research article expression of caspase-1 gene transcript...

Research ArticleExpression of Caspase-1 Gene Transcript Variant mRNA inPeripheral Blood Mononuclear Cells of Patients with PrimaryGout in Different TCM Syndromes

Wan-Tai Dang12 Dan Xu3 Wen-Guang Xie2 and Jing-Guo Zhou2

1School of Clinical Medicine Chengdu University of Traditional Chinese Medicine Chengdu 610075 China2Institute of Rheumatology and Immunology Affiliated Hospital of North Sichuan Medical College Nanchong 637000 China3Nephrology Department Affiliated Hospital of North Sichuan Medical College Nanchong 637000 China

Correspondence should be addressed to Jing-Guo Zhou jgzhounsmceducn

Received 3 January 2015 Revised 15 March 2015 Accepted 16 March 2015

Academic Editor Klaus Heese

Copyright copy 2015 Wan-Tai Dang 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

A large number of studies have shown that cysteinyl aspartate specific protease-1 (CASP1) played an important role in theinflammatory response of primary gout but the decreased expression of different CASP1 transcript variant could inhibit theactivation of IL-1120573 Our study mainly analyzed the expression level and function of CASP1 gene transcript variant mRNA inperipheral blood mononuclear cells of patients with gout in different TCM syndromes The expression of CASP1 gene transcriptvariant and IL-1120573mRNA in PBMCswere detected in patients with PG [acute phase (AP 44 cases) nonacute phase (NAP 52 cases)]and healthy controls (HC 30 cases) by reverse transcription-polymerase chain reaction andor real-time quantitative polymerasechain reaction The expressions of plasma IL-1120573 in patients with PG and HC were detected by enzyme-linked immunosorbentassay Dysregulated expression of the CASP1 gene and its transcript variant plasma proinflammatory cytokines in all patients withprimary gout in different TCM syndromes correlation analysis showed that there was negative correlation between the expressionof CASP1-gamma gene transcript variant mRNA and IL-1120573 protein in APPG group The study suggested that CASP1 gene and itstranscript variantmay play a critical role in the inflammatory response of patients with PG in different phases and TCM syndromes

1 Introduction

Gout is a clinical syndrome which is attributed to precipi-tation and deposition of monosodium urate (MSU) crystalson the tissue or organ caused by purine dysbolism andorexcretion reduction and continuous elevation of uric acidand it belongs tometabolic rheumatism [1] Gout is similar toLijei or severe andmigratory arthralgia in traditional Chinesemedicine its early elaboration is reported in Ge Zhi Yu Lunwritten by Zhu DanXi in which the pathogenesis of goutwas regarded as phlegm wind-heat wind-wet and blooddeficiency Then some doctors classified migratory Bi syn-drome or painful Bi syndrome of Bi syndrome as gout Recentresearches have showed that inflammation and immunity arealso involved in the pathogenesis of gout besides metabolismfactors [2] We know that the MSU released by aging anddeath cells in the body is endogenous danger-associated

molecular patterns (DAMPs) caused by inflammation andapoptosis through innate immunity [3] Cysteinyl aspartatespecific protease-1 (CASP1) is also called IL-1120573 invertase isinvolved mainly in regulation of inflammation and plays animportant role in the inflammatory response [4] Luksch et al[5] found that the decreased expression of different CASP1gene transcript variant could reduce the activation of the IL-1120573 Recent study showed that CASP1 played a key role in thecourse of gout [6 7] However there was no study report-ing the role of CASP1 gene transcript variant in differenttraditional Chinese medicine (TCM) syndromes of primarygout yet In our study the expression level of CASP1 genetranscript variant mRNA in peripheral blood mononuclearcells (PBMCs) of patients with primary gout (PG) in differentTCM syndromes was measured by semiquantitative reversetranscription-polymerase chain reaction (RT-PCR) andorreal-time quantitative polymerase chain reaction (qRT-PCR)

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2015 Article ID 361607 9 pageshttpdxdoiorg1011552015361607

2 Evidence-Based Complementary and Alternative Medicine

in the meantime the interleukin 1120573 was measured to explorethe role of CASP1 gene and its transcript variant in thepathogenesis of gout

2 Methods

21 The Clinical Data All the research objects conformed tothe 1977 American Rheumatism Association (ACR) diagnos-tic criteria [8] excluding the objects which have secondarygout caused by diseases of kidney cardiovascular and bloodsystem or drugs and so on and also excluding coinfectionautoimmune diseases long-term use of hormone therapyor serious condition that may affect the efficacy and safetyof our study [9 10] 96 male patients with PG who visitedthe Department of Rheumatology of the Affiliated Hospitalof North Sichuan Medical College from December 2012 toOctober 2013 were admitted among them 44 patients were inacute phase and 52 patients were in nonacute phase The ageof patients ranged from 23 to 79 years old and the mean agewas 40 plusmn 11 years with their disease course being 9 plusmn 3 years30 healthy people set as healthy controls (HC) were admittedfrom the Department of Physical Examination in the samehospital during the same period whose age ranged from 22 to70 years their mean age was 44plusmn7 years and their laboratoryindexes were normal excluding the people who had diseasesor family history of cardiovascular disease diabetes livergout and so on [10]The age between the two groups showedno statistical significance The study has gained approval andagreement of the local ethics committee and all participantssigned informed consent

22 The TCM Syndromes Based on the gout TCM syn-drome differentiation in Clinical Diagnosis and TreatmentTerminology of Traditional Chinese Medicine-Syndrome TypePart [11] and Guideline of the Study on New TraditionalChinese Medicine [12] and the TCM differentiation accordingto comprehensive analysis by the ldquofour examinationmethodsrdquoldquoEight Principle Pattern Identificationrdquo and ldquoZang-fu patternidentificationrdquo the TCM syndromes differentiation of PGpatients was divided into four types obstruction of damp-ness and heat syndrome (ODHS) intermingled phlegm-stasis blood syndrome (IPSBS) Pi-deficiency induced damp-ness syndrome (PDIDS) and Qi-blood deficiency syndrome(QBDS) [13]

23 Main Reagents and Instruments Human lymphocytesseparation liquid (Batch number LTS10771) was productof Jingyang Tech Co China RNAiso Plus Reagent (Batchnumber A9701-1) PrimeScript RT reagent kit with gDNAeraser (Perfect qRT-PCR) kit (Batch number AK1801) SYBRPremix Ex Taq II (Batch number AK5004) and TaKaRa LATaq kit (CKA4501A) were products of Takara BIO Inc JapanELISA kits specific for human interleukin-1120573 (IL-1120573) (Batchnumber 20131014) was product of Beijing 4A Biotech CoLtd China [7]

