Research Article Increased ZAP70 Is Involved in Dry Skin Pruritus...

Research ArticleIncreased ZAP70 Is Involved in Dry Skin Pruritus in Aged Mice

Nan Zhao1 Min Gu2 Wenxiu Yang1 Man Zhang2 Qi Tian2

Liyan Ru1 Yang Luuml2 and Weihua Yu1

1 Institute of Neuroscience Department of Human Anatomy Chongqing Medical University Chongqing 400016 China2Department of Geriatrics The First Affiliated Hospital of Chongqing Medical University Chongqing 400016 China

Correspondence should be addressed to Weihua Yu 359127112qqcom

Received 7 December 2015 Revised 26 February 2016 Accepted 3 April 2016

Academic Editor Se K Jeong

Copyright copy 2016 Nan Zhao 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

Dry skin pruritus is common in the elderly Recent reports show that T-cell signal path is involved in dry skin pruritus Zeta-chain-associated protein kinase 70 (ZAP70) as a T-cell receptor may induce interleukin 2 (IL-2) secretion and promote nervegrowth factor (NGF) secretion in skin This study aimed to detect the alteration of ZAP70 in a mice model with dry skin pruritusThe C57BL mice with 5 months and 22 months were used as experimental animal Following a 5-day period of treatment of backwith a mixture of acetone-diethyl-ether-water (AEW) mice exhibited a significant increase in spontaneous scratching behaviordirected to the treated back compared to control animals in which back was similarly treated with water only (W) After AEWprocess spontaneous scratching in 22-month AEW mice was increased compared to 5-month AEW mice Western blot and real-time quantitative PCR data analysis showed that ZAP70 expression was significantly increased in 22-month AEWmice comparedwith 5-month AEWmice ELISA data showed that secretions of IL-2 and NGF in 22-month AEWmice were higher than 5-monthAEW mice Our results indicate that increased ZAP70 is involved in dry skin in elderly pruritus Increased secretion of IL-2 andNGF may induce dry skin itch

1 Introduction

Itch is one of the most important symptoms in inflammatoryskin diseases and allergic disorders It is defined as ldquounpleas-ant sensation eliciting the urge to scratchrdquo Dry skin is one ofthemost pruritic skin diseases It is a primary complaint asso-ciated with xerosis and other dermatoses that compromiseskin barrier integrity such as atopic dermatitis and psoriasis[1 2] Elderly people are believed to be particularly prone toprurituswith dry skin these are problems of high relevance inthe aged population especially in the development of irritantcontact dermatitis [3] As the prevalence of atopy continuesto rise it becomes increasingly important that cliniciansunderstand the mechanisms responsible for itch and inparticular dry skin itch [4] However the mechanism of dryskin pruritus is poorly understood All of importance T cellsdirectly communicate with nerves to regulate neurogenicinflammation of pain and are involved in pruritus as well [5ndash7] Thus immune response may explain the mechanism ofdry skin pruritus

Zeta-chain-associated protein kinase 70 (ZAP70) is acytosolic protein and it is recruited at the plasma membraneof T cells following TCR stimulation It regulates thymocytedevelopment as well as motility adhesion and cytokinesecretion of mature T cells including interleukin 2 (IL-2)[8] Also epidermal hyperinnervation is considered as onecause of itch sensitization and is thought to be regulated bykeratinocyte-derived axonal guidance molecules includingnerve growth factor (NGF)These cytokines were explicit andinvolved in dry skin pruritus [9 10]

This paper presumes ZAP70 may be involved in dryskin pruritus via promoting cytokines secretion especiallyin the elderly In our study a mouse model of dry skin itchwas used to investigate presumptive mechanism Behavioralobservation shows that spontaneous scratching in elderlyAEW mice was much serious than young AEW mice Dataanalyses revealed that expression of ZAP70 was significantlyincreased in elderly AEW group compared to young AEWgroup This study shows the alteration of ZAP70 in elderlymice with dry skin pruritus Our result first shows that

