Research Article The Value of Circulating Nogo-B for ...

Research ArticleThe Value of Circulating Nogo-B for Evaluating HepaticFunctional Reserve in Patients with Cirrhosis

Maoyao Wen1 Ruoting Men1 Zongze Yang2 Xuelian Dan1 Wenchao Wu3

Xiaojing Liu3 and Li Yang1

1Division of Digestive Diseases West China Hospital of Sichuan University Chengdu Sichuan 610041 China2Creation and Management of a Tumour Bank West China Hospital of Sichuan University Chengdu Sichuan 610041 China3Laboratory of Cardiovascular Diseases Regenerative Medicine Research Center West China Hospital of Sichuan UniversityChengdu Sichuan 610041 China

Correspondence should be addressed to Xiaojing Liu liuxqscueducn and Li Yang linayang63gmailcom

Received 21 December 2014 Accepted 1 April 2015

Academic Editor George Perry

Copyright copy 2015 Maoyao Wen 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 examine Nogo-B in liver tissues and plasma of patients with liver cirrhosis and associate them with variousclinical parameters Materials and Methods Nogo-B protein expression was examined by immunohistochemistry in 24 humanfibroticcirrhotic liver specimens and 10 healthy controls We determined plasma Nogo-B levels by enzyme-linked immunosorbentassay in 301 patients with liver cirrhosis and 153 healthy controls and then analyzed various clinical parameters Results Nogo-Bwas mainly expressed in nonparenchymal cells in the liver and was marked increased in liver with significant fibrosiscirrhosiscompared to controls Moreover Metavir F4 showed a higher level of expression than F2 Plasma Nogo-B levels were significantlyhigher in cirrhotic patients than in healthy controls and were the highest in Child-Pugh class C patients Plasma Nogo-B levelswere positively correlated with Child-Pugh scores However there was no relationship between plasma Nogo-B levels and etiologyof liver diseases ALT AST platelet counts and the severity of esophagogastric varices Conclusions Nogo-B is mainly expressedin hepatic nonparenchymal cells and is present in plasma Abnormally high plasma levels of Nogo-B are associated with hepaticcirrhosis and Child-Pugh score but not correlated with the grade of liver inflammation or portal hypertension Plasma Nogo-Bmay be a novel surrogate marker to reflect liver function reserve

1 Introduction

Liver fibrosiscirrhosis is a major cause of mortality aroundthe world and the development of cirrhosis has been consid-ered to be an irreversible event [1 2] Prognosis of patientswith liver cirrhosis often depends on their hepatic functionalreserve Blood tests play pivotal roles in the clinical assess-ment of liver function reserve Clinical classification systemssuch as model for end-stage liver disease (MELD) and Child-Pugh scoring systems are widely used Even radiographicexamination of the remnant liver volumes can be helpful [3]It is desirable to have a simple noninvasive blood test thatreflects both the stage of liver fibrosis and functional reserve

Nogo-B is a member of the reticulum (Rtn) family ofproteins localized primarily in the endoplasmic reticulum

(ER) and is widely distributed in cardiac myocyte vascularendothelial cell smooth muscle cell testis and other tissues[4ndash6] Recent studies have shown that Nogo-B levels aresignificantly elevated in rat with cirrhosis whereas low levelsof Nogo-B suggest the absence of liver fibrosis [7] In ourprevious study we found that the plasma Nogo-B levelsin patients with hepatic cirrhosis were significantly higherthan healthy controls [8] As a potential indicator of hepaticcirrhosis the correlation between Nogo-B and clinical char-acteristics of cirrhosis remains unclear Thus we examinedNogo-B protein expression in fibroticcirrhotic liver tissuesby immunohistochemistry and further investigated hepaticcirrhotic patients with different etiology and severity Thenwe analyzed the relationship between plasma Nogo-B levels

Hindawi Publishing CorporationDisease MarkersVolume 2015 Article ID 419124 6 pageshttpdxdoiorg1011552015419124

