Research Article Hepatic Overexpression of GRP94 in a ...downloads.hindawi.com/journals/grp/2015/269831.pdf · Research Article Hepatic Overexpression of GRP94 in a Rabbit Model of

Research ArticleHepatic Overexpression of GRP94 in a Rabbit Model ofParenteral Nutrition-Associated Liver Disease

Xueping Zhu1 Xiaomin Zhang1 Lingling Yu1 Yumin Xu1 Xing Feng1 and Jian Wang2

1Department of Neonatology Childrenrsquos Hospital Affiliated to Soochow University Suzhou Jiangsu 215003 China2Department of Neonatology Surgery Childrenrsquos Hospital Affiliated to Soochow University Suzhou Jiangsu 215003 China

Correspondence should be addressed to Xing Feng xing feng66hotmailcom and Jian Wang wj196312vip163com

Received 14 October 2014 Revised 1 March 2015 Accepted 11 March 2015

Academic Editor Lana Bijelic

Copyright copy 2015 Xueping Zhu 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

Objective To use a rabbit model of parenteral nutrition-associated liver disease (PNALD) to study changes of the endoplasmicreticulum stress (ERS) marker glucose regulatory protein 94 (GRP94) and determine its role in the pathogenesis of PNALDMethods A rabbit PNALDmodel total parenteral nutrition (TPN) group was established A corresponding control group receivedbreast-feeding for one week Serum biochemical parameters were measured and liver histological examinations were performedThe level of GRP94 mRNA and protein were measured Results The results showed that the serum TBIL DBIL and 120574-GT levels inthe TPN group were significantly higher than those in the control group while levels of serumALB in TPN group were significantlylower than those in the control group The immunohistochemistry results showed that the protein expression level of GRP94 inthe liver of TPN group was significantly increased compared with the control group The RT-PCR results showed that the level ofGRP94 mRNA in the liver of the TPN group was significantly higher compared with the control group Conclusions The mRNAand protein levels of GRP94 in the TPN group were both significantly increased indicating that ERS may be directly related to theoccurrence and development of PNALD

1 Introduction

Parenteral nutrition has brought about a revolutionaryimprovement in the care of neonates with growth failuredue to intestinal dysfunction Since the first reported caseof long-term parenteral nutrition supplement in a newborngirl in the United States in the 1960s there have beenmore than 30000 patients whose survival has dependedon parenteral nutrition [1] However long-term (gt2 weeks)parenteral nutrition is associated with a number of problemsParenteral nutrition-related liver disease (PNALD) is oneof the most serious complications of neonatal parenteralnutrition-associated liver disease which usually presentswith steatosis cholestasis and elevated aminotransferasesIt has been reported [2] that about 30ndash60 of childrenon long-term parenteral nutrition develop PNALD Childrenborn prematurely are more likely to suffer from total par-enteral nutrition-associated cholestasis (PNAC) [3 4] andeven severe life-threatening cirrhosis Despite the seriousness

of the disease the specific etiology and pathogenesis remainsunclear

Because hepatocytes have highly active protein syntheticactivity and abundant endoplasmic reticulum it is possiblethat endoplasmic reticulum stress response (ERS) may beinvolved ERS has been shown to be involved in patholog-ical changes of various liver diseases [5 6] ERS has beenimplicated in the development of nonalcoholic steatohepatitis[7] alcoholic liver disease [8] ischemiareperfusion liverinjury [9] and cholestatic disease [10] We hypothesizedthat ERS could also play an important role in developmentof PNALD Glucose-regulated protein 94 (GRP94) is animportant marker protein of ERS and a chaperone localizedin the endoplasmic reticulum [11 12] As one of the knownendoplasmic reticulum stress proteins GRP94 has beenshown to be important contributor to correct protein foldingand processing maintaining the stability of the endoplasmicreticulum under stress and generally protecting cells [12ndash14]

Hindawi Publishing CorporationGastroenterology Research and PracticeVolume 2015 Article ID 269831 8 pageshttpdxdoiorg1011552015269831

2 Gastroenterology Research and Practice

Table 1 Total parenteral nutrition solution formula (total liquid volume 240mLsdotkgminus1sdotdminus1 and total calories 210 kcalsdotkgminus1sdotdminus1)

Ingredient Volume (mL) Calories (kcal) Calories percentage ()20 mediumlong-chain fat emulsion(1) 400 720 343114 compound amino acids 800 364 17350 glucose 360 720 48410 glucose 740 29610 sodium chloride 4010 potassium chloride 3010 calcium gluconate 30Water-soluble vitamins(2) 12 ampouleFat-soluble vitamins(3) 12 ampouleTotal 2400 2100Note (1)mediumlong-chain fat emulsion (250mL) composition soybean oil 125 g medium chain triglycerides 125 g and lecithin 15 g (2)Water-solublevitamins composition vitamin B 106mg vitamin B 2072mg nicotinamide 8mg vitamin B 608mg pantothenic acid 3mg vitamin C 20mg biotin 12120583gfolic acid 80120583g and vitamin B 121 120583g (3)Fat-soluble vitamins composition vitamin A 50120583g (165 IU) vitamin D 2025120583g (10 IU) vitamin E 0455mg (05 IU)and vitamin K1 75 120583g

Thus GRP94 could well be an indicator of ERS in liverdisease The aim of this study was to use a rabbit PNALDmodel to study the changes in expression the ERS markerprotein GRP94 and determine its role in the pathogenesis ofPNALD

