research article peripheral injection of sb203580 inhibits

Research ArticlePeripheral Injection of SB203580 Inhibits theInflammatory-Dependent Synthesis of ProinflammatoryCytokines in the Hypothalamus

Andrzej P Herman1 Agata KrawczyNska1 Joanna Bochenek1 Hanna Antushevich1

Anna Herman2 and Dorota Tomaszewska-Zaremba1

1 Polish Academy of Sciences The Kielanowski Institute of Animal Physiology and Nutrition 05-110 Jabłonna Poland2The Academy of Cosmetics and Health Care 13 Podwale Street 00-252 Warsaw Poland

Correspondence should be addressed to Andrzej P Herman ahermanifzzpanpl

Received 24 February 2014 Revised 9 May 2014 Accepted 20 May 2014 Published 4 June 2014

Academic Editor Kanato Yamagata

Copyright copy 2014 Andrzej P Herman et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The study was designed to determine the effects of peripheral injection of SB203580 on the synthesis of interleukin- (IL-) 1120573 IL-6and tumor necrosis factor (TNF) 120572 in the hypothalamus of ewes during prolonged inflammation Inflammation was induced bythe administration of lipopolysaccharide (LPS) (400 ngkg) over 7 days SB203580 is a selective ATP-competitive inhibitor of thep38 mitogen-activated protein kinase (MAPK) which is involved in the regulation of proinflammatory cytokines IL-1120573 IL-6 andTNF120572 synthesis Intravenous injection of SB203580 successfully inhibited (119875 lt 001) synthesis of IL-1120573 and reduced (119875 lt 001) theproduction of IL-6 in the hypothalamusThe p38 MAPK inhibitor decreased (119875 lt 001) gene expression of TNF120572 but its effect wasnot observed at the level of TNF120572 protein synthesis SB203580 also reduced (119875 lt 001) LPS-stimulated IL-1 receptor type 1 geneexpressionThe conclusion that inhibition of p38MAPK blocks LPS-induced proinflammatory cytokine synthesis seems to initiatenew perspectives in the treatment of chronic inflammatory diseases also within the central nervous system However potentialproinflammatory effects of SB203580 treatment suggest that all therapies using p38 MAPK inhibitors should be introduced verycarefully with analysis of all expected and unexpected consequences of treatment

1 Introduction

Hypothalamus is the brain region responsible for integrationand processing of signals from endocrine nervous andimmune systems It is well known that inflammatory statesinfluence the activity of neurons located in different hypotha-lamic nuclei and affect centrally regulated processes such asthermogenesis [1] food intake [2] reproduction [3 4] andcircadian rhythms of rest-activity and sleep [5] Among themost important mediators that transmit the inflammatorysignals from the periphery into brain parenchyma are proin-flammatory cytokines It is well established that inflamma-tory processes raise the concentration of proinflammatorycytokines such as interleukin- (IL-) 1120573 IL-6 and tumornecrosis factor (TNF) 120572 in the peripheral blood [6 7] During

the development of inflammatory response the increasedconcentration of proinflammatory cytokines is observed bothin the cerebrospinal fluid and in brain parenchyma [6 8 9]The origin of these proinflammatory cytokines in centralnervous system seems to be differentiated the peripheralcytokines can cross the blood-brain barrier through fen-estrated brain capillaries in the choroid plexus organumvasculosum of the lamina terminalis median eminencesubfornical organ and area postrema [10] Moreover it wasdescribed that in response to peripheral bacterial endotoxinproinflammatory IL-1120573 could be synthesized bymacrophage-like cells in the circumventricular organs and choroid plexuswhere the blood-brain barrier is deficient [11] During inflam-mation the cytokines may be also transported into thebrain via blood-brain barrier by saturated self-inhibitable

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 475152 10 pageshttpdxdoiorg1011552014475152

2 BioMed Research International

transport mechanisms [12] However significant sources ofcentral proinflammatory cytokines may generate their ownsynthesis within the brain tissue including hypothalamus[9 13] This brain synthesis of proinflammatory cytokinescould be induced by a fast neural pathway [11] It wasshown that the electrical stimulation of the vagus nerveinduces the expression of brain IL-1 and vagotomy abrogatesthe induction of expression of brain IL-1 in response tointraperitoneal lipopolysaccharide [14 15] The synthesis ofproinflammatory cytokines in brain structures may be alsostimulated and controlled by the same cytokines originatingfrom the periphery or produced by the circumventricularorgans and choroid plexus This is because proinflamma-tory cytokines amplify their own synthesis and secretion[16] However an important role in the transmission andamplification of inflammatory signals in the brain maycause auto- and paracrine stimulation of proinflammatorycytokines in the brain tissue This autostimulatory effect ofIL-1 on its synthesis was observed inmany cytokine-secretingcells including microglia [17 18] It is well established thatcentral proinflammatory cytokines could directly affect theprocesses regulated at the level of hypothalamus via their cor-responding receptors [19] Acting centrally proinflammatorycytokines especially IL-1120573 are considered to be importantmodulator of endocrine system At the level of hypothalamusIL-1120573 was found to stimulate the hypothalamic-pituitary-adrenal stress axis [20] On the other hand the injection ofexogenous IL-1120573 into the region of hypothalamus suppressedsecretion of gonadoliberine (GnRH) leading to downstreaminhibition of the hypothalamic-pituitary-gonadal axis activ-ity [21 22] Therefore the reduction of proinflammatorycytokines synthesis in the hypothalamic areamay profoundlyattenuate the central response on the peripheral inflamma-tion

The study was designed to determine the effect ofperipheral injection of SB203580 on the synthesis of IL-1120573 IL-6 and TNF120572 in the hypothalamus of ewes duringprolonged inflammation SB203580 is pyridinyl imidazolecompound that is a selective ATP-competitive inhibitor ofthe p38 mitogen-activated protein kinase (MAPK) It haspreviously been established that SB 203580 acts primarilyto block the catalytic activity of p38 MAPK but not itsactivation by upstream MAPK [23] However it is worthmentioning that biochemical analysis of SB203580 activityshowed that this inhibitor lost its specificity when it wasused at high concentration and could also suppress activ-ity of other kinases such as lymphocyte kinase glycogensynthase kinase 3 and protein kinase B [24] The p38MAPK is an important mediator of stress-induced geneexpression In particular the p38 kinase is known to playa key role in LPS-induced signal transduction pathwaysleading to cytokine synthesis [25] Previous studies indi-cated that p38 MAPK is involved in the inflammatory-dependent upregulation of proinflammatory cytokines syn-thesis at the translational and posttranslational levels [2326] It was shown that SB203580 strongly inhibited LPS-stimulated synthesis of IL-6 and TNF-120572 in hepatic stellatecells [27]

2 Materials and Methods

21 Animals The studies were performed on adult 3-year-old Polish mountain sheep in the anestrous season (April-May) All animals were in good condition The body con-dition of all animals was estimated at three- on a five-pointscale The animals were maintained indoors in individualpens and exposed to natural daylight The ewes were welladapted to the experimental conditions and always had visualcontact with neighbouring ewes even during the experimen-tal period This avoided the stress of social isolation Theanimals were fed a constant diet of commercial concentrateswith hay and water constantly available

All procedures on animals were performed with the con-sent of the Local Ethics Committee of theWarsawAgricultureUniversity