2 Disease Markers

and the commonly used clinical parameters of liver functionin all patients

2 Methods

21 Subjects Fibroticcirrhotic liver specimens wereobtained from 24 patients who underwent liver surgery fora variety of liver diseases Twelve of the specimens showedMetavir stage 2 (F2) and the other 12 showed stage 4 (F4)fibrosis The 10 healthy liver specimens were from patientswho underwent liver surgery for benign focal lesions [9]All the liver specimens were provided by the Departmentof Pathology West China Hospital Sichuan University Forplasma Nogo-B analysis 301 patients of hepatic cirrhosiswere enrolled in the Division of Digestive Diseases WestChina Hospital Sichuan University from March 2012 toMarch 2014 152 patients had hepatitis B cirrhosis 83 werealcoholic cirrhosis and 66 were primary biliary cirrhosis(PBC) The diagnosis of cirrhosis was made based onimaging studies and impaired liver synthetic function [10]Patients with cardiovascular disease kidney diseases centralnervous system disorder chronic obstructive pulmonarydiseases (COPD) and pulmonary artery hypertension whichmight affect plasma Nogo-B levels were excluded [11 12]The normal control group was composed of 153 healthyvolunteers All subjects provided written informed consentsThis study was approved by the medical ethics committee ofthe West China Hospital Sichuan University

22 Collection of Samples and Clinical Parameters Bloodsamples from patients and healthy controls were obtainedThe plasma was immediately separated by centrifugation at800 g for 10 minutes at room temperature and stored atminus80∘C Parameters of liver function such as alanine amino-transferase (ALT) aspartate aminotransferase (AST) andplatelet (PLT) were collected from the Department of Clin-ical Laboratory West China Hospital Sichuan UniversityThe volume of ascites was observed by ultrasound PatientrsquosChild-Pugh classscore were calculated Esophageal and gas-tric varices were screened by esophagogastroduodenoscopyThe varices were defined as either large (gt5mm) or small(lt5mm) [13]

23 Plasma Nogo-B Assay The plasma Nogo-B levelswere determined by enzyme-linked immunosorbent assay(ELISA) kit (Biolegend Inc USA) and the assays wereperformed by following the manufacturersquos instruction

24 Histological Analyses Five-micrometer sections ofhuman liver tissue were prepared and embedded in paraffinThen these samples were stained with hematoxylin andeosin

25 Immunohistochemistry The sections were deparaffinizedin xylene and dehydrated in ethanol and then treated with001molL citrate buffer of 40 minutes pH 60 at 95∘C inwater bath Endogenous peroxidase activity was blocked byincubation in 3 H

2O2for 15 minutes in the dark Slides

Table 1 Clinical data of hepatic cirrhotic patients

Data Valuedagger

CharacteristicsGender (malefemale) 197104Age 5165 (21ndash80)Etiology (chronic hepatitis Binfectionalcoholic cirrhosisPBC) 1528366

ParametersChild-Pugh category (ABC) 11211574Degrees of esophageal and gastricvarices (absentsmalllarge)Dagger 99102100

Biochemical parametersTotal bilirubin (120583molL) 61ndash6077ALT (IUL) 6ndash389AST (IUL) 9ndash773Albumin (gL) 173ndash453INR 089ndash228PLT (109L)sect 11ndash390

daggerData are presented as the range or as the number of patientsDaggerEsophageal and gastric varices were diagnosed by esophagogastroduo-denoscopy Doi 101002hep21907sectPLT was analyzed in 203 patients who were without splenic embolizationand splenectomyALT alanine aminotransferase AST aspartate aminotransferase INR inter-national normalized ratio PBC primary biliary cirrhosis PLT platelet

were then incubated in 1 200 rabbit polyclonal Nogo-A+Bantibody (Abcam ab47085) for 45 minutes at 37∘C andincubated in 50ndash100120583L ChemMate Envision+HRP (DakoEnvisionTM Detection Kit number K5007) for 45 minutesat 37∘C Next diaminobenzidine (DAB) was added to eachslide for coloration Distilled water was used to end thechromogenic reaction The slides were counterstained withhematoxylin