2 Materials and Methods

21 The PNALD Model and Experimental Groups Sixteen7-day-old breastfed New Zealand White rabbits obtainedfrom Wuxi Huishan Jiangnan Experimental Animal Centerwere randomly divided into a total parenteral nutritiongroup (TPN group 119899 = 8) and a control (breastfed) group(119899 = 8) The PNALD rabbit model was established asdescribed previously [15 16] with some modificationsBriefly the TPN group received continuous total parenteralnutrition 240mLkgsdotd for each rabbit consisting of formulacomposition shown in Table 1 Formula 240mL wasintroduced through a silastic catheter inserted in theright jugular vein TPN group received 40mL of a 20medium- and long-chain fat emulsion (72 kcal 343 oftotal calories) consisting of 2 g soy bean oil 2 g mediumchain triglyceride and 024 g egg phospholipids 80mL of114 pediatric compound amino acid injection-18AA-II(364 kcal 173 of calories) 36mL of 50 glucose (72 kcal484 of calories) 74mL of 10 glucose (296 kcal) 4mL of10 NaCl 3mL of 10 KCl 3mL of 10 calcium gluconatehalf of a water soluble and fat-soluble vitamin ampouleoriginally containing 03mg vitamin B1 036mg vitamin B24mg nicotinamide 04mg vitamin B6 15mg pantothenicacid 10mg vitamin C 6 120583g biotin 40 120583g folic acid 05120583gvitamin B12 25120583g (825 IU) vitamin A 10125 120583g (5 IU)vitamin D 02275mg (025 IU) vitamin E and 375120583gvitamin K1 and trace elements CaCl

2

sdot2H2

O 3925mgMgCl

2

sdot6H2

O 1521mg FeCl3

sdot6H2

O 0675mg ZnCl2

0135mg MnCl

2

sdot4H2

O 0395mg CuCl2

sdot2H2

O 425 120583gNaF 0105mg and KI 85 120583g Each 240mL portion ofTPN comprised 210 kcal and the ratio of sugar to lipid was14 1 The components in the mixture were purchased fromSino-Swed Pharmaceutical China

The control group received breast-feeding Both groupswere treated for one week and housed under conditions ofconstant temperature of 26ndash28∘C relative humidity of 40ndash60 and 12 h light and 12 h dark The study protocol wasapproved by the Animal Care Committee of the ChildrenrsquosHospital Affiliated to Soochow University

22 Specimen Collection and Processing All animals wereanesthetized with 10 chloral hydrate by intraperitonealinjection and fixed on the dissecting table The precordiumwas shaved and disinfected with iodine alcohol The point ofmaximal impulsewas identified and 2mLbloodwas obtainedby cardiac puncture and transferred to the anticoagulanttubes centrifuged at 3500 rpm serum and stored at minus20∘Cfreezer The animals were killed by an overdose of anestheticand the abdominal cavity was quickly opened Liver tissuewas excised and cleaned with normal saline Some tissuewas fixed in 10 paraformaldehyde and 50ndash100mg tissuesamples were placed in tube and stored in liquid nitrogenuntil analyzed

23 Blood Biochemical Tests Serum total bilirubin (TBIL120583M) bilirubin (DBIL 120583M) alanine aminotransferase (ALTIUL) aspartate aminotransferase (AST IUL) total protein(TP gL) albumin (ALB gL) 120574-glutamyl peptidase (120574-GT IUL) alkaline phosphatase (ALP IUL) triglyceride(TG mM) total cholesterol (TC mM) and prealbumin(PA mgL) were measured by a Hitachi 7600 automaticbiochemical analyzer (Japan)

24 Liver Pathology Fresh liver tissue was fixed with 10paraformaldehyde and dehydrated with serial different con-centrations of alcohol The dehydrated liver tissue was clearthrough xylene and embedded by paraffin The paraffin-embedded tissue blocks were cut into 5-micron thick sliceswhich were applied onto glass slides and dried in 45∘Cincubator The glass slides were dewaxed and stained withhematoxylin and eosin (H and E)

Gastroenterology Research and Practice 3

25 Measurement of GRP94 mRNA Levels in Liver Tissueby RT-PCR Frozen liver tissue was thawed at room tem-perature and ground in DEPC-treated mortar Total RNAwas obtained using a Trizol extraction kit RT-PCR wasdone using a Promega reverse transcription kit Primerswere designed using primer 50 software and synthesizedby Shanghai Sangon Biological Engineering Company aftera GenBank Blast search for homology All operations werecarried out according to the kit instructions Rabbit GAPDHwas selected as internal reference whose expected fragmentsize was 497 bp The primers were forward 51015840-GTTTGT-GATGGGCGTGAA-31015840 reverse 51015840-CGAAGGTAGAGG-AGTGGGTG-31015840 GRP94 fragment size was 583 bp andprimers were forward 51015840-AGGAAACACTCTGGGACG-31015840 reverse 51015840-ATTCAGGTACTTAGGCATC-31015840 RT-PCRproducts were observed on a 15 agarose gel electrophoresisSemiquantitative analysis was made in Bio2239 gel imager(Bio-Print Company)