22 Experimental Procedures Venous catheters were im-planted into the jugular vein on the day prior to theexperiment The ewes (119899 = 24) were randomly dividedinto four experimental groups group Imdashcontrol (119899 = 6)group IImdashSB203580 treated (119899 = 6) group IIImdashLPS-treated(119899 = 6) group IVmdashLPS- and SB203580 treated (119899 =6) In treated animals the prolonged immune stress wasinduced by the intravenous (iv) administration of LPS fromEscherichia coli 055B5 (Sigma St LouisMOUSA) dissolvedin saline (09wvNaCl) (Baxter Deerfield IL USA) at aconcentration of 10mgL into the jugular vein (400 ngkg)For six days the animals received a single injection of LPSor saline at 8 am On day 7 of the experiment 30mins priorto salineLPS treatment ewes from groups II and IV receivediv injection of SB203580 hydrochloride (500 120583gkg) (AxonMedchem BV Groningen Netherlands) Concurrently theanimals from the control and LPS treated groups received2mLof saline injectionAnimalswere euthanized three hoursafter the LPSsaline injection and the brains were rapidlyremoved from the skulls The blocks of brains encompassinghypothalamus were sectioned sagittally and dissected fromboth sides according to stereotaxic atlas of the sheep brain[28] Landmarks were the mammillary body median emi-nence and optic chiasm The depth of the cuts was 25 to3mm Following collection all tissue was divided into twoand then frozen in liquid nitrogen and stored at minus80∘C

23 ELISA Assay for IL-1120573 IL-6 and TNF120572 Concentrationin the Hypothalamus The concentrations of IL-1120573 IL-6and TNF120572 in the hypothalamus were determined using acommercial IL-1120573 IL-6 and TNF120572 ELISA kit (BlueGeneBiotech CO LTD China) designed for sheep The tissueswere homogenized in 1mL of cold phosphate bufferedsaline (002M) then homogenates were subjected to twofreeze-thaw cycles to further break the cell membranesHomogenates were then centrifuged for 15min at 1500timesg at4∘C The supernatants were aliquoted and stored until assayat minus80∘C All steps in the assays were performed according tothe manufacturerrsquos instructionsThe incubation of plates andabsorbance measurement at 450 nm were performed usinga VersaMax reader (Molecular Devices LLC Sunnyvale

BioMed Research International 3

Table 1 All genes analyzed by real-time PCR are listed with their full name and abbreviation

GenBank accnumber Gene Amplicon size

[bp] Forwardreverse Sequence 51015840 rarr 31015840

NM 001034034

GAPDHGlyceraldehyde-3-phosphatedehydrogenase

134 ForwardReverse

AGAAGGCTGGGGCTCACTGGCATTGCTGACAATCTTGA

U39357 ACTBBeta actin 168 Forward

ReverseCTTCCTTCCTGGGCATGGGGGCAGTGATCTCTTTCTGC

NM 001076910 PPICCyclophilin C 131 Forward

ReverseACGGCCAAGGTCTTCTTTGTATCCTTTCTCTCCCGTTGC

BC1080881HDC1Bos taurus histonedeacetylase 1

115 ForwardReverse

CTGGGGACCTACGGGATATTGACATGACCGGCTTGAAAAT

X547961 IL-1120573Interleukin-1 beta 137 Forward

ReverseCAGCCGTGCAGTCAGTAAAAGAAGCTCATGCAGAACACCA

NM 0012067351IL-1R1Interleukin-1 receptor typeI

124 ForwardReverse

GGGAAGGGTCCACCTGTAACACAATGCTTTCCCCAACGTA

NM 0010093921 IL-6Interleukin-6 165 Forward

ReverseGTTCAATCAGGCGATTTGCTCCTGCGATCTTTTCCTTCAG

NM 001110785 IL-6RInterleukin 6 receptor 149 Forward

ReverseTCAGCGACTCCGGAAACTATCCGAGGACTCCACTCACAAT

NM 001024860 TNF120572Tumor necrosis factor alpha 153 Forward

ReverseCAAATAACAAGCCGGTAGCCAGATGAGGTAAAGCCCGTCA

NM 174674

TNFR1Tumor necrosis factorreceptor superfamilymember 1A

137 ForwardReverse

AGGTGCCGGGATGAAATGTTCAGAGGCTGCAGTTCAGACA

NM 001040490

TNFR2Tumor necrosis factorreceptor superfamilymember 1B

122 ForwardReverse

ACCTTCTTCCTCCTCCCAAAAGAAGCAGACCCAATGCTGT

California USA)The sensitivity for all assays was 10 pgmLThe significance of the differences in the levels of IL-1120573between the experimental groups was assessed by the Mann-Whitney 119880 test Statistical significance was defined as 119875 lt001

24 Determining the Relative Gene Expression Total RNAfrom hypothalamus was isolated using NucleoSpin RNA IIKit (MACHEREY-NAGEL Gmbh amp Co Duren Germany)according to a manufacturerrsquos instruction The purity andconcentration of isolated RNA were spectrophotometricallyquantified by measuring the optical density at 230 260 and280 nm in a NanoDrop 1000 instrument (Thermo FisherScientific Inc Waltham USA) The RNA integrity wasverified by electrophoresis using 1 agarose gel stained withethidium bromide Maxima First Strand cDNA Synthesis Kitfor RT-qPCR (Thermo Fisher Scientific Inc Waltham USA)was used to prepare cDNA synthesis As a starting materialfor this PCR synthesis 2 120583g of total RNA was used

Real-time RT-PCR was carried out using HOT FIREPolEvaGreen qPCR Mix Plus (Solis BioDyne Tartu Esto-nia) components and HPLC-grade oligonucleotide primers

synthesised byGenomed (Poland) Specific primers for deter-mining the expression of housekeeping genes and the genes ofinterest were designed using Primer 3 software (Table 1) Onetube contained 4 120583L PCR Master Mix (5x) 14 120583L RNase-freewater 1 120583L primers (05 120583L each working concentration was025 120583M) and 1 120583L cDNA template The tubes were run onthe Rotor-Gene 6000 (Qiagen Duesseldorf Germany) Thefollowing protocol was used 95∘C in 15mins for activatingHot Star DNA polymerase and finally the PCR including 30cycles at 95∘C in 10 sec for denaturation 60∘C in 20 sec forannealing and 72∘C in 10 sec for extension After the cyclesa final melting curve analysis under continuous fluorescencemeasurementswas performed to confirm the specificity of theamplification

Relative gene expression was calculated using the com-parative quantification option of Rotor Gene 6000 software17 (Qiagen Duesseldorf Germany) The second differentialmaximum method [29] was used to calculate reaction effi-ciencies A set percentage of themaximumfluorescence valuewas used to calculate the beginning of the exponential phaseTo compensate for the variation in cDNA concentrations andthe PCR efficiency between tubes an endogenous controlgene was assayed in each sample and used for normalization


Initially four housekeeping genes GAPDH 120573-actin PPICand HDC1 were tested The BestKeeper was used to deter-mine the most stable housekeeping gene for normalizinggenes of interest expression The BestKeeper is based on thepair-wise correlation analysis of all pairs of candidate genes[30] and calculates variations of all reference genes SD (plusmnCt)GAPDH was selected as the best endogenous control gene Ithad the lowest SD (plusmnCt) value and correlation coefficient withthe remaining analyzed housekeeping genes