The 7900 real-time fluorescence quantitative PCR instru-ment was a product of ABI Company USAThe hypothermichigh-speed centrifugal machine 5417R was a product ofEppendorf Company USA The FlexCycler PCR instrument

CASP1 51 267 63 116 174 235 144 110 13151 267 63 116 174 235 144 110

51 267 116 174 235 144 110

131 849

849

93

51 267 63 116 174 235 144 110 131 93

51 267 116 174 235 144 110 131 935151 116 174 235 144 110 131 93

CASP1-6

CASP1-7

96bp1774bp

1711bp

1149bp1086bp833bp

CASP1-120572

CASP1-120573CASP1-120574

Figure 1 The exon of CASP1 gene and its transcript variant inhuman Notes rectangle exon straight line intron the numberabove the rectangle length of exon bp fragment size

was a product of Analytik Jena AG Germany The FUSION-Fx5 lithographymachine was a product of Oriental Science ampTechnology Development Co Ltd China [7]

24 Primer Design Located in 11q23 the ID of CASP1 geneis 834 with ten exons nine introns seven transcript variantsand two functional domains (Figure 1) According to human120573-actin and CASP1 gene and its transcript variant in Gen-Bank the primerwas designed to be used in themeasurementof RT-PCR or qRT-PCR and synthesized by polyacrylamidegel electrophoresis method in Genscript Biotechnology CoLtd Designed primer sequences (Table 1)

25 Total RNAExtraction and cDNASynthesis 25mLperiph-eral blood was taken and anticoagulated with heparinPBMCs were separated by human lymphocytes separationliquid under sterile condition According to instructionstrictly total RNA was extracted with Trizol reagent andthe RNA was dissolved with 30 120583L no RNA enzymes water5 120583L RNA samples were taken and measured by agarosegel electrophoresis and three bands were showed in 15agarose gel map 28S 18S and 5S (Figures 2(c) and 3(c))The absorbance value (119860 value) of RNA was detected by UVspectrophotometer detection at 260 and 280 nm wavelengthand the 119860

260119860280

ratio was calculated (adopted 18 to 20)[9] According to instruction strictly cDNA was synthesizedwith reverse transcription kits on condition of 37∘C for 15minand 85∘C for 5 sec and then the reaction was terminated60 120583L of the RT system was recorded as follows 6 120583L 5timesgDNA eraser buffer 3 120583L gDNA eraser 5 120583L total RNA 12 120583L5times PrimeScript buffer 2 (qRT-PCR) 3120583L prime script RTenzyme mix I 3 120583L RT primer mix and 28 120583L RNase freedH2O [10] The cDNA product was stored at minus20∘C

26 Measurement of CASP1 Gene Transcript Variant and IL-1120573 by RT-PCR PCR amplification was made in the 25 120583Lreaction system which was created according to cDNA ofHC and patients with PG 025 120583L TaKaRa LA Taq (5U120583L)25 120583L 10times LA PCR buffer II (Mg2+ Free) 25 120583L MgCl

2

(25mM) 4 120583L dNTP mixture (each 25mM) 1 120583L templateDNA (cDNA) 05 120583L primer 1 (upstream 20120583M) 05 120583Lprimer 2 (downstream 20120583M) and 1375 120583L sterilized anddistilled water The reaction condition was initial denatura-tion in 95∘C for 5min 94∘C for 30 sec 55∘C for 30 sec and72∘C for 1min and repeated 35 cycles and extension in 72∘Cfor 5min [9] Amplification products were measured by 1

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Primer sequences of CASP1 gene and its transcript variant

Gene (transcriptvariant) name

Upstream DownstreamGeneticfragment

size

CASP1 51015840-CGCAGATGCCCACCACT-31015840 51015840-TGCCCACAGACATTCATACAG-31015840 96 bpCASP1-6(NM 0012571182)

51015840-TACAGTTATGGATAAGACCCGAGC-31015840 51015840-GCAGACATAATTCCAAAAACCTTTA-31015840 1774 bp

CASP1-7(NM 0012571192)


CASP1-alpha(NM 0332923)


CASP1-beta(NM 0012234)


CASP1-gamma(NM 0332933)


IL-1120573 51015840-ACAGATGAAGTGCTCCTTCCA-31015840 51015840-GTCGGAGATTCGTAGCTGGAT-31015840 73 bp120573-actin 51015840-GAGCTACGAGCTGCCTGACG-31015840 51015840-GTAGTTTCGTGGATGCCACAG-31015840 120 bp

agarose gel electrophoresis and agarose gel was shot withexposure by FUSION-Fx5 lithography machine The grayvalue of the exposed PCR strip images wasmeasured by BIO-RADQuantity-One softwareThe ratio of gray value of targetgene to internal parameters was to reveal the expression levelof target gene and its transcript variant mRNA

27 Measurement of CASP1 and IL-1120573 Gene by qRT-PCRcDNA of HC and patients with PG was measured by qRT-PCR instrument to create 20120583L reaction system 10 120583L SYBRPremix Ex Taq II 04 120583L ROX Reference Dye II 08 120583Lupstream primer (10 120583molL) 08120583L downstream primer(10 120583molL) and 8 120583L sterilized and distilled water The reac-tion condition was 95∘C for 10min 95∘C for 15 s and 60∘Cfor 1min and repeated 40 cycles Each specimen was donewith multiple pores and the Ct value difference between themultiple pores was controlled within 05 All amplificationswere performed on the ABI 7900 real-time PCR instrumentThe melting curve was made after the amplification ΔCtderived fromCt valueminus internal parameters of the targetgene and 2minusΔCt value represented the expression level of thetarget gene mRNA (the amplification of specific primer oftarget gene and its transcript variant)

28 The Purification and Sequencing of the PCR ProductsThe PCR products were purified and recycled by agarose gelpurification The recycled gene fragments were remeasuredby agarose gel electrophoresis After bands were confirmedthe nucleic acid sequence of purified target gene fragmentswas sequenced in Genscript Biotechnology Co Ltd

29 Measurement of the Level of Plasma IL-1120573 by ELISA KitAccording to instructions strictly ELISA kit was operatedand OD values of each hole was measured with a microplatereader at 450 nm The standard curve was made with stan-dard sample of kit The corresponding concentration wasidentified according to the absorbance value of the sample

and the final concentration of the sample was calculated bymultiply the measured concentration by dilution factor

210 Correlation Analysis We analyzed the correlationbetween mRNA expression of CASP1 gene and its transcriptvariant and IL-1120573 in patients with PG in different phases andTCM syndromes and also analyzed the correlation betweenexpression of CASP1 gene and its transcript variant mRNAand IL-1120573 protein in patients with PG in different phases andTCM syndromes

