Therapeutic effects of compound hypertonic saline on rats with sepsis

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

w w w . e l s e v i e r . c o m / l o c a t e / b j i d

Original

article

Therapeutic

effects

of

compound

hypertonic

saline

on

rats

with

sepsis

Fang

Dong

_,

_Wei

_Chen

_,

_Liang

_Xu

_,

_Huabing

_Wang

_,

_Huizhi

_Lu

b_{TheCentralHospitalofWuhan,Wuhan,China}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received22August2013 Accepted27May2014 Availableonline28June2014

Keywords:

Sepsis

Cecalligationandpuncture Immuneregulation Lunginjury

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b

s

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r

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c

t

Sepsisisoneofthemajorcausesofdeathandisthebiggestobstaclepreventing improve-mentofthesuccessrateincuringcriticalillnesses.Currently,isotonicsolutionsareusedin fluidresuscitationtechnique.Severalstudieshaveshownthathypertonicsalineappliedin hemorrhagicshockcanrapidlyincreasetheplasmaosmoticpressure,facilitatetherapid returnofinterstitialfluidintothebloodvessels,andrestoretheeffectivecirculatingblood volume.Here,weestablishedaratmodelofsepsisbyusingthececalligationandpuncture approach.Wefoundthatintravenousinjectionofhypertonicsalinedextran(7.5%NaCl/6% dextran)aftercecalligationandpuncturecanimprovecirculatoryfailureattheonsetof sepsis.Wefoundthatthelevelsoftumornecrosisfactor-␣,interleukin-1␤,interleukin-6 andintracellularadhesionmolecule1levelsinthelungtissueofcecalligationandpuncture ratstreatedwithhypertonicsalinedextranweresignificantlylowerthanthecorresponding levelsin the control group.We inferred thathypertonic saline dextranhas a positive immunoregulatoryeffectandinhibitstheoverexpressionoftheinflammatoryresponsein thetreatmentofsepsis.Thepercentageofneutrophils,lungmyeloperoxidaseactivity,wet todryweightratiooflungtissues,histopathologicalchangesinlungtissues,andindicators ofarterialblood gasanalysiswassignificantly betterin thehypertonic saline dextran-treatedgroupthanintheothergroupsinthisstudy.Hypertonicsalinedextran-treatedrats hadsignificantlyimprovedsurvivalratesat9and18hcomparedtothecontrolgroup.Our resultssuggestthathypertonicsalinedextranplaysaprotectiveroleinacutelunginjury causedaftercecalligationandpuncture.Inconclusion,hypertonic/hyperoncoticsolutions havebeneficialtherapeuticeffectsinthetreatmentofananimalmodelofsepsis.

©2014ElsevierEditoraLtda.Allrightsreserved.

Infectionandsepsiscauseaveryhighdiseaserateand mor-bidityinpatientswithcriticalillness.Clinicalepidemiological data haverevealed that sepsisis one ofthe major causes of death in critical patients and has become the biggest

∗ _{Correspondingauthorat:IntensiveCareUnit,TheThirdHospitalofWuhanCity,Wuhan,China.}

E-mailaddress:[email protected](F.Dong).

obstacle in the successrate for curing high-risk diseases.1

Obtainingabetterunderstandingofsevereinfectious compli-cationsandtheirpreventionandtreatmentareundoubtedly ofconsiderabletheoreticalvalueandclinicalsignificance.The

http://dx.doi.org/10.1016/j.bjid.2014.05.007

pathogenesisofsepsisisverycomplicated.Itisdifficultto treat,andinvolvesaseriesofbasicproblemssuchas infec-tion,inflammation,immunity,bloodcoagulation,andtissue injury;furthermore,itisassociatedwithpathophysiological changesinmultiplebodysystemsandorgans.