The results are presented as a relative gene expression ofthe target gene versus housekeeping gene relative expressionvalue and mean plusmn SEM The average relative quantity ofgene expression in control groups was set to 10 The signif-icance of differences between the experimental groups wasassessed by the Mann-Whitney119880 test Statistical significancewas defined as 119875 lt 001

3 Results

31 Influence of SB203580 and Prolonged LPSTreatment on IL-1120573 IL-6 and TNF120572 Synthesis in theHypothalamus Sevenfoldadministrations of LPS increased (119875 lt 001) the amount ofall analyzed proinflammatory cytokines in the hypothalamuscompared with both control groups SB203580 decreased(119875 lt 001) LPS-induced elevation of IL-1120573 and IL-6 concen-tration However in the group with concomitant SB203580and LPS treatment the level of IL-6 stayed increased (119875 lt001) compared with control groups On the other hand theinjection of SB203580 did not affect the endotoxin-stimulatedTNF120572 expression in the hypothalamus (Figure 1)

32 Effect of SB203580 and Prolonged LPS Treatment on IL-1120573 IL-6 TNF120572 and Their Corresponding Receptors GeneExpression in the Hypothalamus Seven days of LPS admin-istration simulated (119875 lt 001) the gene expression of IL-1120573 (Figure 2) IL-6 (Figure 3) and TNF120572 (Figure 4) in thehypothalamus On the other hand prolonged endotoxintreatment stimulated (119875 lt 001) only the expression of IL-1R1gene but had no effect on IL-6R TNFR1 and TNFR2 mRNAlevel in the hypothalamus SB203580 reduced (119875 lt 001) LPS-dependent elevation of IL-1120573 IL-6 and TNF120572 as well as IL-1Rtranscription However SB203580 did not completely abolishthe effect of inflammation on the gene expression of TNF120572and IL-1R1 which stayed increased (119875 lt 001) compared tothe control groups

4 Discussion

Our study demonstrated that prolonged inflammationinduced through 7 days of LPS treatment elevated localsynthesis of IL-1120573 IL-6 and TNF120572 in the hypothalamus ofsheep The strongest stimulatory effect of inflammation wasdetermined in the case of IL-6 which showed the highestrelative increase in the concentration These results fullysupport the data obtained in previous studies with singleand repeated LPS injections which reported the potentiationof proinflammatory cytokines synthesis in the brain ofmice [6 31] They also determined higher elevation of IL-6

mRNA levels compared with IL-1120573 and TNF120572 in response toperipheral inflammatory stimuli This could have a profoundeffect on hypothalamic activity It was concluded that centralproinflammatory cytokines especially IL-1 are able to inducechanges in the brain neurochemistryThese cytokines inducenorepinephrine release in the brain (most markedly in thehypothalamus) increase brain concentrations of tryptophanand the metabolism of serotonin decrease acetylcholinesecretion and provoke modest changes in brain dopamine[32] Among others acting at the level of hypothalamusproinflammatory cytokines have been identified as beingresponsible for stimulation of thermogenesis [33] thehypothalamic-pituitary-adrenal axis activation [34 35]and the hypothalamic-pituitary-gonadal axis inhibition[21] These actions of proinflammatory cytokines in thehypothalamus enable the existence of their correspondingreceptors in this region of the brain [19] The expression oftheir receptor was determined in microglia astrocytes andeven neurons [36ndash39] In our study prolonged inflammationelevated only IL-1 type I (R1) gene expression but did notinfluence the transcription of cytokines receptor IL-6RTNFR1 and TNFR2 in the hypothalamus The stimulatoryeffect of endotoxin treatment on IL-1R1 expression in thebrain has been previously described in sheep [8 40] andmice [41 42] This could lead to the assumption that inthe hypothalamus peripheral inflammation increases thesensitivity of IL-1R1 expressing cell on the action of IL-1120573Although no affect of peripheral inflammatory stimuli onthe gene expression of IL-6R was found in the hypothalamusit should be pointed out that the amount of mRNA for IL-6corresponding receptor was the highest among transcriptsencoding other analyzed cytokines receptors Thereforebased on our data it is impossible to judge which cytokineis pivotal in the transmission of the inflammatory effects inthis region of the hypothalamus

The p38 MAPK is one of the three groups of mitogen-activated protein kinases which are key enzymes in the signaltransduction cascade from the extracellular environment tothe nucleus of essentially every eukaryotic cell type [43] Thep38 MAPKs are strongly activated in vivo by environmentalstresses and inflammatory cytokines but less by serum andgrowth factors Therefore together with the JNK family p38MAPKs are also known as stress-activated protein kinases[44] The p38 MAPK was first recognized for its role ininflammation in regulating the biosynthesis of proinflam-matory cytokines such as IL-1 and TNF120572 in endotoxin-stimulatedmonocytes [26] Subsequently it was also found tobe involved in regulating the production of IL-8 in responseto IL-1120572 and IL-1120573 [45] and the production of IL-6 in responseto IL-1 and TNF120572 [43 46] The results of the in vitro studiesindicate that p38 MAPK inhibition by SB203580 stronglyreduces the IL-1120573 induced synthesis of IL-6 and indicates thata key element of p38-dependent IL-6 regulation occurs at thelevel of mRNA stability [43] The in vitro experiments alsodemonstrate that SB203580 inhibits at the dose-dependentmanner endotoxin-stimulated expression of IL-1120573 in themonocytic cell lines [25] It should be noted that the resultsof the study performed on rat primary cortical glial cellcultures indicate that inhibition of p38 MAPK by SB203580


0

2

4

6

8

10

14

12

16

18

ab

cC

ontro

l

LPS

IL-1120573

(pg

mg

of to

tal p

rote

in)

SB20

3580

LPS+

SB20

3580

(a)

0

5

15

20

25

10

ab

abc

Con

trol

LPS

IL-6

(pg

mg

of to

tal p

rote

in)

SB20

3580

LPS+

SB20

3580

(b)

0

1

2

3

4

5

Con

trol

LPS

TNF120572

(pg

mg

of to

tal p

rote

in)

abSB

2035

80

LPS+

SB20

3580

(c)

Figure 1 Effect of lipopolysaccharide (LPS) (400 ngkg iv7 days) and SB203580 (500 120583gkg) injections on IL-1120573 (a) IL-6 (b) and TNF120572(c) expression in the hypothalamus on day 7 of the experiment a b cmdash119875 lt 001 (indicate values that differ significantly from the controlSB203580 control and LPS treated groups resp according to the Mann-Whitney 119880 test) Data are presented as a mean value plusmn SEM

did not prevent LPS and IL-1120573 induced expression of IL-1120573but did inhibit the IL-1120573-induced expression of c-fos andinducible nitric oxide synthase [47] It is worth noticingthat the anti-inflammatory effect of SB203580 has been alsodescribed in the in vivo study In the experiment carriedout on mice and rat treatment with SB203580 for 30minprior to the injection of bacterial endotoxin suppressed theplasma level of TNF120572 at the dose-dependent manner [48]In the same study SB203580 also inhibited the circulatingconcentration of both IL-6 and TNF120572 in rat and mice withexperimentally induced arthritis Moreover treatment withSB203580 reducedmortality in amurinemodel of endotoxin-induced shock Anti-inflammatory effects of SB203580 andits possible therapeutic use were also determined in the micewith experimentally induced endometriosis It was shownthat repeated for 24-day injection of SB203580 stronglyreduced the synthesis of IL-1120573 and TNF120572 translation in themice endometrium [49]