211 Statistical Analysis SPSS 160 software package wasused for statistical analysis and all data were presented asmean plusmn standard deviation (119909 plusmn 119904) Comparison of meanvalues among multiple groups was done with ANOVA andcomparison between two groups was done with the LSDtest The correlation of each group was done with spearmananalysis A 119875 lt 005 was considered as significant differenceamong groups

3 Results

31 The Comparison of the Results between Different Phasesand TCM Syndromes of Patients with PG See Table 2

32 The Primers Amplified Results of CASP1 Gene and ItsTranscript Variant in PBMCs of Patients with PG in DifferentPhases and TCM Syndromes See Figures 2 and 3

33 The Expression of CASP1 Gene and Its Transcript VariantmRNA in PBMCs of PG Patients with PG in Different Phasesand TCM Syndromes The expression of CASP1 mRNA inAPPG group was significantly higher than that in HC group(119875 lt 001) the expression of CASP1-6 mRNA in APPGgroup and CASP1-6 and CASP1-7 mRNA in NAPPG groupwas significantly lower than in HC group (119875 lt 001 or 119875 lt005) the expression of CASP1 and CASP1-gamma mRNA in


APPG NAPPG HC

CASP1

Actin

mRNA

CASP10

50

100

150

HCAPPGNAPPG

Expr

essio

n

lowastlowast lowastlowast

(a)

HC

Actin

x

GoutAP NAP

1774bp

1711bp1149bp

1086 bp

955bp

833bp

HCAPPGNAPPG

mRNA

0

50

100

150

Expr

essio

n

lowast

lowastlowast

lowastlowast

CASP1-6 CASP1-7 CASP1-120572CASP1-120573 CASP1-120574

CASP1-6

CASP1-7

CASP1-120572CASP1-120573

CASP1-120574

(b)

28S

18S

5S

HCAPPG NAPPG

(c)

Figure 2ThemRNA expression of CASP1 gene and its transcript variant in PBMCs of patients with PG in different phases Notes HC healthcontrol AP acute phase of primary gout NAP nonacute phase of primary gout (a) CASP1 gene primers were amplified to one fragment(96 bp) (b) common primers of CASP1 gene transcript variants 6 7 beta alpha and gamma were amplified to six fragments 1774 bp wastranscript variant 6 1711 bp was transcript variant 7 1086 bp was transcript variant beta 1149 bp was transcript variant alpha 833 bp wastranscript variant gamma and x was an unknown stripe Delta and epsilon stripe were not observed in designed primers of our researchand we will carry on the design and experiment through different methods (c) The RNA quality electropherogram of PBMCs lowast119875 lt 005lowastlowast119875 lt 001

Table 2 The comparison of the results between the different phases and TCM syndromes of patients with PG

Phases APPG (119899 = 44) NAPPG (119899 = 52)TCM syndromes ODHS IPSBS PDIDS QBDS PDIDS QBDS IPSBS ODHS119899 16 12 10 6 21 14 10 7Percent () 3636 2727 2273 1364 4038 2692 1923 1346Notes APPG acute phase primary gout NAPPG nonacute phase primary gout ODHS obstruction of dampness and heat syndrome IPSBS intermingledphlegm-stasis blood syndrome PDIDS Pi-deficiency induced dampness syndrome QBDS Qi-blood deficiency syndrome

NAPPG group was significantly lower than in APPG group(119875 lt 001 or 119875 lt 005 Figure 2)

The expression of CASP1 gene mRNA in IPSBS andODHS group was significantly higher than in HC group(119875 lt 001) the expression of CASP1-6 and CASP1-7 mRNA

in ODHS and QBDS group and CASP1-6 mRNA in PDIDSgroup all was significantly lower than in HC group (119875 lt005 or 119875 lt 001) the expression of CASP1-6 and CASP1-7mRNA in ODHS group CASP1 mRNA in PDIDS group andCASP1 CASP1-7 and CASP1-gamma mRNA in QBDS group


CASP10

50

100

150

Expr

essio

n

HCIPSBS ODHS PDIDS QBDS

lowastlowastlowastlowast

lowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

IPSBSODHS

PDIDS

QBDSHC

CASP1Actin

Gout

(a)

CASP1-60

20

40

60

80

100

Expr

essio

n

CASP1-70

20

40

60

80

100


Expr

essio

n

0

20

40

60

80

100

Expr

essio

n

0

20

40

60

80

100

Expr

essio

n

0

20

40

60

80

100

Expr

essio

n



lowastlowastlowast

lowastlowast

lowast

lowast

lowastlowastlowast

lowast lowast

IPSBSODHS

PDIDSQBDS

HCIPSBSODHS

PDIDSQBDS

HCIPSBSODHS

PDIDSQBDS

HC

IPSBSODHS

PDIDSQBDS

HCIPSBSODHS

PDIDSQBDS

HC

Gout

Actin

x

CASP1-6CASP1-7

CASP1-120572

CASP1-120572

CASP1-120573

CASP1-120573

CASP1-120574

CASP1-120574

1774bp1711bp1149bp

1086 bp955bp833bp

(b)

Figure 3 Continued



28S

18S

5S

(c)

Figure 3 The mRNA expression of CASP1 gene and its transcript variant in PBMCs of PG patients with different TCM syndromes Noteslowast119875 lt 005 lowastlowast119875 lt 001

mRNA

0

20

40

60

80

Expr

essio

n

lowastlowast

lowastlowast

IL-1120573

HCAPPGNAPPG

APPG NAPPG HC

IL-1120573

Actin

(a)

mRNA

0

20

40

60

80

Expr

essio

n

lowastlowast

lowastlowast

lowastlowast

lowastlowast

IL-1120573

IPSBSODHS

PDIDSQBDS

HC

IPSBS ODHS PDIDS QBDS HC

Gout

IL-1120573

Actin

(b)

Figure 4 The mRNA expression of IL-1120573 in PBMCs of patients with PG in different phases and TCM syndromes Notes (a) and (b) IL-1120573gene primers were amplified to one fragment (73 bp) (a) the mRNA expression of IL-1120573 in PBMCs of patients with PG in different phases(b) The mRNA expression of IL-1120573 in PBMCs of patients with PG in different TCM syndromes lowastlowast119875 lt 001

all was significantly lower than in IPSBS group (119875 lt 005or 119875 lt 001) the expression of CASP1 mRNA in PDIDSand CASP1-7 and CASP1-gamma mRNA in QBDS group allwas significantly lower than in ODHS group (119875 lt 005 or119875 lt 001) the expression of CASP1-6mRNA in PDIDS groupwas significantly higher than in ODHS group (119875 lt 005)the expression of CASP1-beta and CASP1-gamma mRNA inQBDS group was significantly lower than in PDIDS group(119875 lt 005 or 119875 lt 001 Figure 3)