26 GRP94 Protein Levels in Liver Tissue as Determinedby Immunohistochemistry Immunohistochemical analyseswere conducted using a streptomyces avidin-peroxidase linkmethod All slides were pretreated with polylysine (BosterBiological Engineering Co Ltd) Cells with brownish yellowgranules in the cytoplasm were considered to be positiveLiver tissue slices in glass slides were observed by lightmicroscopy after immunohistochemical staining accord-ing to the manufacturerrsquos instructions (Suzhou En MaikeBiotechnology Co Ltd)Three nonoverlapping fields of viewat high magnification (times400) were randomly selected fromeach slide for gray degree scanning using Image-Pro-Plusimage analysis software system The average gray level ofeach group was calculated to reflect the positive intensity ofGRP94

27 Statistical Analysis Statistical analysis was made usingSPSS170 statistical software Quantitative data were describedas mean plusmn standard deviation Comparisons between twogroups of quantitative variables were performed using Stu-dentrsquos 119905-test 119875 values lt 005 were considered to indicatestatistical significance

3 Results

31 Biochemical Parameters in the TPN and Control GroupsThere were statistically significant differences in serum levelsof TBIL (119905 = 4159 119875 lt 001) DBIL (119905 = 3338 119875 lt 001)120574-GT (119905 = 3907 119875 lt 001) and ALB (119905 = minus1236 119875 lt 001)between the two groups However there were no statisticallysignificant differences in serum TP ALT AST ALP TGTC or PA (119875 gt 005) Compared to the control groupserum TBIL DBIL and 120574-GT levels in the TPN group weresignificantly higher (119875 lt 001) while ALB was significantlylower (119875 lt 001) These results are shown in Table 2 andFigure 1

32 Liver Histology in the TPN and Control Groups The livertissue of the control group showed morphological normalhepatocytes with no bile duct abnormalities inflammatory

160

140

120

100

80

60

40

20

0

minus20

TBIL

DBI

L TP ALB AST

ALT

GG

T

ALP TG TC PA

Control groupTPN group

Serum values

Com

paris

on o

f bio

chem

ical

inde

xes

Figure 1 A comparison of serum biochemical data from theTPN and control groups Note each value is the mean plusmn SD ofassays using 8 independent samples Compared with the controlgroup

119875 lt 001 TBIL total bilirubin DBIL direct bilirubinTP total protein ALB albumin AST aspartate aminotransferaseALT alanine aminotransferase r-GT r-glutamyl GGT ALP alka-line phosphatase TG triglycerides TC total cholesterol and PAprealbumin

cell infiltration or hepatocyte degeneration and necrosis asshown in Figures 2(a) and 2(b) In contrast in liver tissueof the TPN there were inflammatory cell infiltration diffusesteatosis and liver cell cord structural disorder Howeverthere were no bile duct dilatation or epithelial hyperplasiano significant cholestasis and no visible fibrosis group withlobular structure as shown in Figures 2(c) and 2(d)

33 Levels of Liver GRP94 Protein in the TPN and ControlGroups Immunohistochemistry showed that GRP94 proteinexpression gray values in the TPN group and the controlgroup were 133838 plusmn 1366 78138 plusmn 8169 respectivelyGRP94 protein levels in the TPN group were significantlyhigher than those in the controls (119875 lt 001) as shown inFigures 3(a) 3(b) and 3(c) and Table 3

34 Liver GRP94 mRNA Levels in the TPN and ControlGroups RT-PCR in the liver tissue showed that the GRP94mRNA expression gray values in the TPN group and the con-trol group were 1217 plusmn 0112 and 0614 plusmn 0034 respectivelyGRP94mRNA expression gray values in the TPN group weresignificantly higher than those of controls (119875 lt 001) asshown in Table 4 and Figure 4

4 Discussion

Parenteral nutrition has offered powerful nutritional sup-port for critically ill infants including those who fail toget enteral nutrition But at the same time there are alsonegative effects during long-term parenteral nutrition whichare mainly PNALD occurrence [17] However the etiologyand pathogenesis of PNALD are poorly understood [18ndash21]Hepatocytes perform a myriad of metabolic functions andthus are enriched in both smooth and rough ER Recently


Table2Com

paris

onof

biochemicalindicatorsbetweentheg

roup

s

Group

ing

TBIL

DBIL

TPALB

AST

ALT

r-GT

ALP

TGTC

PA(umolL)

(umolL)

(gL)

(gL)

(IUL)

(IUL)

(IUL)

(IUL)

(mmolL)

(mmolL)

(mgL)

Con

trolgroup

228plusmn10

410

5plusmn057

2425plusmn312

2686plusmn283

7275plusmn1033

2193plusmn14

91128plusmn568

7239plusmn1368

174plusmn12

9041plusmn021

8086plusmn2999

TPNgrou

p5025plusmn306

4728plusmn391

2302plusmn19

41173plusmn10

57885plusmn270

2683plusmn657

13047plusmn561

7230plusmn669

286plusmn14

9092plusmn062

8651plusmn

1385

119905value

4159

3338

minus085

minus1236

139

207

3907

001

minus044

minus074

minus083

119875value

lt001

lt001

041

lt001

019

006

lt001

098

067

048

042

Note(1)c

omparedwith

thec

ontro

lgroup

119875lt001

(2)T

BILtotalbilirubinDBILdirectbilirub

inT

PtotalproteinA

LBalbum

inA

STaspartateam

inotransferaseA

LTalanine

aminotransferaser-G

Tr-glutam

ylGGTALP

alkalinep

hosphataseT

Gtrig

lycerid

es

TCtotalcholesterolandPA

prealbu

min


(a) (b)