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 6029538 6 pageshttpdxdoiorg10115520166029538

2 BioMed Research International

increased ZAP70 expression might regulate dry skin pruritusin the elderly mice

2 Materials and Methods

21 Experimental Animals All experimental procedureswere approved by the Ethics Committees of ChongqingMedical University All of C57BL mice with elderly group(22 months 119899 = 10 unlimited gender) and young group (5months 119899 = 10 unlimited gender) from the ExperimentalAnimal Center of Chongqing Medical University were usedin the present study

Acetone-diethyl-ether-water (AEW) model as an inex-pensive and stable model that imitated dry skin conditionaccurately has become an internationally recognized animalmodel of chronic itch It is proved that AEW model hasevidence for the dryness with transepidermal water loss(TEWL) andmeasuring hydration test [2 11ndash15] AEWmodelof dry skin pruritus was performed as follows The hair ofmice was shaved over the rostral part of the back at least3 days before the start of the experiment Then we treatedthe back skin by cotton (3 times 3 cm2) soaked with a 1 1mixture of acetone and diethyl-ether (AE) which was laidupon the shaved area for 15 s After AE treatment cottonsoaked with distilled water was laid upon the same areafor 30 s immediately The treatment was performed twicedaily (900 and 1700) under ether anesthesia such treatmentwas repeated once daily for 5 days Cotton soaked withphysiological saline solution was laid on the shaved area for60 s as control group

22 Behavioral Observation Scratching of mice wasobserved by videotape Briefly six mice were put individuallyinto an acrylic box (26 times 18 times 30 cm3) composed of six cellsThey were acclimated to the experimental environment forat least 30min and then behaviors were videotaped withexperimenters kept out of the observation room in themorning Playback served for counting the scratching ofthe treated area by the hind paws the mouse generally asone bout of scratching Thus we observed scratching of themouse at least 1 h after the treatment for barrier disruptionon the previous day

23 Immunohistochemistry The control and model micewere beheaded after being anesthetized with pentobarbital(1mgg ip) After paraffin embedding the skin tissues werethen sectioned at 5mm thickness for immunohistochemistrySkin was fixed in 4 paraformaldehyde for 1 h blocked by1 BSAPBS for 2 h and then immunostained with a purifiedpolyclonal rabbit antibody ZAP70 (1 100 Sigma) and thensections were washed and incubated with the secondaryantibody directed against rabbit IgG (1 200 Dianova) Fol-lowing tissue sections were incubatedwith Streptavidin-HRPSeveral visual fields images in a tissue section were collectedrandomly by the OLYMPUS PM20 automatic microscope(Olympus Tokyo Japan)

24 Western Blotting Protein concentrations were estimatedby the background-corrected absorbance (BCA) method

(Beyotime Biotechnology) Tissue samples (5 120583Llane) wererun in 10 of Tris-glycine gels depending on the molec-ular weight of the protein of interest and transferred ontoPVDF membranes (Millipore) The PVDF membranes wereblocked in 5 bovine serum albumin (Genview) for 2 hThe PVDF membranes were incubated with primary anti-bodies rabbit anti-ZP70 antibody (1 750 AB319 Sigma-Aldrich Saint Louis MO) and mouse anti-120573-actin as controlantibody (1 1000 Beyotime China) followed by incubationwith anti-mouse (Beyotime Biotechnology) and anti-rabbit(Beyotime Biotechnology) immunoglobulin conjugated tohorseradish peroxidase Finally membranes were incubatedwith enhanced chemiluminescence (ECL) and were analyzedby gelluminescence image analysis system

25 Real-Time Quantitative PCR Analysis of ZAP-70 TotalRNA was purified from cell tissues and used for reversetranscription PCR was performed on ZAP-70 primers(forward primer 51015840-ACGCGCCAGAGTGCATCAA-31015840reverse primer 51015840-CTCGGGGCCCTTCATTTTCTT-31015840) orGAPDH internal control primers (forward primer 51015840-GGC-CGGTGCTGAGTATGTCGTG-31015840 reverse primer 51015840-GGG-GGGCTAAGCAGTTGGTGG-31015840) The real-time qPCR wasperformed on a Strata gene Mx3000P instrument