Recentstudies have madeconsiderable progresson the onsetandclinicalsignificanceofsepsis.However,noresults haveshedlightontheanti-inflammatorytreatmentof sep-sisandmulti-organdysfunction(MODS)syndrome.Currently, thestandard fluidusedinfluidresuscitation isanisotonic solutionsuchas0.9%NaClsolutionandlactatedRinger’s solu-tion.Manystudieshaveshownthathypertonic/hyperoncotic solutions used for fluid resuscitation can rapidly increase theplasmaosmoticpressure,enabletheinterstitialfluidto returntothe blood vesselsrapidlyand thereby restorethe effectivecirculatingbloodvolume,2,3_{andprovidetherapeutic}

effectsforpulmonaryedemacausedbyhemorrhagicshock.4

Zallen et al.5 _{reported that hypertonic saline can improve}

hemodynamicdisordersinhemorrhagicshock,prevent neu-trophilactivation,andinhibitICAM-1expressionbymigrating throughtheintestinallymphaticpathway.Jungeretal.6_found

thathypertonicsalineusedforresuscitationinhemorrhagic shockcaninhibitneutrophilandendothelialcellactivation. Thesestudiessuggestthathypertonicsalinecanfunctionin immuneregulationduringresuscitationfromshock.However, doubtsremainaboutitsusefulnessforthetreatmentofsepsis. Inthepresentstudy,wedevelopedaratmodelofsepsisusing thececalligationandpuncture(CLP)approachtoinvestigate theinfluenceofhypertonic/hyperoncoticsolutionsonmean arterialpressure(MAP)andheartrate,theirpotentialrolein the regulationof serum inflammatorycytokines, and their influenceonlungtissueapoptosisandtissueICAM-1 expres-sioninsepticratsandtodiscusstheirpotentialtherapeutic effectsinthetreatmentofsepsis.

Materials

and

methods

Establishmentofaratsepsismodel

In total, 128 male Wistar rats (10–12 weeks old, weight: 230–280g) were provided by the Institute of Zoology, Chi-nese Academy of Sciences. The rats were anaesthetized with intraperitoneal injection of 3% sodium pentobarbital (30–50mg/kg).TheMAPwasmonitoredbyplacingacatheter inseparated rightcommoncarotidarteryand measuredby connectingapressuretransducer.Arterialbloodwassampled byplacingacatheterinthecarotidartery.HSDand0.9%NaCl solution(NS)wereadministratedbyplacingacatheterinthe separatedleftjugularvein.Aratsepsismodelwasestablished usingtheCLPapproach.Weperformedtraditionaldisinfection oftheabdomenandamedianskinincisionofapproximately 2–3cmtoexposetheabdominalcavity.Thececumwasfreed fromthemesentery;thebaseofthececumwasligatedwitha 3-0suture,andpuncturedattwosites3mmapartbyusinga No.9needle.Theintestinalcanalwasthenrestoredtoensure smoothintestinalpassage,theabdominallayersweresutured, thelostfluidwasreplenished,andthewoundswerebandaged afterdisinfectionwithtamediodine.Operationproceduresfor

theshamoperative(SOP)groupwerethesameas-mentioned above,exceptfortheCLPapproach.

Ratgroups

The rats were randomlydivided into four groups: the SOP group(n=21)receivedasubcutaneousinjectionof30mL/kg 0.9%NaClaftertheoperation;theCLPgroup(n=45)receiveda subcutaneousinjectionof30mL/kg0.9%NaClafterthe oper-ation; the CLP+NS group (n=45) received a subcutaneous injectionof30mL/kg0.9%NaClandajugularveininfusion of5mL/kg0.9%NaClat0.4mL/(kgmin)for3hafterthe oper-ation;andtheCLP+HSD(n=28)groupreceivedsubcutaneous injectionof30mL/kg0.9%NaClandajugularveininfusionof 5mL/kg7.5%NaCl/6%dextranat0.4mL/(kgmin)for3hafter theoperation.

Theexperimentswereperformedinadherencewiththe NationalInstitutesofHealthguidelinesforthetreatmentof animals and ethical animal research. In addition, animals werekilledat0,9,and18haftertheoperation.Blood sam-plesandlungtissueswerecollectedforexamination.Equal volumesof0.9%NaClwereimmediatelyinjecteduponeach bloodsampling.

