de novo endotoxin-induced production of antibodies against...

Research ArticleDe Novo Endotoxin-Induced Production ofAntibodies against the Bile Salt Export Pump Associated withBacterial Infection following Major Hepatectomy

Kun-Ming Chan Chih-Hsien Cheng Tsung-HanWu Chen-Fang Lee Ting-JungWuHong-Shiue Chou andWei-Chen Lee

Department of General Surgery Chang Gung Memorial Hospital at Linkou Chang Gung University College of MedicineTaoyuan Taiwan

Correspondence should be addressed to Kun-Ming Chan chankunmingadmcgmhorgtw

Received 15 November 2017 Revised 1 February 2018 Accepted 18 March 2018 Published 23 April 2018

Academic Editor Hwa-Liang Leo

Copyright copy 2018 Kun-MingChan et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Background Clinically severe infection-related inflammation after major liver resection may cause hyperbilirubinemia Thisstudy aims to clarify the impact of bacterial infection and endotoxins on the hepatobiliary transporter system and to explorepossible mechanisms of endotoxin-related postoperative hyperbilirubinemia Method Mice that underwent major hepatectomywith removal of at least 70 of liver volume were exposed to lipopolysaccharide (LPS) at different dosages Subsequentlyhepatobiliary transporter compounds related to bile salt excretion were further investigatedResultsThe expression of genes relatedto hepatobiliary transporter compounds was not significantly different in the liver tissue of mice after major hepatectomy and LPSexposure However bile salt export pump (BSEP) protein expression within the liver tissue of mice treated with LPS after majorhepatectomy was relatively weaker and was even further reduced in the high-dose LPS group The formation of antibodies againstthe BSEP in response to endotoxin exposure was also detectedConclusionThis study illustrates a possible mechanismwhereby thedysfunction of hepatobiliary transporter systems caused by endotoxin-induced autoantibodies may be involved in the developmentof postoperative jaundice associated with bacterial infection after major hepatectomy

1 Introduction

Liver resection for the removal of tumours is considered bestpractice for treating hepatic malignancy and can nowadaysbe performed safely with low mortality and morbidity [1 2]However up to 75 of patients that undergo liver surgeryhave abnormal postoperative liver function tests resultswhich may be accompanied by jaundice in some patients [3ndash6] Clinically severe postoperative jaundice is rare in patientswith no preexisting underlying liver disease and a sufficientremnant liver volume Nonetheless severe infection-relatedinflammation may result in hyperbilirubinemia after liverresection which might be associated with higher rates ofmorbidity and mortality [7 8] Hence it is of great impor-tance to identify potential mechanisms for the developmentof hyperbilirubinemia related to infection-associated inflam-mation after liver resection

The reasons for the development of postoperative jaun-dice remain unknown however jaundice is known to becaused by defects in the hepatobiliary transporter systemsthat are important for normal bile salt excretion leadingto cholestasis [9ndash11] The bile salt export pump (BSEP) isa crucial hepatobiliary transporter system located on thecanalicular membrane of hepatocytes which is involved inthe excretion of bile salts from the liver into the bile [12]Dysfunction of the BSEP in particular is considered a factorassociated with increased susceptibility for liver injury [1314] However little is known about the relationship betweenpostoperative bacterial infection and the hepatobiliary trans-porter system in the pathogenesis of hyperbilirubinemia afterliver resection Therefore the present study aims to clarifythe impact of bacterial infection and endotoxins on thehepatobiliary transporter system and to explore the possiblemechanism of endotoxin-related postoperative cholestasis

HindawiBioMed Research InternationalVolume 2018 Article ID 6197152 9 pageshttpsdoiorg10115520186197152

2 BioMed Research International

Table 1 List of RNA primers

Gene Forward primer sequenceReverse primer sequence

Abcb11 (BSEP) 51015840-AGACAGGCAACCCGTCATGGACT-3101584051015840-ACGAACGCCGTCGTTTCCCC-31015840

Abcc2 (Mrp2) 51015840-TAATGAGGCGCCGTGGGTGAC-3101584051015840-GTCCTGCCCACCACACCGAC-31015840

Abcc3 (Mrp3) 51015840-GGGCTGCCTTGCCCTGCTAC-3101584051015840-CCGAGGGCCGTCTTGAGCCT-31015840

Abcc4 (Mrp4) 51015840-CCGAGGTGAAACCCAACCCGC-3101584051015840-CGGGTTGAGCCACCAGAAGAACA-31015840

Slc10a1 (Ntcp) 51015840-AATCCAAGCTGCAGACGCACC-3101584051015840-GCATCTTCTGTTGCAGCAGCCTT-31015840

GAPDH 51015840-TGCACCACCAACTGCTTAG-3101584051015840-GATGCAGGGATGATGTTC-31015840

BSEP bile salt export pump Mrp2 multidrug resistance-associated protein 2 Mrp3 multidrug resistance-associated protein 3 Mrp4 multidrug resistance-associated protein 4 Ntcp Na+-dependent taurocholate transporter GAPDH glyceraldehyde-3-phosphate dehydrogenase

2 Materials and Methods

21 Animal Experiments Six- to 8-week-old male C57BL10J(B10) mice were purchased from the National LaboratoryAnimal Center Taipei Center Taiwan and maintained in thepathogen-free facility of Change-Gung Memorial Hospitalin Linkou All experimental procedures for these mice wereapproved by the Animal Care and Use Committee of ChangGung Memorial Hospital The use of animals was also inaccordance with the Animal Protection Law of the Councilof Agriculture Executive Yuan Taiwan and the guidelinesin the Guide for the Care and Use of Laboratory Animalsfrom the Institute of Laboratory Animal Resources of theNational Research Council USA Partial hepatectomy wasperformed by removing certain hepatic lobes as previouslydescribed andmajor hepatectomywas defined as the removalof at least 70 of the liver volume [15] Lipopolysaccharide(LPS Sigma St Louis MO USA) was dissolved in sterilesodium chloride (9 gL) and injected intravenously at theexperimental dosage via the infraorbital venous plexus ortail vein the day following the hepatectomy Mice weresacrificed at different time points for the collection of spec-imens Blood was either collected by cardiac puncture ordrawn from the inferior vena cava The liver was removedand immediately frozen in liquid nitrogen for mRNA andprotein analyses in addition to histological examinationAll experiments were repeated at least three times for eachgroup