(c) (d)

Figure 2 Representative sections of livers from the TPN and control groups obtained on d 7 and stained with H and E Note (a) controlgroup 200x (b) control group 400x (c) TPN group 200x and (d) TPN group 400x

Table 3 GRP94 protein expression gray values in liver tissues (plusmns)

Groups GRP94 protein expression gray values (119909 plusmn 119904)Control group (n = 8) 78138 plusmn 8169TPN group (n = 6) 133838 plusmn 13664

119905 value minus9546119875 value lt001Note compared with the control group 119875 lt 001

Table 4 Comparison of liver tissue GRP94mRNA level gray values(119909 plusmn 119904)

Groups GRP94 mRNAControl group (n = 8) 0614 plusmn 0034TPN group (n = 6) 1217 plusmn 0112


ERS response has been observed in a variety of liver diseasesand ERS response accompanies nearly all forms of acuteand chronic liver disease [5ndash10] Some of these observationsoffer mechanistic insights and present potential therapeutictargets However it is not known whether ERS response alsoplays an important role in PNALDThese associations of ERSresponse and other liver diseases alsomay indicate that a new

hypothesis is required to test the role of the ERS in PNALDTo test the hypothesis the expression changes of GRP94which is one of ERSmarker proteins were analyzed in a rabbitPNALD model

ERS has been shown to be involved in preventing proteinmisfolding and unfolding thus contributing to the mainte-nance of cell survival and normal function [22] HoweverERS of long duration can induce apoptosis [22] Correctprotein folding within the endoplasmic reticulum requiresthe assistance of chaperone proteins such as BipGRP78 andGRP94 and folding enzymes [23] Under normal conditionsthe endoplasmic reticulum chaperones BipGRP78 GRP94and Ire1 ATF6 and PERK combine to form a stable complexand stays in the endoplasmic reticulum lumenWhen proteinunfolding occurs a large number of these unfolded ormisfolded proteins accumulate resulting in dissociation ofIre1 ATF6 PERK and BIP [23] Excessively long or strongERS can increase the levels of ERS-related protein GRP94[24] It is widely accepted that GRP94 is an ERS marker [25]Therefore GRP94 was selected for detecting the occurrenceof ERS in PNALD

In the current study after one week of intravenousnutrition in 7-day-old rabbits serum TBIL DBIL and 120574-GT in the TPN group were significantly higher (119875 lt 001)while ALB was significantly lower (119875 lt 001) than thatin control group Liver pathology showed inflammatory cellinfiltration diffuse steatosis and liver cell cord structuraldisorder in the TPN group while these changes were not


(a) (b)

0

20

40

60

80

100

120

140

160

Control group TPN group

GRP94

(c)

Figure 3 Representative immunohistochemical staining of GRP94 protein in liver tissue from the TPN and control groups Note (a) controlgroup 400x (b) TPN group 400x and (c) a GRP94 protein expression gray value histogram of liver tissue from the two groups Comparedwith the control group 119875 lt 001

observed in the control group Liver damage occurred after1 week of TPN that was consistent with previous researchresults [16 17] The immunohistochemistry results showedthat the protein expression level of GRP94 in the liver ofTPN group was significantly increased compared with thecontrol group (133838 plusmn 13664 versus 78138 plusmn 8169) (119875 lt001)The RT-PCR results showed that the expression level ofGRP94mRNA in the liver of the TPN group was significantlyincreased compared with the control group (1217 plusmn 0113versus 0614 plusmn 0034) (119875 lt 001) Therefore the mRNA andprotein expression of GRP94 in the TPN groupwere both sig-nificantly increased which indicated that ERSmay be directlyrelated to the occurrence and development of PNALD

In the PNALD model 120574-GT activity which is mainlyattributed to the hepatobiliary system [26] has been reportedto be significantly increased While TBIL was also signifi-cantly increased the largest contribution to the elevated TBILwas DBIL but not IBIL Hyperbilirubinemia with mainlyDBIL elevation often suggests bile duct injury or obstructionAn elevation of GGT which has been reported to be closelyrelated to hepatic steatosis [27ndash29] was also observed inliver pathology results of the current study With severe

liver damage synthesis intracellular transport and releaseof ALB can be affected resulting in decreased serum ALB[30] Therefore long-term parenteral nutrition may causeinflammatory cell infiltration diffuse steatosis and liver cellcord structural disorder in liver This liver pathology changemay be accompanied with elevated serum DBIL and 120574-GT and decreased serum ALB Therefore we speculate thatincreases in TBIL DBIL and 120574-GT and the reduction of ALBlevels may be early indicators of the development of PNALD

There are limitations to this study The number of exper-iments was small and the experimental TPN applicationperiod was not long enough to observe cholestatic changesFuture experiments should be done for longer durations oftreatment to observe the relations of relevant biochemicaland the occurrence and development of PNALD There ishowever a problem relatedwith the specificity of these resultsfor parenteral nutrition therapy since controls for the stresswere not included (separation from the mother anesthesiacatheter inserted in the right jugular vein infusion etc)This limitation is likely not significant based on our previousreport that soybean oil parenteral nutrition was associatedwith significant liver dysfunction as indicated by higher


0

02

04

06

08

1

12

14


GRP94

(a)