Determinationofseruminflammatorycytokinelevels PlasmaTNF-␣,IL-1␤,andIL-6levelsweredeterminedusing enzyme-linked immunosorbent assay (ELISA). Blood sam-pleswerecollectedfromthecommoncarotidarteryat0,9, and 18hafter CLP. Bloodaliquots of0.8mLwere added to Eppendorftubescontainingsterilepyrogen-free ethylenedi-aminetetraacetic acid(EDTA)anticoagulantandcentrifuged (3000rpm) for15minat 4◦C.Theplasma samples (100␮L) obtainedat0,9,and18haftertheoperationwereused,and thelevelsofTNF-␣,IL-1␤,andIL-6weremeasuredinduplicate usinganELISAkit(R&DSystems,Minneapolis,MN),according tothemanufacturer’sinstructions.

Arterialbloodgasanalysis

Thecarotidarterybloodofrats ineachgroupwassampled (0.2mL)at0,9,and18haftertheoperation,andthepH,PaO2,

andPaCO2inthebloodsampleswereassayedusingaportable

bloodgasanalyzer(i-STAT,Abbott,Chicago,USA). Percentageofbronchoalveolarlavagefluid(BALF) neutrophils

BALFcollectionandcellsortingmethodswereperformedas reportedbyReisetal.7_{andCalloletal.}8_{Thetracheaandlung}

ofthe sacrificedrats wereexposed at18hafterthe opera-tion.Therightbronchuswasligated,thetracheawasfixedon theannularcartilagewithforceps,andatransverseincision wasmadeunderit.ByremovingtheNo.7needle,an approxi-mately10-cm-longplasticinfusiontubewaskeptremaining. Thetubewasinsertedintothetrachealcavity,suturedatone end, and fixed ona 5-mL syringeatthe other end. Sterile saline(2mL,37◦C)wasslowlyinjectedintothelung,and suc-tionedbackafter30s.Theaboveprocedureswere repeated thriceandatotalof5mLofBALFwascollected.TheBALFwas

centrifugedat3000rpmfor10minandthecellsinthepellet werestainedusingWright’sstaining.Onehundredcellswere countedandclassifiedunderamicroscopeequippedwithan oil-immersionlensaccordingtoleukocytefeatures.The per-centageofneutrophilswascalculatedasfollows:percentage ofneutrophils(%)=numberofneutrophilscounted/numberof totalcellscounted×100%.

Measurementoflungmyeloperoxidase(MPO)activity All the rats were sacrificed 18h after the operation. The frozen right lower lobe lung samples were then weighed andhomogenizedin2mLof50mmol/Lphosphatebufferat pH6.0 containing0.5%hexadecyltrimethylammonium bro-mide(SigmaChemicalCo.,St.Louis,MO).Thesampleswere then frozen on dry ice and thawed at room temperature thrice, after which they were sonicated. The suspensions werethen centrifugedat4000rpmfor15min.MPOactivity inthesupernatantwasassessedbymeasuringthehydrogen peroxide (H2O2)-dependentoxidation ofo-dianisidine

dihy-drochloride (Sigma Chemical Co.), according to a previous report.9 _{We mixed 0.1mL of the supernatant with 2.9mL}

ofphosphatebuffer (50mmol/L,pH6.0)containing0.0005% hydrogen peroxide (Sigma) and 0.167mg/mL o-dianisidine

dihydrochloride(Sigma).Thechangeinabsorbancewas mea-suredat460nmusingaspectrophotometer(U-2000;Hitachi Instruments,Webster,NY).MPOactivitywasderivedfromthe observedchangeinabsorbanceperminute.TheMPO activ-ity was normalized further to the total protein content of the supernatant, whichwas measured bythe micro-Lowry technique.10_{ActivityisexpressedasunitsofMPOactivityper}

milligramofprotein.

Wettodryweightratiooflungissues

Aftertheratsweresacrificed,therightlungtissueswere har-vestedandthewetweightwasmeasuredfirst.Then,thetissue sampleswere driedat80◦Cfor 72hand weighed again to obtain the dry weight. Theratio ofwet to dry weightwas calculated.

