ml, L0Q125 vs. L0Q0, p = 0.020) (Figure 2). Quercetin reduced leptin-induced ERK1/2 phosphoryltion Quntittive mesurement y ELISA reveled 1.5x higher totl phosphorylted ERK1/2 level in leptininduced HUVECs (85.00 ± vs ±22.55 ng/ ml, L0Q0 vs. L500Q0, p = 0.012). Addition of 125 μm quercetin (L500Q125) reduced ERK1/2 phosphoryltion in leptin-induced HUVECs y 0.3x (40.00 ± 1.73 vs ±22.55 ng/ml, L500Q125 vs. L500Q0, p =10-4 ). We oserved tht quercetin lone lso reduced ERK1/2 totl phosphoryltion (43.33 ± 4.93 vs ± ng/ ml, L0Q125 vs. L0Q0, p =0.016)(Figure3). Quercetin inhiited leptin-induced NFκB ctivtion FITC immunoleling ws used to determine the NFκB ctivtion. As shown in Figure 4A signl density ws incresed fter HUVECs ws exposed to 500 ng/ml leptin s compred to control. Quercetin lone did not A B 1 2 Figure 1 O-R ws expressed in HUVECs. (A) Western lot nlysis of O-R in HUVECs showed tht exposure of 500 ng/ml leptin for 48 h incresed O-R expression (without (lne 1) nd with (lne 2) leptin exposure). β-ctin expression in leptin-induced HUVECs ws similr with control (without leptin induction). (B) Confirmtion of incresed O-R expression fter leptin exposure y immunocytochemistry using specific ntiody ginst O-R (without (pnel 1) nd with (pnel 2) leptin exposure).

4 Indr et l. BMC Reserch Notes 2013, 6:275 Pge 4 of 8 c Figure 2 Quercetin down-regultes leptin receptor expression. O-R expression mesured y ELISA ws incresed fter 500 ng/ml leptin exposure, nd 125 μm quercetin ddition ws le to decrese the expression of O-R in leptin-induced HUVECs. L0Q0 control HUVECs without leptin nd quercetin; L0Q125 without leptin, ut with 125 μm quercetin; L500Q0 with 500 ng/ml leptin, ut without quercetin; L500Q125 with 500 ng/ml leptin nd 125 μm quercetin. Mens without common letter differ, t lest p < induce NFκB ctivtion. However, quercetin ddition in leptin-induced HUVECs reduced the density of NFκΒ. This phenomenon ws confirmed y Western lot nlysis (Figure 4B). A further confirmtion y ELISA showed significnt 6.2x incresed of NFκΒ ctivtion in leptin-induced HUVECs when compred to control (111.6 ± vs ± ng/ml, L0Q0 vs. L500Q0, p =10-5 ). After quercetin ddition, NFκB ctivtion ws significntly reduced y 0.4x (304.4 ± vs ± ng/ml, L500Q125 vs. L500Q0, A kD Bet ctin B Totl Phosphorylted ERK1/2 (ng/ml) L0Q0 L0Q125 L500Q0 L500Q125 Figure 3 Quercetin reduced leptin-induced ERK1/2 phosphoryltion. (A) Western lot nlysis of phosphorylted ERK1/2 in HUVECs showed tht 500 ng/ml leptin exposure for 48 h incresed ERK1/2 phosphoryltion (without (lne 1) nd with (lne 3) leptin exposure). Quercetin lone did not show ny effect (lne 2), nevertheless in leptin-induced HUVECs, ERK1/2 phosphoryltion ws reduced (lne 4). (B) Totl phosphorylted ERK1/2 level mesured y ELISA ws reduced fter quercetin tretment on leptin-induced HUVECs. L0Q0 control HUVECs without leptin nd quercetin; L0Q125 without leptin, ut with 125 μm quercetin; L500Q0 with 500 ng/ml leptin, ut without quercetin; L500Q125 with 500 ng/ml leptin nd 125 μm quercetin. Mens without common letter differ, t lest p < c

5 Indr et l. BMC Reserch Notes 2013, 6:275 Pge 5 of 8 A B kd Bet ctin C c Totl NFkB levels (ng/ml) L0Q0 L0Q125 L500Q0 L500Q125 Figure 4 Quercetin inhiits leptin-induced NFκB ctivtion. (A) FITC immunofluorescence leling showed the signl density ws incresed fter 500 ng/ml leptin exposure s compred to control. Quercetin ddition in leptin-induced HUVECs reduced the density of NFκΒ. Pnel 1 control HUVECs without leptin nd quercetin; pnel 2 without leptin, ut with 125 μm quercetin; pnel 3 with 500 ng/ml leptin, ut without quercetin; pnel 4 with 500 ng/ml leptin nd 125 μm quercetin. (B) Western lot nlysis of p65 NFκB in HUVECs confirmed the immunofluorescence leling. L0Q0 control HUVECs without leptin nd quercetin; L0Q125 without leptin, with 125 μm quercetin; L500Q0 with 500 ng/ml leptin, without quercetin; L500Q125 with 500 ng/ml leptin nd 125 μm quercetin. (C) ELISA mesurement showed significnt incresed of NFκΒ ctivtion in leptin-induced HUVECs when compred to control, nd quercetin dministrtion significntly reduced the ctivted NFκB level. L0Q0 control HUVECs without leptin nd quercetin; L0Q125 without leptin, ut with 125 μm quercetin; L500Q0 with 500 ng/ml leptin, ut without quercetin; L500Q125 with 500 ng/ml leptin nd 125 μm quercetin. Mens without common letter differ, p <