26 ELISA IL-2 and NGF values were assayed with anenzyme-linked immunosorbent assay (ELISA) (mice IL-2and NGF high sensitivity ELISA with Signal AmplificationeBioscience) in the skin of AEW model mice and controlsubjects according to the manufacturerrsquos instructions

27 Statistical Analysis Statistical comparisons were per-formed with two-way repeated measured ANOVA or Stu-dentrsquos 119905-test All data are expressed as the mean plusmn SEM 119875 lt005 was considered significant

3 Results

31 Elderly AEW Mice Showed a Serious Pruritus Comparedto Young AEW Mice Behavior data analysis in elderly AEWgroup was significantly increased compared to young AEWgroup which demonstrates that dry skin pruritus in elderlymice was more serious than in young mice The averagenumber in 1 hour of elderly AEW group was 218 bouts andthe young AEW group was 118 bouts (Figure 1)

32 The Expression of ZAP70 Was Significantly Increased inElderly AEW Mice with Dry Skin Pruritus To confirm thelocalization of ZAP70 in skin we performed the ZAP70antibody to label its location in the mice skin by immuno-histochemistry Immunohistochemistry result shows thatZAP70 was ubiquitous localized in corium especially in theroot of hair follicle (Figure 2)

The protein levels of ZAP70 expression were tested bywestern blot Western blot data analysis shows that ZAP70protein immunoreactive band was observed at about 70KDfrom the shaved skin tissue and 120573-actin (42KD) was used asan internal control The ratio of mean optical density (MOD)of total ZAP70 expression in model group was obviously

BioMed Research International 3

0

50

100

150

200

250

Young control Young AEW Elderly control Elderly AEWSp

onta

neou

s scr

atch

ing

lowast

Figure 1 The numbers of spontaneous scratching after 1 hour of AEW treatment The numbers of scratching were 218 bouts in elderly AEWgroup and 118 bouts in young AEW group respectively lowastRepresented statistic difference of numbers of scratching between elderly AEWgroup and young AEW group (119875 lt 005)

(a) (b)

(c) (d)

Figure 2 The expression of ZAP70 in the skin (a) Young control group (b) young AEW group (c) elderly control group and (d) elderlyAEW groupThe arrowmeant enlarged parts of the root of hair follicle In hair follicle blue area represented the negative for ZAP70 in youngcontrol group brown area represented the positive for ZAP70 in other groups Objective magnification in 200

higher than control group The protein expression of ZAP70in elderly AEW group was statistically significantly increasedcompared to youngAEWgroup (184plusmn 0091 versus 17plusmn 006119875 lt 005 Figure 3)

Moreover we used real-time PCR to test themRNA levelsof ZAP70 The results of real-time PCR showed a similartrend with western bolt for ZAP70 The mRNA level ofZAP70 in elderly AEW group was increased compared toyoung AEW group (861 plusmn 020 versus 358 plusmn 018 119875 lt 005Figure 4)

33 IL-2 and NGF Were Increased in the Skin in Elderly Micewith Dry Skin Pruritus Furthermore we used IL-2 and NGFhigh sensitivitymice ELISAKit to test the cytokines secretionin skin between young and elderly mice The ELISA dataanalysis shows that the levels of IL-2 in skin in elderly AEW

group have an obvious increase compared to young AEWgroup (6373 plusmn 192 versus 4047 plusmn 157 119875 lt 005) AlsoNGF secretion in elderly AEW group was increased in skincompared to young AEW group (1638 plusmn 028 versus 1482 plusmn028 119875 lt 005) (Figure 5)