Pathologicalchangesinthelungtissue

Thelungtissueswereharvestedat18hforhistopathological analyses.Theharvestedlungtissueswerefixedinbuffered formaldehyde(10%inPBS,pH7.4) for8h.Thefixedorgans weredehydratedingradedethanolandembeddedinparaffin (Tissue-processor,Japan).Then,4-␮msectionswerecutusing aslidingmicrotome(LeicaJungSM2000,Germany)andthe paraffinwasremovedusingxylene.Thetissuesectionswere thenstainedwithhematoxylinandeosinandvisualizedunder alightmicroscope.

DeterminationoftheICAM-1mRNAlevelinlungtissues The rats were sacrificed at 9 and 18h after the oper-ation. Total RNA was extracted using the one-step approach by adding 1mL Trizol to 100mg of right lung tissue. cDNA was synthesized according to the manu-facturer’s instructions, and 1␮L cDNA was used as the

template for PCR amplification using the following primer sequences: upstream: 5-CGGTAGACACAAGCAAGAGA-3; downstream:5-GCAGGGATTGACCATAATT-3;␤-actin(207bp) upstream:5-CCAACTGGGACGATATGGAG-3;downstream:5 -CAGAGGCATACAGGGACAAC-3.ThePCRreactionconditions wereasfollows:predenaturationat94◦Cfor5minuntilstart ofthecycle;followedby35cyclesat94◦Cfor30s,56◦Cfor 30s,and72◦Cfor1min;andafinalextensionstepat72◦C for7min.For␤-actinamplification,thefollowingconditions were used: predenaturationat94◦Cfor5minuntilstartof thecycle;followedby35cyclesat94◦Cfor30s,56◦Cfor30s, 72◦Cfor1min;andafinalextensionstepat72◦Cfor7min. Afteragarosegelelectrophoresis,theamplifiedproductswere scannedusingagelimagingsystem.ThemRNAexpression level wasexpressedasthe ratioofICAM-1 to␤-actinband luminosity.

Survivalrate

Thesurvivalrateofexperimentalanimalsineachgroupwas givenbypointstatisticsat9and18hpostoperatively. Statisticalmethods

Measurement data were statistically processed using SPSS 11.5 softwareandexpressedasmeans±standarddeviation ( ¯x±s).Meansamongmultiplegroupswerecomparedusing ANOVA,twogroupswerecomparedusingt-test,andp<0.05 wasdeemedstatisticallysignificant.

Results

HemodynamiceffectsofHSD

ThebaselineMAPineachgroupwasnotsignificantly differ-ent(Table1).Duringtheentireresearchperiod,theMAPwas stableintheSOPgroup,butdecreasedsignificantlybetween 9and18hintheCLPandCLP+NSgroups.Hemodynamic dis-ordersweredetectedbuttheMAPwassignificantlybetterin theCLP+HSDgroupthanintheCLPandCLP+NSgroupsat 9h.Thedifferencewasstatisticallysignificantand theHSD showedacontinuedimprovementinbloodpressurefrom9h onwards.

EffectsofHSDonplasmaTNF-˛,IL-1ˇ,andIL-6levels ThebaselineTNF-␣,IL-1␤,andIL-6levelsineachgroupwere notsignificantlydifferent(Table1).TheplasmaTNF-␣,IL-1␤, and IL-6levels intheSOP groupwerenotsignificantly dif-ferentat9and18h,suggestingthatthelaparotomydidnot significantlyinfluencetheexpressionofthesemarkers.The plasmaTNF-␣levelsintheCLPandCLP+NSgroupswere sig-nificantlyincreasedat9handgraduallyincreasedthereafter. Duringtheentireexperimentphase,theplasmaTNF-␣level intheCLP+HSDgroupwassignificantlylowerthanthe cor-respondingvaluesintheCLPandCLP+NSgroups(Fig.1A). Furthermore,intheCLPandCLP+NSgroups,theIL-1␤ expres-sionlevelsincreasedsharplyat9hand18h.TheplasmaIL-1␤ level inthe CLP+HSD groupshowedbiphasic changes and

