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Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 916590 11 pageshttpdxdoiorg1011552013916590

Research ArticleTherapeutic Effect of C-Phycocyanin Extracted fromBlue Green Algae in a Rat Model of Acute Lung InjuryInduced by Lipopolysaccharide

Pak-on Leung1 Hao-Hsien Lee2 Yu-Chien Kung3 Ming-Fan Tsai3 and Tz-Chong Chou4

1 Department of Intensive Care Chi Mei Medical Center Liou Ying Campus Tainan Taiwan2Department of Surgery Chi Mei Medical Center Liou Ying Campus Tainan Taiwan3 Institute of Physiology National Defense Medical Center Taipei Taiwan4Department of Biomedical Engineering National Defense Medical Center Institute of Medical Sciences Tzu Chi University701 Zhongyang Road Hualien Taiwan

Correspondence should be addressed to Tz-Chong Chou tcchoums5hinetnet

Received 3 November 2012 Revised 20 February 2013 Accepted 21 February 2013

Academic Editor Jenny M Wilkinson

Copyright copy 2013 Pak-on Leung et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

C-Phycocyanin (CPC) extracted from blue green algae is a dietary nutritional supplement due to its several beneficialpharmacological effects This study was conducted to evaluate whether CPC protects against lipopolysaccharide- (LPS-) inducedacute lung injury (ALI) in rats Rats were challenged with LPS (5mgkg body weight) intratracheally to induce ALI After 3 h LPSinstillation rats were administrated with CPC (50mgkg body weight ip) for another 3 h Our results showed that posttreatmentwith CPC significantly inhibited LPS-induced elevation of protein concentration nitritenitrate level release of proinflammatorycytokines the number of total polymorphonuclear cells in bronchoalveolar lavage fluid and lung edema evidenced by decreaseof lung wetdry weight ratio accompanied by a remarkable improvement of lung histopathological alterations Furthermore CPCsignificantly attenuated LPS-induced myeloperoxidase activity O2

minus formation expression of inducible nitric oxide synthase andcyclooxygenase-2 as well as nuclear factor-kappa B (NF-120581B) activation in lungs Additionally CPC significantly downregulatedproapoptotic proteins such as caspase-3 and Bax but upregulated antiapoptotic proteins such as Bcl-2 and Bcl-XL in lungs exposedto LPS These findings indicate that CPC could be potentially useful for treatment of LPS-related ALI by inhibiting inflammatoryresponses and apoptosis in lung tissues

1 Introduction

Acute lung injury (ALI) a severe complication often observedin ICU patients has a high mortality [1] ALI is charac-terized by damage to the epithelial and endothelial cells inlungs thereby increasing pulmonary vascular permeabilitypulmonary edema and sequestration of polymorphonuclearneutrophils (PMNs) which finally impairs respiratory func-tion Although the true mechanisms causing ALI remainunclear it is generally accepted that ALI is an excessiveuncontrolled inflammatory response mediated by severalpro-inflammatory mediators [2] Lipopolysaccharide (LPS)a cell wall component of gram-negative bacteria is thoughtto play a key role in the development of ALI through

stimulation of recruitment of inflammatory cells into lungsand production of several inflammatory and chemotacticcytokines [3ndash5] Although there is no ideal animal model ofALI intratracheal (it) administration of LPS into lungs is auseful and applicable experimental ALImodel which directlycauses lung injury without systemic inflammation andmulti-organ failure [6] Currently this model has been usedto investigate the pathological mechanisms of endotoxin-induced ALI and evaluate the potential new therapeuticstrategies

C-Phycocyanin (CPC) a biliprotein occurred in bluegreen algae such as Spirulina platensis is a dietary nutritionalsupplement due to its beneficial values including hep-atoprotective antiplatelet neuroprotective antioxidant and

2 Evidence-Based Complementary and Alternative Medicine

anti-inflammatory activities [7ndash10] Our previous study hasshown that in LPS-stimulated RAW2647macrophages CPCexhibits an anti-inflammatory activity by inhibiting induciblenitric oxide synthase (iNOS) expression and NO productionpossibly by suppressing nuclear transcription factor-120581B (NF-120581B) activation a key transcription factor promoting pro-inflammatory gene expression [11] These findings suggestthat CPC may be a potential reagent to treat inflammatorydiseases However there is little information onwhether CPChas an ability to protect against LPS-induced ALI The aimof this study was to investigate the effects of CPC on LPS(it)-induced ALI in rats and further studied the possiblemechanisms involved Our results have indicated that CPCexhibits a therapeutic effect in LPS-induced ALI

2 Materials and Methods

21 Animal and Reagents Male Sprague-Dawley (SD) rats(8 weeks of age 200ndash230 g) were used for this study Allexperimental procedures were conducted in accordance withthe Guiding Principle in the Care and Use of Animalsand approved by the Institutional Animal Care and UseCommittee of National Defense Medical Center (TaipeiTaiwan)The CPC (with a purity of A

620

A280

gt35) providedby Far East Bio-Tec Co Ltd (Taipei Taiwan) was dissolvedin normal saline Other chemical agents used in the studywere at least analytical grade

22 LPS-Induced ALI Model and Grouping The rats weredivided randomly into three groups (1) phosphate bufferedsaline (PBS) vehicle group (control group) (2) LPS challengegroup (LPS group) (3) LPS and CPC-treated group Forintratracheal instillation rats were anesthetized with a singleintraperitoneal dose of ketamine (90mgkg bodyweight) andxylazine (7mgkg body weight) followed by administrationof LPS (Escherichia coli 055B5 5mgkg body weight it) in02mL PBS through a 24-gauge catheter to induce lung injuryas previously described [12] Control rats were given 02mLPBS intratracheally The treated rats were administrated withCPC (50mgkg body weight) intraperitoneally (ip) 3 h afterinstillation of LPS In our preliminary study we have foundthat the pathological features of LPS-induced ALI weresignificantly improved if rats were treated with CPC at adosage of 50mgkg Therefore the dosage of CPC (50mgkgbody weight ip) was chosen in the following experimentsAll rats were euthanized at 6 h after LPS or PBS instillationand the samples were collected for subsequent analyses

23 Bronchoalveolar Lavage and Cell Counting At the endof the experiment the right lungs were ligated at the rightmain bronchus Then the left lungs were lavaged with 1mLof autoclaved PBS for five times The recovery ratio of thefluid was about 80 (4mL)The bronchoalveolar lavage fluid(BALF) obtained from left lungswas immediately centrifugedat 300 g for 10min at 4∘C and the supernatants were storedat minus70∘C until required for subsequent tests The cell pelletswere resuspended in PBS and the total cell number wascounted using a standard hemocytometer

24 Evans Blue Dye Leakage Thirty minutes before the endof the experiment Evans blue dye (50mgkg Sigma-AldrichSt Louis MO USA) was given intravenously After the lungswere perfused free of blood the Evans blue content in lungtissue was determined using a spectrophotometer at theoptical density of 620 nm

25 Wet-to-Dry Lung Weight Ratio and Protein Content inBALF Six hours after the intratracheal instillation of LPSor PBS right lungs were excised and immediately weighedthe wet weight Dry weight was determined after heating thelungs in an oven at 80∘C for 48 h The wetdry weight ratioof right lungs was measured to assess pulmonary edemaTheprotein content in cell-free BALF obtained from left lungswasdetermined by using a Bio-Rad protein assay kit (Bio-RadLaboratories Hemel Hempstead UK) with bovine serumalbumin (Sigma) as a standard