4 Discussion

Our results reveal that elderly mice have a more seriouspruritus than young mice Subsequently we find that ZAP70expression in elderly AEW group is statistically increasedcompared to young AEW group The higher expression ofZAP70 in skin may explain the mechanism of dry skin pruri-tus in elderly Also IL-2 and NGF secretion are increased inelderly group compared to young group


1 2 3 4

120573-actin (46KDa)

ZAP70 (70KDa)

(a)

0

05

1

15

2

Elderly control Elderly AEW Young control Young AEW

ZAP7

0 ex

pres

sion

lowast

(b)

Figure 3 Western blotting analysis showed the expression of ZAP70 protein in skin (a) showed ZAP70 protein levels Lane 1 representedelderly control group lane 2 represented elderly AEW group lane 3 represented young control group lane 4 represented young AEW group120573-actin was used as a housekeeping protein (b) Histogram represented the expression of ZAP70 protein levels in the skin of young groupand elderly group lowastRepresented statistic difference of protein level between elderly AEW group and young AEW group (119875 lt 005)

0123456789

10

Young control Young AEW Elderly control Elderly AEW

lowast

2(minusΔΔ

Ct)

mea

n va

lue

Figure 4 Real-time quantitative PCR displayed the mRNA levels of ZAP70 expression Histogram revealed the gene levels of ZAP70 in theskin of young group and elderly group lowastRepresented statistic difference of mRNA level between elderly AEW group and young AEW group(119875 lt 005)

The AEW process induced continuous changes in thefunction and lesions of the skin which indeed correlate withincreased activation-regulated chemokine (TARC) level [16]The increased level of TARC contributes to the recruitmentof lymphocytes monocytes and polymorphonuclear cells tothe skin [17] According to recent research TARC is involvedin T-cell migration which is associated with dry skin pruritus[18] With the increased lymphocytes of recruitment theexpression of ZAP70 is increased in skin Then increasedZAP70 activates T-cell signals and promotes cytokines secre-tion

Previous studies demonstrate that ZAP-70 reconstituteinduction of IL-2 promoter activity which regulate for IL-2 secretion and produced high level of IL-2 [19 20] IL-2 isused in the treatment of metastatic cancer but induced severepruritus [21ndash23] Moreover IL-2 promotes T-cell differenti-ation into Th2 cells and suppressed Th1 cells which play animportant pathogenetic role in pruritic dry skin diseases [24]Based on these discussions the increased ZAP70 cause IL-2 increased and severe pruritus As for the increased levelof NGF recent study finds the correlations between levelsof NGF and TARC in cutaneous T-cell lymphoma (CTCL)patients who experience severe pruritus NGF associate withincreased dermal nerve fibers and induce pruritus whichmaybe via TARCThe enhancedNGF expression by keratinocytesand increased dermal nerve fibers are detected in lesion skin

[25] Also hyperinnervation is considered as one cause of itchsensitization

Age has a significant impact on dry skin pruritus Firstlyaging is related to physiological metabolic changes affectsall persons and is inevitable [26] It is characterized by thethinning of the upper cutaneous layers a decrease in theamount of lipids diminished blood flow and fragmentationof collagen [26] Decreased sebaceous and sweat glandactivity combined with impaired skin permeability leads todry skin (xerosis) which is extremely common in elderlysubjects Secondly previous studies demonstrate that afterAEW process treatment the barrier recovered more slowly inaged skin [27] Based on the previous studies elderly micehave more susceptibility to irritant than young mice and thecontinuous changes in lesions may be more serious in agedskin Thus we suppose that elderly skin conditions induce amore serious pruritus and a high level of TARC secretion

In conclusion our study demonstrates that increasedZAP70 participates in the development of dry skin in elderpruritus via secretion of IL-2 and NGF

Competing Interests

The authors declare that they do not have any commercialor associative interests that represent competing interests inconnection with the work submitted


0

10

20

30

40

50

60

70


The l

evels

of I

L-2

(ng

mL)

lowast

(a)

