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Pharmacological Research 58 (2008) 22–31

Contents lists available at ScienceDirect

Pharmacological Research

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lycyrrhizin attenuates the development of carrageenan-induced lung injuryn mice

arta Menegazzi a,1, Rosanna Di Paolab,1, Emanuela Mazzonb, Tiziana Genoveseb,oncetta Crisafulli c, Martina Dal Boscoa, Zhenzhen Zoud, Hisanori Suzukia,alvatore Cuzzocreab,c,∗

Biochemistry Division, Department of Neuroscience and Vision, University of Verona, Verona, ItalyIRCCS Centro Neurolesi “Bonino-Pulejo”, Messina, ItalyDepartment of Clinical and Experimental Medicine and Pharmacology, School of Medicine, University of Messina, Torre Biologica,oliclinico Universitario, Via C. Valeria, 98123 Messina, ItalyMinophagen Pharmaceutical Co. Ltd., Tokyo, Japan

r t i c l e i n f o

rticle history:Accepted 28 May 2008

eywords:lycyrrhizinleurisyytokinesdhesion moleculesitrotyrosineARF-�B

a b s t r a c t

Glycyrrhizin is a triterpene glycoside, a major active constituent of licorice (Glycyrrhiza glabra) root andnumerous pharmacological effects like anti-inflammatory, anti-viral, anti-tumour and hepatoprotectiveactivities has been attributed to it.

In this study we evaluated the anti-inflammatory activities of glycyrrhizin in mice model of acute inflam-mation, carrageenan-induced pleurisy. We report here that glycyrrhizin (given at 10 mg/kg i.p. 5 min priorto carrageenan) exerts potent anti-inflammatory effects in this model. Injection of carrageenan into thepleural cavity of mice elicited an acute inflammatory response characterized by fluid accumulation inthe pleural cavity which contained a large number of neutrophils (PMNs) as well as an infiltration ofPMNs in lung tissues and subsequent lipid peroxidation (as determinated by thiobarbituric acid-reactantsubstances measurement) and increased production of tumour necrosis factor-� (TNF-�) and interleukin-1� (IL-1�). All these parameters were attenuated by glycyrrhizin. Furthermore, carrageenan induced anupregulation of the expression of intercellular cell adhesion molecule (ICAM-1), P-selectin, as well as anincrease in the amounts of nitrotyrosine and poly(ADP-ribose) (PAR), as determined by immunohisto-

chemical analysis of lung tissues. The degree of staining for the ICAM-1, P-selectin, nitrotyrosine and PARwas significantly reduced by glycyrrhizin.

Additionally, we demonstrate that these inflammatory events were associated with the activation ofnuclear factor-�B (NF-�B) and signal transducer and activator transcription-3 (STAT-3) activation in thelung. NF-�B and STAT-3 activation were significantly reduced by glycyrrhizin treatment. Taken together,

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. Introduction

Carrageenan-induced local inflammation is commonly used tovaluate the efficacy of non-steroidal anti-inflammatory drugsNSAIDs). Therefore, carrageenan-induced local inflammationpleurisy) is a useful model to assess the contribution of mediators

∗ Corresponding author at: Department of Clinical and Experimental Medicinend Pharmacology, School of Medicine, University of Messina, Torre Biologica, Poli-linico Universitario, Via C. Valeria, 98123 Messina, Italy. Tel.: +39 090 2213644;ax: +39 090 2213300.

E-mail address: salvator@unime.it (S. Cuzzocrea).1 These authors contributed equally to this work.

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043-6618/$ – see front matter © 2008 Published by Elsevier Ltd.oi:10.1016/j.phrs.2008.05.012

ntion of the activation of NF-�B and STAT-3 by glycyrrhizin reduces theation.

© 2008 Published by Elsevier Ltd.

nvolved in vascular changes associated with acute inflammation.n particular, the initial phase of acute inflammation (0–1 h) whichs not inhibited by NSAID such as indomethacin or aspirin, has beenttributed to the release of histamine, 5-hydroxytryptamine andradykinin, followed by a late phase (1–6 h) mainly sustained byrostaglandin release and more recently has been attributed tohe induction of inducible cyclooxygenase (COX-2) in the tissue1]. It appears that the onset of the carrageenan acute inflamma-ion has been linked to neutrophil infiltration and the production

f neutrophil-derived free radicals, such as hydrogen peroxide,uperoxide and hydroxyl radical, as well as to the release of othereutrophil-derived mediators [2,3].

Oxidative stress is characterized by high intracellular levels ofeactive oxygen species (ROS) and reactive nitrogen species (RNS).

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hey participate as signalling molecules that regulate diverse phys-ological signalling pathways in neutrophils, serving as modulatorsf protein and lipid, kinases and phosphatases, membrane recep-ors and ion channels [4–6].

During the inflammatory response, ROS and RNS modulatehagocytosis, secretion, gene expression, and apoptosis. Indeed,nder pathological circumstances such as acute lung injury andepsis, excess production of neutrophil-derived ROS and RNS maynfluence neighbouring endothelial or epithelial cells, contributingo the amplification of inflammatory tissue injury [7]. Further-

ore, oxidative stress elicits the activation of the redox-sensitiveranscription factors such as NF-�B and AP-1, resulting in a largeutput of proinflammatory cytokines and chemokines, which fur-her enlarge inflammation- and oxidative stress-induced tissueamages [8].

In the lungs, many noxious/inflammatory stimuli have beenhown to activate NF-�B, implicating this pathway as a focal pointor induction of lung inflammation [9,10]. In rodent models of lungnflammation, pretreatment with relatively nonspecific inhibitorsf NF-�B activation has been found to decrease lung inflammation11]. After establishment of an inflammatory response, however, itas been suggested that NF-�B has a role in resolution of inflamma-ion through antiapoptotic effects and expression of proteins thatunction to limit inflammation [12]. The importance of NF-�B inegulating ongoing lung inflammation or progression to lung injuryemains to be elucidated.

STATs are a class of transcription factors bearing SH2 domainshat become activated upon tyrosine phosphorylation. STATs areften activated by members of the signal transducer activatorf transcription Janus kinase (JAK) family of protein-tyrosineinases (PTKs) in response to cytokine stimulation. STAT3, a well-haracterized 92-kDa protein, has been shown to become activatedy both epidermal growth factor and interleukin-6 in human A-431ells [13].