26 Measurement of Cytokines and NitriteNitrate (119873119874119909

)The levels of pro-inflammatory cytokines (TNF-120572 IL-6 andIL-1120573) and cytokine inducible neutrophil chemoattractant-3(CINC-3) in BALF obtained from left lungs were measuredby using enzyme-linked immunosorbent assay (ELISA) kits(GenzymeCorporation CambridgeMA USA) respectivelyTo determine the NO formation the BALF was injectedinto a collection chamber containing 5 VCl

3

In thisstrong reducing environment both nitrate and nitrite wereconverted to NO A constant stream of helium gas was usedto carry the output into an NO analyzer (Sievers 280NOASievers Instruments Inc Boulder CO USA) where the NOreacts with ozone (O

3

) resulting in the emission of lightLight emission is proportional to the NO formed and thelevel of NO

119909

(nitrite + nitrate) reflects the total amount ofNO produced by nitrite + nitrate [13] Standard amounts ofsodium nitrate were used for calibration

27 Myeloperoxidase (MPO) Activity Assay The right lungswere homogenized and sonicated in 50mM potassium phos-phate (pH 6) containing 5mM hexadecyltrimethylammo-nium bromide (Sigma-Aldrich St Louis MO USA) andsubjected to three cycles of freezing and thawing followedby centrifugation at 14000 g for 10min at 4∘C to obtainthe supernatant Then 20120583L tissue supernatant was incu-bated with 40 120583L assay buffer containing 0167mgmL O-dianisidine dihydrochloride (Sigma-Aldrich St Louis MOUSA) and 00005 hydrogen peroxide The MPO activitywas calculated as the change in absorbance at 460 nm over1min and expressed as Ug weight

28 Measurement of 1198742

minus Production in Lungs The freshlyharvested right lungs were cut into pieces of 5 times 5mmand incubated with oxygenated Krebs-Hepes buffer for 5ndash10min Then the pieces of lung tissue were transferred to96-well microplates These microplates containing 125mMlucigenin in 200120583L Krebs-Hepes buffer were placed intoa microplate luminometer (MicroLumat LB96V BertholdGermany) Counts of chemiluminescence signal from lungswere obtained at 1 min intervals at room temperature

Evidence-Based Complementary and Alternative Medicine 3

The counts of plates containing all components without lungtissue were regarded as background and the blank value wassubtracted from the chemiluminescence signal obtained fromthe lung samples The results were expressed as count persecond (CPS) permilligramdryweight (CPSmgdryweight)

29 Immunohistochemistry Staining for Nitrotyrosine Theright lung sections were deparaffinized dehydrated andimmersed in 10mM sodium citrate buffer for 5min at 100∘CAfter blocking with 5 bovine serum albumin at roomtemperature for 20min the slides were incubated at 4∘Cwitha polyclonal nitrotyrosine primary antibody (1 50 dilutionSanta Cruz Biotechnology CA USA) overnight followed byaddition of a goat anti-rabbit immunoglobulin horseradishperoxidase-conjugated secondary antibody (1 50 AbcamCambridge UK) for 1 h at room temperatureThe presence ofnitrotyrosine was indicated by the brown peroxidase reactionproduct and photographed with light micrographs (LeicaDMI6000B Wetzlar Germany)

210 Quantitative Real-Time PCR Assay Total RNA wasextracted from right lung tissues by usingTRIzol reagent (LifeTechnologies Carlsbad CA USA) and RNA was reverse-transcribed to cDNA using RT and amplified by PCR withTaq polymerase (Invitrogen Carlsbad CA USA) accordingto the manufacturerrsquos instructions Relative expressions ofBax Bcl-2 and Bcl-XL were measured with a SYBER greendetection system by using ABI 7300 Real-Time PCR device(Applied Biosystems Foster City CA USA) The threshold(119862119879

) values for each mRNA were subtracted from that of 120573-actin mRNA and converted from log-linear to linear termThe primer sequences used were as follows

Baxforward 51015840-CCAGGATCGAGCAGAGAGG-31015840reverse 51015840-CGGAGGAAGTCCAGTGTCC-31015840Bcl-2forward 51015840-TAATACGACTCACTATAGGCG-GGAGATCGTGATGAAGTA-31015840reverse 51015840-ATTTAGGTGACACTATAGAGAAGG-GCGTCAGGTGC-31015840Bcl-XLforward 51015840-GAGTTTGAGACCCGCTTCC-31015840reverse 51015840-GTCCTCACTGATGCCCAGTT-31015840120573-actinforward 51015840-GACCCAGATCATGTTTGAGACCTT-C-31015840reverse 51015840-GGTGACCGTAACACTACCTGAG-31015840

211 Western Blot Analysis Lung tissues were homogenizedin RIPA lysis buffer (50mM Tris-HCl pH 74 150mMNaCl 025 deoxycholic acid 1 NP-40 and 1mM EDTA)containing EDTA-free protease inhibitor cocktail (ThermoscientificUSA)Theproteinswere extracted at 4∘C for 30minand centrifuged twice at 10000 g for 10min at 4∘C and

protein concentrations were determined by using Bio-Radprotein assay kit The samples (100 120583g proteinlane) wereloaded and separated on 10 sodium dodecyl sulfate (SDS)polyacrylamide (PAGE) The separated proteins were trans-ferred to polyvinylidene fluoride membranes (Immobilon-P Millipore Bedford MA USA) using a Wet transfer tank(Bio-Rad Hercules CA USA) and blocked with 5 skimmilk in TBST (20mM Tris-base pH 75 500mM NaCland 01 Tween 20 vv) for 1 h After blocking the mem-branes were incubated overnight at 4∘Cwith specific primaryantibodies against with cyclooxygenase-2 (COX-2 1 500BD Bioscience Pharmingen San Diego CA USA) iNOS(1 500 Biotechnology Sanata Cruz CA USA) caspase-3(1 1000 Cell Signaling Technology Danvers MA USA) or120573-actin (1 5000) in 5 skim milk After additional washesthe membranes were incubated with a goat anti-rabbitimmunoglobulin horseradish peroxidase-coupled secondaryantibody (Abcam Cambridge UK) at a dilution of 10000in 5 skim milk for 1 h The immuno-reactive bands werevisible after development with a chemiluminescence (ECL)reagent (Amersham International Plc BuckinghamshireUK) and were quantified by densitometry and normalizedwith respective 120573-actin

212 Assay of NF-120581B Translocation The lung tissue slicesmounted on the coverslips were incubated with rabbit mono-clonal phospho-NF-120581B p65 (Ser536) (Cell Signaling Technol-ogy Danvers MA USA) overnight at 4∘C diluted 1 100 in 1BSA in TBS After washing coverslips were incubated witha fluorescein-isothiocyanate- (FITC-) conjugated secondaryantibody (Santa Cruz Biotechnology Santa Cruz CA USA)diluted 1 100 with 1 BSA in TBS All coverslips were alsostained with Hoechst 33258 dye in the nuclei for 10min Afterextensive washings with TBS the coverslips were mountedonto the glass slides and photographed with a fluorescencemicroscope (Leica DMI6000B Wetzlar Germany)