02468

1012141618


The l

evels

of N

GF

(ng

mL)

lowast

(b)

Figure 5 IL-2 and NGF ELISA Kits were used to test the cytokines secretion (a) represented the IL-2 secretion in skin of young and elderlygroup (b) represented the NGF secretion in the skin of young and elderly group lowastRepresented statistic difference of cytokines levels betweenelderly AEW group and young AEW group (119875 lt 005)

Authorsrsquo Contributions

Nan Zhao and Min Gu contributed equally to this workIn this research Nan Zhao was mainly involved in theexperimental operation and writing Min Gu was mainlyinvolved in the data collectionWenxiu Yang andMan Zhangmainly provided technical guidance Qi Tian and Liyan Ruprovided western blot experiment help Professor Weihua Yuand Professor Yang Lu perfected the design of the experimentand provided advice and guidance

Acknowledgments

This study was supported by Graduate Innovation Fund ofChongqing (CYS14128) The authors thank Professor Shan-Quan Sun for providing the experimental facilities and QianFeng for help with image acquisition

References

[1] U Raap S Weiszligmantel M Gehring A M Eisenberg AKapp and R Folster-Holst ldquoIL-31 significantly correlates withdisease activity and Th2 cytokine levels in children with atopicdermatitisrdquo Pediatric Allergy and Immunology vol 23 no 3 pp285ndash288 2012

[2] M V Valtcheva V K Samineni J P Golden R W Gereauand S Davidson ldquoEnhanced nonpeptidergic intraepidermalfiber density and an expanded subset of chloroquine-responsivetrigeminal neurons in a mouse model of dry skin itchrdquo TheJournal of Pain vol 16 no 4 pp 346ndash356 2015

[3] F Seyfarth S Schliemann D Antonov and P Elsner ldquoDry skinbarrier function and irritant contact dermatitis in the elderlyrdquoClinics in Dermatology vol 29 no 1 pp 31ndash36 2011

[4] E Weisshaar and F Dalgard ldquoEpidemiology of itch adding tothe burden of skin morbidityrdquoActa Dermato-Venereologica vol89 no 4 pp 339ndash350 2009

[5] S Bonness and T Bieber ldquoMolecular basis of atopic dermatitisrdquoCurrent Opinion in Allergy and Clinical Immunology vol 7 no5 pp 382ndash386 2007

[6] P Y Ong and D Y M Leung ldquoImmune dysregulation in atopicdermatitisrdquo Current Allergy and Asthma Reports vol 6 no 5pp 384ndash389 2006

[7] G Yosipovitch ldquoAssessment of itch more to be learned andimprovements to be maderdquoThe Journal of Investigative Derma-tology vol 121 no 6 pp 14ndash15 2003

[8] A Fischer C Picard K Chemin S Dogniaux F Le Deist andC Hivroz ldquoZAP70 a master regulator of adaptive immunityrdquoSeminars in Immunopathology vol 32 no 2 pp 107ndash116 2010

[9] A KamoM Tominaga K TanedaHOgawa andK TakamorildquoNeurotropin inhibits the increase in intraepidermal nervedensity in the acetone-treated dry-skin mouse modelrdquo Clinicaland Experimental Dermatology vol 38 no 6 pp 665ndash668 2013

[10] M Nakamura M Toyoda and M Morohashi ldquoPruritogenicmediators in psoriasis vulgaris comparative evaluation of itch-associated cutaneous factorsrdquo British Journal of Dermatologyvol 149 no 4 pp 718ndash730 2003

[11] J A Slade Shantz Y-Y Yu W Andres T Miclau III and RMarcucio ldquoModulation of macrophage activity during fracturerepair has differential effects in young adult and elderly micerdquoJournal of Orthopaedic Trauma vol 28 supplement 1 pp S10ndashS14 2014

[12] C L Krone G Biesbroek K Trzcinski E A M Sandersand D Bogaert ldquoRespiratory microbiota dynamics followingStreptococcus pneumoniae acquisition in young and elderlymicerdquo Infection and Immunity vol 82 no 4 pp 1725ndash1731 2014