6 Indr et l. BMC Reserch Notes 2013, 6:275 Pge 6 of 8 p =10-4 ). Quercetin lone did not ctivte NFκB (Figure 4C). Quercetin prtilly suppressed leptin-induced TNFα secretion Secreted TNFα level ws mrkedly incresed y 3.2x in leptin-induced HUVECs s compred to control (22.39 ± 2.29 vs ± 7.33 ng/ml, L0Q0 vs. L500Q0, p =10-4 ). TNFα secretion ws decresed, leit prtilly (0.8x), fter quercetin ddition in leptin-induced HUVECs (L500Q125) when compred to leptin-induced HUVECs without quercetin (L0Q125) (60.38 ± 1.04 vs ± 7.33 ng/ml, L500Q125 vs. L500Q0, p = 0.022). TNFα secrettion ws lso incresed when HUVECs were exposed to quercetin lone (Figure 5). Discussion We hve demonstrted the presence of the short chin leptin receptor isoform O-R in HUVECs, confirming previous studies [16,17]. We were le to show tht O-R expression ws incresed fter leptin exposure. Leptin hs een reported to differentilly modulte the expression of its receptors in oth dose- nd tissuedependent mnner [18]. When treted with high leptin concentrtion, the level of leptin receptor ws incresed, reported to e due to chnges in receptor numer tht occur prior to gene expression chnges [19], which ws expected to hppen post trnsltionlly [20]. We oserved tht up-regulted O-R expression ws followed y elevted ERK1/2 phosphoryltion, in line with previous reports tht demonstrted ERK1 nd ERK2 ctivtion y the short leptin receptor isoform [21], nd leptin-stimulted pro-inflmmtory cytokine relese rogtion y ERK 1/2 MAPK inhiitor U0126 [22]. As shown from our result, the elevted ERK1/2 phosphoryltion ws followed y incresed NFκB ctivtion nd TNFα secretion, which ws in greement with previous report tht indicted leptin hs proinflmmtory ction, involving pro-inflmmtory cytokines TNFα through NFκB regultion [22]. Quercetin is one of the most widely distriuted flvonoids in fruits, vegetles, te nd wine [23], reported to hve nti-inflmmtory properties, nd might ct s helth-promoting sustnces [24,25]. In this study, we showed tht 125 μm quercetin ws le to downregulte O-R expression, reduce ERK1/2 phosphoryltion, decrese NFκB ctivtion, nd prtilly suppress TNFα secretion in leptin-induced HUVECs. The ntiinflmmtory effect of quercetin hs een descried in vrious reports, mong others were the ility to suppress ERK phosphoryltion in 3 T3-L1 dipocytes [26] nd NCI-H292 cells [27], inhiit NFκB ctivtion in murine J774 mcrophges [28] nd one mrrowderived mcrophges [29], VAT TNFα production in oese Zucker rts [30], reduce the ctivtion of phosphorylted ERK kinse strongly in LPS-induced RAW cells nd inhiit NFκB ctivtion through oth stiliztion of NFκB/IκB complex nd suppression of proinflmmtory cytokines including TNFα [31]. In PBMC, 50% reduction of TNFα gene expression ws oserved fter 24 h exposure to 50 μm quercetin [14]. A met-nlysis of long-term plceo-controlled humn intervention trils reported tht TNFα levels were decresed fter flvonoid consumption, ut only in fixed model, nd higher dose or longer durtion intervention were not ssocited with greter effect size [32]. Although preliminry, our dt suggested tht suppression of TNFα secretion y quercetin ws ssocited with d c Figure 5 Quercetin prtilly suppressed leptin-induced TNFα secretion. TNFα secretion ws mrkedly incresed in leptin-induced HUVECs s compred to control, nd ws prtilly decresed fter quercetin ddition. L0Q0 control HUVECs without leptin nd quercetin; L0Q125 without leptin, ut with 125 μm quercetin; L500Q0 with 500 ng/ml leptin, ut without quercetin; L500Q125 with 500 ng/ml leptin nd 125 μm quercetin. Mens without common letter differ, t lest p < 0.05.