GAPDH

GRP94

100 bp

250bp

500bp

750bp

1000 bp

2000 bp

Marker 1 2 3 4

(b)

Figure 4 (a) A gray value histogram of GRP94mRNA levels in liver tissue from the two groups Compared with the control group 119875 lt 001The bands were quantified as the relative integrated optical density (IOD) values of the ratio of GRP94GAPDH for two groups mean plusmn SD119899 = 8 (b) Liver tissue GRP94 mRNA amplified by RT-PCR and analyzed by electrophoresis Lanes marker DNA marker 1 and 2 controlgroup 3 and 4 TPN group

serum total bilirubin direct bilirubin and 120574-GT and lowerserum albumin levels compared to control These effectswere not observed in the fish oil fat emulsion group (TPN-FO) which was similar to the control Moreover histologicalexamination of liver tissues revealed hepatic damage in thesoybean fat emulsion group (TPN-soy) not seen in the TPN-FO including inflammatory cell infiltration diffuse hepaticsteatosis and disrupted hepatic cord structure [31]

5 Conclusion

In conclusion the current study showed that the mRNAand protein levels of GRP94 in the TPN group were bothsignificantly increased compared with those in the controlgroup The differences were statistically significant (119875 lt005) These results indicate that ERS occurred in the TPNgroup and may be involved in the development of PNALDThis information may provide an important novel basis forthe detection and prevention of PNALD

Conflict of Interests

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

Authorsrsquo Contribution

Xueping Zhu and Xiaomin Zhang contributed equally to thiswork

Acknowledgments

This research was supported by grants from the SuzhouScience and Technology Development Project (SYS201136and SYS201440) and the Jiangsu ProvinceHealthDepartmentsurface scientific research project (H201316) Natural ScienceFoundation Project of Jiangsu Province (no BK20141183)135 Project of Department of Health of Jiangsu Province(no RC2007076) and the Research Project of the SuzhouKey Laboratory of Childrenrsquos Developmental Brain InjuryPrevention and Care (no SZS201108) The authors gratefullyacknowledge all members of the laboratory for sharingreagents and advice The authors thank the pathologist forreviewing the histology slides

References

[1] S J Rangel C M Calkins R A Cowles et al ldquoParenteralnutrition-associated cholestasis an American pediatric surgicalassociation outcomes and clinical trials committee systematicreviewrdquo Journal of Pediatric Surgery vol 47 no 1 pp 225ndash2402012

[2] K M Gura S Lee C Valim et al ldquoSafety and efficacy of a fish-oil based fat emulsion in the treatment of parenteral nutritionassociated liver diseaserdquo Pediatrics vol 121 no 3 pp e678ndashe686 2008

[3] C J Klein T G Havranek M E Revenis Z Hassanali andL M Scavo ldquoPlasma fatty acids in premature infants withhyperbilirubinemia before-and-after nutrition support withfish oil emulsionrdquo Nutrition in Clinical Practice vol 28 no 1pp 87ndash94 2013


[4] A Kubota N Mochizuki J Shiraishi et al ldquoParenteral-nutrition-associated liver disease after intestinal perforationin extremely low-birthweight infants consequent lethal portalhypertensionrdquo Pediatrics International vol 55 no 1 pp 39ndash432013

[5] L Dara C Ji and N Kaplowitz ldquoThe contribution of endoplas-mic reticulum stress to liver diseasesrdquoHepatology vol 53 no 5pp 1752ndash1763 2011

[6] H Malhi and R J Kaufman ldquoEndoplasmic reticulum stress inliver diseaserdquo Journal of Hepatology vol 54 no 4 pp 795ndash8092011

[7] M J Pagliassotti ldquoEndoplasmic reticulum stress in nonalco-holic fatty liver diseaserdquo Annual Review of Nutrition vol 32 pp17ndash33 2012

[8] A Fernandez NMatias R Fucho et al ldquoASMase is required forchronic alcohol induced hepatic endoplasmic reticulum stressand mitochondrial cholesterol loadingrdquo Journal of Hepatologyvol 59 no 4 pp 805ndash813 2013

[9] C D Anderson G Upadhya K D Conzen et al ldquoEndoplasmicreticulum stress is a mediator of posttransplant injury inseverely steatotic liver allograftsrdquo Liver Transplantation vol 17no 2 pp 189ndash200 2011

[10] B-H Dai L Geng YWang et al ldquoMicroRNA-199a-5p protectshepatocytes from bile acid-induced sustained endoplasmicreticulum stressrdquoCell DeathampDisease vol 4 no 4 article e6042013

[11] D Eletto D Dersh and Y Argon ldquoGRP94 in ER quality controland stress responsesrdquo Seminars in Cell and DevelopmentalBiology vol 21 no 5 pp 479ndash485 2010

[12] M Marzec D Eletto and Y Argon ldquoGRP94 an HSP90-likeprotein specialized for protein folding and quality control inthe endoplasmic reticulumrdquo Biochimica et Biophysica ActamdashMolecular Cell Research vol 1823 no 3 pp 774ndash787 2012

[13] K Araki and K Nagata ldquoProtein folding and quality control inthe ERrdquo Cold Spring Harbor Perspectives in Biology vol 4 no 8Article ID a015438 2012

[14] D Morito and K Nagata ldquoER stress proteins in autoimmuneand inflammatory diseasesrdquo Frontiers in Immunology vol 48no 3 pp 1ndash8 2012