[13] P Durairaj and S C Miller ldquoNeoplasm prevention andimmuno-enhancement mediated by daily consumption of aproprietary extract from North American ginseng by elderlymice of a cancer-prone strainrdquo Phytotherapy Research vol 27no 9 pp 1339ndash1344 2013

[14] T Miyamoto H Nojima T Shinkado T Nakahashi and YKuraishi ldquoItch-associated response induced by experimentaldry skin in micerdquo Japanese Journal of Pharmacology vol 88 no3 pp 285ndash292 2002

[15] T Akiyama M I Carstens and E Carstens ldquoSpontaneous itchin the absence of hyperalgesia in a mouse hindpaw dry skinmodelrdquo Neuroscience Letters vol 484 no 1 pp 62ndash65 2010

[16] M Furue T Matsumoto T Yamamoto et al ldquoCorrelationbetween serum thymus and activation-regulated chemokinelevels and stratum corneum barrier function in healthy indi-viduals and patients with mild atopic dermatitisrdquo Journal ofDermatological Science vol 66 no 1 pp 60ndash63 2012

[17] S R Dillon C Sprecher A Hammond et al ldquoInterleukin 31a cytokine produced by activated T cells induces dermatitis inmicerdquo Nature Immunology vol 5 no 7 pp 752ndash760 2004


[18] T Kakinuma K Nakamura M Wakugawa et al ldquoThymusand activation-regulated chemokine in atopic dermatitis serumthymus and activation-regulated chemokine level is closelyrelated with disease activityrdquo Journal of Allergy and ClinicalImmunology vol 107 no 3 pp 535ndash541 2001

[19] A C Chan M Dalton R Johnson et al ldquoActivation of ZAP-70kinase activity by phosphorylation of tyrosine 493 is requiredfor lymphocyte antigen receptor functionrdquoThe EMBO Journalvol 14 no 11 pp 2499ndash2508 1995

[20] A Hussain D K Mohammad M O Gustafsson et al ldquoSig-naling of the ITK (interleukin 2-inducible T cell kinase)-SYK(spleen tyrosine kinase) fusion kinase is dependent on adapterSLP-76 and on the adapter function of the kinases SYK andzap70rdquoThe Journal of Biological Chemistry vol 288 no 10 pp7338ndash7350 2013

[21] A E Kremer J Feramisco P W Reeh U Beuers and RP J Oude Elferink ldquoReceptors cells and circuits involved inpruritus of systemic disordersrdquo Biochimica et Biophysica Acta(BBA)mdashMolecular Basis of Disease vol 1842 no 7 pp 869ndash8922014

[22] R E Lee A A Gaspari M T Lotze A E Chang andS A Rosenberg ldquoInterleukin 2 and psoriasisrdquo Archives ofDermatology vol 124 no 12 pp 1811ndash1815 1988

[23] A A Gaspari M T Lotze S A Rosenberg J B Stern andS I Katz ldquoDermatologic changes associated with interleukin2 administrationrdquo The Journal of the American Medical Associ-ation vol 258 no 12 pp 1624ndash1629 1987

[24] W-J Guo H Qi C-Y Deng H-X Zhou S-P Deng and F-R Li ldquoThe roles of IL-2 and IL-10 enhance anti-CD45RBmAbimmune inhibition in allograft skinrdquo Immunologic Research vol61 no 3 pp 250ndash259 2015

[25] H Suga M Sugaya T Miyagaki et al ldquoAssociation of nervegrowth factor chemokine (C-C motif) ligands and immuno-globulin e with pruritus in cutaneous T-cell lymphomardquo ActaDermato-Venereologica vol 93 no 2 pp 144ndash149 2013

[26] H Hashizume ldquoSkin aging and dry skinrdquo Journal of Dermatol-ogy vol 31 no 8 pp 603ndash609 2004

