International Immunopharmacology 11 (2011) 1606–1612

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International Immunopharmacology

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Naringin attenuates acute lung injury in LPS-treated mice by inhibitingNF-κB pathway

Ying Liu a,1, Hao Wu b,1, Yi-chu Nie a, Jia-ling Chen a, Wei-wei Su a, Pei-bo Li a,⁎a Guangdong Key Laboratory of Plant Resources, School of Life Science, Sun Yat-Sen University, Guangzhou 510275, Chinab China Pharmaceutical University, Nanjing 210009, China

⁎ Corresponding author. Tel.: +86 020 84110808; faE-mail address: lipb73@yahoo.com.cn (P. Li).

1 These authors equally contributed to this work.

1567-5769/$ – see front matter © 2011 Elsevier B.V. Adoi:10.1016/j.intimp.2011.05.022

a b s t r a c t

a r t i c l e i n f o

Article history:Received 22 February 2011Received in revised form 19 May 2011Accepted 19 May 2011Available online 1 June 2011

Keywords:NaringinLipopolysaccharideLungAnti-inflammationNF-κB

Naringin has been reported as an effective anti-inflammatory compound. We previously showed that naringinhad antitussive effect on experimentally induced cough in guinea pigs. However, the effects and mechanism ofnaringin on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice are not fully understood. In thisstudy, our aimwas to evaluate the anti-inflammatory activities of naringin on LPS-induced ALI inmice and clarifyits underlying mechanisms of action. We found that in vivo pretreatment with naringin markedly decreased thelung wet weight to dry weight ratio, and led to significant attenuation of LPS-induced evident lunghistopathological changes. Meanwhile, naringin significantly reduced bronchoalveolar lavage fluid (BALF) totalcell and neutrophil (PMN) counts after LPS challenge. Furthermore, naringin inhibitedmyeloperoxidase (MPO: amarker enzyme of neutrophil granule) and inducible nitric oxide synthase (iNOS) activities in lung tissue andalleviated LPS-induced tumor neurosis factor-α (TNF-α) secretion in BALF in a dose-dependent manner.Additionally, Western blotting showed that naringin efficiently blunt NF-κB activation by inhibiting thedegradation of I B-α and the translocation of p65. Taken together, these results suggest that naringin shows anti-inflammatory effects through inhibiting lung edema, MPO and iNOS activities, TNF-α secretion and pulmonaryneutrophil infiltration by blockade of NF-κB in LPS-induced ALI.

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© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Acute lung injury (ALI) and its more severe form, acute respiratorydistress syndrome (ARDS), are syndromes of acute respiratory failurecharacterized by an intense pulmonary inflammatory response,involving neutrophil recruitment, interstitial edema, a disruption ofepithelial integrity, and lung parenchymal injury [1]. The pathogenesisof ALI/ARDS involves disorders of oxidant/anti-oxidant and inflamma-tion/anti-inflammation, the up-regulation of adhesion molecules, andthe increased production of chemokines [2,3]. Despite recent advancesin clinical management and extensive investigations in new strategiesfor treatment, ALI/ARDS takes responsibility for significant morbidityandmortality. The development of efficient therapeutic approaches thatcould reduce morbidity and mortality from ALI/ARDS is urgentlyneeded. Endotoxin or LPS derived from Gram-negative bacteria hasbeenwell recognized in the pathogenesis of ALI [4]. In vivo intratrachealadministration of LPS has been extensively used as an experimentalmodel of ALI/ARDS characterized by increased levels of neutrophils,protein content, cytokines and chemokines in theBALF, associatingwiththe severity of disease [5,6].