34 The Expression of IL-1120573mRNA in PBMCs of Patients withPG in Different Phases and TCM Syndromes The expression

of IL-1120573mRNA inAPPG andNAPPG groupwas significantlyhigher than in HC group (119875 lt 001 Figure 4(a))

The expression of IL-1120573mRNA in IPSBS ODHS PDIDSand QBDS group was significantly higher than in HC group(119875 lt 001 Figure 4(b))

35 The Expression of Plasma IL-1120573 Protein of Patients withPG in Different Phases and TCM Syndromes The expressionof plasma IL-1120573 protein in APPG and NAPPG group wassignificantly higher than in HC group (119875 lt 001) andthe expression of plasma IL-1120573 protein in APPG group


0

20

40

60

Prot

ein

expr

essio

n (p

gm

L)

IL-1120573

lowastlowast

lowastlowast

lowastlowast

HCAPPGNAPPG

(a)

0

50

100

150

IPSBSODHS

PDIDSQBDS

Prot

ein

expr

essio

n (p

gm

L)

IL-1120573

lowastlowast

lowastlowast


lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

HC

(b)

Figure 5The expression of plasma IL-1120573protein in patientswith PGin different phases and TCM syndromes Notes (a) the expression ofplasma IL-1120573 protein in patients with PG in different phases (b) theexpression of plasma IL-1120573 protein in patients with PG in differentTCM syndromes lowastlowast119875 lt 001

was significantly higher than in NAPPG group (119875 lt 001)(Figure 5(a))

The expression of IL-1120573 protein in IPSBS ODHS PDIDSand QBDS group was significantly higher than in HC group(119875 lt 001) the expression of IL-1120573 protein in IPSBS groupwas significantly higher than in ODHS group (119875 lt 001) theexpression of IL-1120573 protein in IPSBS and ODHS group wassignificantly higher than in PDIDS and QBDS group (119875 lt001) (Figure 5(b))

36 The Results of Correlation Analysis Correlation analysisshowed that there was negative correlation between theexpression of CASP1-gamma gene transcript variant mRNAand IL-1120573 protein in APPG group (119903 = minus04435 119875 =00264 Figure 6) and no significant correlationwas observedbetween the mRNA expression of CASP1 gene and its tran-script variant and IL-1120573 in other groups (119875 gt 005)

4 Discussion

Gout has a strong influence on peoplersquos health The primarygout with certain familial predisposition was caused byboth genetic and environmental factors and the etiologywas unknown except for about 1 due to congenital defectsin purine metabolism enzymes [14] In recent years theincidence of gout in adults in China increases year by yearand also increases with age [14] Chinese medicine believesthat the causes of gout were congenital deficiency beingworn out with age dysfunction of spleen in transportationand with no ability to ascend lucidity or descend turbidityor deficiency of kidney for activation of Qi and not distin-guishing lucidity and turbidity resulted in cereal essence not

Correlations

0 10 20 30 40 500

5

10

15

IL-1120573 (pgmL)

CASP1

-120574 (m

RNA

)

r = minus04435 P = 00264

Figure 6 Correlation analysis of the expression level betweenCASP1-gamma gene transcript variant mRNA and IL-1120573 protein inAPPG group

being reformed alongwith noxious dampnesswhich strandedand accumulated in the body related to the dysfunction ofmiddle jiao and lower jiao By classifying gout patients intoWestern medicine phases and TCM syndrome we foundthat the main TCM syndrome in the acute and nonacutephase of gout patients was IPSBS ODHS PDIDS andQBDSamong which ODHS and IPSBS were mainly in the acutephase of gout while PDIDS and QBDS were in the nonacutephase The results showed that the main syndromes in theacute phase of gout were obstruction of dampness and heatsyndrome and intermingled phlegm-stasis blood syndromewhile in the nonacute phase of gout the main syndromeswere Pi-deficiency induced dampness syndrome and Qi-blood deficiency syndrome

Some researches had shown that inflammation andimmunity played a certain role in the pathogenesis of gout [2]and the IL-1120573 level in peripheral venous blood of gout patientshad significantly increased [15] The bodyrsquos innate immunityuses Toll-like Receptors (TLRs) to recognize the ldquonakedrdquoMSU crystals which can activate myeloid differentiationfactor (MyD88) dependent NF-120581B pathway and lead thegene transcription to produce prointerleukin 1120573 (Pro-IL-1120573)precursor Pro-IL-1120573 is cut intomature IL-1120573 through CASP1By binding to IL-1 receptor IL-1120573 can activate the IL-1 andNF-120581B signal pathway which can cause a large expressionof proinflammatory factor like IL-1120573 tumor necrosis factor-120572 (TNF-120572) and so on producing inflammatory cascadeamplification effect [16]

It was known that an mRNA precursor (pre-mRNA)could produce different mRNA splice variant by selectingdifferent splicing sites and different splice variant playsan important role in the occurrence and development ofdiseases [17ndash19] Studies showed that the immature CASP1mRNAcould be translated into six different subtypes throughvariable shear and transcription alpha beta gamma deltaepsilon and zeta all of which can not onlymediate inflamma-tory response but also play different roles in cell death [20 21]Our study retrieved the already known seven gene transcriptvariants of CASP1 gene fromGenbank After CASP1 gene and


its transcript variant primers were designed and measuredby RT-PCR we found that the expression of CASP1 genemRNA in IPSBS and ODHS group was significantly higherthan inHC group (119875 lt 001) the expressions of CASP1-6 andCASP1-7 mRNA in ODHS group CASP1-6 mRNA in PDIDSgroup and CASP1-6 and CASP1-7 mRNA in QBDS groupwere all significantly lower than in HC group (119875 lt 005 or119875 lt 001) the expression of CASP1 mRNA in APPG groupwas significantly higher than in HC group (119875 lt 001) theexpression of CASP1-6 mRNA in APPG group and CASP1-6andCASP1-7mRNAofNAPPGgroupwas significantly lowerthan in HC group (119875 lt 005 or 119875 lt 001) in the meantimethe expression of IL-1120573mRNA in patientswith PG in differentphases and TCM syndromes was significantly higher than inHC group (119875 lt 001) These results showed that CASP1 geneand its transcript variant are expressed abnormally in patientswith PG in different phases and TCM syndromes and theseresults also suggested that CASP1 gene and its transcriptvariant might play an important role in the regulation ofinflammatory responses in patients with PG The study alsofound that there are differences of the expression of CASP1gene and its transcript variant between APPG and NAPPGgroups The results showed that the change of phases andTCM syndromes of patients with PGmay relate to the changeof the expression of CASP1 gene and its transcript variant