22 RNA and Protein Expression Analyses Total RNA wasextracted from liver tissue using the RNeasy Mini Kit(Qiagen Sciences Valencia CA USA) according to themanufacturerrsquos protocol First-strand cDNA was synthesisedfrom 2 120583g of total RNA using the SuperScript first-strandsynthesis system (Invitrogen Carlsbad CAUSA)Next geneexpression as indicated by mRNA levels was analysed byreal-time polymerase chain reaction (PCR) in which thetarget cDNA was amplified by AmpliTaq DNA polymerase

(Applied Biosystems Foster City CA USA) The thermalcycling parameters for PCR were as follows preamplificationdenaturation for 10min at 95∘C 45ndash50 cycles of 95∘C for 45 s54ndash65∘C annealing for 45 s and extension at 72∘C for 45ndash90 sthen a final extension at 72∘C for 8min Beta-actin was usedas the loading control The primer sequences of RNA used inthis study are listed in Table 1

Protein expressionwas assessed bywestern blotting Totalprotein was extracted from liver tissue in radioimmuno-precipitation assay buffer (RIPA buffer Pierce RockfordIL USA) supplemented with complete protease inhibitorcocktail (Roche Applied Science Indianapolis IN USA)Theextracted protein was loaded onto a 12 sodium dodecylsulfate-polyacrylamide (SDS-PAGE) gel for electrophoresisthen proteins were transferred from the gels onto nitro-cellulose membranes (Amersham Biosciences PiscatawayNJ USA) The membranes were blocked with 5 milkin 01 Tween-20 in Tris-buffered saline and incubatedwith primary antibodies including an anti-BSEP antibody(LifeSpan Biosciences Seattle WA USA) anti-MRP2 (mul-tidrug resistance-associated protein 2) antibody (Cell Sig-naling Technology Beverly MA USA) anti-Ntcp (Na+-dependent taurocholate transporter) antibody and anti-120573-actin antibody (Millipore Billerica MA USA) Protein pat-ternsweremeasuredwith horseradish peroxidase-conjugatedsecondary antibodies (AB Science Taipei Taiwan) andelectrochemiluminescence (Amersham Pharmacia MilanItaly)

An indirect immunoblot assay was performed to test thepresence of BSEP antibodies in the sera after hepatectomyas previously described by Lin et al [16] Briefly membraneprotein lysate (40120583g) from normal liver tissue was subjectedto western blot analysis Proteins were separated by 12SDS-PAGE transferred onto nitrocellulose membranes andthen cut into strips Strips were probed with sera obtainedfrom experimental mice or with a commercial anti-BSEPantibody (positive control) The antibody-protein complexeswere then detected with horseradish peroxidase-conjugated

BioMed Research International 3

goat anti-mouse IgM secondary antibodies (AB ScienceTaipei Taiwan) and the signal was developed with elec-trochemiluminescence (Amersham Pharmacia Milan Italy)(Supplemental Figure 1)

23 Immunopathological Examination Sera levels of immu-noglobulin M (IgM) were measured using a mouse IgMELISA kit according to the manufacturerrsquos instructionsImmunohistochemical staining was performed on paraffin-embedded sections of the liver specimens according to themanufacturerrsquos instructions Immunohistochemical stainingwas performed using polyclonal antibodies against BSEP(LifeSpan Biosciences Seattle WA USA) Immunofluo-rescence staining was performed by staining cryosectionswith DyLight 488 (Bethyl Laboratories Montgomery TXUSA) conjugated anti-BSEP antibodies and DyLight 594(Bethyl Laboratories Montgomery TX USA) conjugatedanti-IgM antibodies Nuclei counterstaining was performedwith TO-PRO3 (Invitrogen Carlsbad CA USA) accordingto themanufacturerrsquos instructionsThe immunofluorescence-stained sections were subsequently visualised under a Bio-Rad Radiance 2100mp multiphotonconfocal microscope(Bio-RadHercules CAUSA) and imageswere captured andanalysed using Image J version 137 software

3 Results

31 Impact of Liver Resection Extent and Endotoxin onSurvival To determine the influence of bacterial infectionand extent of liver resection on the outcome of mice afterhepatectomy mice were subjected to either 50 or 70 liverresection followed by administration of LPS on postoperativeday 1 The results showed that mice tolerated the 50 liverresection well even after administration of LPS However afew mice died after the 70 liver resection and the mortalityrate was higher with increasing dosages of LPS (Figure 1)Administration of a higher dosage of LPS was associated witha poorer survival curve compared to the other groups Theseresults indicate that the postoperative bacterial infection doesindeed affect the outcome of major liver resection and theseverity of infection in regard to the LPS dosage could alsoinfluence survival

32 Expression of Hepatobiliary Transporters Related toBile Salt Excretion after Hepatectomy Previous studies haveshown that numerous hepatobiliary transporter compoundsincluding Ntcp BSEP and multidrug resistance-associatedproteins (MRP2 MRP3 and MRP4) play important rolesin the bile salt excretion of hepatocytes [10 17] ThereforeLPS was administered after major hepatectomy to exam-ine the impact of bacterial endotoxin on the hepatobiliarytransporter during the period of liver regeneration Hepato-biliary transporter compounds related to bile salt excretionwere analysed in the liver tissue and the expression ofhepatobiliary transporters including MRP2 MRP3 MRP4Ntcp and BSEP was found to be similar in the controlmice and the LPS-treated mice after hepatectomy in termsof immunohistochemical staining and mRNA levels The

No LPS (70 HT)

0

20

40

60

80

100

Perc

ent s

urvi

val (

)

4 6 8 10 122After Liver Resection (Days)

LPS 50 ng (70 HT)LPS 500ng (50 HT)LPS 500ng (70 HT)