Table1–Baselinevaluesofparametersineachgroup. SOP(n=10) CLP(n=10) CLP+NS(n=10) CLP+HSD(n=10) MAP(mmHg) 119±5 120±7 117±6 118±7 TNF-␣(pg/mL) 74.72±13.59 74.81±19.13 73.67±13.27 75.08±15.59 IL-1␤(pg/mL) 56.55±12.46 60.91±10.41 58.13±12.35 59.18±11.24 IL-6(pg/mL) 90.14±11.80 88.63±12.76 87.26±13.56 89.23±10.84 pH 7.411±0.034 7.407±0.030 7.419±0.033 7.413±0.029 PaO2(mmHg) 122±11 121±10 123±12 120±11 PaCO2(mmHg) 38±6 37±5 37±5 38±4

Baselinelevelsofmeanarterialbloodpressure(MAP),tumornecrosisfactor-␣(TNF-␣),interleukin-1␤(IL-1␤),interleukin-6(IL-6),arterialblood pH,PaO2,andPaCO2inratsthatreceivedlaparotomyplusvehicle(SOP,n=10),cecalligationandpuncture(CLP;n=45),CLPplusnormalsaline

(CLP+NS,jugularveininjectionof5mL/kg0.9%NaClataninfusionspeedof0.4mL/(kg·min)for3haftertheoperation;n=45),andCLPplus HSD(CLP+HSD,jugularveininfusionof5mL/kg7.5%NaCl/6%dextranataninfusionspeedof0.4mL/(kg·min)for3haftertheoperation;n=28). Dataareexpressedasmeans±SEM.

SOP CLP CLP+NS CLP+HSD 0 9 18 21 -9 0 100 200 300 400 500 600 TNF-α( pg/mL) IL-β( pg/mL) 0 200 400 600 800 SOP CLP CLP+NS CLP+HSD 0 9 18 21 -9 Time(hr) Time(hr) Time(hr) Time(hr) Time(hr) Time(hr) 0 9 18 21 -9 0 9 18 21 -9 0 9 18 21 -9 -9 0 9 18 21 SOP CLP CLP+NS CLP+HSD SOP CLP CLP+NS CLP+HSD SOP CLP CLP+NS CLP+HSD SOP CLP CLP+NS CLP+HSD IL-6(pg/mL) ∆ PH ∆ Paco 2 (mmHg) ∆ Paco 2 (mmHg) 0 200 400 600 800 -20 0 20 40 -40 -60 -80 20 15 10 5 0 -5

F

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Fig.1–EffectofHSDon(A)TNF-␣,(B)IL-1␤,(C)IL-6,(C)pH,(D)PaO2,and(E)PaCO2inratswithsepsisinducedby peritonitis.Thegraphsillustratethechangesduringtheexperimentalperiodinanimalsthatreceivedlaparotomyplus vehicle(SOP;n=10),cecalligationandpuncture(CLP;n=45),CLPplusnormalsaline(CLP+NS,jugularveininjectionof 5mL/kg0.9%NaClataninfusionspeedof0.4mL/(kgmin)for3haftertheoperation;n=45),andCLPplusHSD(CLP+HSD, jugularveininfusionof5mL/kg7.5%NaCl/6%dextranataninfusionspeedof0.4mL/(kgmin)for3haftertheoperation; n=28).Dataareexpressedasmeans±SEM.*p<0.05forallgroupsvs.sham.#_{p<0.05forHSDvs.NS.}

althoughitwassignificantlylowerthanthatintheCLP,the IL-1␤levelinthisgroupwassignificantlydifferentfromthat intheSOPgroupat9and18h(Fig.1B).TheplasmaIL-6levels intheCLPandtheCLP+NSgroupsincreasedsharplyuntil9h anddecreasedslowlythereafter.TheplasmaIL-6levelinthe CLP+HSDgroupwasinitiallylowerthanthatintheCLPand CLP+NSgroups;however,thelevelsweresimilaramongthese groupsat18h(Fig.1C).