[15] S Hata S Kamata R Nezu Y Takagi and A Okada ldquoAnewborn rabbit model for total parenteral nutrition effects ofnutritional components on cholestasisrdquo Journal of Parenteraland Enteral Nutrition vol 13 no 3 pp 265ndash271 1989

[16] J Wu Y F Xu and W Cai ldquoThe establishment of totalparenteral nutrition-related cholestasis infant rabbits modelrdquoClinical Pediatrics vol 22 pp 107ndash109 2004

[17] E M Tillman ldquoReview and clinical update on parenteralnutrition-associated liver diseaserdquoNutrition in Clinical Practicevol 28 no 1 pp 30ndash39 2013

[18] S S Kaufman G E Gondolesi and T M Fishbein ldquoParenteralnutrition associated liver diseaserdquo Seminars in Neonatology vol8 no 5 pp 375ndash381 2003

[19] P Nandivada E Cowan S J Carlson M Chang K M GuraandM Puder ldquoMechanisms for the effects of fish oil lipid emul-sions in the management of parenteral nutrition-associatedliver diseaserdquo Prostaglandins Leukotrienes and Essential FattyAcids vol 89 no 4 pp 153ndash158 2013

[20] M P Cober and D H Teitelbaum ldquoPrevention of parenteralnutrition-associated liver disease lipid minimizationrdquo CurrentOpinion in Organ Transplantation vol 15 no 3 pp 330ndash3332010

[21] D A Kelly ldquoIntestinal failure-associated liver disease what dowe know todayrdquo Gastroenterology vol 130 no 2 supplement1 pp S70ndashS77 2006

[22] E Szczesna-Skorupa C-D Chen H Liu and B KemperldquoGene expression changes associated with the endoplasmicreticulum stress response induced by microsomal cytochromep450 overproductionrdquoThe Journal of Biological Chemistry vol279 no 14 pp 13953ndash13961 2004

[23] D Qing and Z Zhen ldquoThe role of endoplasmic reticulum stressin liver diseaserdquo International Journal of Internal Medicine vol36 no 11 pp 665ndash671 2009

[24] Z Q Li A R Li and C S Tang ldquoThe molecular mechanismstudy of endoplasmic reticulum stress responserdquo Chinese Jour-nal of Biochemistry andMolecular Biology vol 20 no 3 pp 283ndash288 2004

[25] Y Ma and L M Hendershot ldquoER chaperone functions dur-ing normal and stress conditionsrdquo Journal of Chemical Neu-roanatomy vol 28 no 1-2 pp 51ndash65 2004

[26] J B Whitfield ldquoGamma glutamyl transferaserdquo Critical Reviewsin Clinical Laboratory Sciences vol 38 no 4 pp 263ndash355 2001

[27] L P Breitling V Arndt C Drath and H Brenner ldquoLiverenzymes interaction analysis of smoking with alcohol con-sumption or BMI comparing AST and ALT to 120574-GTrdquo PLoSONE vol 6 no 11 Article ID e27951 2011

[28] L P Breitling H Claessen C Drath V Arndt and H BrennerldquoGamma-glutamyltransferase general and cause-specific mor-tality in 19000 construction workers followed over 20 yearsrdquoJournal of Hepatology vol 55 no 3 pp 594ndash601 2011

[29] E G Giannini R Testa and V Savarino ldquoLiver enzymealteration a guide for cliniciansrdquo CanadianMedical AssociationJournal vol 172 no 3 pp 367ndash379 2005

[30] Z Q Sun Y L Mao X Q Chen J X Guo L M Liu and YL Cong ldquoFormulation and application of diagnostic modelsbased on clinical biochemical assays in diagnosis of chronichepatitis and liver cirrhosis associated with viral hepatitisrdquoChinese Journal of Experimental and Clinical Virology vol 21no 3 pp 276ndash278 2007

[31] X Zhu Z Xiao X Chen et al ldquoParenteral nutrition-associatedliver injury and increased GRP94 expression prevented by 120596-3 fish oil-based lipid emulsion supplementationrdquo Journal ofPediatric Gastroenterology and Nutrition vol 59 no 6 pp 708ndash713 2014

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Table 1 Total parenteral nutrition solution formula (total liquid volume 240mLsdotkgminus1sdotdminus1 and total calories 210 kcalsdotkgminus1sdotdminus1)

Ingredient Volume (mL) Calories (kcal) Calories percentage ()20 mediumlong-chain fat emulsion(1) 400 720 343114 compound amino acids 800 364 17350 glucose 360 720 48410 glucose 740 29610 sodium chloride 4010 potassium chloride 3010 calcium gluconate 30Water-soluble vitamins(2) 12 ampouleFat-soluble vitamins(3) 12 ampouleTotal 2400 2100Note (1)mediumlong-chain fat emulsion (250mL) composition soybean oil 125 g medium chain triglycerides 125 g and lecithin 15 g (2)Water-solublevitamins composition vitamin B 106mg vitamin B 2072mg nicotinamide 8mg vitamin B 608mg pantothenic acid 3mg vitamin C 20mg biotin 12120583gfolic acid 80120583g and vitamin B 121 120583g (3)Fat-soluble vitamins composition vitamin A 50120583g (165 IU) vitamin D 2025120583g (10 IU) vitamin E 0455mg (05 IU)and vitamin K1 75 120583g