Glycyrrhiza glabra, also known as licorice and sweetwood, isative to the Mediterranean and certain areas of Asia. A num-er of components have been isolated from licorice, including aater-soluble, biologically active complex that accounts for 40–50%f total dry material weight. This complex is composed of triter-ene saponins, flavonoids, polysaccharides, pectins, simple sugars,mino acids, mineral salts, and various other substances [14]. Gly-yrrhizin, major active constituent of licorice root accounting forhe sweet taste of licorice root, is a triterpene glycoside which con-ists of one molecule of 18�-glycyrrhetinic acid and two moleculesf glucuronic acid having structure, 18�-glycyrrhetinic acid-3-O-�--glucuronopyranosyl-(1 → 2)-�-d-glucuronide [15,13]. Numerousharmacological effects like anti-inflammatory, anti-viral, anti-umour and hepatoprotective activities have been attributed tot [16]. It has been shown that glycyrrhizin and its biologicallyransformed metabolite called 18�-glycyrrhetinic acid, an aglyconomponent of glycyrrhizin, inhibit the passive cutaneous anaphy-axis and skin contact inflammation in mice model of contactypersensitivity. The mechanism of action of glycyrrhizin and itsetabolite against inflammation is unknown, although it could be

ue to steroid-like structure of 18�-glycyrrhetinic acid [17], thusimicking the effect of cortisol that, by inhibiting the catalytic

ctivity of 11�-hydroxysteroid dehydrogenase (11-HSD), enhanceshe accumulation of its inactive metabolite [18].

The present studies were designed to evaluate the effects oflycyrrhizin in mice model of acute inflammation (carrageenan-

nduced pleurisy). In particular, we investigate the effects oflycyrrhizin on the lung injury associated with carrageenan-nduced pleurisy. In order to gain a better insight into the

echanism(s) of action of glycyrrhizin, we have also investigatedhe effects of glycyrrhizin on: (1) NF-�B and STAT-3 activation, (2)

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al Research 58 (2008) 22–31 23

NF-� and IL-1� production, (3) adhesion molecules (ICAM-1 and-selectin) expression, (4) neutrophil infiltration, (5) the nitrationf cellular proteins by peroxynitrite, (6) lipid peroxidation and (7)he activation of the nuclear enzyme poly(ADP-ribose) polymerasePARP).

. Methods

.1. Animals

Male CD mice (weight 20–25 g; Harlan Nossan, Milan, Italy) weresed in these studies. The animals were housed in a controllednvironment and provided with standard rodent chow and water.nimal care was in compliance with Italian regulations on protec-

ion of animals used for experimental and other scientific purposesD.M. 116192) as well as with EEC regulations (O.J. of E.C. L358/12/18/1986).

.2. Experimental groups

Mice were randomly allocated into the following groups: (i)AR + saline group. Mice were subjected to carrageenan-inducedleurisy (N = 10), (ii) Glycyrrhizin group. Same as the CAR + vehicleroup but were administered with glycyrrhizin (10 mg/kg i.p.)min prior to carrageenan (N = 10), (iii) Sham + saline group. Sham-perated group in which identical surgical procedures to the CARroup was performed, except that the saline was administerednstead of carrageenan (N = 10), and (iv) Sham + glycyrrhizin group.ame as the Sham + saline group but glycyrrhizin were adminis-ered (10 mg/kg i.p.) 1 h prior to saline (N = 10). The dose of 10 mg/kgas chosen in agreement with a previous study [19].

.3. Carrageenan-induced pleurisy

Mice were anaesthetised with isoflurane and submitted to a skinncision at the level of the left sixth intercostals space. The underly-ng muscle was dissected and saline (0.1 ml) or saline containing 2%-carrageenan (0.1 ml) was injected into the pleural cavity. The skin

ncision was closed with a suture and the animals were allowed toecover. At 4 h after the injection of carrageenan, the animals wereilled by inhalation of CO2. The chest was carefully opened andhe pleural cavity rinsed with 1 ml of saline solution containingeparin (5 U/ml) and indomethacin (10 �g/ml). The exudate andashing solution were removed by aspiration and the total vol-me measured. Any exudate, which was contaminated with blood,as discarded. The amount of exudate was calculated by subtract-

ng the volume injected (1 ml) from the total volume recovered.he leukocytes in the exudate were suspended in phosphate-bufferaline (PBS) and counted with an optical microscope in a Burker’shamber after Blue Toluidine staining.

.4. Determination of myeloperoxidase activity

Myeloperoxidase (MPO) activity, an indicator of polymorphonu-lear (PMN) leukocyte accumulation, was determined in the pleuralxudates as well as in the lung tissues at 4 h after intrapleu-al injection of carrageenan as previously described [20]. Briefly,ung tissues were weighed and homogenised in a solution con-aining 0.5% hexa-decyl-trimethyl-ammonium bromide dissolvedn 10 mM potassium phosphate buffer (pH 7) and centrifuged for

0 min at 20,000 × g at 4 ◦C. An aliquot of the supernatant was thenllowed to react with a solution of tetra-methyl-benzidine (1.6 mM)nd 0.1 mM H2O2. The rate of change in absorbance was measuredpectrophotometrically at 650 nm. MPO activity was defined ashe quantity of enzyme degrading 1 �mol of peroxide min at 37 ◦C

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4 M. Menegazzi et al. / Pharmac

nd was expressed in milliunits per gram weight of wet tissue.yeloperoxidase, standard preparation, M6908 was obtained from

igma–Aldrich Company, Milan, Italy.

.5. Measurement of TNF-˛ and IL-1ˇ levels

TNF-� and IL-1� levels were evaluated in the exudates and inhe lung tissues at 4 h after the induction of pleurisy by carrageenannjection. The assay was carried out by using a colorimetric, com-

ercial ELISA kit (Calbiochem-Novabiochem Corporation, USA).ower detection limit of the assay was of 5 pg/ml.