Naringin is a kind of bioflavonoid derived fromgrapefruit and relatedcitrus species [7]. Naringin or its metabolite naringenin has beenreported to possess diverse biological and pharmacological propertiesincluding anticarcinogenic [8], lipid-lowering [9], superoxide scavenging[10], anti-apoptotic [11], anti-atherogenic [12], metal chelating [13] andantioxidant activities [14]. Naringin has recently received considerableattention as an antioxidant dietary supplement. Recently, growingevidence has indicated that naringin or naringenin displays anti-inflammatory effects both in vitro and in vivo [15–19]. However, thereis no information regarding the effects of naringin on ALI. Moreover, ourlaboratory has recently reported that naringin may exert peripheralantitussive effect and naringenin can inhibit the LPS-induced mucinincrease in the rat tracheal ring explants [20,21].We therefore evaluatedthe preventive effects of naringin on ALI induced by the intratrachealinstillation of LPS in vivo, and tried to clarify the mechanism involved.

2. Material and methods

2.1. Animals

Male and female KM mice (6–7 weeks) weighing 18–22 g werepurchased fromGuangdong Experimental Animal Center, kept in a 12 hdark/12 h light cycle in a temperature- and humidity-controlled roomand fed on standard laboratory diet and water. All experimentalprocedures were approved by the Animal Care and Use Committee of

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the School of Life Sciences, Sun Yat-Sen University, PR China. Adequatemeasures were taken to minimize pain of experimental animals.

2.2. Chemicals

Naringin (extracted from Citrus grandis ‘Tomentosa’ by water,deposited in ethanol, with concentrated filtrate obtained after one toten times of recrystallization, purityN98.3%, determined by peak areanormalization). Lipopolysaccharides (from Escherichia coli 055:B5)were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-p65, anti-I B-α and anti-β-actin monoclonal antibodies were pur-chased from Beyotime Institute of Biotechnology (Jiangsu, China).Dexamethasone (DEX) Sodium Phosphate Injection was purchasedfrom Baiyunshan Medicine Co. (Batch No.100304, China). The kits fordetermination of myeloperoxidase and nitric oxide synthetase werepurchased from Jiancheng Bioengineering Institute (Nanjing, China).The ELISA kits for TNF-α were purchased from R&D, America. Thepurity of all chemical reagents was at least analytical grade.

2.3. Development of mouse model of ALI and grouping

After adjustment to the environment, micewere randomly dividedinto seven groups, i.e., naïve group (n=32), normal saline (NS, i.e.,sham-operated control) group (n=32), LPS group (n=32), naringin(15 mg/kg)+LPS group(n=32), naringin (30 mg/kg)+LPS group(n=32), naringin (60 mg/kg)+LPS group (n=32) and dexameth-asone (DEX)+LPS group (n=32). Mice were anesthetized withchloral hydrate (3.5%) and fixed on a board at angle of 50° in thesupine position. 50 μl PBS containing 40 μg LPS was instilled into themouse trachea with a microliter injector. After intratracheal instilla-tion, the mouse was placed in a vertical position and spinned for0.5 min tomake sure that the instillation distributes evenly within thelungs [22]. Naringin was orally administrated 1 h before LPSchallenge. DEX (5 mg/kg) was used as a positive control in thisexperiment and intraperitoneally injected 1 h before LPS challenge.

2.4. Lung wet-to-dry weight (W/D) ratio

The mice were killed by exsanguinations 24 h after LPS challenge.The whole lungs were removed, each lung was blotted dry, weighed,and then placed in an oven at 80 °C for 48 h to obtain the “dry”weight.The ratio of thewet lung to the dry lungwas calculated to assess tissueedema [23].

2.5. Myeloperoxidase assay

The mice were killed by exsanguinations 24 h after LPS challenge.The whole lungs were homogenized and sonicated in 5% HTAB bufferaccording to the protocol. After incubation in a 50 mM KPO4 buffercontaining the substrate H2O2 (1.5 M) and o-dianisidine dihy-drochloride (167 mg/ml) for 30 min, the activity of MPO enzyme inthe homogenates was determined spectrophotometrically by mea-suring the absorbance at 460 nm [23].

2.6. iNOS assay

The mice were killed by exsanguination 24 h after LPS challengeand then the whole lungs were homogenized and sonicated in 10%PBS. The rest of the determinations were carried out according to theprotocol.