Thus GRP94 could well be an indicator of ERS in liverdisease The aim of this study was to use a rabbit PNALDmodel to study the changes in expression the ERS markerprotein GRP94 and determine its role in the pathogenesis ofPNALD

2 Materials and Methods

21 The PNALD Model and Experimental Groups Sixteen7-day-old breastfed New Zealand White rabbits obtainedfrom Wuxi Huishan Jiangnan Experimental Animal Centerwere randomly divided into a total parenteral nutritiongroup (TPN group 119899 = 8) and a control (breastfed) group(119899 = 8) The PNALD rabbit model was established asdescribed previously [15 16] with some modificationsBriefly the TPN group received continuous total parenteralnutrition 240mLkgsdotd for each rabbit consisting of formulacomposition shown in Table 1 Formula 240mL wasintroduced through a silastic catheter inserted in theright jugular vein TPN group received 40mL of a 20medium- and long-chain fat emulsion (72 kcal 343 oftotal calories) consisting of 2 g soy bean oil 2 g mediumchain triglyceride and 024 g egg phospholipids 80mL of114 pediatric compound amino acid injection-18AA-II(364 kcal 173 of calories) 36mL of 50 glucose (72 kcal484 of calories) 74mL of 10 glucose (296 kcal) 4mL of10 NaCl 3mL of 10 KCl 3mL of 10 calcium gluconatehalf of a water soluble and fat-soluble vitamin ampouleoriginally containing 03mg vitamin B1 036mg vitamin B24mg nicotinamide 04mg vitamin B6 15mg pantothenicacid 10mg vitamin C 6 120583g biotin 40 120583g folic acid 05120583gvitamin B12 25120583g (825 IU) vitamin A 10125 120583g (5 IU)vitamin D 02275mg (025 IU) vitamin E and 375120583gvitamin K1 and trace elements CaCl

2

sdot2H2

O 3925mgMgCl

2

sdot6H2

O 1521mg FeCl3

sdot6H2

O 0675mg ZnCl2

0135mg MnCl

2

sdot4H2

O 0395mg CuCl2

sdot2H2

O 425 120583gNaF 0105mg and KI 85 120583g Each 240mL portion ofTPN comprised 210 kcal and the ratio of sugar to lipid was14 1 The components in the mixture were purchased fromSino-Swed Pharmaceutical China

The control group received breast-feeding Both groupswere treated for one week and housed under conditions ofconstant temperature of 26ndash28∘C relative humidity of 40ndash60 and 12 h light and 12 h dark The study protocol wasapproved by the Animal Care Committee of the ChildrenrsquosHospital Affiliated to Soochow University

22 Specimen Collection and Processing All animals wereanesthetized with 10 chloral hydrate by intraperitonealinjection and fixed on the dissecting table The precordiumwas shaved and disinfected with iodine alcohol The point ofmaximal impulsewas identified and 2mLbloodwas obtainedby cardiac puncture and transferred to the anticoagulanttubes centrifuged at 3500 rpm serum and stored at minus20∘Cfreezer The animals were killed by an overdose of anestheticand the abdominal cavity was quickly opened Liver tissuewas excised and cleaned with normal saline Some tissuewas fixed in 10 paraformaldehyde and 50ndash100mg tissuesamples were placed in tube and stored in liquid nitrogenuntil analyzed

23 Blood Biochemical Tests Serum total bilirubin (TBIL120583M) bilirubin (DBIL 120583M) alanine aminotransferase (ALTIUL) aspartate aminotransferase (AST IUL) total protein(TP gL) albumin (ALB gL) 120574-glutamyl peptidase (120574-GT IUL) alkaline phosphatase (ALP IUL) triglyceride(TG mM) total cholesterol (TC mM) and prealbumin(PA mgL) were measured by a Hitachi 7600 automaticbiochemical analyzer (Japan)

24 Liver Pathology Fresh liver tissue was fixed with 10paraformaldehyde and dehydrated with serial different con-centrations of alcohol The dehydrated liver tissue was clearthrough xylene and embedded by paraffin The paraffin-embedded tissue blocks were cut into 5-micron thick sliceswhich were applied onto glass slides and dried in 45∘Cincubator The glass slides were dewaxed and stained withhematoxylin and eosin (H and E)





3 Results



160

140

120

100

80

60

40

20

0

minus20

TBIL

DBI

L TP ALB AST

ALT

GG

T

ALP TG TC PA


Serum values

Com

paris

on o

f bio

chem

ical

inde

xes






4 Discussion



Table2Com

paris

onof


roup

s

Group

ing

TBIL

DBIL

TPALB

AST

ALT

r-GT

ALP

TGTC

PA(umolL)

(umolL)

(gL)

(gL)

(IUL)

(IUL)

(IUL)

(IUL)

(mmolL)

(mmolL)

(mgL)

Con

trolgroup

228plusmn10

410

5plusmn057

2425plusmn312

2686plusmn283

7275plusmn1033

2193plusmn14

91128plusmn568

7239plusmn1368

174plusmn12

9041plusmn021

8086plusmn2999

TPNgrou

p5025plusmn306

4728plusmn391

2302plusmn19

41173plusmn10

57885plusmn270

2683plusmn657

13047plusmn561

7230plusmn669

286plusmn14

9092plusmn062

8651plusmn

1385

119905value

4159

3338

minus085

minus1236

139

207

3907

001

minus044

minus074

minus083

119875value

lt001

lt001

041

lt001

019

006

lt001

098

067

048

042

Note(1)c

omparedwith

thec

ontro

lgroup

119875lt001

(2)T


inT

PtotalproteinA

LBalbum

inA

STaspartateam

inotransferaseA

LTalanine

aminotransferaser-G

Tr-glutam

ylGGTALP

alkalinep

hosphataseT

Gtrig

lycerid

es


prealbu

min


(a) (b)