.6. Immunohistochemical localization of ICAM-1, P-selectin, PARnd nitrotyrosine

At 4 h after carrageenan administration, the lungs were fixedn 10% buffered formaldehyde and 8-�m sections were preparedrom paraffin-embedded tissues. After deparaffinization, endoge-ous peroxidase was quenched with 0.3% H2O2 in 60% methanol

or 30 min. The sections were permeabilized with 0.1% Triton X-100n PBS for 20 min. Nonspecific adsorption was minimised by incu-ating the section in 2% normal goat serum in phosphate-bufferedaline for 20 min. Endogenous biotin or avidin binding sites werelocked by sequential incubation for 15 min with avidin and biotin.he sections were then incubated overnight with primary anti-CAM-1 antibody (1:500), anti-P-selectin antibody (1:500), with:1000 dilution of primary anti-nitrotyrosine antibody (DBA), andnti-PAR antibody (1:500) or with control solutions. Controlsncluded buffer alone or nonspecific purified rabbit IgG.

To confirm that the immunoreaction for the nitrotyrosine waspecific, some sections were also incubated with the primary anti-ody (anti-nitrotyrosine) in the presence of excess nitrotyrosine10 mM) to verify the binding specificity. To verify the bindingpecificity for PAR, ICAM-1 and P-selectin sections were also incu-ated with only the primary antibody (no secondary) or withnly the secondary antibody (no primary). In these situations, noositive staining was found in the sections, indicating that the

mmunoreaction was positive in all the experiments carried out.mmunocytochemistry photographs (n = 5) were assessed by den-itometry. The assay was carried out by using Optilab Graftekoftware on a Macintosh personal computer (CPU G3-266).

.7. Histological examination

Lung biopsies were taken at 4 h after injection of carrageenan.he biopsies were fixed for 1 week in buffered formaldehyde solu-ion (10% in PBS) at room temperature, dehydrated by gradedthanol and embedded in Paraplast (Sherwood Medical, Mahwah,J). Tissue sections (thickness 7 �m) were deparaffinized withylene, stained with haematoxylin/eosin, and studied using lighticroscopy. The following morphological criteria were used for

coring: 0, normal lung; grade 1, minimal oedema or infiltrationf alveolar or bronchiolar walls; grade 3, moderate oedema andnflammatory cells infiltration without obvious damage to lungrchitecture; grade 4, severe inflammatory cells infiltration withbvious damage to lung architecture. All the histological studiesere performed in a blinded fashion.

.8. Thiobarbituric acid-reactant substances measurement

Thiobarbituric acid-reactant substances measurement, whichs considered a good indicator of lipid peroxidation in the lungissues. Tissues, collected 4 h after carrageenan administration,ere homogenized in 1.15% KCl solution. An aliquot (100 �l) of theomogenate was added to a reaction mixture containing 200 �l

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al Research 58 (2008) 22–31

f 8.1% SDS, 1500 �l of 20% acetic acid (pH 3.5), 1500 �l of 0.8%hiobarbituric acid and 700 �l distilled water. Samples were thenoiled for 1 h at 95 ◦C and centrifuged at 3000 × g for 10 min.he optical density at 650 nm (OD650) was measured using ELISAicroplate reader (SLT-Labinstruments, Salzburg, Austria). Thio-

arbituric acid-reactant substances were calculated by comparisonith OD650 of standard solutions of 1,1,3,3-tetramethoxypropan

9% malondialdehyde bis(dimethyl acetal) 99% (Sigma, Milan). Thebsorbance of the supernatant was measured by spectrophotome-ry at 650 nm.

.9. Electrophoretic mobility shift assay

The lung samples have been collected in liquid nitrogen andtored at −80 ◦C until use. Nuclear extracts have been preparedccording to [21] in the presence of 10 �g/ml leupeptin, 5 �g/mlntipain and pepstain, and 1 mM PMSF (Sigma–Aldrich Com-any, Milan, Italy). Protein concentration in the nuclear extractsas determined using the method of Bradford [22]. Ten micro-

rams of nuclear extract have been incubated at room temperatureor 20 min with (2–5 × 104 cpm) of 32P-labeled double strandedligonucleotide, containing the NF-�B-binding site from theL-6 promoter (5′-GATCATGTGGGATTTTCCCATGT-3′) and the STAT-inding site (sis-inducible factor-binding recognition element,IE/M67) from the c-Fos promoter (5′-GTCGACATTTCCCGTAAATCG-′) in a 15 �l of binding reaction buffer (20 mM HEPES, pH.9, 50 mM KCl, 10% glycerol, 0.5 mM DTT, 0.1 mM EDTA, 2 �goly(dI–dC), 1 �g salmon sperm DNA). Products have beenractioned on a non-denaturing 5% polyacrylamide gel in TBETris–Borate–EDTA buffer, 0.5×). The intensity of the retardedands has been measured with a Phosphorimager (Molecularynamic, Milan, Italy). Supershift assay was performed by incubat-

ng the nuclear extracts in binding buffer for 1 h at 4 ◦C with 1 �lf antibody before addiction of labelled oligonucleotide. Polyclonalntibodies against the p65, p50, p52, rel-B and c-rel subunits of NF-B, and STAT-1/3 were purchased from Santa Cruz Biotechnology

Santa Cruz, CA).

.10. Materials

Unless otherwise stated, all compounds were obtained fromigma–Aldrich Company (Milan, Italy). Glycyrrhizin was providedy Minophagen Pharmaceutical Co., Ltd., Tokyo, Japan. Primaryonoclonal ICAM-1 (CD54) for immunohistochemistry was pur-

hased by Pharmingen. Reagents and secondary and nonspecificgG antibody for immunohistochemical analysis were from Vectoraboratories Inc. Primary monoclonal anti-poly(ADP-ribose) anti-ody (PAR) was purchased by Alexis. All other chemicals were of theighest commercial grade available. All stock solutions were pre-ared in non-pyrogenic saline (0.9% NaCl; Baxter Healthcare Ltd.,hetford, Norfolk, UK).

.11. Data analysis

All values in the figures and text are expressed asean ± standard error (S.E.M.) of the mean of n observations.

or the in vivo studies n represents the number of animals studied.n the experiments involving histology or immunohistochemistry,he figures shown are representative of at least three experiments

histological or immunohistochemistry coloration) performedn different experimental days on the tissues section collectedrom all the animals in each group. The results were analysed byne-way ANOVA followed by a Bonferroni post hoc test for multipleomparisons. A p-value less than 0.05 was considered significant.