2.7. BALF total cell and PMN counting

The mice were anesthetized and provided with a plastic cannulainserted into the trachea. BALF was performed with three aliquotsof 0.5 ml PBS (pH7.2) instilled up to a total volume of 1.5 ml, and

withdrawn three times each. The recovery rate of BALF was 95%. BALFsamples were centrifuged (3000 rpm, 4 °C) for 10 min. The sedimentcells were resuspended in 50 μl PBS. The total BALF cells were counteddouble-blindly using a hemocytometer followed by the differentialcounting of leukocytes (Giemsa staining; two counts per slide, 300cells per count) [24]. The supernatants were stored at −80 °C untilanalysis for TNF-α by enzyme-linked immunosorbent assay (ELISA)assay.

2.8. TNF-α ELISA assay

The levels of TNF-α in the BALF supernatant were determined byELISA assay using commercially available kits according to the manufac-turer's instructions. The levels of TNF-α in the samples were calculatedbased on the standard curve generated from recombinant mice TNF-α.The detection range of this assay for TNF-α is 12.25–720 pg/ml. Sampleswith a concentration exceeding the limits of the standard curve weremeasured after dilution.

2.9. Histopathologic evaluation

The mice were killed by exsanguinations 24 h after LPS challenge.Afterwards, the lungs were removed and stored in the fixativecontaining of 10% paraformaldehyde in 0.1 M PBS (pH7.4) for 48 h at4 °C. The hematoxylin and eosin staining was carried out according tothe regular staining method, and the slides were histopathologicallyevaluated using a semiquantitative scoring method. Lung injury wasgraded from 0 (normal) to 4 (severe) in four categories: interstitialinflammation, inflammatory cell infiltration, congestion, and edema.The total lung injury score was calculated by adding up the individualscores of each category [23].

2.10. Western blotting

Proteins were extracted from the lungs using Nuclear andCytoplasmic Protein Extraction Kit (Beyotime Biotechnology, China).The extracts were boiled for 5 min with loading buffer. Proteins weresubjected to sodium dodecyl sulfate polyacrylamide gel electropho-resis (SDS-PAGE) in a 12% gel and transferred onto polyvinylidenedifluoride sheets. The membranes were washed with PBST and 5%skim milk for 1 h at room temperature. Following three washes withPBST, the membranes were incubated with primary antibody diluted1:1000 in PBST overnight at room temperature. After three furtherwashes, secondary antibody was added at 1/1000 dilution in PBST andincubated for 1 h at room temperature. After three final washes, theblots were developed using BeyoECL Plus reagent (BeyotimeBiotechnology, China) and then exposed to film for 1 min and putinto developing and fixing solutions for 1 min respectively [24].

2.11. Statistical analysis

Data are expressed as the mean±SD. Statistical analyses werecarried out using SPSS 16.0. One-way ANOVA followed by theStudent–Newman–Keuls test were used for comparing the resultsamong treatments. Differences were considered to be statisticallysignificant when Pb0.05.

3. Results

3.1. Naringin reduced pulmonary edema in mice challenged with LPS

As illustrated in Fig. 1, there was no significant difference in W/Dratio, a parameter of pulmonary edema, between normal saline groupand naïve group (PN0.05), indicating that the protocol of intratrachealadministration didn't cause additional inflammation response in thismodel. The lung W/D ratio in LPS group increased significantly when

Fig. 1. Effects of naringin on LPS-induced lung edema. Mice were challenged by LPS(2 mg/kg) to induce lung edema with or without naringin (15, 30 and 60 mg/kg)pretreatment 1 h before LPS. Lung wet weight was marked 24 h after LPS challenge.Lungswere put in an oven at 80 °C for 48 h to determine the dryweight (n=6). ▲▲Pb0.01vs. naïve group. ##Pb0.01 vs. normal saline treated group. ⁎Pb0.05 vs. LPS group.