Our study also demonstrated that iv injection ofSB203580 attenuates endotoxin-dependent synthesis of theproinflammatory cytokines in the hypothalamusTheperiph-eral administration of the p38 MAPK inhibitor restoredsynthesis of IL-1120573 to the control value and reduced IL-6 expression in the hypothalamus However the effect ofSB203580 on the synthesis of hypothalamic TNF120572 wasobserved only in the case of transcriptomic analysis Thelack of effect of p38 MAPK inhibition on TNF120572 synthesisat the level of protein expression could be connected withrelatively low elevation of this cytokine in the hypothalamusand limited sensitivity of ELISA assay However it shouldbe emphasized that the effectiveness of anti-inflammatorytreatment did not require blockade of all proinflammatorycytokinesThere is evidence that cytokines exist in ldquocascadesrdquoand that interrupting one cytokine interrupts the cascade[50] for example blocking TNF120572 reduces the activity of IL-6and IL-1120573 [51] while blocking IL-1120573 reduces IL-6 [52]


0

1

2

3

ab

c

Con

trol

LPS

LPS+

SB20

3580

Rela

tive I

L-1120573

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

(a)

3

4

2

1

0

ab

abc

Con

trol

LPS

Rela

tive I

L-1

R1ve

rsus

GA

PDH

mRN

A le

vel

SB20

3580

LPS+

SB20

3580

(b)

Figure 2 Influence of lipopolysaccharide (LPS) (400 ngkg iv7 days) and SB203580 (500 120583gkg) injections on the gene expression of IL-1120573(a) and IL-1R1 (b) in the hypothalamus on day 7 of the experiment a b cmdash119875 lt 001 (indicate values that differ significantly from the controlSB203580 control and LPS treated groups resp according to the Mann-Whitney 119880 test) Data are presented as a mean value plusmn SEM

0

3

4

5

1

2

ab

bc

Con

trol

LPS

Rela

tive I

L-6

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

LPS+

SB20

3580

(a)

0

1

2

Con

trol

LPS

Rela

tive I

L-6

R ve

rsus

GA

PDH

mRN

A le

vel

SB20

3580

LPS+

SB20

3580

(b)

Figure 3 Effect of lipopolysaccharide (LPS) (400 ngkg iv7 days) and SB203580 (500 120583gkg) injections on the gene expression of IL-6 (a)and IL-6R (b) in the hypothalamus on day 7 of the experiment a b cmdash119875 lt 001 (indicate values that differ significantly from the controlSB203580 control and LPS treated groups resp according to the Mann-Whitney 119880 test) Data are presented as a mean value plusmn SEM

Our study determined that changes in the synthesis ofcytokines and their receptors after peripheral administrationof SB203580 do not have to be an effect of direct inhibitionof p38 MAPK in the hypothalamic tissue It is possible thatSB203580 attenuates the peripheral inflammatory responsereflecting on reduced inflammatory signal and reducedproinflammatory cytokines synthesis in the hypothalamusThe study conducted on mice demonstrated that reductionof the peripheral inflammatory response using the anti-inflammatory drug which did not cross the blood-brainbarrier was enough to suppress the brain synthesis of IL-1120573[53] This suggested that even a relatively small reduction ofperipheral cytokines particularly IL-1120573 may translate into

greater attenuation of cytokines synthesis in the brain Thismay be because LPS-induced production of IL-1120573 in theperiphery must exceed a fairly high threshold to increasethe levels of IL-1120573 in the brain [53] One of the endogenousmechanisms controlling the synthesis of proinflammatorycytokines is effect of choline It was reported that directstimulation of the vagus nerve or pharmacological inhibi-tion of acetylcholinesterase activated the cholinergic anti-inflammatory pathway which suppressed systemic produc-tion of cytokines such as TNF120572 IL-1 and IL-6 [54] Thestudies carried out on rats showed that intrathecal injectionof SB203580 caused a 3-fold rise in the high-frequencypower spectral component of heart rate variability which


0

3

1

2

ab

c

Con

trol

LPS

Rela

tive T

NF120572

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

LPS+

SB20

3580

(a)

0

1

2

Con

trol

LPS

Relat

ive T

NFR1

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

LPS+

SB20

3580

(b)

0

1

2

Con

trol

LPS

Relat

ive T

NFR2

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

LPS+

SB20

3580

(c)

Figure 4 Effect of lipopolysaccharide (LPS) (400 ngkg iv7 days) and SB203580 (500 120583gkg) injections on the gene expression of TNF120572 (a)TNFR1 (b) and TNFR2 (c) in the hypothalamus on day 7 of the experiment a b cmdash119875 lt 001 (indicate values that differ significantly fromthe control SB203580 control and LPS treated groups resp according to the Mann-Whitney 119880 test) Data are presented as a mean value plusmnSEM

is a widely used parameter of parasympathetic activity thatdirectly correlates with activation of the cholinergic anti-inflammatory pathway [55] Thus the ability of SB203580inhibitors to activate the vagus nerve could at least partiallyexplain observed reduction of proinflammatory cytokinessynthesis in our study

The findings that inhibiting p38 MAPK blocks LPS-induced proinflammatory cytokine production seem to ini-tiate new perspectives in the treatment of chronic inflamma-tory diseases also in the central nervous system Howeverrecent studies suggest that the role of p38 MAPK duringinflammation may be more complex and ambiguous [56] Itwas reported that p38MPAK is also involved in the activationof anti-inflammatory processes In myeloid cells activation ofp38MAPK signaling pathway limited inflammation in a UV-induced irradiation model [57] This immunomodulatoryeffect of p38 MAPK seems to be mediated by the induction

of the anti-inflammatory cytokine IL-10 and the inhibitionof proinflammatory IL-12 [56ndash59] It should be noted thatthe results of in vitro study on primary human monocytesdemonstrated that SB203580 produced profound inhibitionof spontaneous production of IL-1 and TNF120572 Unfortunatelyit was also determined that SB203580 significantly increasesLPS-stimulated IL-6 production and decreases the synthesisof anti-inflammatory IL-10 in primary human monocytes[60] These potential proinflammatory effects of SB203580treatment suggest that all therapy using p38MAPK inhibitorsshould be performed cautiously and with analysis conductedon expected and unexpected consequences of the treatment

Conflict of Interests

All of the authors have declared there is no conflict of interestsregarding the publication of this work


Acknowledgment

This research was supported by Grant MNiSW ldquoIuventusPlusrdquo no IP2011 017371

References

[1] M J Kluger ldquoFever role of pyrogens and cryogensrdquo Physiolog-ical Reviews vol 71 no 1 pp 93ndash127 1991

[2] C B Lawrence and N J Rothwell ldquoAnorexic but not pyrogenicactions of interleukin-1 aremodulated by central melanocortin-34 receptors in the ratrdquo Journal of Neuroendocrinology vol 13no 6 pp 490ndash495 2001

[3] A P Herman and D Tomaszewska-Zaremba ldquoEffect of endo-toxin on the expression of GnRH and GnRHR genes in thehypothalamus and anterior pituitary gland of anestrous ewesrdquoAnimal Reproduction Science vol 120 no 1ndash4 pp 105ndash111 2010

[4] O Ebisui J Fukata T Tominaga et al ldquoRoles of interleukin-1120572 and -1120573 in endotoxin-induced suppression of plasmagonadotropin levels in ratsrdquo Endocrinology vol 130 no 6 pp3307ndash3313 1992