26 Statistical Analysis Statistical analyses were performedusing SPSS 170 119875 lt 005 was considered significant Datawere presented as mean plusmn standard deviations (SD) or rangeCorrelations were analyzed with the Spearman correlationcoefficient Multiple comparisons for different groups werecarried out using unpaired 119905-test or one-way analysis ofvariance (ANOVA) followed by SNK test IPP 60 was usedto analyze the results of immunohistochemistry

3 Results

31 Nogo-B Is Highly Expressed in Liver Tissues with MetavirStage F4 Nogo-B was mainly expressed in hepatic non-parenchymal cells and highly expressed in fibroticcirrhotictissues Nogo-B expression in the liver positively correlatedwith the stage of liver fibrosis (Figure 1 F4 versus F2 119875 lt005)

32 Circulating Nogo-B Levels Are Significantly Increased inPatients with Hepatic Cirrhosis Clinical characteristics ofpatients in our study were summarized in Table 1 Our results

Disease Markers 3

HE F0 HE F2 HE F4

IHC-F0 IHC-F2 IHC-F4

Figure 1 Nogo-B is associated withMetavir fibrosis scoring Human liver tissues were stained with hematoxylin and eosin (200x) and Nogo-B is highly expressed in cirrhotic human livers (F4) and localizes primarily in nonparenchymal cells F0 Metavir stage 0 F2 Metavir stageF2 and F4 Metavir stage F4

showed that plasma Nogo-B levels were significantly higherin cirrhotic patients (42984 plusmn 22666 pgmL) than in healthycontrols (22204 plusmn 7454 pgmL 119875 lt 005 Figure 2(a))The mean plasma Nogo-B levels in patients with hepatitisB cirrhosis (44118 plusmn 22269 pgmL) alcoholic cirrhosis(43578 plusmn 23368 pgmL) and PBC (39628 plusmn 22700 pgmL)were not significantly different However they were all muchhigher than healthy controls (22204plusmn7454 pgmL119875 lt 005Figure 2(b))

33 Circulating Nogo-B Is Correlated with Child-Pugh Classi-fication in Liver Cirrhotic Patients Child-Pugh classificationis considered an important indicator to reflect the severityof liver diseases [13] To investigate whether plasma Nogo-B levels correlate with Child-Pugh classification we dividedall 301 patients into three groups according to their Child-Pugh scores (112 patients were Child-Pugh A 115 were Child-Pugh B and 74 were Child-Pugh C Table 1) The resultsshowed that plasma Nogo-B levels were significantly higherin Child-Pugh A B and C groups compared with healthycontrols (32844plusmn14117 pgmL 40251plusmn17933 pgmL and62579 plusmn 27336 pgmL versus 22204 plusmn 7454 pgmL resp119875 lt 005) In Child-Pugh class C group the plasma Nogo-B levels were significantly higher than Child-Pugh A and Bgroups (62579plusmn27336 pgmL versus 32844plusmn14117 pgmL40251 plusmn 17933 pgmL resp 119875 lt 005) And Nogo-B levelswere significantly increased in Child-Pugh class B patientscompared with Child-Pugh class A (119875 lt 005 Figure 3(a))

Furthermore the Child-Pugh scores were positively corre-lated with plasma Nogo-B levels (119903 = 0570 119875 lt 001Figure 3(b))

34 Association of Circulating Nogo-B Levels with Ala-nine Aminotransferase and Aspartate Aminotransferase Weexaminedwhether plasmaNogo-B levels correlatedwith liverinflammatory markers The results showed no significantcorrelation between plasma Nogo-B concentrations and ALT(119903 = 0062 119875 = 0285 Figure 4(a)) or AST (119903 = 0112119875 = 0052 Figure 4(b))