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. Results

.1. Effects of glycyrrhizin on tissue damage inarrageenan-induced pleurisy

When compared with lung sections taken from saline-treatednimals (data not shown), histological examination showed thatarrageenan-induced oedema, tissue injury and infiltration ofhe tissue with inflammatory cells (Fig. 1A). The degree of theissue injury (on an arbitrary score ranging from 0 to 4) was.52 ± 0.09. The damage score for carrageenan-treated mice whichave received glycyrrhizin was significantly lower (1.07 ± 0.08) asell as the histological observation (Fig. 1B). Furthermore, injec-

ion of carrageenan into the pleural cavity of mice elicited an acutenflammatory response characterized by the accumulation of fluidoedema) that contained large amounts of PMNs (Fig. 1C and D).xudate formation and PMNs amounts were significantly attenu-ted by the i.p. injection of glycyrrhizin (Fig. 1C and D).

.2. Effects of glycyrrhizin on MPO activity

The above histological pattern of lung injury appeared to beorrelated with the influx of leukocytes into the pleural exudatess well as in the lung tissue. Therefore, we investigate the effectf glycyrrhizin on the neutrophils infiltration by measurementf the activity of myeloperoxidase. Myeloperoxidase activity inhe pleural exudates as well as in the lung tissues was signifi-

antly elevated (p < 0.001) at 4 h after carrageenan administrationn vehicle-treated mice (Fig. 2). In mice treated with glycyrrhizinung MPO activity in the pleural exudates as well as in the lung tis-ues was significantly reduced (p < 0.01) in comparison to those ofehicle-treated mice (Fig. 2).

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ig. 1. Effect of glycyrrhizin on lung injury and carrageenan-induced inflammation. Lung snfiltration of the tissue with inflammatory cells (see arrows A1). Lung sections from a carnflammatory cell infiltration (B). The increase in volume exudate (C) and accumulation olycyrrhizin. Figure is representative of all the animals in each group. Data are means ± Sr: bronchioles, PV: perivascular space; OE: oedema.

al Research 58 (2008) 22–31 25

.3. Effect of glycyrrhizin on TNF-˛ and IL-1ˇ levels

A substantial increase in TNF-� and IL-1� production wasound in pleural exudates (Fig. 3A and B, respectively) and inhe lung tissues (Fig. 3C and D, respectively) collected from

ice at 4 h after carrageenan injection. In contrast, the levelsf these pro-inflammatory cytokines were significantly lower inarrageenan-treated mice treated with glycyrrhizin (Fig. 3). No sig-ificant increase of TNF-� and IL-1� levels was observed in thexudates as well as in the lung of sham-treated mice.

.4. Effects of glycyrrhizin on the expression of adhesionolecules (ICAM-1 and P-selectin)

At 4 h after carrageenan injection, the staining intensity of lungissue sections obtained from carrageenan-treated mice with anti-CAM-1 and P-selectin antibodies substantially increased along theessels (Fig. 4A and C, see densitometry analysis Fig. 6). Sectionsrom glycyrrhizin-treated mice did not reveal any up-regulation ofxpressed ICAM-1 and P-selectin (Fig. 4B, see densitometry anal-sis Fig. 6). It is important to underline that constitutive stainingor ICAM-1 and no positive staining for P-selectin was observed inungs of sham-treated mice (data not shown).

.5. Effects of glycyrrhizin on nitrotyrosine formation and PARormation

At 4 h after carrageenan injection, lung sections were taken inrder to determine the immunohistological staining for nitroty-osine or PAR. A positive staining for nitrotyrosine was localizedrimarily in the vessels and in the alveolar epithelium (Fig. 5A, seeensitometry analysis Fig. 6). At 4 h after carrageenan injection, the

ections from carrageenan-treated mice (A) demonstrate oedema, tissue injury andrageenan-treated mice that received glycyrrhizin exhibit reduced tissue injury andf PMNs (D) in pleural cavity 4 h after carrageenan (CAR) injection was inhibited by.E.M. of 10 mice for each group. *P < 0.01 vs. SHAM. ◦P < 0.01 vs. carrageenan (CAR).

26 M. Menegazzi et al. / Pharmacological Research 58 (2008) 22–31

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ig. 2. Effect of glycyrrhizin on MPO activity. Within 4 h, pleural injection of carragen the pleural exudates (A) as well as in the lung (B). Glycyrrhizin treatment significare means ± S.E.M. of 10 mice for each group. *P < 0.01 vs. SHAM. ◦P < 0.01 vs. CAR.

taining for PAR was substantially increased (Fig. 5C, see densito-etry analysis Fig. 6), mainly localized along the alveolar nuclei.lycyrrhizin reduced the staining for both nitrotyrosine (Fig. 5B,ee densitometry analysis Fig. 6) and PAR (Fig. 5D, see densitometrynalysis Fig. 6). It is important to underline that no positive stain-ng for nitrotyrosine and PAR was observed in lungs of sham-treated

ice (data not shown).

.6. Effects of glycyrrhizin on lipid peroxidation

At 4 h after carrageenan administration, lungs were investi-ated for thiobarbituric acid-reactant substances, indicative ofipid peroxidation. As shown in Fig. 7, thiobarbituric acid-reactant

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ig. 3. Effect of glycyrrhizin on TNF-�, and IL-1�. TNF-� and IL-1� production was evanjection using a ELISA kit. A significant production TNF-� (A) and IL-1� (B) was observed i

ith glycyrrhizin significantly reduced the pleural exudate production of TNF-� (A) and ILhe lung tissues from carrageenan-injected mice at 4 h after carrageenan. In the lung tissuL-1� (D) levels were significantly reduced in comparison to those of vehicle-treated miceAR.

(CAR) led to an increase in neutrophil accumulation (as measured by MPO activity)nhibited the MPO activity in the pleural exudates (A) as well as in the lung (B). Data

ubstances levels were significantly increased in the lung ofarrageenan-treated mice. In mice treated with glycyrrhizin lunghiobarbituric acid-reactant substances levels were significantlyeduced in comparison to those of vehicle-treated mice (Fig. 7).