[5] M Palomba andM Bentivoglio ldquoChronic inflammation affectsthe photic response of the suprachiasmatic nucleusrdquo Journal ofNeuroimmunology vol 193 no 1-2 pp 24ndash27 2008

[6] M A Erickson and W A Banks ldquoCytokine and chemokineresponses in serum and brain after single and repeated injec-tions of lipopolysaccharide multiplex quantification with pathanalysisrdquo Brain Behavior and Immunity vol 25 no 8 pp 1637ndash1648 2011

[7] A A Creasey P Stevens J Kenney et al ldquoEndotoxin andcytokine profile in plasmaof baboons challengedwith lethal andsublethal Escherichia colirdquo Circulatory Shock vol 33 no 2 pp84ndash91 1991

[8] A P Herman T Misztal A Herman and D Tomaszewska-Zaremba ldquoExpression of Interleukin (IL)-1120573 and IL-1 receptorsgenes in the hypothalamus of anoestrous ewes after lipopolysac-charide treatmentrdquo Reproduction in Domestic Animals vol 45no 6 pp e426ndashe433 2010

[9] M G de Simoni L Terreni R Chiesa F Mangiarotti and G LForloni ldquoInteferon-120574 potentiates interleukin (IL)-6 and tumornecrosis factor- 120572 but not IL-1120573 induced by endotoxin in thebrainrdquo Endocrinology vol 138 no 12 pp 5220ndash5226 1997

[10] W A Banks ldquoCytokines and the blood-brain barrierrdquo in TheNeuroimmunological Basis of Behavior and Mental Disorderspp 3ndash17 Springer 2009

[11] R Dantzer ldquoCytokine sickness behavior and depressionrdquoNeurologic Clinics vol 24 no 3 pp 441ndash460 2006

[12] W A Banks A J Kastin and R D Broadwell ldquoPassage ofcytokines across the blood-brain barrierrdquo NeuroImmunoMod-ulation vol 2 no 4 pp 241ndash248 1995

[13] Y Kakizaki H Watanobe A Kohsaka and T Suda ldquoTemporalprofiles of interleukin-1120573 interleukin-6 and tumor necro-sis factor-120572 in the plasma and hypothalamic paraventricularnucleus after intravenous or intraperitoneal administration oflipopolysaccharide in the rat estimation by push-pull perfu-sionrdquo Endocrine Journal vol 46 no 4 pp 487ndash496 1999

[14] M K Hansen K T Nguyen L E Goehler et al ldquoEffects ofvagotomy on lipopolysaccharide-induced brain interleukin-1120573protein in ratsrdquoAutonomic Neuroscience Basic and Clinical vol85 no 1ndash3 pp 119ndash126 2000

[15] S Laye R-M Bluthe S Kent et al ldquoSubdiaphragmatic vago-tomyblocks induction of IL-1120573mRNA inmice brain in responseto peripheral LPSrdquo American Journal of Physiology RegulatoryIntegrative and Comparative Physiology vol 268 no 5 ppR1327ndashR1331 1995

[16] G P Chrousos ldquoThe stress response and immune functionclinical implications The 1999 Novera H Spector lecturerdquoAnnals of the New York Academy of Sciences vol 917 pp 38ndash672000

[17] P Taishi L Churchill A De F Obal Jr and J M KruegerldquoCytokine mRNA induction by interleukin-1120573 or tumor necro-sis factor 120572 in vitro and in vivordquo Brain Research vol 1226 pp89ndash98 2008

[18] R E Mrak andW S T Griffin ldquoInterleukin-1 and the immuno-genetics of Alzheimer diseaserdquo Journal of Neuropathology andExperimental Neurology vol 59 no 6 pp 471ndash476 2000

[19] L Vitkovic J Bockaert and C Jacque ldquolsquoInflammatoryrsquo cytok-inesrsquo neuromodulators in normal brainrdquo Journal of Neurochem-istry vol 74 no 2 pp 457ndash471 2000

[20] S V Vellucci R F Parrott A C Da Costa S Ohkura and KM Kendrick ldquoIncreased body temperature cortisol secretionand hypothalamic expression of c-fos corticotrophin releas-ing hormone and interleukin-1120573 mRNAs following centraladministration of interleukin-1120573 in the sheeprdquoMolecular BrainResearch vol 29 no 1 pp 64ndash70 1995

[21] A PHerman TMisztal K Romanowicz andDTomaszewska-Zaremba ldquoCentral injection of exogenous IL-1120573 in the controlactivities of hypothalamic-pituitary-gonadal axis in anestrousewesrdquo Reproduction in Domestic Animals vol 47 no 1 pp 44ndash52 2012

[22] S Rivest and C Rivier ldquoCentrally injected interleukin-1 betainhibits the hypothalamic LHRH secretion and circulating LHlevels via prostaglandins in ratsrdquo Journal of Neuroendocrinologyvol 5 no 4 pp 445ndash450 1993

[23] S Kumar M S Jiang J L Adams and J C Lee ldquoPyridinylim-idazole compound SB 203580 inhibits the activity but not theactivation of p38 mitogen-activated protein kinaserdquo Biochem-ical and Biophysical Research Communications vol 263 no 3pp 825ndash831 1999

[24] S P Davies H Reddy M Caivano and P Cohen ldquoSpecificityand mechanism of action of some commonly used proteinkinase inhibitorsrdquo Biochemical Journal vol 351 no 1 pp 95ndash105 2000

[25] J J Baldassare Y Bi andC J Bellone ldquoThe role of p38mitogen-activated protein kinase in IL-1120573 transcriptionrdquo Journal ofImmunology vol 162 no 9 pp 5367ndash5373 1999

[26] J C Lee J T Laydon P C McDonnell et al ldquoA proteinkinase involved in the regulation of inflammatory cytokinebiosynthesisrdquo Nature vol 372 no 6508 pp 739ndash746 1994

[27] C Thirunavukkarasu S C Watkins and C R Gandhi ldquoMech-anisms of endotoxin-induced NO IL-6 and TNF-120572 productionin activated rat hepatic stellate cells role of p38 MAPKrdquoHepatology vol 44 no 2 pp 389ndash398 2006

[28] J Welento S Szteyn and Z Milart ldquoObservations on thestereotaxic configuration of the hypothalamus nuclei in thesheeprdquo Anatomischer Anzeiger vol 124 no 1 pp 1ndash27 1969

[29] R Rasmussen ldquoQuantification on the LightCyclerrdquo in RapidCycle Real-Time PCR Methods and Applications S Meuer CWittwer and K Nakagawara Eds Berlin Germany 2001


[30] M W Pfaffl A Tichopad C Prgomet and T P NeuviansldquoDetermination of stable housekeeping genes differentiallyregulated target genes and sample integrity bestKeeperndashexcel-based tool using pair-wise correlationsrdquo Biotechnology Lettersvol 26 no 6 pp 509ndash515 2004

[31] S Laye G Gheusi S Cremona et al ldquoEndogenous brain IL-1 mediates LPS-induced anorexia and hypothalamic cytokineexpressionrdquoAmerican Journal of Physiology Regulatory Integra-tive and Comparative Physiology vol 279 no 1 pp R93ndashR982000

[32] A J Dunn ldquoEffects of cytokines and infections on brain neu-rochemistryrdquo Clinical Neuroscience Research vol 6 no 1-2 pp52ndash68 2006