35 Circulating Nogo-B and the Relationship with the PlateletCounts and the Degree of Esophageal and Gastric Varices Weanalyzed the relationship between plasma Nogo-B levels andPLT counts (excluding patients with splenic embolizationand splenectomy) the degree of esophageal and gastricvaricesThis analysis showed no significant change in patientswith different degrees of esophageal and gastric varices(Figure 5(a)) PLT counts in patients who did not receivesplenic embolization or splenectomy were analyzed as welland similarly no correlationwas found (119903 = minus024119875 = 0739Figure 5(b))

4 Discussion

Our study confirmed that Nogo-B protein was mainlyexpressed in nonparenchymal cell in liver tissues [7] and

4 Disease Markers

P lt 005

Healthy control Patients0

200

400

600

800

1000

1200

1400

1600

Rang

e

(a)

800

700

600

500

400

300

200

100

0

Hea

lthy

cont

rol

Hep

atiti

s B ci

rrho

sis

Alc

ohol

ic ci

rrho

sis

PBC

Plas

ma N

ogo-

B le

vel (

pgm

L)

lowastP lt 005 versus healthy control

lowast

lowastlowast

(b)

Figure 2 (a) Plasma Nogo-B levels were significantly higher in cirrhotic patients than in healthy controls (119875 lt 005) (b) Plasma Nogo-Blevels in patients with hepatitis B cirrhosis alcoholic cirrhosis and primary biliary cirrhosis were significantly higher than healthy controlswhile there is no significant difference among patients with different etiologies PBC primary biliary cirrhosis

1000

900

800

700

600

500

400

300

200

100

0Healthy control Child-Pugh A Child-Pugh B Child-Pugh C

Plas

ma N

ogo-

B le

vel (

pgm

L)


P lt 005 versus Child-Pugh A

ampP lt 005 versus Child-Pugh B

lowast

lowast

lowast

amp

(a)

1500

1200

900

600

300

04 6 8 10 12 14

Child-Pugh score

Nog

o-B

(pg

mL)

r = 0570 P lt 001

(b)

Figure 3 (a) Plasma Nogo-B levels were significantly higher in Child-Pugh C than Child-Pugh A and B groups (119875 lt 005) And Nogo-Blevels were significantly increased in Child-Pugh class B compared with Child-Pugh class A (119875 lt 005) (b) Child-Pugh scores were positivelycorrelated with plasma Nogo-B levels (119903 = 0570 119875 lt 001)

showed that the expression of Nogo-B in liver tissues posi-tively correlatedwithMetavir fibrosis scoringThis result sug-gested that Nogo-B correlates with the histological severity ofhepatic cirrhosis

In order to further examine the relationship of Nogo-Band hepatic cirrhosis we measured the plasma Nogo-B levelsin 301 patients with hepatic cirrhosis and 153 healthy controlsWe found that plasmaNogo-B levels were significantly higher

in the cirrhotic patients than in controls [8] Howeverthere was no significant difference of plasma Nogo-B amongcirrhotic patients of different etiologies (hepatitis B alcoholiccirrhosis and PBC) We also found a positive correlationbetween the plasma Nogo-B levels and the Child-Pughscores Child-Pugh scores were considered an importantindicator of the liver functional reserve Thus the plasmaNogo-B might be a good surrogate marker for evaluation of

Disease Markers 5

1500

1200

900

600

300

0450400350300250200150100500

ALT (IUL)

Nog

o-B

(pg

mL)

r = 0062 P = 0285

(a)

1500

1200

900

600

300

08007006005004003002001000

AST (IUL)

Nog

o-B

(pg

mL)

r = 0112 P = 0052

(b)

Figure 4 (a) Plasma Nogo-B levels were not in correlation with alanine aminotransferase (119903 = 0062 119903 = 0570 119875 = 0285) and (b) aspartateaminotransferase (119903 = 0112 119875 = 0052) ALT alanine aminotransferase and AST aspartate aminotransferase