7 Indr et l. BMC Reserch Notes 2013, 6:275 Pge 7 of 8 inhiition of NFκB ctivtion, s reported previously [28]. However, our result showed tht quercetin suppression of TNFα secretion ppered to e prtil, which we elieve might e due to the short durtion of quercetin exposure period. Although essentil, unfortuntely we cn not perform further confirmtion of TNFα suppression y TNFα mrna ssessment, since our smples were not prepred nd stored for RNA nlysis. This is the limittion of our study, esides tht we only pply one quercetin concentrtion in our study, which might not e the optimum concentrtion. Further studies, such s mesurements of TNFα mrna nd ppliction of NFkB ctivtion inhiitor, re still needed to clrify this discrepncy. The suppression of inflmmtion y quercetin my hve clinicl significnce in preventing crdiovsculr disese induced y leptin-resistnt in oesity. As reported previously, quercetin dministrtion to oese Zucker rts improved dyslipidemi, hypertension, nd hyperinsulinemi, ll of which re crdiovsculr risk fctors [30]. A deeper ssessements on the moleculr mechnisms of quercetin ction in suppressing leptin-induced inflmmtion re needed s high intke of plnt-derived food rich in quercetin or the use of supplements of this flvonoid might e protective nd could reduce crdiovsculr risks. It might lso e promising re for the development of flvonoid-sed neutrphrmceuticl gents for the tretment of chronic inflmmtory disese. Conclusion Leptin exposure of HUVECs resulted in up-regulted O-R expression nd elevted ERK1/2 phosphoryltion nd NFkB ctivtion, nd incresed TNF-lph secretion. Despite strong suppression effect of quercetin on the incresed O-R expression, ERK1/2 phosphoryltion nd NFkB ctivtion, however, TNFα secretion ws only prtilly suppressed. Therefore, further studies re still needed to elucidte the sis of this exception. Arevitions HUVECs: Humn umilicl vein endothelil cells; TNFα: Tumor necrosis fctor lph; O-R: Leptin receptor; MAPK: Mitogen ctivted protein kinse; ERK1/ 2: Extrcellulr regulted kinse 1/2; NFκΒ: Nucler fctor kpp ; L: Leptin; Q: Quercetin; ELISA: Enzyme linked immunosorent ssy; FITC: Fluorescence isothiocynte; ANOVA: Anlysis of vrince. Competing interest The uthors declre tht they hve no competing interest. Authors contriutions MRI designed the study, performed dt nlysis, wrote nd revised the finl mnuscript. SK crried out ll ssys nd performed dt nlysis. RR designed the study, performed dt nlysis, wrote nd revised the finl mnuscript. SGM provided direction, performed dt nlysis nd revised the finl mnuscript. All uthors hve red nd pproved the finl mnuscript. Acknowledgement This study ws fully funded y Indonesin Dnone Institute Foundtion (Grnt numer I ). The views expressed herein re those of the individul uthors, nd do not necessrily reflect those of Indonesin Dnone Institute Foundtion. The uthors would like to thnk Dr. Budi Siswnto, MD, SpOG who prepred humn fetl umilicls, Ms. Umi Slmh nd Mr. Budi Wicksono for their technicl ssistnce. Author detils 1 Deprtment of Physiology, Fculty of Medicine, Brwijy University, Jl. Vetern, Mlng 65145, Est Jv, Indonesi. 