Fig. 2. Effects of naringin on LPS-induced neutrophil and macrophage infiltration.A: Myeloperoxidase activity in lung homogenate (n=6). Myeloperoxidase activity wasperformed according to the manufacturer's instruction 24 h after LPS challenge. Itsactivity reflects the neutrophil infiltration in the lungs. B: iNOS activity in lunghomogenate (n=6). iNOS activity was performed according to the manufacturer'sinstruction 24 h after LPS challenge. #Pb0.01 vs. normal saline treated group. ⁎Pb0.05vs. LPS group. ⁎⁎Pb0.01 vs. LPS group.

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compared with normal saline group (Pb0.01) and naïve group(Pb0.01), whereas the increase was significantly attenuated in thenaringin (15, 30 and 60 mg/kg) and DEX groups, suggesting that theLPS-induced pulmonary edema was suppressed by different doses ofnaringin.

3.2. Naringin reduced MPO and iNOS levels in mice challenged with LPS

As illustrated in Fig. 2, there was no significant difference in MPOand iNOS levels between normal saline group and naïve group(PN0.05). LPS markedly increased MPO and iNOS activities ascompared with normal saline group (Pb0.01). In the groupspretreated with naringin (15, 30 and 60 mg/kg) and DEX, the up-regulations of MPO and iNOS activities were significantly inhibited ascompared with LPS group (Pb0.05).

3.3. Naringin reduced BALF total cell and PMN counts in mice challengedwith LPS

As illustrated in Fig. 3, there's no significant difference in total celland PMN counts between normal saline group and naïve group(PN0.05). The LPS group showed a remarkably higher BALF total celland PMN counts when compared with normal saline treated group(Pb0.01). In naringin (15, 30 and 60 mg/kg)-treated groups, total celland PMN counts were significantly decreased when compared withLPS group (Pb0.01). The data indicated that naringin reduced PMNinfiltration, which was caused by LPS-induced acute lung injury.

3.4. Naringin reduced the production of TNF-α in mice challenged withLPS

As illustrated in Fig. 4, the TNF-α level was found to be significantlyincreased in the LPS group compared with the naïve group (Pb0.01),whereas the naringin (30 and 60 mg/kg)-treated groups and DEX-treated group showed significant decrease when compared with theLPS group (Pb0.05). The results indicated that naringin attenuatedLPS-induced TNF-α secretion in BALF.

3.5. Naringin reduced morphologic damages in mice challenged with LPS

As illustrated in Fig. 5A and B, the mice treated with normal salineshowed no significant difference in morphologic damages compared

to naïve group mice (PN0.05), which indicated that intratrachealadministration didn't provoke additional inflammation response inthis protocol. Compared with the naïve group, the lungs of LPS groupmice showed marked inflammatory alterations characterized by thepresence of alveolar hemorrhage, edema, inflammation, remarkablerecruitment of neutrophils and leukocytes into the alveolar spaces. Incontrast, histological damage was less pronounced in naringin (15, 30and 60 mg/kg)-treated mice in a dose-dependent manner. The resultsindicated that naringin ameliorated many of the symptoms of LPS-induced ALI in mice.

3.6. Naringin inhibited NF-κB signal transduction

As showed in Fig. 6, LPS group showed significant I B-αdegradation in cytoplasm protein extracts when compared to naïvegroup. On the contrary, I B-α degradation in naringin-treated groups(15 and 60 mg/kg) were reduced significantly when compared withLPS group, which indicated that naringin inhibited I B-α degradation.LPS-treated mice also showed decrease in cytoplasm but increase innuclear extracts of p65 subunit of NF- B. In contrast, naringin at a doseof 60 mg/kg inhibited translocation of p65 from cytoplasm to nuclearsince naringin-treated groups stayed almost the same level as naïvegroup for cytoplasm and nuclear p65.