Figure 1Kaplan-Meier survival of mice that underwent hepatectomy(HT) Mice that underwent 70 HT that were subsequently treatedwith high-dose lipopolysaccharide (LPS) had higher postoperativemortality than the other groups

immunohistochemical staining of BSEP expression showedno difference in the liver tissue of mice that were subjected tohepatectomy when compared to control livers (Figure 2(a))The gene expression of mRNA for MRP2 MRP3 MRP4Ntcp and BSEP was also examined and no significantdifferences were observed (Figure 2(b))

Because the BSEP is the main transport pathway forbile efflux we hypothesized that BSEP may be destroyedor dysfunctional as a result of endotoxin administrationduring the course of liver regeneration after major hepa-tectomy Therefore we tested the expression of the BSEPprotein within the liver tissue of mice treated with LPSafter major hepatectomy and the results showed that BSEPprotein expression was relatively lower in groups treated withLPS and was even further reduced as the dosage increased(Figure 3) These results suggest that bacterial endotoxinmight lead to impaired BSEP protein expression and reducedfunctional capacity of BSEP in the hepatocytes during theinitial period of liver regeneration after major hepatectomywhich might be further reduced by severe bacterial infection

33 Identification of Endotoxin-Induced Antibodies afterMajor Hepatectomy The newly formed antibodies inresponse to exposure to endotoxin were examined Thedynamic expression of IgM in mice subjected to 70hepatectomy and LPS injection was investigated Theresults showed that the serum level of IgM increasedgradually after LPS treatment reaching its highest levelat day 4 Additionally the increments of increased IgMappeared to be correlated with the LPS dosage (Figure 4)Furthermore an indirect immunoblot assay was performedto test the presence of the BSEP antibody in the sera afterhepatectomy Membrane protein lysate from normal livertissue which reasonably contains BSEP protein was subjected


(a)

BSEP

MRP2

MRP3

MRP4Ntcp

00

05

10

15

Arb

itrar

y ex

pres

sion

of m

RNA

（４

+20

0ng

200

ng

（４

+50

0ng

（４

+ＨＩ

LPS

500

ng

Nor

mal

500

ng

200

ng

（４

+ＨＩ

LPS

（４

+20

0ng

（４

+50

0ng

Nor

mal

00

05

10

15

Arb

itrar

y ex

pres

sion

of m

RNA

00

05

10

15

20

Arb

itrar

y ex

pres

sion

of m

RNA

200

ng

（４

+20

0ng

（４

+ＨＩ

LPS

（４

+50

0ng

Nor

mal

500

ng

00

05

10

15

20

Arb

itrar

y ex

pres

sion

of m

RNA

500

ng

200

ng

（４

+ＨＩ

LPS

（４

+20

0ng

（４

+50

0ng

Nor

mal

00

05

10

15

Arb

itrar

y ex

pres

sion

of m

RNA

200

ng

（４

+20

0ng

（４

+ＨＩ

LPS

（４

+50

0ng

Nor

mal

500

ng

(b)

Figure 2 Examination of hepatobiliary transporter compounds (a) Immunohistochemical staining of bile salt export pump (BSEP) in livertissue showed no difference even when compared with normal control liver (20x) From left to right normal liver tissue without hepatectomyand LPS exposure liver tissue with hepatectomy plus LPS 200 ng at postoperative day 4 liver tissue with hepatectomy plus LPS 500 ng atpostoperative day 2 and liver tissue with hepatectomy plus 500 ng LPS at postoperative day 4 (b) The mRNA expression levels of MRP2MRP3 MRP4 BSEP and Ntcp were examined and no significant differences were observed between groups

to western blot for gel electrophoresis of BSEP proteinAfter electroblotting of nitrocellulose membrane from thegel electrophoresis anti-BSEP antibodies (positive control)or sera obtained from experimental mice were applied tothe nitrocellulose membrane as a primary antibodies fortesting the existence of BSEP antibody Antibodies againstBSEP were detectable in the sera obtained from mice thatunderwent 70 hepatectomy followed by LPS treatment(Figure 5(a)) However antibodies against BSEP were notdetected in the sera from normal mice nor in control micethat had undergone 70 hepatectomy without LPS treatment(Figure 5(b)) These results suggest that bacterial infectionmight lead to the production of antibodies specifically

against BSEP during the initial period of liver regenerationas well as impaired BSEP function

34 De Novo Production of Antibodies against the Bile SaltExport Pump To determine whether BSEP was targetedby the newly produced BSEP antibodies we further exam-ined BSEP and IgM expression within the liver tissue byimmunofluorescence staining (Figure 6) Positive stainingfor both IgM and BSEP was observed in certain areas ofliver tissue from mice that had undergone 70 hepatectomyfollowed by 500 ng LPS treatment whereas very weak orbarely detectable staining for IgM and BSEP was noted inthe liver tissue of mice that had not undergone hepatectomy


BSEP170

130100

(kDa) Nor

mal

live

r

No

LPS

40-actin

LPS

50ng

LPS20

0ng

LPS50

0ng

Figure 3 Bile salt export pump (BSEP) protein expression on thehepatocyte membrane Western blot of BSEP protein expression inliver tissue at postoperative day 4 showed a relative decrease in BSEPexpression in the hepatocyte membrane of mice that had undergone70 hepatectomy and subsequently treated with lipopolysaccharide(LPS) Arrow indicates BSEP protein expression (146 kDa)

No LPS

LPS

0

200

400

600

800

IgM

(ng

ml)

minus1 1 2 3 4 5 6 7 80After Liver Resection (Days)

LPS 50 ngLPS 200 ngLPS 500 ng

Figure 4 Serum IgM level of mice that underwent 70 hepatec-tomy followed by lipopolysaccharide (LPS) treatment The serumIgM level progressively increased after LPS injection and reached thehighest level at postoperative day 6The increase in IgM is correlatedwith the LPS dosage

or those that had undergone 70 hepatectomy but were nottreated with LPSThese findings together with the precedingdata suggest that LPS-induced de novo antibody productionagainst BSEP occurred Therefore the main transport path-way of bile efflux BSEP could potentially be targeted by theLPS-induced antibodies leading to BSEP dysfunction andhyperbilirubinemia after major hepatectomy