213 Lung Histological Analysis The right lung tissues wereremoved and fixed with 4 (wv) paraformaldehyde dehy-drated in graded ethanol embedded in paraffin and tissuesections (4ndash6120583m thick) were stained with hematoxylin andeosin (HampE) and photographed by a light microscopy Todetermine the score of the lung injury the histologicalimages were evaluated by an investigator who was initiallyblinded to these research groups The lung injury was scoredaccording to the following principle (1) alveolar congestion(2) hemorrhage (3) infiltration or aggregation of neutrophilsin the airspace or vessel wall and (4) thickness of the alveolarwallhyaline membrane formation Each item was gradedaccording to a four-point scale from 0 to 3 as follows 0 =no damage l = mild damage 2 = moderate damage and 3 =severe damage [14]

214 Statistical Analysis The experimental data were ex-pressed as the mean plusmn SEM One-way ANOVAwith post hocBonferroni test was used for statistical analysis Results wereconsidered significant difference at a value of 119875 lt 005


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Figure 1 Effects of CPC on LPS-induced lung edema lung MPO activity and total cells in BALF Rats were administrated with CPC(50mgkg ip) 3 h after LPS was instilled intratracheally The protein concentration and total cell numbers in the BALF Evans blue levellung wetdry weigh ratio and lung MPO activity were determined 6 h after LPS challenge Data are presented as the mean plusmn SEM (119899 = 5)lowast

119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001 compared with control group 119875 lt 005 119875 lt 0001 compared with LPS group


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Figure 2 Effects of CPC on LPS-induced secretion of pro-inflammatory cytokines in BALF BALF was prepared from rats 6 h after LPSinstillation and the levels of TNF-120572 IL-1120573 IL-6 and CINC-3 in the BALF were measured by ELISA kits respectively Data are presented asthe mean plusmn SEM (119899 = 5) lowastlowastlowast119875 lt 0001 compared with control group 119875 lt 0001 compared with LPS group

3 Results

31 Effect of CPC on Lung Edema MPO Activity and CellAccumulation in LPS-Induced ALI Rats Intratracheal instil-lation of LPS resulted in a significant increase in proteinlevel and the number of total cells in BALF Evans bluelevel and lung edema evidenced by elevation of lung wetdryweight ratio as compared to that of control ratsThe elevationcaused by LPS was markedly inhibited by posttreatment withCPC (50mgkg ip) (Figure 1) The increased neutrophilinfiltration is known to play an important role in thedevelopment of LPS-induced ALI and the MPO activity wasconsidered as a marker of influx of neutrophils Our data alsoshowed that elevation of lung MPO activity stimulated byLPS instillation was significantly inhibited by posttreatmentof CPC (Figure 1)

32 Effect of CPC on the Concentrations of Proinflamma-tory Cytokines and CINC-3 in BALF The levels of pro-inflammatory cytokines including TNF-120572 IL-1120573 IL-6 andCINC-3 a chemotactic cytokine in BALF were markedlyincreased in rats received LPS instillation compared tocontrol group The augmentation of these inflammatorymediators induced by LPS was all significantly attenuatedby posttreatment of CPC as compared to LPS-instilled alonegroup (Figure 2)

33 Effect of CPC on NO119909

1198742

minus Production and Nitrotyro-sine Expression in Lungs The levels of NO

119909

in BALF andO2

minus production (Figures 3(a) and 3(b)) and nitrotyrosineexpression (Figure 3(c)) were significantly increased afterLPS instillation and these levels were significantly reducedby posttreatment with CPC


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(c)

Figure 3 Effects of CPC on superoxide production and nitrotyrosine expression in lungs and NO119909

formation in BALF in LPS-induced ALIrats Rats were administrated with CPC (50mgkg ip) 3 h after LPS was instilled intratracheally BALF was prepared from rats 6 h after LPSchallenge and the level of NO

119909

was determined (a) Lung homogenates were used to measure superoxide production (b) and nitrotyrosineexpression (c) Data are presented as the mean plusmn SEM (119899 = 5) lowastlowastlowast119875 lt 0001 compared with control group 119875 lt 001 119875 lt 0001 comparedwith LPS group


34 Effect of CPC on NF-120581B Activation and iNOS andCOX-2 Expressions Activation of NF-120581B is known criticalfor inducing several inflammatory gene expression such asiNOS and COX-2 in the response to LPS [15] Our resultsshowed that posttreatment with CPC greatly inhibited LPS-induced NF-120581B activation confirmed by the attenuationof nuclear translocation of NF-120581B and phosphorylation-p65NF-120581B expression in nuclei as compared to LPS group(Figure 4(a)) As expected the LPS-induced protein expres-sions of iNOS and COX-2 in lungs were significantly inhib-ited by CPC (Figure 4(b)) These findings suggest that theinhibitory effects of CPC on iNOS COX-2 expressionsand pro-inflammatory mediator formation in LPS-inducedALI model may be associated with suppression of NF-120581Bactivation

35 Effect of CPC on Apoptosis-Related Gene ExpressionThe degree of cleaved caspase-3 protein expression a keymediator for cell apoptosis in lungs was markedly higher inLPS-instilled rats than in control rats and the increase wassignificantly inhibited by CPC (Figure 5) To further examinethe mechanisms underlying the anti-apoptotic activity ofCPC the mRNA expression of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2 Bcl-XL) proteins was measured As shownin Figure 5 CPC markedly inhibited Bax mRNA expres-sion and up-regulated Bcl-2 and Bcl-XL mRNA expressionsas compared to LPS-instilled alone group Therefore theinhibitory effect of CPC on apoptosis may be at least in partattributed to the reduction of pro-apoptoticanti-apoptoticprotein ratio Additionally we observed that there are notoxic signs found in organs including lung in rats treated withCPC (50mgkg ip) alone compared to the control groupsuggesting that CPC is safe at the dose used Moreover theparameters measured in this study were all not affected byCPC treatment in the absence of LPS

36 Effects of CPC on Lung Histopathological Changes Thelungs exposed to LPS instillation caused several histopatho-logicial alterations characterized by lung edema alveolar wallthickening and neutrophil infiltration into the lung intersti-tium and alveolar space Similarly posttreatment with CPCgreatly improved these abnormal features of ALI confirmedby the lower injury score of lungs (Figure 6) indicating thatCPC is a potential therapeutic drug for LPS-induced ALI

4 Discussion

In this ALI model instillation of LPS resulted in an inflam-matory cascade and in turn increasing the endothelial andepithelial permeability as well as pulmonary edema whichmeets the characteristics of ALI We demonstrated for thefirst time that posttreatment with CPC significantly reducesthe level of Evans blue an index of tissue permeability andprotein concentration in BALF and improves pulmonaryhistological alterations These results support that CPC pro-tects against LPS-induced ALI Next the precise mechanismsaccounting for the protective effect of CPC were investigated