.7. Effects of glycyrrhizin on NF-�B and STAT-3 activation

To examine the molecular mechanisms responsible for medi-

ting the anti-inflammatory effects of glycyrrhizin we measured,y EMSA, the changes in activation of the transcription factors NF-B and STAT-3. In an effort to identify the subunit composition ofifferent complexes that bind to the �B element, we used poly-lonal antibodies directed against members of the NF-�B family

luated in the pleural exudates and lung tissues collected at 4 h after carrageenann pleural exudates collected from carrageenan-treated mice. The treatment of mice-1� (B). Similarly, a significant increase in TNF-� (C) and IL-1� (D) was observed ines from carrageenan-injected mice which have received glycyrrhizin TNF-� (C) and. Data are means ± S.E.M. of 10 mice for each group. *P < 0.01 vs. SHAM. ◦P < 0.01 vs.

M. Menegazzi et al. / Pharmacological Research 58 (2008) 22–31 27

Fig. 4. Immunohistochemical localization of ICAM-1 and P-selectin in the lung. Section obtained from carrageenan-treated mice showed intense positive staining for ICAM-1(A see arrows) and P-selectin (C see arrows) mainly localized along the vessels. The degree of positive staining for ICAM-1 (B) and P-selectin (D) was markedly reduced intissue section obtained from glycyrrhizin-treated mice. Figure is representative of all the animals in each group.

Fig. 5. Immunohistochemical localization for nitrotyrosine and PAR in the lung. Immunohistochemistry for nitrotyrosine show positive staining (A see arrows) mainlylocalized along the alveolar nuclei from a carrageenan-treated mice. The intensity of the positive staining for nitrotyrosine was significantly reduced in the lung fromglycyrrhizin-treated mice (B). Moreover, at 4 h after carrageenan immunohistochemistry for PAR show positive staining (C see arrows) mainly localized along the alveolarnuclei from a carrageenan-treated mice. The intensity of the positive staining for PAR was significantly reduced in the lung from glycyrrhizin-treated mice (D). Figure isrepresentative of all the animals in each group.

28 M. Menegazzi et al. / Pharmacologic

Fig. 6. Typical densitometry evaluation. Densitometry analysis of immunocyto-clMa

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hemistry photographs (n = 5) for ICAM-1, P-selectin, nitrotyrosine and PAR fromung was assessed. The assay was carried out by using Optilab Graftek software on a

acintosh personal computer (CPU G3-266). Data are expressed as % of total tissuerea. ND: not detectable. *P < 0.01 vs. SHAM. ◦P < 0.01 vs. carrageenan (CAR).

roteins in a supershift assay. The anti-p65 antibody produced aupershift of the faster mobility complex (Fig. 8B). The anti-p50ntibody leads to immunodepletion of the slower DNA–proteinomplex and, in minor extend, the faster one (Fig. 8B). Differentubunits, such as p52, rel-B, c-rel, seem not to be included in theseranscriptional complexes. Thus, it is reasonable to conclude thathe faster mobility complex might be p65 homodimer, even if we doot exclude the possibility that it might be p65/p50 heterodimer,nd the slower mobility complex is p50 homodimer. The third bandhown in Fig. 8A do not belong to NF-�B, it represents the DNAinding to the constitutively express CBF-1 transcription factor to

ts DNA-binding site, that is located very near to the �B side in theL-6 gene promoter. The intensity of that band might be used as aontrol of the total amount of nuclear extract loaded onto the gel.

Our data showed the presence of the p65 subunit only in

arrageenan-treated mice (Fig. 8A). This faster mobility band waslmost undetectable in the glycyrrhizin pre-treated mice (Fig. 8And C). Significant differences were also shown for the slowerobility complex (Fig. 8A and C).

ig. 7. Effect of glycyrrhizin on lipid peroxidation in the lung. Thiobarbituriccid-reactant substances, a good indicator of lipid peroxidation, were determinedn the lung from carrageenan-treated mice. Within 4 h, pleural injection of car-ageenan (CAR) led to increased levels of thiobarbituric acid-reactant substancesn comparison to sham mice. Glycyrrhizin treatment significantly reduced thearrageenan-induced lipid peroxidation. Data are means ± S.E.M. of 10 mice for eachroup. *P < 0.01 vs. SHAM. ◦P < 0.01 vs. CAR.

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Supershift experiment with anti-STAT-1 and anti-STAT-3 anti-odies showed a presence of shifted band only in the samplere-treated with anti-STAT-3 antibody (data not shown). DNA-inding activity of STAT-3 was significantly elevated at 4 h afterarrageenan administration in vehicle-treated mice (Fig. 8A). Inice treated with glycyrrhizin STAT-3 activity was similar to those

f sham-operated group and significantly reduced in comparisono those of vehicle-treated mice (Fig. 8A and B and 9).

. Discussion

This study provides the first evidence that glycyrrhizin attenu-tes: (i) the development of carrageenan-induced pleurisy, (ii) thenfiltration of the lung with PMNs, (iii) the degree of P-selectin andCAM-1 production, (iv) NF-�B activation, (v) STAT-3 activation andvi) the degree of lung injury caused by injection of carrageenan.ll these findings support the view that glycyrrhizin attenuates theegree of acute inflammation in the mouse.

What, then, is the mechanism by which glycyrrhizin protectsgainst inflammatory injury?

The beneficial effects of licorice can be attributed to a number ofechanisms. Glycyrrhizin and glycyrrhizic acid have been shown

o inhibit growth and cytopathology of numerous RNA and DNAiruses, including hepatitis A and C [23,24], Herpes zoster [25], HIV26,27], Herpes simplex [28,29] and Cytomegalovirus (CMV) [30].

Glycyrrhizin and its metabolites inhibit hepatic metabolism ofldosterone and suppress 5-[�]-reductase, properties responsibleor the well-documented pseudoaldosterone syndrome. The sim-lar structure of glycyrrhetinic acid and glycyrrhizic acid to thetructure of hormones secreted by the adrenal cortex seems toccount for their mineralocorticoid and glucocorticoid activity [31].

Licorice constituents also exhibit hydrocortisone-like anti-nflammatory activity. This is due, in part, to inhibition ofhospholipase A2 activity, an enzyme critical to numerous inflam-atory processes [19]. In vitro research has also demonstrated that

lycyrrhizic acid inhibits cyclooxygenase activity and prostaglandinormation (specifically prostaglandin [E2]), as well as indirectlynhibiting platelet aggregation, all factors playing a critical role inhe inflammatory process [32,33].