4 Discussion

Clinically severe postoperative bacterial infection may leadto hyperbilirubinemia after partial hepatectomy related to

a higher risk of morbidity and mortality The reasons forthe development of postoperative jaundice remain unknownbut it is known that jaundice can be caused by defectsin hepatobiliary transporter systems that are important fornormal bile salt excretionThis study intended to characterizefactors that may induce posthepatectomy jaundice and liverfailure associated with bacterial infectionThe results showedthat the hepatobiliary transporter system as indicated byBSEP expression was mildly decreased under the high-doseLPS endotoxin challenge Furthermore de novo anti-BSEPantibodies were detected in posthepatectomy livers afteradministration of bacterial endotoxin and antibodies againstBSEPwere also found in the liver tissueTherefore the resultsof the current study suggest that a possible mechanism mayinclude an interaction between endotoxin and the bile salttransporter system in the liver after major hepatectomy withsevere bacterial infection

The liver is a quiescent organ that has the unique capacityto regulate its growth and mass in both humans and animalsThere are many conditions under which a change in livermass may occur for example deficits may result from cellloss caused by chemicals viruses or other toxic agents orfrom surgical removal of liver tissue Under each of theseconditions quiescent hepatocytes become proliferative andreplicate to restore the functional capacity and mass ofthe liver The set point for growth regulation is weightedagainst the balance between the liver mass and body massOptimisation of this balance occurs when the liver reachesa state in which the metabolic activity meets the functionalrequirements of the body As a result segmental or lobarresections are applied in clinical settings for the removal oftumours or for living-donor liver transplantation It has beenreported that at least 25 of the future remnant liver volumeor at least 30 of standard liver volume should bemaintainedin order to meet the metabolic demand of the patient afterliver resection [18ndash20] However an accurate estimation ofthe smallest liver volume that meets the functional demandsof the body has not yet been established The higher the ratioof liver volume removed the higher the risk of postoperativemorbidity and mortality in terms of hepatic dysfunctionIn line with previous reports the results of the currentstudy suggest that removal of a greater liver volume wouldset the host in a vulnerable situation leading to a higherchance of lethal complications and mortality related to liverdysfunction

Although the existence of an underlying liver diseaseprior to liver surgery might cause abnormal postopera-tive liver function test results various factors may also beresponsible for the development of postoperative jaundiceincluding intraoperative blood loss with consecutive bloodtransfusionsmedication sepsis and oxidative stress even fora healthy liver Inflammatory cholestasis has been identifiedas one the most frequent causes of jaundice in hospitalisedpatients [21 22] Specifically sepsis-associated cholestasisis frequently observed in critically ill patients the mostcommon cause of which is the inhibition of hepatobiliarytransporter expression and function due to bacterial endo-toxins [23 24] Although the immunohistochemical stainingshowed no overt downregulated expression of BSEP within


170

130

(kDa)

BSEP AbSerum from mice

without HT

Serum from mice with 70 HT

Serum from mice with 70 HT +

-actin

LPS 500 ng+ LPS 200 ng

(a)

00

01

02

03

04

Expr

essio

n ra

tio (B

SEP

-a

ctin

)

No

HT

70

HT

BSEP

Ab

70

H

T+20

0ＨＡ

70

H

T+50

0ＨＡ

(b)

Figure 5 Indirect immunoblot assay to test the presence of antibodies against bile salt export pump (BSEP) in the sera after hepatectomy (HT)(a) Membrane protein lysate from normal liver tissue was subjected to western blot analysis with control anti-BSEP antibodies (BSEP Ab)or sera obtained from experimental mice Blots with sera as primary antibodies and with peroxidase-labeled anti-mouse IgM as secondaryantibodies showed that the presence of BSEP antibodies was detected at postoperative day 6 in mice that had undergone 70 HT and LPStreatmentThe experiments were done by triple lanes with the same protein sample from normal liver tissue Each stripe represented intensityof BSEP antibodywithin sera from experimentalmouse as shownby legend in the left-hand side (b)Quantification intensity of BSEP antibodyexpression within sera Mice that had not undergone HT and mice that had undergone HT without LPS treatment had no detectable BSEPantibodies in sera at the same time point Arrow indicates BSEP (146 kDa)

the liver tissue of mice in the groups that received LPS treat-ment BSEP protein expression was found to be decreasedby LPS treatment in the western blotting analysis Theseresults indicate that BSEP might be inhibited or functionallyimpaired by LPS and other bacterial endotoxins

Bile salt export pumps are exclusively expressed onhepatocytes There are a few hereditary diseases that causeBSEP deficiency which may result in severe cholestasis andend-stage liver disease [25] Moreover previous studies haveshown that anti-BSEP antibodies are induced in certaincircumstances or diseases which can lead to cholestasis[16 26ndash28] Based on the indirect immunoblot assay BSEPantibodies were detected in the sera of mice that had under-gone major hepatectomy followed by LPS treatment but noantibodies were detected in the sera of control mice or mice

that underwent hepatectomy without subsequent LPS treat-ment The results suggest that BSEP autoantibodies mightbe induced by the endotoxin challenge However the exactmechanism that triggered the formation of autoantibodiesagainst BSEP remains largely unclear and cannot be explainedby the results of the present study

Usually many proteins are present in bile in whichproteins are mostly come from plasma through blood tobile transfer by simple diffusion across the semipermeabletight junction of paracellular pathway [29 30] Howeverfew proteins such as secretory IgA IgM ceruloplasmin andhaemoglobin presumably enter bile as content of intracellularvesicles that fuse with the canalicular membrane [31] More-over immunoglobulin J chain that is a protein component ofthe antibodies IgM and IgA has previously been identified