[27] P M Elias and R Ghadially ldquoThe aged epidermal permeabilitybarrier basis for functional abnormalitiesrdquo Clinics in GeriatricMedicine vol 18 no 1 pp 103ndash120 2002

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


increased ZAP70 expression might regulate dry skin pruritusin the elderly mice

2 Materials and Methods

21 Experimental Animals All experimental procedureswere approved by the Ethics Committees of ChongqingMedical University All of C57BL mice with elderly group(22 months 119899 = 10 unlimited gender) and young group (5months 119899 = 10 unlimited gender) from the ExperimentalAnimal Center of Chongqing Medical University were usedin the present study

Acetone-diethyl-ether-water (AEW) model as an inex-pensive and stable model that imitated dry skin conditionaccurately has become an internationally recognized animalmodel of chronic itch It is proved that AEW model hasevidence for the dryness with transepidermal water loss(TEWL) andmeasuring hydration test [2 11ndash15] AEWmodelof dry skin pruritus was performed as follows The hair ofmice was shaved over the rostral part of the back at least3 days before the start of the experiment Then we treatedthe back skin by cotton (3 times 3 cm2) soaked with a 1 1mixture of acetone and diethyl-ether (AE) which was laidupon the shaved area for 15 s After AE treatment cottonsoaked with distilled water was laid upon the same areafor 30 s immediately The treatment was performed twicedaily (900 and 1700) under ether anesthesia such treatmentwas repeated once daily for 5 days Cotton soaked withphysiological saline solution was laid on the shaved area for60 s as control group

22 Behavioral Observation Scratching of mice wasobserved by videotape Briefly six mice were put individuallyinto an acrylic box (26 times 18 times 30 cm3) composed of six cellsThey were acclimated to the experimental environment forat least 30min and then behaviors were videotaped withexperimenters kept out of the observation room in themorning Playback served for counting the scratching ofthe treated area by the hind paws the mouse generally asone bout of scratching Thus we observed scratching of themouse at least 1 h after the treatment for barrier disruptionon the previous day

23 Immunohistochemistry The control and model micewere beheaded after being anesthetized with pentobarbital(1mgg ip) After paraffin embedding the skin tissues werethen sectioned at 5mm thickness for immunohistochemistrySkin was fixed in 4 paraformaldehyde for 1 h blocked by1 BSAPBS for 2 h and then immunostained with a purifiedpolyclonal rabbit antibody ZAP70 (1 100 Sigma) and thensections were washed and incubated with the secondaryantibody directed against rabbit IgG (1 200 Dianova) Fol-lowing tissue sections were incubatedwith Streptavidin-HRPSeveral visual fields images in a tissue section were collectedrandomly by the OLYMPUS PM20 automatic microscope(Olympus Tokyo Japan)

24 Western Blotting Protein concentrations were estimatedby the background-corrected absorbance (BCA) method

(Beyotime Biotechnology) Tissue samples (5 120583Llane) wererun in 10 of Tris-glycine gels depending on the molec-ular weight of the protein of interest and transferred ontoPVDF membranes (Millipore) The PVDF membranes wereblocked in 5 bovine serum albumin (Genview) for 2 hThe PVDF membranes were incubated with primary anti-bodies rabbit anti-ZP70 antibody (1 750 AB319 Sigma-Aldrich Saint Louis MO) and mouse anti-120573-actin as controlantibody (1 1000 Beyotime China) followed by incubationwith anti-mouse (Beyotime Biotechnology) and anti-rabbit(Beyotime Biotechnology) immunoglobulin conjugated tohorseradish peroxidase Finally membranes were incubatedwith enhanced chemiluminescence (ECL) and were analyzedby gelluminescence image analysis system