4. Discussion

Recent advances have been made in the understanding of theepidemiology, pathogenesis, and treatment of ALI. However, ALI

Fig. 3. Effects of naringin on LPS-induced total cell and PMN counts. A: total cell countsin lung BALF (n=8). BALF was performed 24 h after LPS challenge and total cells werecounted. B: PMN counts in lung BALF (n=8). The cells were stained with Giemsa andcounted double blind under a microscope. ##Pb0.01 vs. normal saline treated group.⁎⁎Pb0.01 vs. LPS group.

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remains a significant source of morbidity and mortality in thecritically ill patient population [25]. Novel therapies need to bedeveloped to further improve clinical outcomes. LPS can enter thebloodstream and elicit inflammatory responses thatmay lead to shockand ultimately death. Experimental LPS administration via thetracheal route has been extensively used to study the biological andpathophysiologic pathways of inflammation [26]. Intratracheal LPSadministration triggers the optimal ALI which is characterized by

Fig. 4. Effects of naringin on LPS-induced TNF-α secretion. TNF-α secretion in lung BALFsupernatant (n=8). BALF was prepared 24 h after LPS challenge and centrifuged at700 g for 10 min and TNF-α level in the supernatant was evaluated. ##Pb0.01 vs.normal saline treated group. ⁎Pb0.05 vs. LPS group.

increased capillary permeability, alveolar edema and an influx ofcirculating inflammatory cells [26,27]. At present, glucocorticoids arethe most frequently used anti-inflammatory drugs in the clinicaltreatment of ALI/ARDS because they modulate most steps of theinflammatory process [28,29]. Therefore, DEX was used as a positivecontrol to evaluate the anti-inflammatory efficiency of naringin inLPS-induced ALI. Naringin has been empirically proved to have no sideeffects, as historically humans have been ingesting citrus fruits for along time [30].

Naringin is one of the main components of Chinese herbal Citrusgrandis ‘Tomentosa’, which has been commonly used in respiratorydiseases for relieving cough and reducing sputum for thousands ofyears. Our previous studies have shown that naringin may exertperipheral antitussive effect and naringenin can inhibit the LPS-inducedmucin increase in the rat tracheal ring explants [20,21]. Recently,growing evidence has indicated that naringin or naringenin displaysanti-inflammatory effects both in vitro and in vivo [15–18]. In vitro studyusing RAW264.7 cells showed that naringin inhibited LPS-inducedproduction of NO by suppressing the activation of NF-κB [17]. Naringincould repress PI3K/AKT/mTOR/p70S6K pathway, invasion and migra-tion, and subsequently inhibited MMP-9 expression through thetranscription factors NF-κB and activator protein-1 in TNF-alpha-induced VSMC [19]. Naringin was also reported to show anti-inflammatory activity in a model of DSS-induced colitis revealing byintestine dry/wet weight ratio, nitrates/nitrites, and tissue malondial-dehyde levels [16]. Moreover, a recent study reported that naringenininhibited ovalbumin-induced airway inflammation by inhibiting NF-κBactivity inmurine asthmamodel [18]. However, there is no informationregarding the effects of naringin on LPS-induced lung inflammation.Therefore, we hypothesized that naringin plays a pivotal role in lunginflammation and would inhibit neutrophil migration into the lungs,pro-inflammatory cytokine production and subsequent lung disease. Totest this hypothesis, we investigated the preventive effects of naringinon ALI induced by the intratracheal instillation of LPS in vivo and tried toclarify the mechanism involved.

In the present study, we demonstrated that pretreatment withnaringin could protect the mice from LPS-induced ALI. Naringinmarkedly improved lung morphology, inhibited pulmonary edemaformation and prevented infiltration of activated PMN into lungtissue. The increases in lung tissue MPO and iNOS activity and BALFTNF-α level induced by LPS were also significantly suppressed bynaringin. Moreover, we found that naringin effectively repressed theactivation of transcription factor NF-κB, and this may be part of themechanisms whereby naringin elicits its salutary effects.