[33] N J Busbridge M J Dascombe F J H Tilders J W A MVan Oers E A Linton and N J Rothwell ldquoCentral activationof thermogenesis and fever by interleukin-1120573 and interleukin-1120572 involves different mechanismsrdquo Biochemical and BiophysicalResearch Communications vol 162 no 2 pp 591ndash596 1989

[34] A J Dunn ldquoCytokine activation of the HPA axisrdquo Annals of theNew York Academy of Sciences vol 917 pp 608ndash617 2000

[35] T M OrsquoConnor D J OrsquoHalloran and F Shanahan ldquoThe stressresponse and the hypothalamic-pituitary-adrenal axis frommolecule tomelancholiardquoQJM vol 93 no 6 pp 323ndash333 2000

[36] L Yang K Lindholm Y Konishi R Li and Y Shen ldquoTargetdepletion of distinct tumor necrosis factor receptor subtypesreveals hippocampal neuron death and survival through differ-ent signal transduction pathwaysrdquo Journal of Neuroscience vol22 no 8 pp 3025ndash3032 2002

[37] R A French R W Vanhoy R Chizzonite et al ldquoExpressionand localization of p80 and p68 interleukin-1 receptor proteinsin the brain of adult micerdquo Journal of Neuroimmunology vol 93no 1-2 pp 194ndash202 1999

[38] P Marz K Heese B Dimitriades-Schmutz S Rose-John andU Otten ldquoRole of interleukin-6 and soluble IL-6 receptorin region-specific induction of astrocytic differentiation andneurotrophin expressionrdquo Glia vol 26 no 3 pp 191ndash200 1999

[39] E M-H Ban ldquoInterleukin-1 receptors in the brain character-ization by quantitative in situ autoradiographyrdquo ImmunoMeth-ods vol 5 no 1 pp 31ndash40 1994

[40] A P Herman A Krawczynska J Bochenek et al ldquoInhibi-tion of acetylcholinesterase activity by rivastigmine decreaseslipopolysaccharide-induced IL-1120573 expression in the hypothala-mus of ewesrdquoDomestic Animal Endocrinology vol 44 no 3 pp109ndash114 2013

[41] M-M Gabellec R Griffais G Fillion and F HaourldquoInterleukin-1 receptors type I and type II in the mouse brainkinetics of mRNA expressions after peripheral administrationof bacterial lipopolysacchariderdquo Journal of Neuroimmunologyvol 66 no 1-2 pp 65ndash70 1996

[42] T Takao K Hashimoto and E B de Souza ldquoInterleukin-1 receptors in the brain-endocrine-immune axismdashmodulationby stress and infectionrdquo Annals of the New York Academy ofSciences vol 771 pp 372ndash385 1995

[43] C Patil X Zhu C Rossa Jr Y J Kim and K L Kirkwood ldquop38MAPK regulates IL-1120573 induced IL-6 expression throughmRNAstability in osteoblastsrdquo Immunological Investigations vol 33no 2 pp 213ndash233 2004

[44] A Cuenda and S Rousseau ldquop38 MAP-Kinases pathway reg-ulation function and role in human diseasesrdquo Biochimica etBiophysica Acta Molecular Cell Research vol 1773 no 8 pp1358ndash1375 2007

[45] L Shapiro and C A Dinarello ldquoOsmotic regulation of cytokinesynthesis in vitrordquo Proceedings of the National Academy ofSciences of the United States of America vol 92 no 26 pp12230ndash12234 1995

[46] R Beyaert A Cuenda W V Berghe et al ldquoThe p38RKmitogen-activated protein kinase pathway regulates inter-leukin-6 synthesis in response to tumour necrosis factorrdquoEMBO Journal vol 15 no 8 pp 1914ndash1923 1996

[47] A Simi M Porsmyr-Palmertz A Hjerten M Ingelman-Sundberg and N Tindberg ldquoThe neuroprotective agentschlomethiazole and SB203580 inhibit IL-1120573 signalling but not itsbiosynthesis in rat cortical glial cellsrdquo Journal ofNeurochemistryvol 83 no 3 pp 727ndash737 2002

[48] A M Badger J N Bradbeer B Votta J C Lee J L Adamsand D E Griswold ldquoPharmacological profile of SB 203580 aselective inhibitor of cytokine suppressive binding proteinp38kinase in animal models of arthritis bone resorption endo-toxin shock and immune functionrdquo Journal of Pharmacologyand Experimental Therapeutics vol 279 no 3 pp 1453ndash14611996

[49] W-D Zhou H-M Yang Q Wang et al ldquoSB203580 ap38 mitogen-activated protein kinase inhibitor suppresses thedevelopment of endometriosis by down-regulating proinflam-matory cytokines and proteolytic factors in a mouse modelrdquoHuman Reproduction vol 25 no 12 pp 3110ndash3116 2010

[50] C A Dinarello ldquoAnti-inflammatory agents present and futurerdquoCell vol 140 no 6 pp 935ndash950 2010

[51] Y Fong K J Tracey L L Moldawer et al ldquoAntibodies tocachectintumor necrosis factor reduce interleukin 1120573 andinterleukin 6 appearance during lethal bacteremiardquo Journal ofExperimental Medicine vol 170 no 5 pp 1627ndash1633 1989

[52] R Goldbach-Mansky N J Dailey S W Canna et alldquoNeonatal-onset multisystem inflammatory disease responsiveto interleukin-1120573 inhibitionrdquo New England Journal of Medicinevol 355 no 6 pp 581ndash592 2006

[53] Y Pollak A Gilboa O Ben-Menachem T Ben-Hur H Soreqand R Yirmiya ldquoAcetylcholinesterase inhibitors reduce brainand blood interleukin-1120573 productionrdquoAnnals of Neurology vol57 no 5 pp 741ndash745 2005

[54] L V Borovikova S Ivanova M Zhang et al ldquoVagus nervestimulation attenuates the systemic inflammatory response toendotoxinrdquo Nature vol 405 no 6785 pp 458ndash462 2000

[55] J-MWaldburger D L Boyle M Edgar et al ldquoSpinal p38MAPkinase regulates peripheral cholinergic outflowrdquo Arthritis andRheumatism vol 58 no 9 pp 2919ndash2921 2008

[56] K Katholnig C C Kaltenecker H Hayakawa et al ldquoP38120572senses environmental stress to control innate immuneresponses via mechanistic target of rapamycinrdquo Journal ofImmunology vol 190 no 4 pp 1519ndash1527 2013

[57] C Kim Y Sano K Todorova et al ldquoThe kinase p38120572 serves celltype-specific inflammatory functions in skin injury and coor-dinates pro- and anti-inflammatory gene expressionrdquo NatureImmunology vol 9 no 9 pp 1019ndash1027 2008

[58] Z Yang X Zhang P A Darrah and D M Mosser ldquoTheregulation of Th1 responses by the p38 MAPKrdquo Journal ofImmunology vol 185 no 10 pp 6205ndash6213 2010

[59] X Guo R E Gerl and J W Schrader ldquoDefining the involve-ment of p38120572 MAPK in the production of anti- and proin-flammatory cytokines using an SB 203580-resistant form of thekinaserdquo Journal of Biological Chemistry vol 278 no 25 pp22237ndash22242 2003