Accumulation of inflammatory cells including neu-trophils in BALF has been implicated in the poor prognosis inseptic ALI and blocking recruitment of neutrophilsmitigatesthe pathological symptoms of ALI [16] Consistent withhistological analysis of the lungs rats challenged by LPSmarkedly increased the numbers of total cells in BALF andpulmonary MPO activity a marker of neutrophil influxFurthermore it is known that LPS-induced acute pulmonaryinflammation is characterized by release of various pro-inflammatory cytokines including TNF-120572 IL-1120573 and IL-6mainly driven by neutrophils and other inflammatory cellswhich subsequently amplify the inflammatory cascade andrecruit neutrophils into the lungs leading to lung injury [17]Clinical study has confirmed that in patients with ALI theconcentrations of pro-inflammatory cytokines in BALF wereelevated and this is closely related to their severity and pooroutcome [18 19] Posttreatment with CPC could significantlyattenuate lung tissue neutrophilia and levels of TNF-120572 IL-1120573IL-6 and CINC-3 a chemokine in BALF in rats challengedby LPS instillation Therefore inhibition of inflammatorycell sequestration and infiltration into the lungs as well asproduction of pro-inflammatory cytokines all contribute tothe beneficial effect of CPC in LPS-induced ALI

NO synthesized by NOS is an important regulatorymediator in modulating several physiological functionsHowever under stimulation of LPS andor inflammatorymediators the iNOS-derived NO overproduction by alveolarinflammatory cells results in the dysfunction of alveolar-capillary barrier and pulmonary edema in LPS-induced ALI[20 21] Clinical study has reported that the pulmonaryiNOS expression and concentration of NO

119909

in BALF frompatients with acute respiratory distress syndrome (ARDS)are significantly higher than those in normal subjects [22]As expected LPS-induced iNOS expression and NO forma-tion were inhibited by CPC Moreover several studies haverevealed that upregulation of COX-2 in response to LPS isthought to play an important role in the pathogenesis ofinflammatory diseases such as ALI [23 24] Similarly LPS-induced COX-2 expression in lungs was significantly inhib-ited by posttreatment with CPC Collectively these findingsindicate that the mechanisms by which CPC mitigates LPS-induced lung injury may be related to effective abrogation ofiNOS and COX-2 induction

It has been demonstrated that overproduction of reactiveoxygen species (ROS) is crucial in the development of lunginjury in response to LPS possibly through decrease ofsuperoxide dismutase (SOD) activity an antioxidant enzymescavenging superoxide [25 26] During the inflammatoryprocesses of ALI induced by LPS neutrophils located inlungs and airways have been regarded as a major source toproduce superoxide by undergoing a respiratory burst Whatis more worse is that the overproduction of NO and O

2

minus canrapidly generate peroxynitrite (ONOOminus) a highly cytotoxicproduct and subsequently causes severe damage to cellsand tissues It was found that the superoxide formation andnitrotyrosine expression a marker of peroxynitrite in lungsexposed to LPS were attenuated by CPC administrationTherefore it is likely that the antioxidative effect of CPC maybe mediated by decreased neutrophil infiltration as a result


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Figure 4 Effects of CPC on LPS-induced NF-120581B activation iNOS and COX-2 expressions in lungs The expression of nuclearphosphorylation-p65NF-120581B (a) iNOS and COX-2 (b) in lungs was determined by Western blot 6 h after LPS challenge For NF-120581B nucleartranslocation assay the nuclei of these cells visualized by Hoechst 33285 staining (blue) and the p65NF-120581B expression (green) were detectedin lungs The merged figures indicated the translocation of p65NF-120581B to nuclei The figures are representative of four similar experimentsData are presented as the mean plusmn SEM (119899 = 4) lowast119875 lt 005 lowastlowastlowast119875 lt 0001 compared with control group 119875 lt 005 compared with LPS group


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Figure 5 Effects of CPC on apoptosis-related protein expression in lungs from rats challenged by LPS The cleaved caspase-3 proteinexpression Bax Bcl-2 and Bcl-XL mRNA expression levels in lungs were determined 6 h after LPS instillation The relative expression ofmRNA was expressed as folds of the control group The expression levels from control group were assigned as 1 Data are presented as themean plusmn SEM (119899 = 4) lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 compared with control group 119875 lt 005 119875 lt 001 compared with LPS group

of the suppression of pro-inflammatory cytokine productionHowever we cannot exclude the possibility that the inhibitionof ROS formation by CPC may be due to its direct activationof the antioxidant enzymes including SOD and glutathioneperoxidase (GSH-Px) [27]

There are two types of cells to form the alveolarcapillarybarriers the microvascular endothelium and the alveo-lar epithelium The importance of endothelial injury andincreased vascular permeability in the formation of pul-monary edema is well established To date the alveolar

and distal airway epithelium have been believed to play acritical role in regulation of alveolar fluid clearance throughthe actions of transepithelial Na+ and Clminus transports [28]The disruption of the alveolar-capillary membranes leadsto alveolar flooding with serum proteins and edema fluidA lot of evidence supports that apoptosis of endothelialand epithelial cells accompanied by increased pulmonaryvascular permeability caused by a variety of inflammatorystimuli is a crucial factor resulting in pulmonary edemain ALI [4] Administration of CPC significantly inhibited


Control

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Figure 6 Effect of CPC on LPS-induced lung histopathological changes Six hours after LPS instillation the lung tissues were fixed andstained with HampE (200x magnification) The lung injury score was used to evaluate the histological alterations Data are presented as themean plusmn SEM (119899 = 4) lowastlowast119875 lt 001 compared with control group 119875 lt 005 compared with LPS group

LPS-induced caspase-3 and Bax expression but up-regulatedBcl-2 and Bcl-XL proteins suggesting that reduction of pro-apoptoticanti-apoptotic protein ratio contributes to its anti-apoptotic activity and subsequently maintains the alveolar-capillary barrier intact Collectively the protective effectof CPC against LPS-induced ALI may be associated withinhibition of LPS-dependent inflammatory responses andapoptosis

Under inflammatory condition NF-120581B is activated byphosphorylation and enters into the nucleus leading to up-regulation of downstream target genes such as iNOS COX-2and pro-inflammatory cytokines as well as ROS formationMoreover ROS also function as secondmessenger regulatingseveral downstream signaling molecules including NF-120581Bto amplify the inflammatory responses [29] Importantlyactivated NF-120581B also causes alveolar epithelial cell apoptosisby regulating gene expression related to cell death [30] Ithas been confirmed that NF-120581B activation plays a crucialrole in the pathogenesis of ALI [31] Thus we furtherexamined whether CPC affects the NF-120581B activation Asexpected posttreatment with CPC significantly inhibitedLPS-induced NF-120581B activation in lungs evidenced by atten-uation of nuclear expression of phosphorylation-p65NF-120581B

and nuclear translocation of NF-120581B Although the true targetof CPC is unclear we propose that suppression of inflamma-tory cell infiltration ROS generation pulmonary apoptosisand NF-120581B-mediated inflammatory responses may accountfor the protective effect of CPC However the possible molec-ular mechanisms involved need further investigation In con-clusion this is the first study to demonstrate that CPC pos-sesses a therapeutic effect in LPS-induced ALI through sup-pressing pulmonary apoptosis and NF-120581B-mediated inflam-matory responses Accordingly CPC can be considered apotential drug to improve pulmonary inflammatory diseasesinduced by direct injury or systemic infection

Conflict of Interests

The authors report no conflict of interests

Authorsrsquo Contribution

P-o Leung and H-H Lee contributed equally to the work


Acknowledgment

This study was partly supported by a research grant from theNational Science Council of Taiwan Republic of China (NSC97-2320-B-016-008-MY3)