BSEP Merge

Normal liver

70 HT No LPS

70 HT LPS 500 ng

IgM Ab

Figure 6 Immunofluorescence analysis of newly formed antibodies against bile salt export pump (BSEP) protein and IgM antibodies Positivestaining of both IgM and BSEP was detected in certain areas of the liver tissue of mice that had undergone 70 hepatectomy plus high-doselipopolysaccharide (LPS) treatment However weak or barely detectable double positive for IgM and BSEP was noted in the liver tissue ofmice that had not undergone hepatectomy or in those that had undergone 70 hepatectomy without LPS treatment

in bile suggesting that IgA and IgM are capable of enteringthe canalicular space [30] The inhibitory effect of BSEPautoantibodies could result from interferencewith the assem-bly of microdomains and disturbing the coordinated actionof canalicular transporters or potentially direct mechanicalocclusion of the BSEP pore by the antibodies [26] Apartfrom that a purified de novo BSEP antibody as well as acell line transfected with BSEP to test the inhibitory effect ofautoantibodiesmight be able to clarify the pathophysiologicalmechanism in the future

Although these antibodies were identified against theBSEP protein no overt cholestasis was noted in mice thatunderwentmajor hepatectomy plus LPS treatment in the cur-rent study The phenomenon may be explained by the quan-tity of antibody possibly being too low to induce cholestasisand the antibody titre threshold that could lead to cholestasisor hyperbilirubinemia needs to be confirmed by a moredetailed scientific study Similarly the mRNA expression ofhepatobiliary transporter compounds was unchanged afterLPS treatment andmight also be related to no overt cholesta-sis of mice in this study However in contrast to study thathad showed downregulation of hepatobiliary transporter ininflammatory cholestasis [32 33] few reports also indicatedthat some unchanged or even upregulated transport systems

such as MRP2 and MRP3 in endotoxin challenged livertissue [23 34 35] Additionally species differences regardingexpression of hepatobiliary transporter systems might alsoexist The animal model of partial hepatectomy through therodent liver ofmultilobulated character perhaps is not similarto human hepatectomy that is characterized by relativelymore invasive procedure and severe inflammatory reaction

Taken together extensivemajor hepatectomywith a smallremnant liver mass is usually very risky and subsequentdevelopment of a severe bacterial infection could result inthe hepatic failure or mortality In general hyperbilirubine-mia and jaundice are the most common presentations ofhepatic dysfunction Although numerous causes may beresponsible for the development of postoperative jaundicethe current study illustrates a possible mechanism wherebydysfunction of hepatobiliary transporter systems due toendotoxin-induced autoantibody production may play a rolein the development of jaundice after major hepatectomy withconcurrent bacterial infection

Conflicts of Interest

The authors have no conflicts of interest


Acknowledgments

This work was supported by grants from the Chang GungMedical Research Program (CMRPG3F0211sim2) and TaiwanNational Science Council (103-2314-B-182-048) to K-MChan

Supplementary Materials

Supplemental Figure 1 schematic illustration of an indirectimmunoblot assay to test the presence of BSEP antibodies in thesera Membrane protein lysate from normal liver tissue wassubjected to western blot for gel electrophoresis of BSEP pro-tein After electroblotting of nitrocellulose membrane fromthe gel electrophoresis anti-BSEP antibodies (positive con-trol) or sera obtained from experimental mice were appliedto the nitrocellulose membrane as the primary antibodies fortesting the existence of BSEP antibody The antibody-proteincomplexes were then detected with horseradish peroxidase-conjugated goat anti-mouse IgM secondary antibodies andthe signal was developed with electrochemiluminescence asshown in Figure 5(a)The experimentwas done by triple laneswith the same protein sample from normal liver tissue Eachstripe represented intensity of BSEP antibody of sera fromexperimental mouse (Supplementary Materials)

References

[1] K-M Chan Y-C Wang T-H Wu et al ldquoThe Preferencefor Anterior Approach Major Hepatectomy Experience over3 Decades and a Propensity Score-Matching Analysis in RightHepatectomy for Hepatocellular CarcinomardquoMedicine (UnitedStates) vol 94 no 34 p e1385 2015

[2] D Dahiya T-J Wu C-F Lee K-M Chan W-C Lee andM-F Chen ldquoMinor versus major hepatic resection for smallhepatocellular carcinoma (HCC) in cirrhotic patients A 20-year experiencerdquo Surgery vol 147 no 5 pp 676ndash685 2010

[3] T W Faust and K R Reddy ldquoPostoperative jaundicerdquo Clinics inLiver Disease vol 8 no 1 pp 151ndash166 2004

[4] C Pauli-Magnus and P J Meier ldquoHepatobiliary transportersand drug-induced cholestasisrdquo Hepatology vol 44 no 4 pp778ndash787 2006

[5] O Hyder C Pulitano A Firoozmand et al ldquoA risk model topredict 90-day mortality among patients undergoing hepaticresectionrdquo Journal of the American College of Surgeons vol 216no 6 pp 1049ndash1056 2013

[6] K-M Chan C-F Lee T-J Wu et al ldquoAdverse outcomesin patients with postoperative ascites after liver resection forhepatocellular carcinomardquoWorld Journal of Surgery vol 36 no2 pp 392ndash400 2012

[7] M Fuchs and A J Sanyal ldquoSepsis and Cholestasisrdquo Clinics inLiver Disease vol 12 no 1 pp 151ndash172 2008

[8] A Kosters and S J Karpen ldquoThe role of inflammation incholestasis Clinical and basic aspectsrdquo Seminars in Liver Dis-ease vol 30 no 2 pp 186ndash194 2010

[9] G A Bernhardt G Zollner H Cerwenka et al ldquoHepatobiliarytransporter expression and post-operative jaundice in patientsundergoing partial hepatectomyrdquo Liver International vol 32no 1 pp 119ndash127 2012

[10] M Trauner M Wagner P Fickert and G Zollner ldquoMolecularregulation of hepatobiliary transport systems clinical impli-cations for understanding and treating cholestasisrdquo Journal ofClinical Gastroenterology vol 39 no 4 supplement 2 pp S111ndashS124 2005

[11] M Wagner and M Trauner ldquoTranscriptional regulation ofhepatobiliary transport systems in health and disease implica-tions for a rationale approach to the treatment of intrahepaticcholestasisrdquo Annals of Hepatology vol 4 pp 77ndash99 2005