[60] T H Page A Brown E M Timms B M J Foxwell andK P Ray ldquoInhibitors of p38 suppress cytokine production inrheumatoid arthritis synovial membranes does variable inhi-bition of interleukin-6 production limit effectiveness in vivordquoArthritis and Rheumatism vol 62 no 11 pp 3221ndash3231 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


transport mechanisms [12] However significant sources ofcentral proinflammatory cytokines may generate their ownsynthesis within the brain tissue including hypothalamus[9 13] This brain synthesis of proinflammatory cytokinescould be induced by a fast neural pathway [11] It wasshown that the electrical stimulation of the vagus nerveinduces the expression of brain IL-1 and vagotomy abrogatesthe induction of expression of brain IL-1 in response tointraperitoneal lipopolysaccharide [14 15] The synthesis ofproinflammatory cytokines in brain structures may be alsostimulated and controlled by the same cytokines originatingfrom the periphery or produced by the circumventricularorgans and choroid plexus This is because proinflamma-tory cytokines amplify their own synthesis and secretion[16] However an important role in the transmission andamplification of inflammatory signals in the brain maycause auto- and paracrine stimulation of proinflammatorycytokines in the brain tissue This autostimulatory effect ofIL-1 on its synthesis was observed inmany cytokine-secretingcells including microglia [17 18] It is well established thatcentral proinflammatory cytokines could directly affect theprocesses regulated at the level of hypothalamus via their cor-responding receptors [19] Acting centrally proinflammatorycytokines especially IL-1120573 are considered to be importantmodulator of endocrine system At the level of hypothalamusIL-1120573 was found to stimulate the hypothalamic-pituitary-adrenal stress axis [20] On the other hand the injection ofexogenous IL-1120573 into the region of hypothalamus suppressedsecretion of gonadoliberine (GnRH) leading to downstreaminhibition of the hypothalamic-pituitary-gonadal axis activ-ity [21 22] Therefore the reduction of proinflammatorycytokines synthesis in the hypothalamic areamay profoundlyattenuate the central response on the peripheral inflamma-tion

The study was designed to determine the effect ofperipheral injection of SB203580 on the synthesis of IL-1120573 IL-6 and TNF120572 in the hypothalamus of ewes duringprolonged inflammation SB203580 is pyridinyl imidazolecompound that is a selective ATP-competitive inhibitor ofthe p38 mitogen-activated protein kinase (MAPK) It haspreviously been established that SB 203580 acts primarilyto block the catalytic activity of p38 MAPK but not itsactivation by upstream MAPK [23] However it is worthmentioning that biochemical analysis of SB203580 activityshowed that this inhibitor lost its specificity when it wasused at high concentration and could also suppress activ-ity of other kinases such as lymphocyte kinase glycogensynthase kinase 3 and protein kinase B [24] The p38MAPK is an important mediator of stress-induced geneexpression In particular the p38 kinase is known to playa key role in LPS-induced signal transduction pathwaysleading to cytokine synthesis [25] Previous studies indi-cated that p38 MAPK is involved in the inflammatory-dependent upregulation of proinflammatory cytokines syn-thesis at the translational and posttranslational levels [2326] It was shown that SB203580 strongly inhibited LPS-stimulated synthesis of IL-6 and TNF-120572 in hepatic stellatecells [27]

2 Materials and Methods

21 Animals The studies were performed on adult 3-year-old Polish mountain sheep in the anestrous season (April-May) All animals were in good condition The body con-dition of all animals was estimated at three- on a five-pointscale The animals were maintained indoors in individualpens and exposed to natural daylight The ewes were welladapted to the experimental conditions and always had visualcontact with neighbouring ewes even during the experimen-tal period This avoided the stress of social isolation Theanimals were fed a constant diet of commercial concentrateswith hay and water constantly available

All procedures on animals were performed with the con-sent of the Local Ethics Committee of theWarsawAgricultureUniversity

22 Experimental Procedures Venous catheters were im-planted into the jugular vein on the day prior to theexperiment The ewes (119899 = 24) were randomly dividedinto four experimental groups group Imdashcontrol (119899 = 6)group IImdashSB203580 treated (119899 = 6) group IIImdashLPS-treated(119899 = 6) group IVmdashLPS- and SB203580 treated (119899 =6) In treated animals the prolonged immune stress wasinduced by the intravenous (iv) administration of LPS fromEscherichia coli 055B5 (Sigma St LouisMOUSA) dissolvedin saline (09wvNaCl) (Baxter Deerfield IL USA) at aconcentration of 10mgL into the jugular vein (400 ngkg)For six days the animals received a single injection of LPSor saline at 8 am On day 7 of the experiment 30mins priorto salineLPS treatment ewes from groups II and IV receivediv injection of SB203580 hydrochloride (500 120583gkg) (AxonMedchem BV Groningen Netherlands) Concurrently theanimals from the control and LPS treated groups received2mLof saline injectionAnimalswere euthanized three hoursafter the LPSsaline injection and the brains were rapidlyremoved from the skulls The blocks of brains encompassinghypothalamus were sectioned sagittally and dissected fromboth sides according to stereotaxic atlas of the sheep brain[28] Landmarks were the mammillary body median emi-nence and optic chiasm The depth of the cuts was 25 to3mm Following collection all tissue was divided into twoand then frozen in liquid nitrogen and stored at minus80∘C

23 ELISA Assay for IL-1120573 IL-6 and TNF120572 Concentrationin the Hypothalamus The concentrations of IL-1120573 IL-6and TNF120572 in the hypothalamus were determined using acommercial IL-1120573 IL-6 and TNF120572 ELISA kit (BlueGeneBiotech CO LTD China) designed for sheep The tissueswere homogenized in 1mL of cold phosphate bufferedsaline (002M) then homogenates were subjected to twofreeze-thaw cycles to further break the cell membranesHomogenates were then centrifuged for 15min at 1500timesg at4∘C The supernatants were aliquoted and stored until assayat minus80∘C All steps in the assays were performed according tothe manufacturerrsquos instructionsThe incubation of plates andabsorbance measurement at 450 nm were performed usinga VersaMax reader (Molecular Devices LLC Sunnyvale





NM 001034034


134 ForwardReverse







115 ForwardReverse





124 ForwardReverse








NM 174674


137 ForwardReverse


NM 001040490


122 ForwardReverse










3 Results



4 Discussion





0

2

4

6

8

10

14

12

16

18

ab

cC

ontro

l

LPS

IL-1120573

(pg

mg

of to

tal p

rote

in)

SB20

3580

LPS+

SB20

3580

(a)

0

5

15

20

25

10

ab

abc

Con

trol

LPS

IL-6

(pg

mg

of to

tal p

rote

in)

SB20

3580

LPS+

SB20

3580

(b)

0

1

2

3

4

5

Con

trol

LPS

TNF120572

(pg

mg

of to

tal p

rote

in)

abSB

2035

80

LPS+

SB20

3580

(c)





0

1

2

3

ab

c

Con

trol

LPS

LPS+

SB20

3580

Rela

tive I

L-1120573

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

(a)

3

4

2

1

0

ab

abc

Con

trol

LPS

Rela

tive I

L-1

R1ve

rsus

GA

PDH

mRN

A le

vel

SB20

3580

LPS+

SB20

3580

(b)


0

3

4

5

1

2

ab

bc

Con

trol

LPS

Rela

tive I

L-6

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

LPS+

SB20

3580

(a)