References

[1] S E Erickson G S Martin J L Davis M A Matthay and MD Eisner ldquoRecent trends in acute lung injury mortality 1996ndash2005rdquo Critical Care Medicine vol 37 no 5 pp 1574ndash1579 2009

[2] K Tsushima L S King and N R Aggarwal ldquoAcute lung injuryreviewrdquo Internal Medicine vol 48 no 9 pp 621ndash631 2009

[3] D Togbe S Schnyder-Candrian B Schnyder et al ldquoToll-likereceptor and tumour necrosis factor dependent endotoxin-induced acute lung injuryrdquo International Journal of Experimen-tal Pathology vol 88 no 6 pp 387ndash391 2007

[4] M Chopra J S Reuben and A C Sharma ldquoAcute lung injuryapoptosis and signalingmechanismsrdquoExperimental Biology andMedicine vol 234 no 4 pp 361ndash371 2009

[5] S Copeland H Shaw Warren S F Lowry S E Galvano andD Remick ldquoAcute inflammatory response to endotoxin in miceand humansrdquo Clinical and Diagnostic Laboratory Immunologyvol 12 no 1 pp 60ndash67 2005

[6] H Chen C Bai and X Wang ldquoThe value of the lipopoly-saccharide-induced acute lung injury model in respiratorymedicinerdquo Expert Review of Respiratory Medicine vol 4 no 6pp 773ndash783 2010

[7] V B Bhat and K M Madyastha ldquoC-Phycocyanin a potentperoxyl radical scavenger in vivo and in vitrordquo Biochemical andBiophysical Research Communications vol 275 no 1 pp 20ndash252000

[8] B B Vadiraja N W Gaikwad and K M Madyastha ldquoHep-atoprotective effect of C-phycocyanin protection for carbontetrachloride and R-(+)-pulegone-mediated hepatotoxicty inratsrdquo Biochemical and Biophysical Research Communicationsvol 249 no 2 pp 428ndash431 1998

[9] H F Chiu S P Yang Y L Kuo Y S Lai and T C Chou ldquoMech-anisms involved in the antiplatelet effect of C-phycocyaninrdquoBritish Journal of Nutrition vol 95 no 2 pp 434ndash439 2006

[10] C Romay R Gonzalez N Ledon D Remirez and V Rim-bau ldquoC-Phycocyanin a biliprotein with antioxidant anti-inflammatory and neuroprotective effectsrdquo Current Protein andPeptide Science vol 4 no 3 pp 207ndash216 2003

[11] S C Cherng S N Cheng A Tarn and T C Chou ldquoAnti-inflammatory activity of c-phycocyanin in lipopolysaccharide-stimulated RAW 2647 macrophagesrdquo Life Sciences vol 81 no19-20 pp 1431ndash1435 2007

[12] J L Kang H W Lee H S Lee et al ldquoGenistein preventsnuclear factor-kappa B activation and acute lung injury inducedby lipopolysacchariderdquo American Journal of Respiratory andCritical Care Medicine vol 164 no 12 pp 2206ndash2212 2002

[13] J H Yeh H F Chiu J S Wang J K Lee and T C Chou ldquoPro-tective effect of baicalein extracted from Scutellaria baicalen-sis against lipopolysaccharide-induced glomerulonephritis inmicerdquo International Journal of Pharmacology vol 6 no 2 pp81ndash88 2010

[14] B Xu X Gao J Xu et al ldquoIschemic postconditioning attenuateslung reperfusion injury and reduces systemic proinflammatorycytokine release via heme oxygenase 1rdquo Journal of SurgicalResearch vol 166 no 2 pp e157ndashe164 2011

[15] P P Tak and G S Firestein ldquoNF-120581B a key role in inflammatorydiseasesrdquo Journal of Clinical Investigation vol 107 no 1 pp 7ndash112001

[16] W L Lee and G P Downey ldquoNeutrophil activation and acutelung injuryrdquo Current Opinion in Critical Care vol 7 no 1 pp1ndash7 2001

[17] R B Goodman J Pugin J S Lee and M A MatthayldquoCytokine-mediated inflammation in acute lung injuryrdquoCytokine and Growth Factor Reviews vol 14 no 6 pp 523ndash5352003

[18] P Agouridakis D Kyriakou M G Alexandrakis et al ldquoThepredictive role of serum and bronchoalveolar lavage cytokinesand adhesionmolecules for acute respiratory distress syndromedevelopment and outcomerdquo Respiratory Research vol 3 article25 2002

[19] G U Meduri G Kohler S Headley E Tolley F Stentzand A Postlethwaite ldquoInflammatory cytokines in the BAL ofpatients with ARDS persistent elevation over time predictspoor outcomerdquo Chest vol 108 no 5 pp 1303ndash1314 1995

[20] S Mehta ldquoThe effects of nitric oxide in acute lung injuryrdquoVascular Pharmacology vol 43 no 6 pp 390ndash403 2005

[21] L F Wang M Patel H M Razavi et al ldquoRole of induciblenitric oxide synthase in pulmonary microvascular protein leakin murine sepsisrdquo American Journal of Respiratory and CriticalCare Medicine vol 165 no 12 pp 1634ndash1639 2002

[22] C Sittipunt K P Steinberg J T Ruzinski et al ldquoNitric oxideand nitrotyrosine in the lungs of patients with acute respiratorydistress syndromerdquoAmerican Journal of Respiratory and CriticalCare Medicine vol 163 no 2 pp 503ndash510 2001

[23] K Ejima M D Layne I M Carvajal et al ldquoCyclooxygenase-2-deficient mice are resistant to endotoxin-induced inflammationand deathrdquo The FASEB Journal vol 17 no 10 pp 1325ndash13272003

[24] R Gust J K Kozlowski A H Stephenson and D P SchusterldquoRole of cyclooxygenase-2 in oleic acid-induced acute lunginjuryrdquo American Journal of Respiratory and Critical CareMedicine vol 160 no 4 pp 1165ndash1170 1999

[25] Y C Xie X W Dong X M Wu X F Yan and Q MXie ldquoInhibitory effects of flavonoids extracted from licorice onlipopolysaccharide-induced acute pulmonary inflammation inmicerdquo International Immunopharmacology vol 9 no 2 pp 194ndash200 2009

[26] H S Park S R Kim and Y C Lee ldquoImpact of oxidative stresson lung diseasesrdquo Respirology vol 14 no 1 pp 27ndash38 2009

[27] Y Ou S Zheng L Lin Q Jiang and X Yang ldquoProtectiveeffect of C-phycocyanin against carbon tetrachloride-inducedhepatocyte damage in vitro and in vivordquo Chemico-BiologicalInteractions vol 185 no 2 pp 94ndash100 2010

[28] Y Berthiaume and M A Matthay ldquoAlveolar edema fluidclearance and acute lung injuryrdquo Respiratory Physiology andNeurobiology vol 159 no 3 pp 350ndash359 2007

[29] M J Morgan and Z G Liu ldquoCrosstalk of reactive oxygenspecies and NF-120581B signalingrdquo Cell Research vol 21 no 1 pp103ndash115 2010

[30] J G Wright and J W Christman ldquoThe role of nuclear factor120581B in the pathogenesis of pulmonary diseases implications fortherapyrdquoAmerican Journal of Respiratory Medicine vol 2 no 3pp 211ndash219 2003