25 Real-Time Quantitative PCR Analysis of ZAP-70 TotalRNA was purified from cell tissues and used for reversetranscription PCR was performed on ZAP-70 primers(forward primer 51015840-ACGCGCCAGAGTGCATCAA-31015840reverse primer 51015840-CTCGGGGCCCTTCATTTTCTT-31015840) orGAPDH internal control primers (forward primer 51015840-GGC-CGGTGCTGAGTATGTCGTG-31015840 reverse primer 51015840-GGG-GGGCTAAGCAGTTGGTGG-31015840) The real-time qPCR wasperformed on a Strata gene Mx3000P instrument

26 ELISA IL-2 and NGF values were assayed with anenzyme-linked immunosorbent assay (ELISA) (mice IL-2and NGF high sensitivity ELISA with Signal AmplificationeBioscience) in the skin of AEW model mice and controlsubjects according to the manufacturerrsquos instructions

27 Statistical Analysis Statistical comparisons were per-formed with two-way repeated measured ANOVA or Stu-dentrsquos 119905-test All data are expressed as the mean plusmn SEM 119875 lt005 was considered significant

3 Results

31 Elderly AEW Mice Showed a Serious Pruritus Comparedto Young AEW Mice Behavior data analysis in elderly AEWgroup was significantly increased compared to young AEWgroup which demonstrates that dry skin pruritus in elderlymice was more serious than in young mice The averagenumber in 1 hour of elderly AEW group was 218 bouts andthe young AEW group was 118 bouts (Figure 1)

32 The Expression of ZAP70 Was Significantly Increased inElderly AEW Mice with Dry Skin Pruritus To confirm thelocalization of ZAP70 in skin we performed the ZAP70antibody to label its location in the mice skin by immuno-histochemistry Immunohistochemistry result shows thatZAP70 was ubiquitous localized in corium especially in theroot of hair follicle (Figure 2)

The protein levels of ZAP70 expression were tested bywestern blot Western blot data analysis shows that ZAP70protein immunoreactive band was observed at about 70KDfrom the shaved skin tissue and 120573-actin (42KD) was used asan internal control The ratio of mean optical density (MOD)of total ZAP70 expression in model group was obviously


0

50

100

150

200

250


onta

neou

s scr

atch

ing

lowast


(a) (b)

(c) (d)






4 Discussion



1 2 3 4


ZAP70 (70KDa)

(a)

0

05

1

15

2


ZAP7

0 ex

pres

sion

lowast

(b)


0123456789

10


lowast

2(minusΔΔ

Ct)

mea

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lue







Competing Interests



0

10

20

30

40

50

60

70


The l

evels

of I

L-2

(ng

mL)

lowast

(a)

02468

1012141618


The l

evels

of N

GF

(ng

mL)

lowast

(b)




Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

150

200

250


onta

neou

s scr

atch

ing

lowast


(a) (b)

(c) (d)






4 Discussion



1 2 3 4


ZAP70 (70KDa)

(a)

0

05

1

15

2


ZAP7

0 ex

pres

sion

lowast

(b)


0123456789

10


lowast

2(minusΔΔ

Ct)

mea

n va

lue







Competing Interests



0

10

20

30

40

50

60

70


The l

evels

of I

L-2

(ng

mL)

lowast

(a)

02468

1012141618


The l

evels

of N

GF

(ng

mL)

lowast

(b)




Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















1 2 3 4


ZAP70 (70KDa)

(a)

0

05

1

15

2


ZAP7

0 ex

pres

sion

lowast

(b)


0123456789

10


lowast

2(minusΔΔ

Ct)

mea

n va

lue







Competing Interests



0

10

20

30

40

50

60

70


The l

evels

of I

L-2

(ng

mL)

lowast

(a)

02468

1012141618


The l

evels

of N

GF

(ng

mL)

lowast

(b)




Acknowledgments


References


































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

10

20

30

40

50

60

70


The l

evels

of I

L-2

(ng

mL)

lowast

(a)

02468

1012141618


The l

evels

of N

GF

(ng

mL)

lowast

(b)




Acknowledgments


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 Increased ZAP70 Is Involved in Dry Skin Pruritus...

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