EffectofHSDonindicatorsofarterialbloodgasanalysis ThebaselinelevelsofarterialbloodpH,partialpressureof oxy-gen,partialpressureofcarbondioxideineachgroupwerenot significantlydifferent(Table1).ThearterialbloodpHineach groupwasdecreasedat9h,butitfurtherdecreasedat18hin theCLPandCLP+NSgroups,whereasthebloodpHshowed thesamegradualincreasingtrendintheSOPandCLP+HSD groups(Fig.1E).

Thearterialpartialpressureofoxygendidnotsignificantly change in the SOP group, but it showed asharp decrease at9h followed byagradual decrease until18h inthe CLP andCLP+NSgroups.Thearterialpartialoxygenpressurein theCLP+HSDgroupdecreasedat9h,butitwassignificantly higherthanthecorrespondingvaluesintheCLPandCLP+NS groups.Sucheffectscontinueduntil18h(Fig.1F).

Thearterialpartialpressure ofcarbon dioxideincreased slightlyat9hintheCLP,CLP+NS,andCLP+HSDgroups,but showedacontinuousrisingtrendevenat18hintheCLPand CLP+NSgroups.Thearterialpartialpressureofcarbon diox-ideinthesetwogroupsshowedahigherincreasingtrendthan thatintheCLP+HSDgroup,whichwasslightlyincreased dur-ingthe studyduration butdidnotshowsignificantchange comparedtothebaselinevalueat0hintheSOPgroup(Fig.1G). EffectofHSDonthenumberofBALFneutrophils,lung myeloperoxidase(MPO)activityandwettodryweight ratiooflungtissue

ThepercentageofBALFneutrophilswashigherintheCLPand CLP+NSgroupsthanintheSOPgroup.ThereweremoreBALF neutrophilsinthe CLP+HSD groupthan inthe SOPgroup; however,thisvaluewasstillsignificantlylowerthanthe cor-respondingvaluesintheCLPandCLP+NSgroups(Fig.2A). TheMPOactivitiesintheCLPandCLP+NSgroupswere sig-nificantlyincreasedascomparedtotheSOPgroup;however, theMPOactivityinthelungtissueintheCLP+HSDgroupwas lowerthanthatintheCLPandCLP+NSgroups(Fig.2B).The CLPandCLP+NSgroupshadahigherwettodryweightratio oflungtissuesascomparedtotheSOPandCLP+HSDgroups (Fig.2C).

Histopathologicalchangesinlungtissues

Therewerenosignificantpathologicalchangesinratlung tis-suesectionsintheSOPgroupat18h(Fig.3A).Thelungtissues inthe CLPand CLP+NSgroups showedsignificant inflam-matory pathological changessuch ashemorrhage, alveolar congestion, thickening ofalveolar walls/hyalinemembrane formations,andinfiltrationandaggregationofneutrophilsin airspacesorvesselwalls(Fig.3BandC).TheCLP+HSDgroup

60.0 50.0 40.0 30.0 20.0 10.0 0.0

Neutropbil counts in BALF (n)

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B

Lung MPO activity (Units/g lung tissue)

Lung wet/dry weight ratio

0.0 2.0 4.0 6.0 8.0 #

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Fig.2–EffectofHSDon(A)BALFneutrophilnumber,(B) lungtissueMPOand(C)wet/dryweightratiooflungtissue activitiesinratswithsepsisinducedbyperitonitis.The graphillustrateschangesduringtheexperimentalperiod inanimalsthatreceivedlaparotomyplusvehicle(SOP;

n=6),cecalligationandpuncture(CLP;n=6),CLPplusNS (CLP+NS;n=6),andCLPplusHSD(CLP+HSD;n=6).Data areexpressedasmeans±SEM.*p<0.05forallgroupsvs. sham.#_{p<0.05forHSDvs.NS.}

Fig.3–Histopathologicalstudiesoflungtissue.Lightmicroscopyshowedlungsectionsofratsinthe(A)sham-operated (SOP)group,(B)cecalligationandpuncture(CLP)group,(C)CLP+NSgroup,and(D)CLP+HSDgroup.Sectionswerestained withhematoxylinandeosin(originalmagnification,200×).

hadsignificantlyfewerinflammatorychangesascomparedto theCLPandCLP+NSgroups(Fig.3D).