[31] A A Imanifooladi S Yazdani and M R Nourani ldquoThe role ofnuclear factor-120581B in inflammatory lung diseaserdquo Inflammationand Allergy Drug Targets vol 9 no 3 pp 197ndash205 2010


anti-inflammatory activities [7ndash10] Our previous study hasshown that in LPS-stimulated RAW2647macrophages CPCexhibits an anti-inflammatory activity by inhibiting induciblenitric oxide synthase (iNOS) expression and NO productionpossibly by suppressing nuclear transcription factor-120581B (NF-120581B) activation a key transcription factor promoting pro-inflammatory gene expression [11] These findings suggestthat CPC may be a potential reagent to treat inflammatorydiseases However there is little information onwhether CPChas an ability to protect against LPS-induced ALI The aimof this study was to investigate the effects of CPC on LPS(it)-induced ALI in rats and further studied the possiblemechanisms involved Our results have indicated that CPCexhibits a therapeutic effect in LPS-induced ALI

2 Materials and Methods

21 Animal and Reagents Male Sprague-Dawley (SD) rats(8 weeks of age 200ndash230 g) were used for this study Allexperimental procedures were conducted in accordance withthe Guiding Principle in the Care and Use of Animalsand approved by the Institutional Animal Care and UseCommittee of National Defense Medical Center (TaipeiTaiwan)The CPC (with a purity of A

620

A280

gt35) providedby Far East Bio-Tec Co Ltd (Taipei Taiwan) was dissolvedin normal saline Other chemical agents used in the studywere at least analytical grade

22 LPS-Induced ALI Model and Grouping The rats weredivided randomly into three groups (1) phosphate bufferedsaline (PBS) vehicle group (control group) (2) LPS challengegroup (LPS group) (3) LPS and CPC-treated group Forintratracheal instillation rats were anesthetized with a singleintraperitoneal dose of ketamine (90mgkg bodyweight) andxylazine (7mgkg body weight) followed by administrationof LPS (Escherichia coli 055B5 5mgkg body weight it) in02mL PBS through a 24-gauge catheter to induce lung injuryas previously described [12] Control rats were given 02mLPBS intratracheally The treated rats were administrated withCPC (50mgkg body weight) intraperitoneally (ip) 3 h afterinstillation of LPS In our preliminary study we have foundthat the pathological features of LPS-induced ALI weresignificantly improved if rats were treated with CPC at adosage of 50mgkg Therefore the dosage of CPC (50mgkgbody weight ip) was chosen in the following experimentsAll rats were euthanized at 6 h after LPS or PBS instillationand the samples were collected for subsequent analyses

23 Bronchoalveolar Lavage and Cell Counting At the endof the experiment the right lungs were ligated at the rightmain bronchus Then the left lungs were lavaged with 1mLof autoclaved PBS for five times The recovery ratio of thefluid was about 80 (4mL)The bronchoalveolar lavage fluid(BALF) obtained from left lungswas immediately centrifugedat 300 g for 10min at 4∘C and the supernatants were storedat minus70∘C until required for subsequent tests The cell pelletswere resuspended in PBS and the total cell number wascounted using a standard hemocytometer

24 Evans Blue Dye Leakage Thirty minutes before the endof the experiment Evans blue dye (50mgkg Sigma-AldrichSt Louis MO USA) was given intravenously After the lungswere perfused free of blood the Evans blue content in lungtissue was determined using a spectrophotometer at theoptical density of 620 nm

25 Wet-to-Dry Lung Weight Ratio and Protein Content inBALF Six hours after the intratracheal instillation of LPSor PBS right lungs were excised and immediately weighedthe wet weight Dry weight was determined after heating thelungs in an oven at 80∘C for 48 h The wetdry weight ratioof right lungs was measured to assess pulmonary edemaTheprotein content in cell-free BALF obtained from left lungswasdetermined by using a Bio-Rad protein assay kit (Bio-RadLaboratories Hemel Hempstead UK) with bovine serumalbumin (Sigma) as a standard

26 Measurement of Cytokines and NitriteNitrate (119873119874119909

)The levels of pro-inflammatory cytokines (TNF-120572 IL-6 andIL-1120573) and cytokine inducible neutrophil chemoattractant-3(CINC-3) in BALF obtained from left lungs were measuredby using enzyme-linked immunosorbent assay (ELISA) kits(GenzymeCorporation CambridgeMA USA) respectivelyTo determine the NO formation the BALF was injectedinto a collection chamber containing 5 VCl

3

In thisstrong reducing environment both nitrate and nitrite wereconverted to NO A constant stream of helium gas was usedto carry the output into an NO analyzer (Sievers 280NOASievers Instruments Inc Boulder CO USA) where the NOreacts with ozone (O

3

) resulting in the emission of lightLight emission is proportional to the NO formed and thelevel of NO

119909

(nitrite + nitrate) reflects the total amount ofNO produced by nitrite + nitrate [13] Standard amounts ofsodium nitrate were used for calibration

27 Myeloperoxidase (MPO) Activity Assay The right lungswere homogenized and sonicated in 50mM potassium phos-phate (pH 6) containing 5mM hexadecyltrimethylammo-nium bromide (Sigma-Aldrich St Louis MO USA) andsubjected to three cycles of freezing and thawing followedby centrifugation at 14000 g for 10min at 4∘C to obtainthe supernatant Then 20120583L tissue supernatant was incu-bated with 40 120583L assay buffer containing 0167mgmL O-dianisidine dihydrochloride (Sigma-Aldrich St Louis MOUSA) and 00005 hydrogen peroxide The MPO activitywas calculated as the change in absorbance at 460 nm over1min and expressed as Ug weight

28 Measurement of 1198742

minus Production in Lungs The freshlyharvested right lungs were cut into pieces of 5 times 5mmand incubated with oxygenated Krebs-Hepes buffer for 5ndash10min Then the pieces of lung tissue were transferred to96-well microplates These microplates containing 125mMlucigenin in 200120583L Krebs-Hepes buffer were placed intoa microplate luminometer (MicroLumat LB96V BertholdGermany) Counts of chemiluminescence signal from lungswere obtained at 1 min intervals at room temperature













1800

1600

1400

1200

1000

800

600

400

200

0


otei

n co

ncen

trat

ion

of B

ALF

(120583g

mL)

Control 0 50

CPC (mgkg)LPS

(a)

40

30

20

10

0

lowast

Evan

s blu

e (m

gg

of lu

ng ti

ssue

)

Control 0 50

CPC (mgkg)LPS

(b)

52

5

48

46

44

42

4

Wet

dry

wei

ght r

atio

of l

ung

lowastlowast

Control 0 50

CPC (mgkg)LPS

(c)

25

2

15

1

05

0


tal c

ell c

ount

s (times10

3m

L)

Control 0 50

CPC (mgkg)LPS

(d)


8

6

4

2

0

Lung

MPO

activ

ity (U

g ti

ssue

)

Control 0 50

CPC (mgkg)

LPS

(e)




3500

3000

2500

2000

1500

1000

500

0Control 0 50

lowastlowastlowast

TNF-120572

(pg

mL

BA

LF)

CPC (mgkg)LPS

(a)

lowastlowastlowast

2500

2000

1500

1000

500

0

IL-1120573

(pg

mL

BA

LF)