Certain licorice constituents possess significant antioxidant andepatoprotective properties. Glycyrrhizin and glabridin inhibit theeneration of ROS by neutrophils at the site of inflammation34,35]. In vitro studies have demonstrated licorice isoflavones,ispaglabridin A and B, inhibit [Fe3+]-induced mitochondrial lipideroxidation in rat liver cells [36]. Other research indicates thatlycyrrhizin lowers lipid peroxide values in animal models of livernjury caused by ischemia reperfusion [37]. Licorice constituentslso exhibit hepatoprotective activity by lowering serum livernzyme levels and improving tissue pathology in hepatitis patients38].

ROS including superoxide anion radical (O2−), hydrogen perox-

de (H2O2), hydroxyl radical (OH−), and hypochlorous acids suchs HOCl [39,40] and RNS such as peroxynitrate (ONOO−) [41] haveong been recognized to significantly contribute to the pathogene-es of various inflammatory lung injuries and diseases. The effectsf these products on lung tissue are multiple. In particular, they cannitiate lipid peroxidation in cellular membranes with formationf vasoactive and proinflammatory molecules such as thrombox-ne. Excessive lipid peroxidation in membranes destroys vascularndothelial cells. In the present study, we indicated that gly-

yrrhizin treatment prevents the formation of tissue thiobarbituriccid-reactant substances which are considered a good indicator ofipid peroxidation

RNS production may also be catalyzed by neutrophil MPO.ypochlorous acid (HOCl), formed by the MPO-catalyzed reac-

M. Menegazzi et al. / Pharmacological Research 58 (2008) 22–31 29

Fig. 8. Effect of glycyrrhizin on NF-�B activation. DNA binding activity of NF-�B in sham-operated (SHAM), carrageenan-treated (CAR) and glycyrrhizin-treated mice(CAR + GLY). Nuclear extracts (10 �g) from lung sample were incubated with a 32P-labeled double-stranded oligonucleotide containing binding sequence for NF-�B andseparated by nondenaturing PAGE. The specificity of the retarded bands was demonstrated by competition with 100-fold excess of specific unlabeled oligonucleotide (notshown). Two specific bands are indicated by arrows. (A) In the supershift experiment nuclear extracts from a carrageenan-treated mice sample were incubated with 1 �l ofantibody against p65, p50, p52, rel-B, or c-rel subunit of NF-�B before addiction of labelled oligonucleotide. The anti-p65 antibody produced a supershift of the faster mobilityband. The anti-p50 antibody immunodepleted the slower DNA–protein complex. (B) The intensity of retarded bands (measured by phosphoimager) in carrageenan-treatedmice was significantly increased vs. SHAM group. Glycyrrhizin treatment significantly reduced the carrageenan-induced NF-�B activation. *P < 0.01 vs. SHAM. ◦P < 0.01 vs.CAR (C).

Fig. 9. Effect of glycyrrhizin on STAT-3 activation. DNA-binding activity of STAT-3 (indicated by an arrow) in sham-operated (SHAM), carrageenan-treated (CAR) andglycyrrhizin-treated mice (CAR + GLY). Nuclear extracts (10 �g) from lung sample were incubated with a 32P-labeled double-stranded oligonucleotide containing bindingsequence for STAT-3 and separated by nondenaturing PAGE. (A) The intensity of retarded bands (measured by phosphoimager) in carrageenan-treated mice was significantlyincreased vs. SHAM group. Glycyrrhizin treatment significantly reduced the carrageenan-induced elevation of STAT-3 activity *P < 0.01 vs. SHAM. ◦P < 0.01 vs. CAR (B).

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ion of H2O2 and chloride, reacts whit nitrite to form reactiventermediates capable of nitrating and chlorinating proteins [39].

e demonstrated here that nitrotyrosine levels (an indicationf protein nitration) and MPO level were reduced significantlyn glycyrrhizin-treated mice. Peroxynitrite elicits cellular injuryia multiple mechanisms including activation of poly(ADP-ribose)olymerase (PARP). Upon activation by DNA single-strand breaks,ARP initiates an energy-consuming cycle by transferring ADP-ibose moieties from NAD+ to nuclear proteins including itself. Thisrocess results in rapid depletion of the intracellular NAD+ and ATPools, slowing the rate of glycolysis and mitochondrial respirationnd, eventually, leading to cellular dysfunction and death. More-ver various studies have clearly demonstrated an important rolef PARP activation in acute inflammation [40]. We demonstrate herehat glycyrrhizin attenuates the increase in ROS, PARP activity in theung from carrageenan-treated mice.

The transcription factor nuclear factor kappa B (NF-�B) has beenhown to be a major regulator of many functionally diverse proin-ammatory mediators. NF-�B is a general term used to describenumber of dimeric combinations of members of the Rel family

f gene regulatory proteins that possess transcriptional activatingroperties [41].

In the NF-�B pathway, the presence of inhibitory factor kappa BI�B) and the I�B/NF-�B interactions prevent nuclear translocationf NF-�B by masking nuclear localization sequences of the NF-�Bimers. Degradation of I�B leads to “activation” of NF-�B, which isefined as translocation of the NF-�B complex from the cytoplasmo the nucleus. Once in the nucleus, NF-�B binds specific promoterlements of DNA and induces transcription of relevant genes. Theesults of this study demonstrate that glycyrrhizin inhibits, in par-icular, the DNA binding of the �B dimer containing p65 subunit,omplex that is transcriptionally active in comparison to the p50omodimer. Although the exact mechanisms by which glycyrrhizinuppresses NF-�B activation in inflammation remain to be furtherlucidated, its antioxidant property should be responsible for thebility to inhibit NF-�B. In particular, we have recently demon-trated that inhibition of ROS formation prevents NF-�B activationn the lung tissues from mice subjected to carrageenan-inducedung injury [42]. In the present study, we further demonstrated thathe dose of glycyrrhizin used (10 mg/kg i.p.) is sufficient to inhibithe activation of NF-�B in vivo.

The secretion of pro-inflammatory cytokines is under the con-rol of the transcription factors of the nuclear factor-�B family.hese cytokines are interleukin (IL)-8, IL-1�, and TNF-�. The acti-ation of the transcription of these genes has been demonstrateduring acute lung injury [43].