DeterminationofICAM-1mRNAlevelsinlungtissue The ICAM-1 levels in the CLP and CLP+NS groups were increasedat9handfurtherincreasedat18h.TheICAM-1 lev-elsintheCLP+HSDgroupweresignificantlylowerthanthose intheCLPandCLP+NSgroups(Fig.4).

EffectofHSDonsurvivalrates

TheratsintheSOPgroupdidnotdiewithin18h.Thesurvival ratesofanimalsintheCLPgroupat9and18hwere62.2%and 31.1%,respectively.ThesurvivalratesofratsintheCLP+NS groupat9and18hwere57.8%and35.6%,respectively.The survivalratesoftheCLP+HSDgroupat9and18hwere85.7% and64.3%,respectively.Thus,intravenousinfusionofHSDcan improvetheearlysurvivalrate(within18h)inCLPrats. How-ever,thesurvivalrateoftheCLP+HSDgroupwasdecreasedat 9h.Therefore,additionaltherapeuticmeansmustbe imple-mentedinordertoimprovethesurvivalrateofCLPratsusing HSD.

Discussion

Clinically,many cytokines,including tumornecrosisfactor, interleukins, andadhesion molecules,are produced during sepsisasaresultofstimulibybacteriaandbacterial endo-toxins.Interactionsbetween thesevarious cytokines result inthe activation ofa cascade reaction.11 Thesepticshock

progresses andpatientsfrequentlydiefrom multipleorgan failuresbecauseofinflammatoryreactionimbalance, inflam-matory cell infiltration, micro-thrombosis, microcirculation disorders, tissue hypoperfusion, lactic acidosis, imbalance betweenoxygensupplyanddemand,anddeclineof myocar-dialcontractionforce.

In a rat model with CLP-induced septicshock, bacteria leakedto the internalorgans, infectedthe abdominal cav-ity, and causedsystemicinfection and septicshock.In the present study,therats that underwentCLPexhibited signs suchaspiloerection,curling,fatigue,andhypokinesia,eating less,weightloss,shortnessofbreath,severebleedinginsnouts andinnercanthi,andtrembling.RatsintheSOPgroupmoved freely,respondedsensitively,andexhibitednormaldrinking behavior.Thehemodynamicchanges,times,andpatternsof changes inthe cytokinelevelsintheCLPgroupwere simi-lartothoseobservedinclinicalhumanpatientswithsepsis whilelungedema,congestion,andinfiltrativeinflammatory changesoccurredinthelungtissuesofrats.Thesesymptoms aresimilartothoseobservedinhumanpatientswithsepsis. Theseresultsdemonstratedthatasepticshockratmodelcan beeffectivelyestablishedusingtheCLPapproach.

ThepresentstudyfoundthattheCLP+HSDgroupshowed significanthemodynamicimprovement.Thisissupposedto be relatedtoblood volume expansionowingtotransferof interstitialfluidandintracellularfluidinto bloodvesselsby osmotic pressure gradients after injectionof HSD into the bloodvessels.12_{Thehypertonicsalinecanenhance}

myocar-dialcontractionforces,improvecardiaccapacity,andregulate angiostasis under the body’sinfectious state,thus helping improvethecirculatoryfailureinsepsis.

CLP CLP+NS CLP+HSD SOP 9h 18h 0 10 20 30 40 50 60 70 80 90 100 Ratio (%of β -actim) SOP 18h SOP 9h CLP+HSD 18h CLP+HSD 9h CLP+NS 18h CLP+NS 9h CLP 18h CLP 9h ICAM-1 517bp B-actin 207bp

A

B

Fig.4–ExpressionofICAM-1mRNAinlungtissues.The expressionofICAM-1wasthelowestintheSOPgroup.The expressionlevelintheCLPandCLP+NSgroupsincreased at9hand18h.TheICAM-1levelintheCLP+HSDgroup wassignificantlylowerthanthatintheCLPandCLP+NS groups(p<0.05).