Control 0 50

CPC (mgkg)LPS

(b)

3000

2500

2000

1500

1000

500

0

lowastlowastlowast

IL-6

(pg

mL

BA

LF)

Control 0 50

CPC (mgkg)LPS

(c)

Control 0 50

CPC (mgkg)LPS

lowastlowastlowast

5000

4000

3000

2000

1000

0

CIN

C-3

(pg

mL

BA

LF)

(d)


3 Results




1198742


119909

in BALF andO2



Control 0 50

CPC (mgkg)LPS

lowastlowastlowast

30

25

20

15

10

5

0

NO119909

(120583M

)

(a)

lowastlowastlowast

0

10000

8000

6000

4000

2000Supe

roxi

de (C

PCm

g dr

y w

eigh

t)

Control 0 50

CPC (mgkg)LPS

(b)

Control

LPS LPS + CPC

(c)



119909






4 Discussion




119909



2



p65 NF-120581B

NF120581B

Control 0 50

CPC (mgkg)LPS

Control

LPS

LPS + CPC


(a)

Control 0 50

CPC (mgkg)LPS

Control 0 50

CPC (mgkg)LPS

iNOS


COX-2

12

1

08

06

04

02

0

iNO

S120573

-act

in ra

tio

lowast

12

1

08

06

04

02

0

COX-

2120573

-act

in ra

tio

lowastlowastlowast

(b)



Cleaved caspase-3

120573-actin

07

06

05

04

03

02

01

0Control 0 50

CPC (mgkg)LPS

lowastlowast

Clea

ved

casp

ase-

3120573

-act

in ra

tio

(a)

Control 0 50

CPC (mgkg)LPS

lowastlowastlowast

Relat

ive B

ax m

RNA

expr

essio

n

8

6

4

2

0

(b)

14

12

1

08

06

04

02

0

lowast

Relat

ive B

cl-2

mRN

A ex

pres

sion

lowast

Control 0 50

CPC (mgkg)LPS

(c)

lowast

8

6

4

2

0

Relat

ive B

cl-XL

mRN

A ex

pres

sion

Control 0 50

CPC (mgkg)LPS

(d)






Control

(a)

LPS

(b)

LPS + CPC

(c)

Lung

inju

ry sc

ore

7

6

5

4

3

2

1

0Control 0 50

CPC (mgkg)LPS

lowastlowast

(d)










Acknowledgment


References












































1800

1600

1400

1200

1000

800

600

400

200

0


otei

n co

ncen

trat

ion

of B

ALF

(120583g

mL)

Control 0 50

CPC (mgkg)LPS

(a)

40

30

20

10

0

lowast

Evan

s blu

e (m

gg

of lu

ng ti

ssue

)

Control 0 50

CPC (mgkg)LPS

(b)

52

5

48

46

44

42

4

Wet

dry

wei

ght r

atio

of l

ung

lowastlowast

Control 0 50

CPC (mgkg)LPS

(c)

25

2

15

1

05

0


tal c

ell c

ount

s (times10

3m

L)

Control 0 50

CPC (mgkg)LPS

(d)


8

6

4

2

0

Lung

MPO

activ

ity (U

g ti

ssue

)

Control 0 50

CPC (mgkg)

LPS

(e)




3500

3000

2500

2000

1500

1000

500

0Control 0 50

lowastlowastlowast

TNF-120572

(pg

mL

BA

LF)

CPC (mgkg)LPS

(a)

lowastlowastlowast

2500

2000

1500

1000

500

0

IL-1120573

(pg

mL

BA

LF)

Control 0 50

CPC (mgkg)LPS

(b)

3000

2500

2000

1500

1000

500

0

lowastlowastlowast

IL-6

(pg

mL

BA

LF)

Control 0 50

CPC (mgkg)LPS

(c)

Control 0 50

CPC (mgkg)LPS

lowastlowastlowast

5000

4000

3000

2000

1000

0

CIN

C-3

(pg

mL

BA

LF)

(d)


3 Results




1198742


119909

in BALF andO2



Control 0 50

CPC (mgkg)LPS

lowastlowastlowast

30

25

20

15

10

5

0

NO119909

(120583M

)

(a)

lowastlowastlowast

0

10000

8000

6000

4000

2000Supe

roxi

de (C

PCm

g dr

y w

eigh

t)

Control 0 50

CPC (mgkg)LPS

(b)

Control

LPS LPS + CPC

(c)



119909






4 Discussion




119909



2



p65 NF-120581B

NF120581B

Control 0 50

CPC (mgkg)LPS

Control

LPS

LPS + CPC


(a)

Control 0 50

CPC (mgkg)LPS

Control 0 50

CPC (mgkg)LPS

iNOS


COX-2

12

1

08

06

04

02

0

iNO

S120573

-act

in ra

tio

lowast

12

1

08

06

04

02

0

COX-

2120573

-act

in ra

tio

lowastlowastlowast

(b)



Cleaved caspase-3

120573-actin

07

06

05

04

03

02

01

0Control 0 50

CPC (mgkg)LPS

lowastlowast

Clea

ved

casp

ase-

3120573

-act

in ra

tio

(a)

Control 0 50

CPC (mgkg)LPS

lowastlowastlowast

Relat

ive B

ax m

RNA

expr

essio

n

8

6

4

2

0

(b)

14

12

1

08

06

04

02

0

lowast

Relat

ive B

cl-2

mRN

A ex

pres

sion

lowast

Control 0 50

CPC (mgkg)LPS

(c)

lowast

8

6

4

2

0

Relat

ive B

cl-XL

mRN

A ex

pres

sion

Control 0 50

CPC (mgkg)LPS

(d)






Control

(a)

LPS

(b)

LPS + CPC

(c)

Lung

inju

ry sc

ore

7

6

5

4

3

2

1

0Control 0 50

CPC (mgkg)LPS

lowastlowast

(d)










Acknowledgment


References

































3500

3000

2500

2000

1500

1000

500

0Control 0 50

lowastlowastlowast

TNF-120572

(pg

mL

BA

LF)

CPC (mgkg)LPS

(a)

lowastlowastlowast

2500

2000

1500

1000

500

0

IL-1120573

(pg

mL

BA

LF)

Control 0 50

CPC (mgkg)LPS

(b)

3000

2500

2000

1500

1000

500

0

lowastlowastlowast

IL-6

(pg

mL

BA

LF)

Control 0 50

CPC (mgkg)LPS

(c)

Control 0 50

CPC (mgkg)LPS

lowastlowastlowast

5000

4000

3000

2000

1000

0

CIN

C-3

(pg

mL

BA

LF)

(d)


3 Results




1198742


119909

in BALF andO2



Control 0 50

CPC (mgkg)LPS

lowastlowastlowast

30

25

20

15

10

5

0

NO119909

(120583M

)

(a)

lowastlowastlowast

0

10000

8000

6000

4000

2000Supe

roxi

de (C

PCm

g dr

y w

eigh

t)

Control 0 50

CPC (mgkg)LPS

(b)

Control

LPS LPS + CPC

(c)



119909






4 Discussion




119909



2



p65 NF-120581B

NF120581B

Control 0 50

CPC (mgkg)LPS

Control

LPS

LPS + CPC


(a)