[12] B Stieger Y Meier and P J Meier ldquoThe bile salt export pumprdquoPflugers Archiv - European Journal of Physiology vol 453 no 5pp 611ndash620 2007

[13] J A Byrne S S Strautnieks GMieliVergani C F Higgins K JLinton and R JThompson ldquoThe human bile salt export pumpCharacterization of substrate specificity and identification ofinhibitorsrdquo Gastroenterology vol 123 no 5 pp 1649ndash16582002

[14] B Stieger K Fattinger J Madon G A Kullak-Ublick andP J Meier ldquoDrug- and estrogen-induced cholestasis throughinhibition of the hepatocellular bile salt export pump (Bsep) ofrat liverrdquo Gastroenterology vol 118 no 2 pp 422ndash430 2000

[15] M Nikfarjam C Malcontenti-Wilson M Fanartzis JDaruwalla and C Christophi ldquoA model of partial hepatectomyin micerdquo Journal of Investigative Surgery vol 17 no 5 pp291ndash294 2004

[16] H C Lin L Alvarez G Laroche et al ldquoRituximab as therapyfor the recurrence of bile salt export pump deficiency after livertransplantationrdquo Liver Transplantation vol 19 no 12 pp 1403ndash1410 2013

[17] R Kubitz C Droge J Stindt K Weissenberger and DHaussinger ldquoThe bile salt export pump (BSEP) in health anddiseaserdquo Clinics and Research in Hepatology and Gastroenterol-ogy vol 36 no 6 pp 536ndash553 2012

[18] C-L Chen S-T Fan S-G Lee M Makuuchi and K TanakaldquoLiving-donor liver transplantation 12 Years of experience inAsiardquo Transplantation vol 75 no 3 pp S6ndashS11 2003

[19] H Chisuwa Y Hashikura A Mita et al ldquoLiving liver donationPreoperative assessment anatomic considerations and long-term outcomerdquo Transplantation vol 75 no 10 pp 1670ndash16762003

[20] S Hwang T-Y Ha G-W Song et al ldquoQuantified RiskAssessment for Major Hepatectomy via the Indocyanine GreenClearance Rate and Liver Volumetry Combined with StandardLiver Volumerdquo Journal of Gastrointestinal Surgery vol 19 no 7pp 1305ndash1314 2015

[21] M W Whitehead I Hainsworth and J G C Kingham ldquoThecauses of obvious jaundice in South West Wales Perceptionsversus realityrdquo Gut vol 48 no 3 pp 409ndash413 2001

[22] A S DeLemos and L S Friedman ldquoSystemic Causes ofCholestasisrdquo Clinics in Liver Disease vol 17 no 2 pp 301ndash3172013

[23] M G L Elferink P Olinga A L Draaisma et al ldquoLPS-induceddownreguiation of MRP2 and BSEP in human liver is due toa posttranscriptional processrdquo American Journal of Physiology-Gastrointestinal and Liver Physiology vol 287 no 5 pp G1008ndashG1016 2004

[24] H Yang T Plosch T Lisman et al ldquoInflammation mediateddown-regulation of hepatobiliary transporters contributes tointrahepatic cholestasis and liver damage in murine biliaryatresiardquo Pediatric Research vol 66 no 4 pp 380ndash385 2009

[25] S S Strautnieks J A Byrne L Pawlikowska et al ldquoSevere BileSalt Export Pump Deficiency 82 Different ABCB11 Mutations


in 109 Familiesrdquo Gastroenterology vol 134 no 4 pp 1203ndash12142008

[26] V Keitel M Burdelski Z Vojnisek L Schmitt D HaussingerandRKubitz ldquoDe novo bile salt transporter antibodies as a pos-sible cause of recurrent graft failure after liver transplantationA novel mechanism of cholestasisrdquo Hepatology vol 50 no 2pp 510ndash517 2009

[27] P Jara L Hierro P Martınez-Fernandez et al ldquoRecurrence ofbile salt export pump deficiency after liver transplantationrdquoTheNew England Journal of Medicine vol 361 no 14 pp 1359ndash13672009

[28] G Maggiore E Gonzales M Sciveres et al ldquoRelapsing featuresof bile salt export pump deficiency after liver transplantation intwo patients with progressive familial intrahepatic cholestasistype 2rdquo Journal of Hepatology vol 53 no 5 pp 981ndash986 2010

[29] M A Barbhuiya N A Sahasrabuddhe S M Pinto et alldquoComprehensive proteomic analysis of human bilerdquo Proteomicsvol 11 no 23 pp 4443ndash4453 2011

[30] T Z Kristiansen J Bunkenborg M Gronborg et al ldquoA pro-teomic analysis of humanbilerdquoMolecularampCellular Proteomicsvol 3 no 7 pp 715ndash728 2004

[31] J L Boyer ldquoBile formation and secretionrdquo ComprehensivePhysiology vol 3 pp 1035ndash1078 2013

[32] R Kubitz M Wettstein U Warskulat and D HaussingerldquoRegulation of the multidrug resistance protein 2 in the rat liverby lipopolysaccharide and dexamethasonerdquo Gastroenterologyvol 116 no 2 pp 401ndash410 1999

[33] J M Lee M Trauner C J Soroka B Stieger P J Meier and JL Boyer ldquoExpression of the bile salt export pump is maintainedafter chronic cholestasis in the ratrdquo Gastroenterology vol 118no 1 pp 163ndash172 2000

[34] C G Dietrich A Geier N Salein et al ldquoConsequences ofBile Duct Obstruction on Intestinal Expression and Function ofMultidrug Resistance-Associated Protein 2rdquo Gastroenterologyvol 126 no 4 pp 1044ndash1053 2004

[35] G Zollner P Fickert R Zenz et al ldquoHepatobiliary transporterexpression in percutaneous liver biopsies of patients withcholestatic liver diseasesrdquo Hepatology vol 33 no 3 pp 633ndash646 2001