Edema is a typical symptom of inflammation not only in systemicinflammation, but also in local inflammation. To quantify themagnitudeof pulmonary edema,wefirst evaluated the lungW/D ratio.Widespreaddestruction of alveolar epithelium and flooding of the alveolar spaceswith proteinaceous exudates containing large amounts of neutrophilsrepresent the typical lesion in ALI [31]. In our study,mice exposed to LPSshowed all these features, attesting to the development of high-permeability pulmonary edema. In naringin-treated group, the edemaand congestion in the lungs were significantly reduced. These findingsindicated that naringin effectively decreased the lung vascular perme-ability and promoted the resolution of lung edema. Therefore, naringinmight exert a salutary effect on the integrity of the alveolocapillarymembrane, thereby markedly improving lung morphology.

The hallmark of pulmonary inflammation is the presence ofinfiltrating leukocytes. In ALI, the predominant inflammatory cells arethe neutrophils, which play an important role in the development ofmost cases of ALI [32]. Infiltrationof neutrophils in the alveolar space is akey phase in ALI [33]. MPO is an enzyme located mainly in the primarygranules of neutrophils, thus MPO activity in the parenchyma reflectsthe adhesion and margination of neutrophils in the lungs [34]. In thisstudy, as expected,miceexposed to LPS exhibitedamassive recruitmentof inflammatory cells includingneutrophils in the airways and increased

Fig. 5. A: Effects of naringin on LPS-induced lung morphology. The mice were killed by exsanguinations 24 h after LPS challenge. Afterwards, the lungs were removed and storedin the fixative containing of 10% paraformaldehyde in 0.1 M PBS (pH7.4) for 48 h at 4 °C. The hematoxylin and eosin staining was carried out according to the regular stainingmethod. a–g (n=8): naïve group;NSgroup; LPSgroup; naringin (15 mg/kg)+LPS; naringin (30 mg/kg)+LPS;naringin (60 mg/kg)+LPS;DEX+LPS. Lungswere removed24 h after LPSchallenge, fixed in 10% formalin and stained with hematoxylin and eosin. B: Effects of naringin on LPS-induced lung morphology. The slides were histopathologically evaluated using asemiquantitative scoringmethod. Lung injurywas graded from0 (normal) to 4 (severe) in four categories: interstitial inflammation, inflammatory cell infiltration, congestion, and edema.The total lung injury score was calculated by adding up the individual scores of each category. ##Pb0.01 vs. normal saline treated group. ⁎⁎Pb0.01 vs. LPS group.

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MPO activity in the lungs. In contrast, pretreatment with naringinmarkedly reduced the inflammatory histological changes in lung tissuesproduced by LPS challenge, as well as suppressed LPS-inducedneutrophil migration into the lung and MPO activity. These results

Fig. 6. Naringin inhibited NF-κB signal transduction when mice were pretreated withnaringin following LPS challenge for 24 h. Nuclear and cytoplasm extracts wereprepared from the lung tissues and subjected to western blotting. Assay shown isrepresentative of three experiments with similar results.

thus confirm that the protective effect of naringin on ALI induced by LPSis related to an attenuation of inflammatory cell sequestration andmigration into the lung tissue.

ALI/ARDS is associated with the development of interconnectedinflammatory cascades, where pro-inflammatory cytokines play acentral role in the initiation and propagation of the inflammation andALI [35]. Studies in both humans and animals indicate that a networkof pro-inflammatory cytokines, such as TNF-α and IL-6, andchemokines, such as IL-8, are critical for initiating, amplifying, andperpetuating lung injury induced by diverse stimuli [36]. ALI patientshad high BALF TNF-α levels and the level was significantly higher innon-survivors than survivors [37]. Cytokines have been considered astherapeutic targets for LPS-induced ALI and many treatments havehad inhibitory effects on the gene expression of cytokines in the lung(i.e., glucocorticoids) [38]. LPS triggers monocytes and macrophagesto produce several inflammatory cytokines and mediators as well asbiological mediators involved in the control of pathogens. Exacerbat-ed responses to LPS induce an overproduction of inflammatorycytokines and mediators that are responsible for severe systemicdysfunctions [39]. LPS-induced ALI has a complex mechanism inhumans and a single mediator such as TNF-a might play a partial butfundamental role in the pathogenesis [40]. In line with these concepts,we found that mice challenged with LPS expressed very largeamounts of TNF-α in their BALF. In contrast, pretreatment with