Our study also found that the protein expression ofplasma IL-1120573 of patients with PG in different phases andTCM syndromes was significantly higher than in HC group(119875 lt 001) there were differences between the expression ofplasma IL-1120573 protein in patients with PG in different phasesand TCM syndromes and the correlation analysis showedthat there was negative correlation between the expressionof CASP1-gamma gene transcript variant mRNA and IL-1120573protein inAPPGgroup (119903 = minus04435119875 = 00264)The resultssuggested indirectly that the CASP1 gene transcript variantmay play an important role in the inflammatory responses ofpatients with PG and the mechanism needs further and in-depth study [22 23]

In conclusion obstruction of dampness and heat syn-drome and intermingled phlegm-stasis blood syndrome tendto occur in the acute phase of gout while Pi-deficiencyinduced dampness syndrome and Qi-blood deficiency syn-drome tend to occur in the nonacute phase of gout andthe mechanism may relate to the dysregulated expressionof CASP1 gene and its transcript variant the expressionchange of CASP1 gene and its transcript variant may beassociated with the onset of gout Therefore further studyfor the mechanism of CASP1 gene transcript variant in PG isexpected to provide new method for the effective preventionand treatment of PG

5 Limitations

A small sample size may be a limitation for the present studyCASP1 transcript variant primers are not specific and cannotbe detected by qRT-PCR there may have been an error in theresults detected by RT-PCR Hence every transcript variantspecific primer of CASP1 should be redesigned later and

CASP1 gene transcript variant should be detected by qRT-PCR

6 Conclusions

In summary through our research we initially demonstratedthe existence of CASP1-6 CASP1-7 CASP1-alpha CASP1-beta and CASP1-gamma transcript variant in PBMCs of goutpatients and the expression of each transcript variant mRNAshowed difference between patients with gout in differentTCM syndromes and health controls combined with rel-evant laboratory index the results preliminarily indicatedthat CASP1 gene and its transcript variant might play animportant regulating role in the pathogenesis of gout

Abbreviations

ACR American Rheumatism AssociationAP Acute phaseAPPG Acute phase primary goutCASP1 Caspase-1DAMPs Danger-associated molecular patternsHC Healthy controlIPSBS Intermingled phlegm-stasis blood syndromeLPS LipopolysaccharidesLSD Least significant differenceMSU Monosodium urateNAP Nonacute phaseNAPPG Non-acute phase primary goutODHS Obstruction of dampness and heat syndromePBMCs Peripheral blood mononuclear cellsPDIDS Pi-deficiency induced dampness syndromePG Primary goutPro-IL-1120573 Prointerleukin 1120573QBDS Qi-blood deficiency syndromeTCM Traditional Chinese medicineTLRs Toll-like receptor-specific

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

The authors contributed equally to this work

Acknowledgments

This work was partly supported by the Surface Project ofNational Natural Science Foundation of China (81272047)and Technology Innovation Talent Funding Project ofSichuan Province of China (2014-087)

References

[1] P Richette and T Bardin ldquoGoutrdquoThe Lancet vol 375 no 9711pp 318ndash328 2010


[2] Z Miao C Li Y Chen et al ldquoDietary and lifestyle changesassociated with high prevalence of hyperuricemia and goutin the Shandong coastal cities of Eastern Chinardquo Journal ofRheumatology vol 35 no 9 pp 1859ndash1864 2008

[3] Y Shi J E Evans and K L Rock ldquoMolecular identification ofa danger signal that alerts the immune system to dying cellsrdquoNature vol 425 no 6957 pp 516ndash521 2003

[4] R R Schumann C Belka D Reuter et al ldquoLipopolysaccha-ride activates caspase-1 (interleukin-1-converting enzyme) incultured monocytic and endothelial cellsrdquo Blood vol 91 no 2pp 577ndash584 1998

[5] H LukschM J Romanowski O Chara et al ldquoNaturally occur-ring genetic variants of human caspase-1 differ considerablyin structure and the ability to activate interleukin-1120573rdquo HumanMutation vol 34 no 1 pp 122ndash131 2013

[6] N Busso and H-K Ea ldquoThe mechanisms of inflammation ingout and pseudogout (CPP-induced arthritis)rdquo Reumatismovol 63 no 4 pp 230ndash237 2012

[7] W T Dang J G Zhou W G Xie et al ldquoExpression ofcaspase-1 gene transcript variant mRNA in peripheral bloodmonocytes of patients with primary goutrdquo Chinese Journal ofRheumatology vol 18 no 6 p 400 2014

[8] S L Wallace H Robinson A T Masi J L Decker D JMcCarty andT F Yu ldquoPreliminary criteria for the classificationof the acute arthritis of primary goutrdquo Arthritis and Rheuma-tism vol 20 no 3 pp 895ndash900 1977

[9] W T Dang J G Zhou W G Xie et al ldquoExpression ofNLRP3 gene transcript variant mRNA in the peripheral bloodmononuclear cells of patients with primary goutrdquo ChineseJournal of Rheumatology (China) vol 18 no 2 pp 76ndash81 2014

[10] W T Dang J G Zhou W G Xie et al ldquoMechanism of NLRP3inflammasome in inflammatory response with gouty arthritisrdquoChinese Journal of Immunology vol 30 no 3 pp 373ndash377 2014

[11] State Bureau of Technical SupervisionTheType of Part ClinicalDiagnosis and Treatment of Traditional ChineseMedicine Termi-nology Standards Press of China Beijing China 1997

[12] X Y ZhengGuiding Principle of Clinical Research onNewDrugsof Traditional Chinese Medicine China Medical Science andTechnology Press Beijing China 1995

[13] W T Dang J G ZhouWG Xie et al ldquoComparative analysis ofclinical indicators of gout patients of different syndrome typesand its significancerdquo Chinese Journal of Integrated Traditionaland Western Medicine vol 33 no 10 pp 1323ndash1327 2013

[14] Q Zeng R Chen J Darmawan et al ldquoRheumatic diseases inChinardquo Arthritis Research ampTherapy vol 10 no 1 p R17 2008

[15] R M Pope and J Tschopp ldquoThe role of interleukin-1 and theinflammasome in gout implications for therapyrdquo Arthritis ampRheumatism vol 56 no 10 pp 3183ndash3188 2007

[16] S R Kingsbury P G Conaghan and M F McDermottldquoThe role of the NLRP3 inflammasome in goutrdquo Journal ofInflammation Research vol 4 no 1 pp 39ndash49 2011

[17] E T Wang R Sandberg S Luo et al ldquoAlternative isoformregulation in human tissue transcriptomesrdquoNature vol 456 no7221 pp 470ndash476 2008