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


Table 1 List of RNA primers

Gene Forward primer sequenceReverse primer sequence

Abcb11 (BSEP) 51015840-AGACAGGCAACCCGTCATGGACT-3101584051015840-ACGAACGCCGTCGTTTCCCC-31015840

Abcc2 (Mrp2) 51015840-TAATGAGGCGCCGTGGGTGAC-3101584051015840-GTCCTGCCCACCACACCGAC-31015840

Abcc3 (Mrp3) 51015840-GGGCTGCCTTGCCCTGCTAC-3101584051015840-CCGAGGGCCGTCTTGAGCCT-31015840

Abcc4 (Mrp4) 51015840-CCGAGGTGAAACCCAACCCGC-3101584051015840-CGGGTTGAGCCACCAGAAGAACA-31015840

Slc10a1 (Ntcp) 51015840-AATCCAAGCTGCAGACGCACC-3101584051015840-GCATCTTCTGTTGCAGCAGCCTT-31015840

GAPDH 51015840-TGCACCACCAACTGCTTAG-3101584051015840-GATGCAGGGATGATGTTC-31015840

BSEP bile salt export pump Mrp2 multidrug resistance-associated protein 2 Mrp3 multidrug resistance-associated protein 3 Mrp4 multidrug resistance-associated protein 4 Ntcp Na+-dependent taurocholate transporter GAPDH glyceraldehyde-3-phosphate dehydrogenase

2 Materials and Methods

21 Animal Experiments Six- to 8-week-old male C57BL10J(B10) mice were purchased from the National LaboratoryAnimal Center Taipei Center Taiwan and maintained in thepathogen-free facility of Change-Gung Memorial Hospitalin Linkou All experimental procedures for these mice wereapproved by the Animal Care and Use Committee of ChangGung Memorial Hospital The use of animals was also inaccordance with the Animal Protection Law of the Councilof Agriculture Executive Yuan Taiwan and the guidelinesin the Guide for the Care and Use of Laboratory Animalsfrom the Institute of Laboratory Animal Resources of theNational Research Council USA Partial hepatectomy wasperformed by removing certain hepatic lobes as previouslydescribed andmajor hepatectomywas defined as the removalof at least 70 of the liver volume [15] Lipopolysaccharide(LPS Sigma St Louis MO USA) was dissolved in sterilesodium chloride (9 gL) and injected intravenously at theexperimental dosage via the infraorbital venous plexus ortail vein the day following the hepatectomy Mice weresacrificed at different time points for the collection of spec-imens Blood was either collected by cardiac puncture ordrawn from the inferior vena cava The liver was removedand immediately frozen in liquid nitrogen for mRNA andprotein analyses in addition to histological examinationAll experiments were repeated at least three times for eachgroup

22 RNA and Protein Expression Analyses Total RNA wasextracted from liver tissue using the RNeasy Mini Kit(Qiagen Sciences Valencia CA USA) according to themanufacturerrsquos protocol First-strand cDNA was synthesisedfrom 2 120583g of total RNA using the SuperScript first-strandsynthesis system (Invitrogen Carlsbad CAUSA)Next geneexpression as indicated by mRNA levels was analysed byreal-time polymerase chain reaction (PCR) in which thetarget cDNA was amplified by AmpliTaq DNA polymerase

(Applied Biosystems Foster City CA USA) The thermalcycling parameters for PCR were as follows preamplificationdenaturation for 10min at 95∘C 45ndash50 cycles of 95∘C for 45 s54ndash65∘C annealing for 45 s and extension at 72∘C for 45ndash90 sthen a final extension at 72∘C for 8min Beta-actin was usedas the loading control The primer sequences of RNA used inthis study are listed in Table 1

Protein expressionwas assessed bywestern blotting Totalprotein was extracted from liver tissue in radioimmuno-precipitation assay buffer (RIPA buffer Pierce RockfordIL USA) supplemented with complete protease inhibitorcocktail (Roche Applied Science Indianapolis IN USA)Theextracted protein was loaded onto a 12 sodium dodecylsulfate-polyacrylamide (SDS-PAGE) gel for electrophoresisthen proteins were transferred from the gels onto nitro-cellulose membranes (Amersham Biosciences PiscatawayNJ USA) The membranes were blocked with 5 milkin 01 Tween-20 in Tris-buffered saline and incubatedwith primary antibodies including an anti-BSEP antibody(LifeSpan Biosciences Seattle WA USA) anti-MRP2 (mul-tidrug resistance-associated protein 2) antibody (Cell Sig-naling Technology Beverly MA USA) anti-Ntcp (Na+-dependent taurocholate transporter) antibody and anti-120573-actin antibody (Millipore Billerica MA USA) Protein pat-ternsweremeasuredwith horseradish peroxidase-conjugatedsecondary antibodies (AB Science Taipei Taiwan) andelectrochemiluminescence (Amersham Pharmacia MilanItaly)

An indirect immunoblot assay was performed to test thepresence of BSEP antibodies in the sera after hepatectomyas previously described by Lin et al [16] Briefly membraneprotein lysate (40120583g) from normal liver tissue was subjectedto western blot analysis Proteins were separated by 12SDS-PAGE transferred onto nitrocellulose membranes andthen cut into strips Strips were probed with sera obtainedfrom experimental mice or with a commercial anti-BSEPantibody (positive control) The antibody-protein complexeswere then detected with horseradish peroxidase-conjugated




3 Results



No LPS (70 HT)

0

20

40

60

80

100

Perc

ent s

urvi

val (

)








(a)

BSEP

MRP2

MRP3

MRP4Ntcp

00

05

10

15

Arb

itrar

y ex

pres

sion

of m

RNA

（４

+20

0ng

200

ng

（４

+50

0ng

（４

+ＨＩ

LPS

500

ng

Nor

mal

500

ng

200

ng

（４

+ＨＩ

LPS

（４

+20

0ng

（４

+50

0ng

Nor

mal

00

05

10

15

Arb

itrar

y ex

pres

sion

of m

RNA

00

05

10

15

20

Arb

itrar

y ex

pres

sion

of m

RNA

200

ng

（４

+20

0ng

（４

+ＨＩ

LPS

（４

+50

0ng

Nor

mal

500

ng

00

05

10

15

20

Arb

itrar

y ex

pres

sion

of m

RNA

500

ng

200

ng

（４

+ＨＩ

LPS

（４

+20

0ng

（４

+50

0ng

Nor

mal

00

05

10

15

Arb

itrar

y ex

pres

sion

of m

RNA

200

ng

（４

+20

0ng

（４

+ＨＩ

LPS

（４

+50

0ng

Nor

mal

500

ng

(b)