0

1

2

Con

trol

LPS

Rela

tive I

L-6

R ve

rsus

GA

PDH

mRN

A le

vel

SB20

3580

LPS+

SB20

3580

(b)





0

3

1

2

ab

c

Con

trol

LPS

Rela

tive T

NF120572

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

LPS+

SB20

3580

(a)

0

1

2

Con

trol

LPS

Relat

ive T

NFR1

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

LPS+

SB20

3580

(b)

0

1

2

Con

trol

LPS

Relat

ive T

NFR2

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

LPS+

SB20

3580

(c)








Acknowledgment


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















NM 001034034


134 ForwardReverse







115 ForwardReverse





124 ForwardReverse








NM 174674


137 ForwardReverse


NM 001040490


122 ForwardReverse










3 Results



4 Discussion





0

2

4

6

8

10

14

12

16

18

ab

cC

ontro

l

LPS

IL-1120573

(pg

mg

of to

tal p

rote

in)

SB20

3580

LPS+

SB20

3580

(a)

0

5

15

20

25

10

ab

abc

Con

trol

LPS

IL-6

(pg

mg

of to

tal p

rote

in)

SB20

3580

LPS+

SB20

3580

(b)

0

1

2

3

4

5

Con

trol

LPS

TNF120572

(pg

mg

of to

tal p

rote

in)

abSB

2035

80

LPS+

SB20

3580

(c)





0

1

2

3

ab

c

Con

trol

LPS

LPS+

SB20

3580

Rela

tive I

L-1120573

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

(a)

3

4

2

1

0

ab

abc

Con

trol

LPS

Rela

tive I

L-1

R1ve

rsus

GA

PDH

mRN

A le

vel

SB20

3580

LPS+

SB20

3580

(b)


0

3

4

5

1

2

ab

bc

Con

trol

LPS

Rela

tive I

L-6

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

LPS+

SB20

3580

(a)

0

1

2

Con

trol

LPS

Rela

tive I

L-6

R ve

rsus

GA

PDH

mRN

A le

vel

SB20

3580

LPS+

SB20

3580

(b)





0

3

1

2

ab

c

Con

trol

LPS

Rela

tive T

NF120572

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

LPS+

SB20

3580

(a)

0

1

2

Con

trol

LPS

Relat

ive T

NFR1

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

LPS+

SB20

3580

(b)

0

1

2

Con

trol

LPS

Relat

ive T

NFR2

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

LPS+

SB20

3580

(c)








Acknowledgment


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















3 Results



4 Discussion





0

2

4

6

8

10

14

12

16

18

ab

cC

ontro

l

LPS

IL-1120573

(pg

mg

of to

tal p

rote

in)

SB20

3580

LPS+

SB20

3580

(a)

0

5

15

20

25

10

ab

abc

Con

trol

LPS

IL-6

(pg

mg

of to

tal p

rote

in)

SB20

3580

LPS+

SB20

3580

(b)

0

1

2

3

4

5

Con

trol

LPS

TNF120572

(pg

mg

of to

tal p

rote

in)

abSB

2035

80

LPS+

SB20

3580

(c)





0

1

2

3

ab

c

Con

trol

LPS

LPS+

SB20

3580

Rela

tive I

L-1120573

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

(a)

3

4

2

1

0

ab

abc

Con

trol

LPS

Rela

tive I

L-1

R1ve

rsus

GA

PDH

mRN

A le

vel

SB20

3580

LPS+

SB20

3580

(b)


0

3

4

5

1

2

ab

bc

Con

trol

LPS

Rela

tive I

L-6

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

LPS+

SB20

3580

(a)

0

1

2

Con

trol

LPS

Rela

tive I

L-6

R ve

rsus

GA

PDH

mRN

A le

vel

SB20

3580

LPS+

SB20

3580

(b)





0

3

1

2

ab

c

Con

trol

LPS

Rela

tive T

NF120572

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

LPS+

SB20

3580

(a)

0

1

2

Con

trol

LPS

Relat

ive T

NFR1

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

LPS+

SB20

3580

(b)

0

1

2

Con

trol

LPS

Relat

ive T

NFR2

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

LPS+

SB20

3580

(c)








Acknowledgment


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

2

4

6

8

10

14

12

16

18

ab

cC

ontro

l

LPS

IL-1120573

(pg

mg

of to

tal p

rote

in)

SB20

3580

LPS+

SB20

3580

(a)

0

5

15

20

25

10

ab

abc

Con

trol

LPS

IL-6

(pg

mg

of to

tal p

rote

in)

SB20

3580

LPS+

SB20

3580

(b)

0

1

2

3

4

5

Con

trol

LPS

TNF120572

(pg

mg

of to

tal p

rote

in)

abSB

2035

80

LPS+

SB20

3580

(c)





0

1

2

3

ab

c

Con

trol

LPS

LPS+

SB20

3580

Rela

tive I

L-1120573

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

(a)

3

4

2

1

0

ab

abc

Con

trol

LPS

Rela

tive I

L-1

R1ve

rsus

GA

PDH

mRN

A le

vel

SB20

3580

LPS+

SB20

3580

(b)


0

3

4

5

1

2

ab

bc

Con

trol

LPS

Rela

tive I

L-6

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

LPS+

SB20

3580

(a)

0

1

2

Con

trol

LPS

Rela

tive I

L-6

R ve

rsus

GA

PDH

mRN

A le

vel

SB20

3580

LPS+

SB20

3580

(b)





0

3

1

2

ab

c

Con

trol

LPS

Rela

tive T

NF120572

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

LPS+

SB20

3580

(a)

0

1

2

Con

trol

LPS

Relat

ive T

NFR1

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

LPS+

SB20

3580

(b)

0

1

2

Con

trol

LPS

Relat

ive T

NFR2

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

LPS+

SB20

3580

(c)








Acknowledgment


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

1

2

3

ab

c

Con

trol

LPS

LPS+

SB20

3580

Rela

tive I

L-1120573

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

(a)

3

4

2

1

0

ab

abc

Con

trol

LPS

Rela

tive I

L-1

R1ve

rsus

GA

PDH

mRN

A le

vel

SB20

3580

LPS+

SB20

3580

(b)


0

3

4

5

1

2

ab

bc

Con

trol

LPS

Rela

tive I

L-6

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

LPS+

SB20

3580

(a)

0

1

2

Con

trol

LPS

Rela

tive I

L-6

R ve

rsus

GA

PDH

mRN

A le

vel

SB20

3580

LPS+

SB20

3580

(b)





0

3

1

2

ab

c

Con

trol

LPS

Rela

tive T

NF120572

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

LPS+

SB20

3580

(a)

0

1

2

Con

trol

LPS

Relat

ive T

NFR1

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

LPS+

SB20

3580

(b)

0

1

2

Con

trol

LPS

Relat

ive T

NFR2

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

LPS+

SB20

3580

(c)








Acknowledgment


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

3

1

2

ab

c

Con

trol

LPS

Rela

tive T

NF120572

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

LPS+

SB20

3580

(a)

0

1

2

Con

trol

LPS

Relat

ive T

NFR1

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

LPS+

SB20

3580

(b)

0

1

2

Con

trol

LPS

Relat

ive T

NFR2

vers

us G

APD

Hm

RNA

leve

l

SB20

3580

LPS+

SB20

3580

(c)








Acknowledgment


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Acknowledgment


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article peripheral injection of sb203580 inhibits

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