[18] M J Moore Q Wang C J Kennedy and P A Silver ldquoAn alter-native splicing network links cell-cycle control to apoptosisrdquoCell vol 142 no 4 pp 625ndash636 2010

[19] Y Barash J A Calarco W Gao et al ldquoDeciphering the splicingcoderdquo Nature vol 465 no 7294 pp 53ndash59 2010

[20] E S Alnemri T Fernandes-Alnemri and G Litwack ldquoCloningand expression of four novel isoforms of human interleukin-1120573

converting enzymewith different apoptotic activitiesrdquoThe Jour-nal of Biological Chemistry vol 270 no 9 pp 4312ndash4317 1995

[21] Q Feng P Li P C K Leung and N Auersperg ldquoCaspase-1120577 anew splice variant of the caspase-1 generdquo Genomics vol 84 no3 pp 587ndash591 2004

[22] C-N Son S-Y Bang J H Kim C-B Choi T-H Kim and J-B Jun ldquoCaspase-1 level in synovial fluid is high in patients withspondyloarthropathy but not in patients with goutrdquo Journal ofKorean Medical Science vol 28 no 9 pp 1289ndash1292 2013

[23] R C Coll A A Robertson J J Chae et al ldquoA small-moleculeinhibitor of the NLRP3 inflammasome for the treatment ofinflammatory diseasesrdquoNatureMedicine vol 21 no 3 pp 248ndash255 2015

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


in the meantime the interleukin 1120573 was measured to explorethe role of CASP1 gene and its transcript variant in thepathogenesis of gout

2 Methods

21 The Clinical Data All the research objects conformed tothe 1977 American Rheumatism Association (ACR) diagnos-tic criteria [8] excluding the objects which have secondarygout caused by diseases of kidney cardiovascular and bloodsystem or drugs and so on and also excluding coinfectionautoimmune diseases long-term use of hormone therapyor serious condition that may affect the efficacy and safetyof our study [9 10] 96 male patients with PG who visitedthe Department of Rheumatology of the Affiliated Hospitalof North Sichuan Medical College from December 2012 toOctober 2013 were admitted among them 44 patients were inacute phase and 52 patients were in nonacute phase The ageof patients ranged from 23 to 79 years old and the mean agewas 40 plusmn 11 years with their disease course being 9 plusmn 3 years30 healthy people set as healthy controls (HC) were admittedfrom the Department of Physical Examination in the samehospital during the same period whose age ranged from 22 to70 years their mean age was 44plusmn7 years and their laboratoryindexes were normal excluding the people who had diseasesor family history of cardiovascular disease diabetes livergout and so on [10]The age between the two groups showedno statistical significance The study has gained approval andagreement of the local ethics committee and all participantssigned informed consent

22 The TCM Syndromes Based on the gout TCM syn-drome differentiation in Clinical Diagnosis and TreatmentTerminology of Traditional Chinese Medicine-Syndrome TypePart [11] and Guideline of the Study on New TraditionalChinese Medicine [12] and the TCM differentiation accordingto comprehensive analysis by the ldquofour examinationmethodsrdquoldquoEight Principle Pattern Identificationrdquo and ldquoZang-fu patternidentificationrdquo the TCM syndromes differentiation of PGpatients was divided into four types obstruction of damp-ness and heat syndrome (ODHS) intermingled phlegm-stasis blood syndrome (IPSBS) Pi-deficiency induced damp-ness syndrome (PDIDS) and Qi-blood deficiency syndrome(QBDS) [13]

23 Main Reagents and Instruments Human lymphocytesseparation liquid (Batch number LTS10771) was productof Jingyang Tech Co China RNAiso Plus Reagent (Batchnumber A9701-1) PrimeScript RT reagent kit with gDNAeraser (Perfect qRT-PCR) kit (Batch number AK1801) SYBRPremix Ex Taq II (Batch number AK5004) and TaKaRa LATaq kit (CKA4501A) were products of Takara BIO Inc JapanELISA kits specific for human interleukin-1120573 (IL-1120573) (Batchnumber 20131014) was product of Beijing 4A Biotech CoLtd China [7]

The 7900 real-time fluorescence quantitative PCR instru-ment was a product of ABI Company USAThe hypothermichigh-speed centrifugal machine 5417R was a product ofEppendorf Company USA The FlexCycler PCR instrument

CASP1 51 267 63 116 174 235 144 110 13151 267 63 116 174 235 144 110

51 267 116 174 235 144 110

131 849

849

93

51 267 63 116 174 235 144 110 131 93

51 267 116 174 235 144 110 131 935151 116 174 235 144 110 131 93

CASP1-6

CASP1-7

96bp1774bp

1711bp

1149bp1086bp833bp

CASP1-120572

CASP1-120573CASP1-120574

Figure 1 The exon of CASP1 gene and its transcript variant inhuman Notes rectangle exon straight line intron the numberabove the rectangle length of exon bp fragment size

was a product of Analytik Jena AG Germany The FUSION-Fx5 lithographymachine was a product of Oriental Science ampTechnology Development Co Ltd China [7]

24 Primer Design Located in 11q23 the ID of CASP1 geneis 834 with ten exons nine introns seven transcript variantsand two functional domains (Figure 1) According to human120573-actin and CASP1 gene and its transcript variant in Gen-Bank the primerwas designed to be used in themeasurementof RT-PCR or qRT-PCR and synthesized by polyacrylamidegel electrophoresis method in Genscript Biotechnology CoLtd Designed primer sequences (Table 1)

25 Total RNAExtraction and cDNASynthesis 25mLperiph-eral blood was taken and anticoagulated with heparinPBMCs were separated by human lymphocytes separationliquid under sterile condition According to instructionstrictly total RNA was extracted with Trizol reagent andthe RNA was dissolved with 30 120583L no RNA enzymes water5 120583L RNA samples were taken and measured by agarosegel electrophoresis and three bands were showed in 15agarose gel map 28S 18S and 5S (Figures 2(c) and 3(c))The absorbance value (119860 value) of RNA was detected by UVspectrophotometer detection at 260 and 280 nm wavelengthand the 119860

260119860280

ratio was calculated (adopted 18 to 20)[9] According to instruction strictly cDNA was synthesizedwith reverse transcription kits on condition of 37∘C for 15minand 85∘C for 5 sec and then the reaction was terminated60 120583L of the RT system was recorded as follows 6 120583L 5timesgDNA eraser buffer 3 120583L gDNA eraser 5 120583L total RNA 12 120583L5times PrimeScript buffer 2 (qRT-PCR) 3120583L prime script RTenzyme mix I 3 120583L RT primer mix and 28 120583L RNase freedH2O [10] The cDNA product was stored at minus20∘C