(c) (d)













(a) (b)

0

20

40

60

80

100

120

140

160


GRP94

(c)







0

02

04

06

08

1

12

14


GRP94

(a)

GAPDH

GRP94

100 bp

250bp

500bp

750bp

1000 bp

2000 bp

Marker 1 2 3 4

(b)



5 Conclusion






Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















3 Results



160

140

120

100

80

60

40

20

0

minus20

TBIL

DBI

L TP ALB AST

ALT

GG

T

ALP TG TC PA


Serum values

Com

paris

on o

f bio

chem

ical

inde

xes






4 Discussion



Table2Com

paris

onof


roup

s

Group

ing

TBIL

DBIL

TPALB

AST

ALT

r-GT

ALP

TGTC

PA(umolL)

(umolL)

(gL)

(gL)

(IUL)

(IUL)

(IUL)

(IUL)

(mmolL)

(mmolL)

(mgL)

Con

trolgroup

228plusmn10

410

5plusmn057

2425plusmn312

2686plusmn283

7275plusmn1033

2193plusmn14

91128plusmn568

7239plusmn1368

174plusmn12

9041plusmn021

8086plusmn2999

TPNgrou

p5025plusmn306

4728plusmn391

2302plusmn19

41173plusmn10

57885plusmn270

2683plusmn657

13047plusmn561

7230plusmn669

286plusmn14

9092plusmn062

8651plusmn

1385

119905value

4159

3338

minus085

minus1236

139

207

3907

001

minus044

minus074

minus083

119875value

lt001

lt001

041

lt001

019

006

lt001

098

067

048

042

Note(1)c

omparedwith

thec

ontro

lgroup

119875lt001

(2)T


inT

PtotalproteinA

LBalbum

inA

STaspartateam

inotransferaseA

LTalanine

aminotransferaser-G

Tr-glutam

ylGGTALP

alkalinep

hosphataseT

Gtrig

lycerid

es


prealbu

min


(a) (b)

(c) (d)













(a) (b)

0

20

40

60

80

100

120

140

160


GRP94

(c)







0

02

04

06

08

1

12

14


GRP94

(a)

GAPDH

GRP94

100 bp

250bp

500bp

750bp

1000 bp

2000 bp

Marker 1 2 3 4

(b)



5 Conclusion






Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table2Com

paris

onof


roup

s

Group

ing

TBIL

DBIL

TPALB

AST

ALT

r-GT

ALP

TGTC

PA(umolL)

(umolL)

(gL)

(gL)

(IUL)

(IUL)

(IUL)

(IUL)

(mmolL)

(mmolL)

(mgL)

Con

trolgroup

228plusmn10

410

5plusmn057

2425plusmn312

2686plusmn283

7275plusmn1033

2193plusmn14

91128plusmn568

7239plusmn1368

174plusmn12

9041plusmn021

8086plusmn2999

TPNgrou

p5025plusmn306

4728plusmn391

2302plusmn19

41173plusmn10

57885plusmn270

2683plusmn657

13047plusmn561

7230plusmn669

286plusmn14

9092plusmn062

8651plusmn

1385

119905value

4159

3338

minus085

minus1236

139

207

3907

001

minus044

minus074

minus083

119875value

lt001

lt001

041

lt001

019

006

lt001

098

067

048

042

Note(1)c

omparedwith

thec

ontro

lgroup

119875lt001

(2)T


inT

PtotalproteinA

LBalbum

inA

STaspartateam

inotransferaseA

LTalanine

aminotransferaser-G

Tr-glutam

ylGGTALP

alkalinep

hosphataseT

Gtrig

lycerid

es


prealbu

min


(a) (b)

(c) (d)













(a) (b)

0

20

40

60

80

100

120

140

160


GRP94

(c)







0

02

04

06

08

1

12

14


GRP94

(a)

GAPDH

GRP94

100 bp

250bp

500bp

750bp

1000 bp

2000 bp

Marker 1 2 3 4

(b)



5 Conclusion






Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)













(a) (b)

0

20

40

60

80

100

120

140

160


GRP94

(c)







0

02

04

06

08

1

12

14


GRP94

(a)

GAPDH

GRP94

100 bp

250bp

500bp

750bp

1000 bp

2000 bp

Marker 1 2 3 4

(b)



5 Conclusion






Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

0

20

40

60

80

100

120

140

160


GRP94

(c)







0

02

04

06

08

1

12

14


GRP94

(a)

GAPDH

GRP94

100 bp

250bp

500bp

750bp

1000 bp

2000 bp

Marker 1 2 3 4

(b)



5 Conclusion






Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

02

04

06

08

1

12

14


GRP94

(a)

GAPDH

GRP94

100 bp

250bp

500bp

750bp

1000 bp

2000 bp

Marker 1 2 3 4

(b)



5 Conclusion






Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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Research Article Hepatic Overexpression of GRP94 in a ...downloads.hindawi.com/journals/grp/2015/269831.pdf · Research Article Hepatic Overexpression of GRP94 in a Rabbit Model of