2 Eijkmn Institute for Moleculr Biology, Jl. Diponegoro 69, Jkrt 10430, Indonesi. Received: 6 Jnury 2012 Accepted: 24 June 2013 Pulished: 16 July 2013 References 1. Bdn Penelitin dn Pengemngn Kesehtn, Deprtemen Kesehtn Repulik Indonesi: Lporn hsil riset kesehtn dsr (RISKESDAS) nsionl Jkrt; de Ferrnti S, Mozffrin D: The perfect storm, dipocyte dysfunction, nd metolic consequences. Clin Chem 2008, 54: Friedmn JM: Leptin t 14 yers of ge: n ongoing story. Am J Clin Nutr 2009, 89(suppl):973S 979S. 4. Lu DCW, Dhillon B, Yn H, Szmitko PE, Verm S: Adipokines: moleculr links etween oesity nd therosclerosis. 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8 Indr et l. BMC Reserch Notes 2013, 6:275 Pge 8 of 8 plcent nd mternl dipose tissue vi nucler fctor-κb, peroxisoml prolifertor-ctivted receptor-γ nd extrcellulrly regulted kinse 1/2. Endocrinology 2005, 146: Perez-Vizcino F, Durte J: Flvonols nd crdiovsculr disese. Mol Aspects Med 2010, 31: Ruiz PA, Brune A, Holzlwimmer G, Quintnill-Fend L, Hller D: Quercetin inhiits TNF-induced NF- κβ trnscription fctor recruitment to proinflmmtory gene promoters in murine intestinl epithelil cells. J Nutr 2007, 137: Middleton E Jr, Kndswmi C, Theohrides TC: The effects of plnt flvonoids on mmmlin cells: implictions for inflmmtion, hert disese, nd cncer. Phrmcol Rev 2000, 52: Ahn J, Lee H, Kim S, Prk J, H T: The nti-oesity effect of quercetin is medited y the AMPK nd MAPK signling pthwys. Biochem Biophys Res Commun 2008, 373: Kwon SH, Nm JI, Kim SH, Kim JH, Yoon JH, Kim KS: Kempferol nd quercetin, essentil ingredients in Ginkgo ilo extrct, inhiit interleukin-1et-induced MUC5AC gene expression in humn irwy epithelil cells. Phytother Res 2009, 23: Comld M, Cmuesco D, Sierr S, Bllester I, Xus J, Glvez J, Zrzuelo A: In vivo quercitrin nti-inflmmtory effect involves relese of quercetin, which inhiits inflmmtion through down-regultion of the NF-kppB pthwy. Eur J Immunol 2005, 35: Hämäläinen M, Nieminen R, Vuorel P, Heinonen M, Moilnen E: Anti-inflmmtory effects of flvonoids: genistein, kempferol, quercetin, nd didzein inhiit STAT-1 nd NF-kppB ctivtions, wheres flvone, isorhmnetin, nringenin, nd pelrgonidin inhiit only NF-kppB ctivtion long with their inhiitory effect on inos expression nd NO production in ctivted mcrophges. Meditors Inflmm 2007, 2007: River L, Morón R, Sánchez M, Zrzuelo A, Glisteo M: Quercetin meliortes metolic syndrome nd improves the inflmmtory sttus in oese Zucker rts. Oesity (Silver Spring) 2008, 16: Cho SY, Prk SJ, Kwon MJ, Jeong TS, Bok SH, Choi WY, Jeong WI, Ryu SY, Do SH, Lee CS, Song JC, Jeong KS: Quercetin suppresses proinflmmtory cytokines production through MAP kinses ndnf-kppb pthwy in lipopolyscchride-stimulted mcrophge. Mol Cell Biochem 2003, 243: Peluso I, Rguzzini A, Serfini M: Effect of flvonoids on circulting levels of TNF-α nd IL-6 in humns: systemtic review nd met-nlysis. Mol Nutr Food Res doi: /mnfr [Epu hed of print]. doi: / Cite this rticle s: Indr et l.: Quercetin suppresses inflmmtion y reducing ERK1/2 phosphoryltion nd NF kpp B ctivtion in Leptininduced Humn Umilicl Vein Endothelil Cells (HUVECs). BMC Reserch Notes :275. Sumit your next mnuscript to BioMed Centrl nd tke full dvntge of: Convenient online sumission Thorough peer review No spce constrints or color figure chrges Immedite puliction on cceptnce Inclusion in PuMed, CAS, Scopus nd Google Scholr Reserch which is freely ville for redistriution Sumit your mnuscript t