26 Measurement of CASP1 Gene Transcript Variant and IL-1120573 by RT-PCR PCR amplification was made in the 25 120583Lreaction system which was created according to cDNA ofHC and patients with PG 025 120583L TaKaRa LA Taq (5U120583L)25 120583L 10times LA PCR buffer II (Mg2+ Free) 25 120583L MgCl

2

(25mM) 4 120583L dNTP mixture (each 25mM) 1 120583L templateDNA (cDNA) 05 120583L primer 1 (upstream 20120583M) 05 120583Lprimer 2 (downstream 20120583M) and 1375 120583L sterilized anddistilled water The reaction condition was initial denatura-tion in 95∘C for 5min 94∘C for 30 sec 55∘C for 30 sec and72∘C for 1min and repeated 35 cycles and extension in 72∘Cfor 5min [9] Amplification products were measured by 1





size



CASP1-7(NM 0012571192)
















3 Results





APPG NAPPG HC

CASP1

Actin

mRNA

CASP10

50

100

150

HCAPPGNAPPG

Expr

essio

n


(a)

HC

Actin

x

GoutAP NAP

1774bp

1711bp1149bp

1086 bp

955bp

833bp

HCAPPGNAPPG

mRNA

0

50

100

150

Expr

essio

n

lowast

lowastlowast

lowastlowast


CASP1-6

CASP1-7

CASP1-120572CASP1-120573

CASP1-120574

(b)

28S

18S

5S

HCAPPG NAPPG

(c)








CASP10

50

100

150

Expr

essio

n




IPSBSODHS

PDIDS

QBDSHC

CASP1Actin

Gout

(a)

CASP1-60

20

40

60

80

100

Expr

essio

n

CASP1-70

20

40

60

80

100


Expr

essio

n

0

20

40

60

80

100

Expr

essio

n

0

20

40

60

80

100

Expr

essio

n

0

20

40

60

80

100

Expr

essio

n



lowastlowastlowast

lowastlowast

lowast

lowast

lowastlowastlowast

lowast lowast

IPSBSODHS

PDIDSQBDS

HCIPSBSODHS

PDIDSQBDS

HCIPSBSODHS

PDIDSQBDS

HC

IPSBSODHS

PDIDSQBDS

HCIPSBSODHS

PDIDSQBDS

HC

Gout

Actin

x

CASP1-6CASP1-7

CASP1-120572

CASP1-120572

CASP1-120573

CASP1-120573

CASP1-120574

CASP1-120574

1774bp1711bp1149bp

1086 bp955bp833bp

(b)

Figure 3 Continued



28S

18S

5S

(c)


mRNA

0

20

40

60

80

Expr

essio

n

lowastlowast

lowastlowast

IL-1120573

HCAPPGNAPPG

APPG NAPPG HC

IL-1120573

Actin

(a)

mRNA

0

20

40

60

80

Expr

essio

n

lowastlowast

lowastlowast

lowastlowast

lowastlowast

IL-1120573

IPSBSODHS

PDIDSQBDS

HC


Gout

IL-1120573

Actin

(b)








0

20

40

60

Prot

ein

expr

essio

n (p

gm

L)

IL-1120573

lowastlowast

lowastlowast

lowastlowast

HCAPPGNAPPG

(a)

0

50

100

150

IPSBSODHS

PDIDSQBDS

Prot

ein

expr

essio

n (p

gm

L)

IL-1120573

lowastlowast

lowastlowast


lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

HC

(b)





4 Discussion


Correlations

0 10 20 30 40 500

5

10

15

IL-1120573 (pgmL)

CASP1

-120574 (m

RNA

)

r = minus04435 P = 00264









5 Limitations



6 Conclusions


Abbreviations






Acknowledgments


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















size



CASP1-7(NM 0012571192)
















3 Results





APPG NAPPG HC

CASP1

Actin

mRNA

CASP10

50

100

150

HCAPPGNAPPG

Expr

essio

n


(a)

HC

Actin

x

GoutAP NAP

1774bp

1711bp1149bp

1086 bp

955bp

833bp

HCAPPGNAPPG

mRNA

0

50

100

150

Expr

essio

n

lowast

lowastlowast

lowastlowast


CASP1-6

CASP1-7

CASP1-120572CASP1-120573

CASP1-120574

(b)

28S

18S

5S

HCAPPG NAPPG

(c)








CASP10

50

100

150

Expr

essio

n




IPSBSODHS

PDIDS

QBDSHC

CASP1Actin

Gout

(a)

CASP1-60

20

40

60

80

100

Expr

essio

n

CASP1-70

20

40

60

80

100


Expr

essio

n

0

20

40

60

80

100

Expr

essio

n

0

20

40

60

80

100

Expr

essio

n

0

20

40

60

80

100

Expr

essio

n



lowastlowastlowast

lowastlowast

lowast

lowast

lowastlowastlowast

lowast lowast

IPSBSODHS

PDIDSQBDS

HCIPSBSODHS

PDIDSQBDS

HCIPSBSODHS

PDIDSQBDS

HC

IPSBSODHS

PDIDSQBDS

HCIPSBSODHS

PDIDSQBDS

HC

Gout

Actin

x

CASP1-6CASP1-7

CASP1-120572

CASP1-120572

CASP1-120573

CASP1-120573

CASP1-120574

CASP1-120574

1774bp1711bp1149bp

1086 bp955bp833bp

(b)

Figure 3 Continued



28S

18S

5S

(c)


mRNA

0

20

40

60

80

Expr

essio

n

lowastlowast

lowastlowast

IL-1120573

HCAPPGNAPPG

APPG NAPPG HC

IL-1120573

Actin

(a)

mRNA

0

20

40

60

80

Expr

essio

n

lowastlowast

lowastlowast

lowastlowast

lowastlowast

IL-1120573

IPSBSODHS

PDIDSQBDS

HC


Gout

IL-1120573

Actin

(b)








0

20

40

60

Prot

ein

expr

essio

n (p

gm

L)

IL-1120573

lowastlowast

lowastlowast

lowastlowast

HCAPPGNAPPG

(a)

0

50

100

150

IPSBSODHS

PDIDSQBDS

Prot

ein

expr

essio

n (p

gm

L)

IL-1120573

lowastlowast

lowastlowast


lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

HC

(b)





4 Discussion


Correlations

0 10 20 30 40 500

5

10

15

IL-1120573 (pgmL)

CASP1

-120574 (m

RNA

)

r = minus04435 P = 00264









5 Limitations



6 Conclusions


Abbreviations






Acknowledgments


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















APPG NAPPG HC

CASP1

Actin

mRNA

CASP10

50

100

150

HCAPPGNAPPG

Expr

essio

n


(a)