The present study demonstrated that glycyrrhizin attenuateshe TNF-� and IL-1�, production in the pleural exudates and in theung tissues from carrageenan-treated mice. Therefore, the inhibi-ion of the production of TNF-� and IL-1� by glycyrrhizin describedn the present study is most likely attributed to the inhibitory effectoward the activation of NF-�B. Moreover cytokine-dependentctivation and expression of adhesion molecules allows for thedhesion and extravasation (emigration) of the neutrophil that maynduce local injury and participate in the orchestration of systemicnflammation and all of its consequences [44].

In accordance with these findings, we observed that glycyrrhizineduces the upregulation of the expression of ICAM-1 on endothe-ial cells and prevents the infiltration of neutrophils at inflammedites. In addition, in response to cytokines and growth factors, STAT

amily members are phosphorylated by the receptor-associatedinases, and then form dimers that translocate to the cell nucleushere they act as transcription activators.

Recent study indicated that STAT-3 is activated in the lungollowing acute lung injury [45]. Moreover, Gao and colleagues

[

al Research 58 (2008) 22–31

eported that STAT-3 protein synthesis in rat lungs after tis-ue injuries was induced by IgG immune complexes. They alsohowed that STAT-3 activation is dependent on the presencef macrophages, neutrophils and cytokines [45]. In the presenttudy, we demonstrated that glycyrrhizin pretreatment signifi-antly reduced STAT-3 activation in carrageenan-treated mice.

In conclusion, our results indicate that in carrageenan-inducedleurisy model glycyrrhizin has strong anti-inflammatory proper-ies resulting in a reduced: (1) TNF-� and IL-1� production, (2) PMNnfiltration, (3) upregulation of ICAM-1 and P-selectin expressionnd formation of PAR and nitrotyrosine, (4) NF-�B and STAT-3 acti-ation, and ultimately the degree of peroxynitrite formation andissue injury.

cknowledgements

This study was supported by grant from MURST (40%) to S.C.,.M. and H.S. and Cariverona to H.S. The authors would like to

hank Giovanni Pergolizzi and Carmelo La Spada for their excel-ent technical assistance during this study, Mrs Caterina Cutronaor secretarial assistance and Miss Valentina Malvagni for editorialssistance with the manuscript.

eferences

[1] Nantel F, Denis D, Gordon R, Northey A, Cirino M, Metters KM, et al. Distributionand regulation of cyclooxygenase-2 in carrageenan-induced inflammation. BrJ Pharmacol 1999;128:853–9.

[2] Xinmin D, Yunyou D, Chaosheng P, Huasong F, Pingkun Z, Jiguang M, et al. Dex-amethasone treatment attenuates early seawater instillation-induced acutelung injury in rabbits. Pharmacol Res 2006;53:372–9.

[3] Mariotto S, Esposito E, Di Paola R, Ciampa A, Mazzon E, de Prati AC, et al. Pro-tective effect of Arbutus unedo aqueous extract in carrageenan-induced lunginflammation in mice. Pharmacol Res 2008;57:110–24.

[4] Schreckand R, Baeuerle PA. A role for oxygen radicals as second messengers.Trends Cell Biol 1991;1:39–42.

[5] Schreck R, Rieber P, Baeuerle PA. Reactive oxygen intermediates as apparentlywidely used messengers in the activation of the NF-kappa B transcription factorand HIV-1. EMBO J 1991;10:2247–58.

[6] Brumell JH, Burkhardt AL, Bolen JB, Grinstein S. Endogenous reactive oxygenintermediates activate tyrosine kinases in human neutrophils. J Biol Chem1996;271:1455–61.

[7] Fialkow L, Wang Y, Downey GP. Reactive oxygen and nitrogen species assignaling molecules regulating neutrophil function. Free Radic Biol Med2007;42:153–64.

[8] Guoand RF, Ward PA. Role of oxidants in lung injury during sepsis. AntioxidRedox Signal 2007;9:1991–2002.

[9] Mizgerd JP, Peschon JJ, Doerschuk CM. Roles of tumor necrosis factor receptorsignaling during murine Escherichia coli pneumonia. Am J Respir Cell Mol Biol2000;22:85–91.

10] Mizgerd JP, Spieker MR, Doerschuk CM. Early response cytokines and innateimmunity: essential roles for TNF receptor 1 and type I IL-1 receptor duringEscherichia coli pneumonia in mice. J Immunol 2001;166:4042–8.

11] Lauzurica P, Martinez-Martinez S, Marazuela M, Gomez del Arco P, Martinez C,Sanchez-Madrid F, et al. Pyrrolidine dithiocarbamate protects mice from lethalshock induced by LPS or TNF-alpha. Eur J Immunol 1999;29:1890–900.

12] Lawrence T, Gilroy DW, Colville-Nash PR, Willoughby DA. Possible new role forNF-kappaB in the resolution of inflammation. Nat Med 2001;7:1291–7.

13] Zhong Z, Wen Z, Darnell Jr JE. Stat3: a STAT family member activated by tyro-sine phosphorylation in response to epidermal growth factor and interleukin-6.Science 1994;264:95–8.

14] Litvinenkoand VI, Obolentseva GV. Chemical and pharmaceutical research onflavanoids of glycyrrhiza Glabra L. and Glycyrrhiza uralensis Fisch. Med PromSSSR 1964;18:20–3.

15] Matsui S, Matsumoto H, Sonoda Y, Ando K, Aizu-Yokota E, Sato T, et al.Glycyrrhizin and related compounds down-regulate production of inflamma-tory chemokines IL-8 and eotaxin 1 in a human lung fibroblast cell line. IntImmunopharmacol 2004;4:1633–44.

16] Sato H, Goto W, Yamamura J, Kurokawa M, Kageyama S, Takahara T, et al. Thera-peutic basis of glycyrrhizin on chronic hepatitis B. Antiviral Res 1996;30:171–7.

17] Tamaya T, Sato S, Okada HH. Possible mechanism of steroid action of the plant

herb extracts glycyrrhizin, glycyrrhetinic acid, and paeoniflorin: inhibition byplant herb extracts of steroid protein binding in the rabbit. Am J Obstet Gynecol1986;155:1134–9.