900

800

700

600

500

400

300

200

100

0Without varices Small varices Large varices

Plas

ma N

ogo-

B le

vel (

pgm

L)

(a)

1500

1200

900

600

300

05004003002001000

PLT (109L)sect

Nog

o-B

(pg

mL)

r = minus024 P = 0739

(b)

Figure 5 (a) PlasmaNogo-B levels have no significant changes in patients with different degrees of esophageal and gastric varices (b) Plateletcounts in patients who did not receive splenic embolization or splenectomy were not correlated with plasma Nogo-B (119903 = minus024 119875 = 0739)PLT platelet

liver functional reserve We found no correlation betweenplasma Nogo-B and ALT and AST suggesting that plasmaNogo-B concentrations do reflect inflammation of liver Thisis understandable as Nogo-B is not produced in hepatocytesAlso Nogo-B levels were not correlated with thrombocy-topenia or the severity of esophagogastric varices suggestingthat plasma Nogo-B does not reflect the severity of portalhypertension These findings suggest that plasma Nogo-Bwas closely related to the degree of liver fibrosis and liverreserve function but not to the active inflammation of theliver Due to the easy ELISA assay Nogo-B may be used asa simple clinical test for staging liver fibrosis and assessing

liver functional reserve in both liver transplantation andhepatology clinics

In conclusion our study showed Nogo-B was mainlyexpressed in nonparenchymal cells in the liver and thetissue Nogo-B levels correlated with the severity of liverfibrosis scores histologically The plasma Nogo-B levels weresignificantly higher in cirrhosis patients and there was amoderate degree of correlation between the plasma Nogo-B levels and the Child-Pugh scores Nogo-B levels howeverdo not correlate with inflammation of liver and the severityof portal hypertension These findings suggest that plasmaNogo-B may be a very useful surrogate marker for clinical

6 Disease Markers

assessment of liver fibrosis and liver function reserve inpatients with chronic liver diseases with or without portalhypertension and thrombocytopenia

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

The authors thank Dr James Mu for his critical review andinsightful discussion of the research data Dr James Mu is apracticing physician in the Gastroenterology Department ofWesternWashingtonMedical Group EverettWA USAThisstudy was funded by grants from Science and TechnologyDepartment of Sichuan Province of China (no 2013FZ0085and no 2014FZ0002) Chengdu City Science and TechnologyBureau of Sichuan Province of China (13PPYB994SF-014)and Natural Science Foundation (no 11072163) This workwas carried out in Laboratory of Cardiovascular DiseasesRegenerativeMedicineResearchCenterWestChinaHospitalof Sichuan University Chengdu Sichuan 610041 China

References

[1] E S L Chan M C Montesinos P Fernandez et al ldquoAdenosineA2119860

receptors play a role in the pathogenesis of hepaticcirrhosisrdquo British Journal of Pharmacology vol 148 no 8 pp1144ndash1155 2006

[2] S L Friedman ldquoHepatic stellate cells protean multifunctionaland enigmatic cells of the liverrdquo Physiological Reviews vol 88no 1 pp 125ndash172 2008

[3] P Ge S Du and Y Mao ldquoAdvances in preoperative assessmentof liver functionrdquo Hepatobiliary amp Pancreatic Diseases Interna-tional vol 13 no 4 pp 361ndash370 2014

[4] A Di Lorenzo T D Manes A Davalos P L Wright and WC Sessa ldquoEndothelial reticulon-4B (Nogo-B) regulates ICAM-1-mediated leukocyte transmigration and acute inflammationrdquoBlood vol 117 no 7 pp 2284ndash2295 2011

[5] A B Huber O Weinmann C Brosamle T Oertle and M ESchwab ldquoPatterns of NogomRNA and protein expression in thedeveloping and adult rat and after CNS lesionsrdquo The Journal ofNeuroscience vol 22 no 9 pp 3553ndash3567 2002