Control 0 50

CPC (mgkg)LPS

Control 0 50

CPC (mgkg)LPS

iNOS


COX-2

12

1

08

06

04

02

0

iNO

S120573

-act

in ra

tio

lowast

12

1

08

06

04

02

0

COX-

2120573

-act

in ra

tio

lowastlowastlowast

(b)



Cleaved caspase-3

120573-actin

07

06

05

04

03

02

01

0Control 0 50

CPC (mgkg)LPS

lowastlowast

Clea

ved

casp

ase-

3120573

-act

in ra

tio

(a)

Control 0 50

CPC (mgkg)LPS

lowastlowastlowast

Relat

ive B

ax m

RNA

expr

essio

n

8

6

4

2

0

(b)

14

12

1

08

06

04

02

0

lowast

Relat

ive B

cl-2

mRN

A ex

pres

sion

lowast

Control 0 50

CPC (mgkg)LPS

(c)

lowast

8

6

4

2

0

Relat

ive B

cl-XL

mRN

A ex

pres

sion

Control 0 50

CPC (mgkg)LPS

(d)






Control

(a)

LPS

(b)

LPS + CPC

(c)

Lung

inju

ry sc

ore

7

6

5

4

3

2

1

0Control 0 50

CPC (mgkg)LPS

lowastlowast

(d)










Acknowledgment


References

































Control 0 50

CPC (mgkg)LPS

lowastlowastlowast

30

25

20

15

10

5

0

NO119909

(120583M

)

(a)

lowastlowastlowast

0

10000

8000

6000

4000

2000Supe

roxi

de (C

PCm

g dr

y w

eigh

t)

Control 0 50

CPC (mgkg)LPS

(b)

Control

LPS LPS + CPC

(c)



119909






4 Discussion




119909



2



p65 NF-120581B

NF120581B

Control 0 50

CPC (mgkg)LPS

Control

LPS

LPS + CPC


(a)

Control 0 50

CPC (mgkg)LPS

Control 0 50

CPC (mgkg)LPS

iNOS


COX-2

12

1

08

06

04

02

0

iNO

S120573

-act

in ra

tio

lowast

12

1

08

06

04

02

0

COX-

2120573

-act

in ra

tio

lowastlowastlowast

(b)



Cleaved caspase-3

120573-actin

07

06

05

04

03

02

01

0Control 0 50

CPC (mgkg)LPS

lowastlowast

Clea

ved

casp

ase-

3120573

-act

in ra

tio

(a)

Control 0 50

CPC (mgkg)LPS

lowastlowastlowast

Relat

ive B

ax m

RNA

expr

essio

n

8

6

4

2

0

(b)

14

12

1

08

06

04

02

0

lowast

Relat

ive B

cl-2

mRN

A ex

pres

sion

lowast

Control 0 50

CPC (mgkg)LPS

(c)

lowast

8

6

4

2

0

Relat

ive B

cl-XL

mRN

A ex

pres

sion

Control 0 50

CPC (mgkg)LPS

(d)






Control

(a)

LPS

(b)

LPS + CPC

(c)

Lung

inju

ry sc

ore

7

6

5

4

3

2

1

0Control 0 50

CPC (mgkg)LPS

lowastlowast

(d)










Acknowledgment


References




































4 Discussion




119909



2



p65 NF-120581B

NF120581B

Control 0 50

CPC (mgkg)LPS

Control

LPS

LPS + CPC


(a)

Control 0 50

CPC (mgkg)LPS

Control 0 50

CPC (mgkg)LPS

iNOS


COX-2

12

1

08

06

04

02

0

iNO

S120573

-act

in ra

tio

lowast

12

1

08

06

04

02

0

COX-

2120573

-act

in ra

tio

lowastlowastlowast

(b)



Cleaved caspase-3

120573-actin

07

06

05

04

03

02

01

0Control 0 50

CPC (mgkg)LPS

lowastlowast

Clea

ved

casp

ase-

3120573

-act

in ra

tio

(a)

Control 0 50

CPC (mgkg)LPS

lowastlowastlowast

Relat

ive B

ax m

RNA

expr

essio

n

8

6

4

2

0

(b)

14

12

1

08

06

04

02

0

lowast

Relat

ive B

cl-2

mRN

A ex

pres

sion

lowast

Control 0 50

CPC (mgkg)LPS

(c)

lowast

8

6

4

2

0

Relat

ive B

cl-XL

mRN

A ex

pres

sion

Control 0 50

CPC (mgkg)LPS

(d)






Control

(a)

LPS

(b)

LPS + CPC

(c)

Lung

inju

ry sc

ore

7

6

5

4

3

2

1

0Control 0 50

CPC (mgkg)LPS

lowastlowast

(d)










Acknowledgment


References

































p65 NF-120581B

NF120581B

Control 0 50

CPC (mgkg)LPS

Control

LPS

LPS + CPC


(a)

Control 0 50

CPC (mgkg)LPS

Control 0 50

CPC (mgkg)LPS

iNOS


COX-2

12

1

08

06

04

02

0

iNO

S120573

-act

in ra

tio

lowast

12

1

08

06

04

02

0

COX-

2120573

-act

in ra

tio

lowastlowastlowast

(b)



Cleaved caspase-3

120573-actin

07

06

05

04

03

02

01

0Control 0 50

CPC (mgkg)LPS

lowastlowast

Clea

ved

casp

ase-

3120573

-act

in ra

tio

(a)

Control 0 50

CPC (mgkg)LPS

lowastlowastlowast

Relat

ive B

ax m

RNA

expr

essio

n

8

6

4

2

0

(b)

14

12

1

08

06

04

02

0

lowast

Relat

ive B

cl-2

mRN

A ex

pres

sion

lowast

Control 0 50

CPC (mgkg)LPS

(c)

lowast

8

6

4

2

0

Relat

ive B

cl-XL

mRN

A ex

pres

sion

Control 0 50

CPC (mgkg)LPS

(d)






Control

(a)

LPS

(b)

LPS + CPC

(c)

Lung

inju

ry sc

ore

7

6

5

4

3

2

1

0Control 0 50

CPC (mgkg)LPS

lowastlowast

(d)










Acknowledgment


References

































Cleaved caspase-3

120573-actin

07

06

05

04

03

02

01

0Control 0 50

CPC (mgkg)LPS

lowastlowast

Clea

ved

casp

ase-

3120573

-act

in ra

tio

(a)

Control 0 50

CPC (mgkg)LPS

lowastlowastlowast

Relat

ive B

ax m

RNA

expr

essio

n

8

6

4

2

0

(b)

14

12

1

08

06

04

02

0

lowast

Relat

ive B

cl-2

mRN

A ex

pres

sion

lowast

Control 0 50

CPC (mgkg)LPS

(c)

lowast

8

6

4

2

0

Relat

ive B

cl-XL

mRN

A ex

pres

sion

Control 0 50

CPC (mgkg)LPS

(d)






Control

(a)

LPS

(b)

LPS + CPC

(c)

Lung

inju

ry sc

ore

7

6

5

4

3

2

1

0Control 0 50

CPC (mgkg)LPS

lowastlowast

(d)










Acknowledgment


References

































Control

(a)

LPS

(b)

LPS + CPC

(c)

Lung

inju

ry sc

ore

7

6

5

4

3

2

1

0Control 0 50

CPC (mgkg)LPS

lowastlowast

(d)










Acknowledgment


References

































Acknowledgment


References
































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