BSEP170

130100

(kDa) Nor

mal

live

r

No

LPS

40-actin

LPS

50ng

LPS20

0ng

LPS50

0ng


No LPS

LPS

0

200

400

600

800

IgM

(ng

ml)





4 Discussion






170

130

(kDa)


without HT



-actin


(a)

00

01

02

03

04

Expr

essio

n ra

tio (B

SEP

-a

ctin

)

No

HT

70

HT

BSEP

Ab

70

H

T+20

0ＨＡ

70

H

T+50

0ＨＡ

(b)







BSEP Merge

Normal liver

70 HT No LPS

70 HT LPS 500 ng

IgM Ab









Acknowledgments




References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






















3 Results



No LPS (70 HT)

0

20

40

60

80

100

Perc

ent s

urvi

val (

)








(a)

BSEP

MRP2

MRP3

MRP4Ntcp

00

05

10

15

Arb

itrar

y ex

pres

sion

of m

RNA

（４

+20

0ng

200

ng

（４

+50

0ng

（４

+ＨＩ

LPS

500

ng

Nor

mal

500

ng

200

ng

（４

+ＨＩ

LPS

（４

+20

0ng

（４

+50

0ng

Nor

mal

00

05

10

15

Arb

itrar

y ex

pres

sion

of m

RNA

00

05

10

15

20

Arb

itrar

y ex

pres

sion

of m

RNA

200

ng

（４

+20

0ng

（４

+ＨＩ

LPS

（４

+50

0ng

Nor

mal

500

ng

00

05

10

15

20

Arb

itrar

y ex

pres

sion

of m

RNA

500

ng

200

ng

（４

+ＨＩ

LPS

（４

+20

0ng

（４

+50

0ng

Nor

mal

00

05

10

15

Arb

itrar

y ex

pres

sion

of m

RNA

200

ng

（４

+20

0ng

（４

+ＨＩ

LPS

（４

+50

0ng

Nor

mal

500

ng

(b)






BSEP170

130100

(kDa) Nor

mal

live

r

No

LPS

40-actin

LPS

50ng

LPS20

0ng

LPS50

0ng


No LPS

LPS

0

200

400

600

800

IgM

(ng

ml)





4 Discussion






170

130

(kDa)


without HT



-actin


(a)

00

01

02

03

04

Expr

essio

n ra

tio (B

SEP

-a

ctin

)

No

HT

70

HT

BSEP

Ab

70

H

T+20

0ＨＡ

70

H

T+50

0ＨＡ

(b)







BSEP Merge

Normal liver

70 HT No LPS

70 HT LPS 500 ng

IgM Ab









Acknowledgments




References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















(a)

BSEP

MRP2

MRP3

MRP4Ntcp

00

05

10

15

Arb

itrar

y ex

pres

sion

of m

RNA

（４

+20

0ng

200

ng

（４

+50

0ng

（４

+ＨＩ

LPS

500

ng

Nor

mal

500

ng

200

ng

（４

+ＨＩ

LPS

（４

+20

0ng

（４

+50

0ng

Nor

mal

00

05

10

15

Arb

itrar

y ex

pres

sion

of m

RNA

00

05

10

15

20

Arb

itrar

y ex

pres

sion

of m

RNA

200

ng

（４

+20

0ng

（４

+ＨＩ

LPS

（４

+50

0ng

Nor

mal

500

ng

00

05

10

15

20

Arb

itrar

y ex

pres

sion

of m

RNA

500

ng

200

ng

（４

+ＨＩ

LPS

（４

+20

0ng

（４

+50

0ng

Nor

mal

00

05

10

15

Arb

itrar

y ex

pres

sion

of m

RNA

200

ng

（４

+20

0ng

（４

+ＨＩ

LPS

（４

+50

0ng

Nor

mal

500

ng

(b)






BSEP170

130100

(kDa) Nor

mal

live

r

No

LPS

40-actin

LPS

50ng

LPS20

0ng

LPS50

0ng


No LPS

LPS

0

200

400

600

800

IgM

(ng

ml)





4 Discussion






170

130

(kDa)


without HT



-actin


(a)

00

01

02

03

04

Expr

essio

n ra

tio (B

SEP

-a

ctin

)

No

HT

70

HT

BSEP

Ab

70

H

T+20

0ＨＡ

70

H

T+50

0ＨＡ

(b)







BSEP Merge

Normal liver

70 HT No LPS

70 HT LPS 500 ng

IgM Ab









Acknowledgments




References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















BSEP170

130100

(kDa) Nor

mal

live

r

No

LPS

40-actin

LPS

50ng

LPS20

0ng

LPS50

0ng


No LPS

LPS

0

200

400

600

800

IgM

(ng

ml)





4 Discussion






170

130

(kDa)


without HT



-actin


(a)

00

01

02

03

04

Expr

essio

n ra

tio (B

SEP

-a

ctin

)

No

HT

70

HT

BSEP

Ab

70

H

T+20

0ＨＡ

70

H

T+50

0ＨＡ

(b)







BSEP Merge

Normal liver

70 HT No LPS

70 HT LPS 500 ng

IgM Ab









Acknowledgments




References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















170

130

(kDa)


without HT



-actin


(a)

00

01

02

03

04

Expr

essio

n ra

tio (B

SEP

-a

ctin

)

No

HT

70

HT

BSEP

Ab

70

H

T+20

0ＨＡ

70

H

T+50

0ＨＡ

(b)







BSEP Merge

Normal liver

70 HT No LPS

70 HT LPS 500 ng

IgM Ab









Acknowledgments




References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















BSEP Merge

Normal liver

70 HT No LPS

70 HT LPS 500 ng

IgM Ab









Acknowledgments




References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















Acknowledgments




References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















de novo endotoxin-induced production of antibodies against...

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