[6] R Prinjha S E Moore M Vinson et al ldquoNeurobiologyinhibitor of neurite outgrowth in humansrdquoNature vol 403 no6768 pp 383ndash384 2000

[7] D Zhang T Utsumi H-C Huang et al ldquoReticulon 4B (Nogo-B) is a novel regulator of hepatic fibrosisrdquo Hepatology vol 53no 4 pp 1306ndash1315 2011

[8] RMenMWenXDan et al ldquoNogo-B a potential indicator forhepatic cirrhosis and regulator in hepatic stellate cell activationrdquoHepatology Research vol 45 no 1 pp 113ndash122 2015

[9] Z D Goodman ldquoGrading and staging systems for inflamma-tion andfibrosis in chronic liver diseasesrdquo Journal ofHepatologyvol 47 no 4 pp 598ndash607 2007

[10] EA Tsochatzis J Bosch andAK Burroughs ldquoLiver cirrhosisrdquoThe Lancet vol 383 no 9930 pp 1749ndash1761 2014

[11] G Novak and T Tallerico ldquoNogo A B and C expression inschizophrenia depression and bipolar frontal cortex and cor-relation of Nogo expression with CAATATC polymorphism in31015840-UTRrdquo Brain Research vol 1120 no 1 pp 161ndash171 2006

[12] E P Marin G Moeckel R Al-Lamki et al ldquoIdentification andregulation of reticulon 4B (Nogo-B) in renal tubular epithelialcellsrdquo The American Journal of Pathology vol 177 no 6 pp2765ndash2773 2010

[13] G Garcia-Tsao A J Sanyal N D Grace et al ldquoPreventionand management of gastroesophageal varices and varicealhemorrhage in cirrhosisrdquo Hepatology vol 46 no 3 pp 922ndash938 2007

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OncologyJournal of



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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



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OphthalmologyJournal of


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Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Disease Markers

and the commonly used clinical parameters of liver functionin all patients

2 Methods

21 Subjects Fibroticcirrhotic liver specimens wereobtained from 24 patients who underwent liver surgery fora variety of liver diseases Twelve of the specimens showedMetavir stage 2 (F2) and the other 12 showed stage 4 (F4)fibrosis The 10 healthy liver specimens were from patientswho underwent liver surgery for benign focal lesions [9]All the liver specimens were provided by the Departmentof Pathology West China Hospital Sichuan University Forplasma Nogo-B analysis 301 patients of hepatic cirrhosiswere enrolled in the Division of Digestive Diseases WestChina Hospital Sichuan University from March 2012 toMarch 2014 152 patients had hepatitis B cirrhosis 83 werealcoholic cirrhosis and 66 were primary biliary cirrhosis(PBC) The diagnosis of cirrhosis was made based onimaging studies and impaired liver synthetic function [10]Patients with cardiovascular disease kidney diseases centralnervous system disorder chronic obstructive pulmonarydiseases (COPD) and pulmonary artery hypertension whichmight affect plasma Nogo-B levels were excluded [11 12]The normal control group was composed of 153 healthyvolunteers All subjects provided written informed consentsThis study was approved by the medical ethics committee ofthe West China Hospital Sichuan University

22 Collection of Samples and Clinical Parameters Bloodsamples from patients and healthy controls were obtainedThe plasma was immediately separated by centrifugation at800 g for 10 minutes at room temperature and stored atminus80∘C Parameters of liver function such as alanine amino-transferase (ALT) aspartate aminotransferase (AST) andplatelet (PLT) were collected from the Department of Clin-ical Laboratory West China Hospital Sichuan UniversityThe volume of ascites was observed by ultrasound PatientrsquosChild-Pugh classscore were calculated Esophageal and gas-tric varices were screened by esophagogastroduodenoscopyThe varices were defined as either large (gt5mm) or small(lt5mm) [13]