HC

Actin

x

GoutAP NAP

1774bp

1711bp1149bp

1086 bp

955bp

833bp

HCAPPGNAPPG

mRNA

0

50

100

150

Expr

essio

n

lowast

lowastlowast

lowastlowast


CASP1-6

CASP1-7

CASP1-120572CASP1-120573

CASP1-120574

(b)

28S

18S

5S

HCAPPG NAPPG

(c)








CASP10

50

100

150

Expr

essio

n




IPSBSODHS

PDIDS

QBDSHC

CASP1Actin

Gout

(a)

CASP1-60

20

40

60

80

100

Expr

essio

n

CASP1-70

20

40

60

80

100


Expr

essio

n

0

20

40

60

80

100

Expr

essio

n

0

20

40

60

80

100

Expr

essio

n

0

20

40

60

80

100

Expr

essio

n



lowastlowastlowast

lowastlowast

lowast

lowast

lowastlowastlowast

lowast lowast

IPSBSODHS

PDIDSQBDS

HCIPSBSODHS

PDIDSQBDS

HCIPSBSODHS

PDIDSQBDS

HC

IPSBSODHS

PDIDSQBDS

HCIPSBSODHS

PDIDSQBDS

HC

Gout

Actin

x

CASP1-6CASP1-7

CASP1-120572

CASP1-120572

CASP1-120573

CASP1-120573

CASP1-120574

CASP1-120574

1774bp1711bp1149bp

1086 bp955bp833bp

(b)

Figure 3 Continued



28S

18S

5S

(c)


mRNA

0

20

40

60

80

Expr

essio

n

lowastlowast

lowastlowast

IL-1120573

HCAPPGNAPPG

APPG NAPPG HC

IL-1120573

Actin

(a)

mRNA

0

20

40

60

80

Expr

essio

n

lowastlowast

lowastlowast

lowastlowast

lowastlowast

IL-1120573

IPSBSODHS

PDIDSQBDS

HC


Gout

IL-1120573

Actin

(b)








0

20

40

60

Prot

ein

expr

essio

n (p

gm

L)

IL-1120573

lowastlowast

lowastlowast

lowastlowast

HCAPPGNAPPG

(a)

0

50

100

150

IPSBSODHS

PDIDSQBDS

Prot

ein

expr

essio

n (p

gm

L)

IL-1120573

lowastlowast

lowastlowast


lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

HC

(b)





4 Discussion


Correlations

0 10 20 30 40 500

5

10

15

IL-1120573 (pgmL)

CASP1

-120574 (m

RNA

)

r = minus04435 P = 00264









5 Limitations



6 Conclusions


Abbreviations






Acknowledgments


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















CASP10

50

100

150

Expr

essio

n




IPSBSODHS

PDIDS

QBDSHC

CASP1Actin

Gout

(a)

CASP1-60

20

40

60

80

100

Expr

essio

n

CASP1-70

20

40

60

80

100


Expr

essio

n

0

20

40

60

80

100

Expr

essio

n

0

20

40

60

80

100

Expr

essio

n

0

20

40

60

80

100

Expr

essio

n



lowastlowastlowast

lowastlowast

lowast

lowast

lowastlowastlowast

lowast lowast

IPSBSODHS

PDIDSQBDS

HCIPSBSODHS

PDIDSQBDS

HCIPSBSODHS

PDIDSQBDS

HC

IPSBSODHS

PDIDSQBDS

HCIPSBSODHS

PDIDSQBDS

HC

Gout

Actin

x

CASP1-6CASP1-7

CASP1-120572

CASP1-120572

CASP1-120573

CASP1-120573

CASP1-120574

CASP1-120574

1774bp1711bp1149bp

1086 bp955bp833bp

(b)

Figure 3 Continued



28S

18S

5S

(c)


mRNA

0

20

40

60

80

Expr

essio

n

lowastlowast

lowastlowast

IL-1120573

HCAPPGNAPPG

APPG NAPPG HC

IL-1120573

Actin

(a)

mRNA

0

20

40

60

80

Expr

essio

n

lowastlowast

lowastlowast

lowastlowast

lowastlowast

IL-1120573

IPSBSODHS

PDIDSQBDS

HC


Gout

IL-1120573

Actin

(b)








0

20

40

60

Prot

ein

expr

essio

n (p

gm

L)

IL-1120573

lowastlowast

lowastlowast

lowastlowast

HCAPPGNAPPG

(a)

0

50

100

150

IPSBSODHS

PDIDSQBDS

Prot

ein

expr

essio

n (p

gm

L)

IL-1120573

lowastlowast

lowastlowast


lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

HC

(b)





4 Discussion


Correlations

0 10 20 30 40 500

5

10

15

IL-1120573 (pgmL)

CASP1

-120574 (m

RNA

)

r = minus04435 P = 00264









5 Limitations



6 Conclusions


Abbreviations






Acknowledgments


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















28S

18S

5S

(c)


mRNA

0

20

40

60

80

Expr

essio

n

lowastlowast

lowastlowast

IL-1120573

HCAPPGNAPPG

APPG NAPPG HC

IL-1120573

Actin

(a)

mRNA

0

20

40

60

80

Expr

essio

n

lowastlowast

lowastlowast

lowastlowast

lowastlowast

IL-1120573

IPSBSODHS

PDIDSQBDS

HC


Gout

IL-1120573

Actin

(b)








0

20

40

60

Prot

ein

expr

essio

n (p

gm

L)

IL-1120573

lowastlowast

lowastlowast

lowastlowast

HCAPPGNAPPG

(a)

0

50

100

150

IPSBSODHS

PDIDSQBDS

Prot

ein

expr

essio

n (p

gm

L)

IL-1120573

lowastlowast

lowastlowast


lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

HC

(b)





4 Discussion


Correlations

0 10 20 30 40 500

5

10

15

IL-1120573 (pgmL)

CASP1

-120574 (m

RNA

)

r = minus04435 P = 00264









5 Limitations



6 Conclusions


Abbreviations






Acknowledgments


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

Prot

ein

expr

essio

n (p

gm

L)

IL-1120573

lowastlowast

lowastlowast

lowastlowast

HCAPPGNAPPG

(a)

0

50

100

150

IPSBSODHS

PDIDSQBDS

Prot

ein

expr

essio

n (p

gm

L)

IL-1120573

lowastlowast

lowastlowast


lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

HC

(b)





4 Discussion


Correlations

0 10 20 30 40 500

5

10

15

IL-1120573 (pgmL)

CASP1

-120574 (m

RNA

)

r = minus04435 P = 00264









5 Limitations



6 Conclusions


Abbreviations






Acknowledgments


References































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















5 Limitations



6 Conclusions


Abbreviations






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 expression of caspase-1 gene transcript...

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