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naringin was associated with a significant reduction in the BALF levelsof TNF-α. These results show that the anti-inflammatory effect ofnaringin on LPS-induced acute lung injury depends on a decrease inthe production of TNF-α, in linewith other studies, which suggest thatnaringin might inhibit TNF-α secretion in cellular and in vivo models[41–43].

It has been well known that LPS is a strong stimulator of iNOSexpression and NO over-production. ALI in both humans and animalmodels is associated with pulmonary overexpression of iNOS andenhanced NO production [44,45]. Wang LF and his colleagues foundthat both pulmonary parenchymal cells and inflammatory cellscontribute to the increased lung iNOS activity in endotoxemia,pulmonary parenchymal cells contribute to a significantly greaterdegree [46]. Accumulating evidence suggest that inhibitors of iNOSmay be potential therapeutic agents for clinical application in patientswith ALI/ARDS because the inflammatory responses and ALI followinginfusion of LPS are due to the production of NO, free radicals and pro-inflammatory cytokines through the iNOS system [47–50]. Here, ourfinding revealed that naringin significantly reduced LPS-inducedincrement of iNOS activity. This result demonstrates that naringinconfers powerful protection against LPS-induced acute lung injury byiNOS activity.

In most unstimulated cells, NF-κB covalently bound to inhibitorprotein IκB is sequestered in the cytoplasm [51]. Exposure of the cellsto diverse stimuli, such as inflammatory cytokines, oxidative stress,ultraviolet irradiation, or bacterial endotoxins, results in activation ofNF-κB through the stimulation of phosphorylation and degradation ofIκBα [52,53]. The activated NF-κB is then translocated to the nucleus,where it binds to the cis-acting κB enhancer element of target genesand activates the expression of pro-inflammatory mediators [54].NF-κB plays an important role in inflammatory phenotypic changes invarious pathophysiological conditions [55]. Like other members of theNF-κB family, NF-κB p65 resides in the cytoplasm in an inactive formbound to inhibitory IκB proteins. Cellular activation results in thenuclear translocation of NF-κB p65 for initiating gene transcription.The translocation of NF-κB p65 from cytoplasm to nuclear is oftentaken as an indication of NF-κB activation and is related to the cellularresponse to oxidants or to the inflammatory and acute immuneresponse [56]. The degree of NF-κB activation was reported to increasein patients with sepsis or acute lung injury [57], and the nucleartranslocation of NF-κB p65 was observed in alveolar macrophages frompatientswith acute lung injury caused by severe infection, in contrast toalveolar macrophages from control patients [58]. It has been documen-ted that inhibition of nuclear translocation of NF-κB decreases theexpression of NF-κB-dependent pro-inflammatory mediators anddiminishes the severity of endotoxemia-induced acute lung injury[59]. In the present study, we are the first to demonstrate thatdegradation of IκB and NF-κB p65 subunit nuclear translocation inlung tissue was inhibited by naringin pretreatment in LPS-inducedmurine model. It might be suggested that naringin could exert potentanti-inflammatory effects in lungs exposed to LPS, most likely byinhibiting the activation of NF-κB.

On the basis of the results presented herein, we showed for thefirst time that pretreatment of naringin significantly attenuatedpulmonary inflammation in mice with LPS-induced ALI, and theprotective effect of naringin in ALI may be related to its suppression ofNF-κB activation, and subsequently leads to a remarkable reduction ininflammatory cell infiltration, and pro-inflammatory cytokine secre-tion in lung tissues. Our work defines, for the first time, a potentialrole of naringin in treating LPS-associated acute lung injury patients.

Acknowledgements

This work was supported by the National Natural ScienceFoundation of China (No. 30873422).

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