In sepsis, multiple inflammatory pathwayssuch as the cytokinenetwork and coagulationcascadeare triggered as aresultofthecomplexinteractions betweenthe bodyand infectiousfactors suchasbacteria.13 _{TNF-␣,IL-1␤,andIL-6}

arereleasedinmassiveamountsandinappropriateimmune activation is induced in the body, resulting in organ- and cellular-levelinjuries.Fiorentinoetal.14_foundthat

appropri-ateexpressionlevelsofinflammatorycytokinesarebeneficial forabody’sresistancetoinfection.Ourstudyalsoverifiedthat theTNF-␣,IL-1␤,andIL-6levelsinCLP+HSDratswere signifi-cantlydecreasedascomparedtotheCLPandCLP+NSgroups. Furthermore,weobservedthatHSDdidnotcompletelyinhibit thesecytokines;rather,theirexpressionlevelswere consider-ablyreduced.ThisfindingsuggestedthatHSDplaysapositive immunoregulatoryroleinthetreatmentofsepsis.Weinferred that HSD can play this role via multiple pathways: during severeinfections;immunoparalysiscausedbythepresenceof

immunosuppressivemediatorssuchasPGE2,TGF-␤,and IL-10;lateMODS;inhibitionofTcellproliferationbyPGE2.Insuch cases,theinjectedhypertonicsalinemayhelptoimprovethe inhibitionofTcellactivationandproliferationbyexpressing additionallevelsofmigrationinhibitoryfactor.

Insepsis,proinflammatorycytokinessuchasTNF-␣and IL-1activateendothelialcellsandupregulateICAM-1expression. ICAM-1 is attached to the pulmonary capillary endothe-lium.Neutrophilsadheretotheendothelialcellsandbecome trappedinlungtissuescontinuouslyundertheeffectsof ␤2-integrinsandICAM-1,andthelungisinjuredbytherelease of reduced oxygen-free radicals and proteases.15 _Fahrner

et al.16 _{observedthatHSDcanreducethenumberofwhite}

bloodcellsincreasedbyformyl-methionyleucylphenylalanine (FMLP)stimulus,causerespiratoryburstsand polymorphonu-clear cell aggregation, and increase the expression level of ␤2-integrin (CD18) in the normalhuman body. Wealso observed that HSD plays a positive regulatory role on the expressionlevelsofTNF-␣,IL-1␤,andIL-6;theICAM-1mRNA levelsinlungtissueswerelowerintheCLP+HSDgroupthan intheothertwoCLPgroups.Furthermore,thevarious indi-catorsweresignificantlybetterintheCLP+HSDgroupthan intheCLPandCLP+NSgroups.Takentogether,theseresults suggesttheprotectiveroleofHSDinacutelunginjurycaused afterCLP.

Conclusion

Inthepresentstudy,wecreatedratmodelsofsepticshock usingtheCLPapproach.Ratsthatwereintravenouslyinjected with7.5%NaCl/6%dextran(HSD)showedimprovementsin thecirculatoryfailureduringthestudyperiodafterCLP.Our studyalsoconfirmedthattheTNF-␣,IL-1␤,andIL-6expression levelsandtheICAM-1levelswerelowerintheHSD-treatedrats thaninthecontrolrats.WeinferredthatHSDplaysapositive immunoregulatory role in sepsis, thus inhibiting the over-expression of inflammatory response factors. Investigated parameterssuchasneutrophilpercentage,MPOactivity,wet todryweightratiooflungtissues,histopathologicalchanges inthelungtissues,arterialbloodgasindicatorssuchaspH andpartialpressuresofoxygenandcarbondioxidewere sig-nificantlybetterintheHSD-treatedratsthanintheotherCLP groups. Taken together,these findings suggested that HSD playsaprotectiveroleinacutelunginjurycausedbyCLP.Our studyshowedthatthehypertonic/hyperoncoticsolutionhas bettertherapeuticeffectinthetreatmentofsepticshockand sepsis.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgement

ThisstudywassupportedbyagrantfromtheWuhan Munic-ipalHealthBureauFund(grantnumberWX10C15).

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