18] Krahenbuhl S, Hasler F, Frey BM, Frey FJ, Brenneisen R, Krapf R. Kinetics anddynamics of orally administered 18 beta-glycyrrhetinic acid in humans. J ClinEndocrinol Metab 1994;78:581–5.

ologic

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[[

[

[

[

[

[

[

[

[44] Wyman TH, Bjornsen AJ, Elzi DJ, Smith CW, England KM, Kelher M, etal. A two-insult in vitro model of PMN-mediated pulmonary endothelial

M. Menegazzi et al. / Pharmac

19] Takei M, Kobayashi M, Li XD, Pollard RB, Suzuki F. Glycyrrhizin inhibits R5 HIVreplication in peripheral blood monocytes treated with 1-methyladenosine.Pathobiology 2005;72:117–23.

20] Mullane KM, Kraemer R, Smith B. Myeloperoxidase activity as a quantitativeassessment of neutrophil infiltration into ischemic myocardium. J Pharm Meth-ods 1985;14:157–67.

21] Schreiber E, Matthias P, Muller MM, Schaffner W. Rapid detection of octamerbinding proteins with ‘mini-extracts’, prepared from a small number of cells.Nucleic Acids Res 1989;17:6419.

22] Bradford MM. A rapid and sensitive method for the quantitation of micro-gram quantities of protein utilizing the principle of protein-dye binding. AnalBiochem 1976;72:248–54.

23] van Rossum TG, Vulto AG, Hop WC, Brouwer JT, Niesters HG, Schalm SW.Intravenous glycyrrhizin for the treatment of chronic hepatitis C: a double-blind, randomized, placebo-controlled phase I/II trial. J Gastroenterol Hepatol1999;14:1093–9.

24] Su XS, Chen HM, Wang LH, Jiang CF, Liu JH, Zhao MQ, et al. Clinical and laboratoryobservation on the effect of glycyrrhizin in acute and chronic viral hepatitis. JTradit Chin Med 1984;4:127–32.

25] Babaand M, Shigeta S. Antiviral activity of glycyrrhizin against varicella-zostervirus in vitro. Antiviral Res 1987;7:99–107.

26] Hattori T, Ikematsu S, Koito A, Matsushita S, Maeda Y, Hada M, et al. Preliminaryevidence for inhibitory effect of glycyrrhizin on HIV replication in patients withAIDS. Antiviral Res 1989;11:255–61.

27] Ito M, Sato A, Hirabayashi K, Tanabe F, Shigeta S, Baba M, et al. Mechanismof inhibitory effect of glycyrrhizin on replication of human immunodeficiencyvirus (HIV). Antiviral Res 1988;10:289–98.

28] Pompei R, Flore O, Marccialis MA, Pani A, Loddo B. Glycyrrhizic acid inhibitsvirus growth and inactivates virus particles. Nature 1979;281:689–90.

29] Partridgeand M, Poswillo DE. Topical carbenoxolone sodium in the manage-ment of herpes simplex infection. Br J Oral Maxillofac Surg 1984;22:138–45.

30] Numazaki K, Umetsu M, Chiba S. Effect of glycyrrhizin in children with liverdysfunction associated with cytomegalovirus infection. Tohoku J Exp Med

1994;172:147–53.

31] Armanini D, Karbowiak I, Funder JW. Affinity of liquorice derivatives for min-eralocorticoid and glucocorticoid receptors. Clin Endocrinol 1983;19:609–12.

32] Okimasu E, Moromizato Y, Watanabe S, Sasaki J, Shiraishi N, Morimoto YM, etal. Inhibition of phospholipase A2 and platelet aggregation by glycyrrhizin, anantiinflammation drug. Acta Med Okayama 1983;37:385–91.

[

al Research 58 (2008) 22–31 31

33] Ohuchi K, Kamada Y, Levine L, Tsurufuji S. Glycyrrhizin inhibits prostaglandinE2 production by activated peritoneal macrophages from rats. ProstaglandinsMed 1981;7:457–63.

34] Akamatsu H, Komura J, Asada Y, Niwa Y. Mechanism of anti-inflammatoryaction of glycyrrhizin: effect on neutrophil functions including reactive oxygenspecies generation. Planta Med 1991;57:119–21.

35] Wangand ZY, Nixon DW. Licorice and cancer. Nutr Cancer 2001;39:1–11.36] Haraguchi H, Yoshida N, Ishikawa H, Tamura Y, Mizutani K, Kinoshita T. Protec-

tion of mitochondrial functions against oxidative stresses by isoflavans fromGlycyrrhiza glabra. J Pharm Pharmacol 2000;52:219–23.

37] Nagai T, Egashira T, Yamanaka Y, Kohno M. The protective effect of glycyrrhizinagainst injury of the liver caused by ischemia-reperfusion. Arch Environ ContamToxicol 1991;20:432–6.

38] van Rossum TG, Vulto AG, Hop WC, Schalm SW. Glycyrrhizin-induced reduc-tion of ALT in European patients with chronic hepatitis C. Am J Gastroenterol2001;96:2432–7.

39] Eiserich JP, Cross CE, Jones AD, Halliwell B, van der Vliet A. Formation of nitrat-ing and chlorinating species by reaction of nitrite with hypochlorous acid Anovel mechanism for nitric oxide-mediated protein modification. J Biol Chem1996;271:19199–208.

40] Cuzzocrea S, Wang ZQ. Role of poly(ADP-ribose) glycohydrolase (PARG) inshock, ischemia and reperfusion. Pharmacol Res 2005;52:100–8.

41] Ghosh S, May MJ, Kopp EB. NF-kappa B. Rel proteins: evolutionarily con-served mediators of immune responses. Annu Rev Immunol 1998;16:225–60.

42] Cuzzocrea S, Pisano B, Dugo L, Ianaro A, Patel NS, Caputi AP, et al. Tempol reducesthe activation of nuclear factor-kappaB in acute inflammation. Free Radic Res2004;38:813–9.

43] Yeh CC, Kao SJ, Lin CC, Wang SD, Liu CJ, Kao ST. The immunomodulation ofendotoxin-induced acute lung injury by hesperidin in vivo and in vitro. Life Sci2007;80:1821–31.

damage: requirements for adherence and chemokine release. Am J Physiol2002;283:C1592–1603.

45] Gao H, Guo RF, Speyer CL, Reuben J, Neff TA, Hoesel LM, et al. Stat3 activationin acute lung injury. J Immunol 2004;172:7703–12.