23 Plasma Nogo-B Assay The plasma Nogo-B levelswere determined by enzyme-linked immunosorbent assay(ELISA) kit (Biolegend Inc USA) and the assays wereperformed by following the manufacturersquos instruction

24 Histological Analyses Five-micrometer sections ofhuman liver tissue were prepared and embedded in paraffinThen these samples were stained with hematoxylin andeosin

25 Immunohistochemistry The sections were deparaffinizedin xylene and dehydrated in ethanol and then treated with001molL citrate buffer of 40 minutes pH 60 at 95∘C inwater bath Endogenous peroxidase activity was blocked byincubation in 3 H

2O2for 15 minutes in the dark Slides

Table 1 Clinical data of hepatic cirrhotic patients

Data Valuedagger

CharacteristicsGender (malefemale) 197104Age 5165 (21ndash80)Etiology (chronic hepatitis Binfectionalcoholic cirrhosisPBC) 1528366

ParametersChild-Pugh category (ABC) 11211574Degrees of esophageal and gastricvarices (absentsmalllarge)Dagger 99102100

Biochemical parametersTotal bilirubin (120583molL) 61ndash6077ALT (IUL) 6ndash389AST (IUL) 9ndash773Albumin (gL) 173ndash453INR 089ndash228PLT (109L)sect 11ndash390

daggerData are presented as the range or as the number of patientsDaggerEsophageal and gastric varices were diagnosed by esophagogastroduo-denoscopy Doi 101002hep21907sectPLT was analyzed in 203 patients who were without splenic embolizationand splenectomyALT alanine aminotransferase AST aspartate aminotransferase INR inter-national normalized ratio PBC primary biliary cirrhosis PLT platelet

were then incubated in 1 200 rabbit polyclonal Nogo-A+Bantibody (Abcam ab47085) for 45 minutes at 37∘C andincubated in 50ndash100120583L ChemMate Envision+HRP (DakoEnvisionTM Detection Kit number K5007) for 45 minutesat 37∘C Next diaminobenzidine (DAB) was added to eachslide for coloration Distilled water was used to end thechromogenic reaction The slides were counterstained withhematoxylin

26 Statistical Analysis Statistical analyses were performedusing SPSS 170 119875 lt 005 was considered significant Datawere presented as mean plusmn standard deviations (SD) or rangeCorrelations were analyzed with the Spearman correlationcoefficient Multiple comparisons for different groups werecarried out using unpaired 119905-test or one-way analysis ofvariance (ANOVA) followed by SNK test IPP 60 was usedto analyze the results of immunohistochemistry

3 Results

31 Nogo-B Is Highly Expressed in Liver Tissues with MetavirStage F4 Nogo-B was mainly expressed in hepatic non-parenchymal cells and highly expressed in fibroticcirrhotictissues Nogo-B expression in the liver positively correlatedwith the stage of liver fibrosis (Figure 1 F4 versus F2 119875 lt005)

32 Circulating Nogo-B Levels Are Significantly Increased inPatients with Hepatic Cirrhosis Clinical characteristics ofpatients in our study were summarized in Table 1 Our results

Disease Markers 3

HE F0 HE F2 HE F4








4 Discussion


4 Disease Markers

P lt 005


200

400

600

800

1000

1200

1400

1600

Rang

e

(a)

800

700

600

500

400

300

200

100

0

Hea

lthy

cont

rol

Hep

atiti

s B ci

rrho

sis

Alc

ohol

ic ci

rrho

sis

PBC

Plas

ma N

ogo-

B le

vel (

pgm

L)


lowast

lowastlowast

(b)


1000

900

800

700

600

500

400

300

200

100


Plas

ma N

ogo-

B le

vel (

pgm

L)




lowast

lowast

lowast

amp

(a)

1500

1200

900

600

300

04 6 8 10 12 14

Child-Pugh score

Nog

o-B

(pg

mL)

r = 0570 P lt 001

(b)





Disease Markers 5

1500

1200

900

600

300

0450400350300250200150100500

ALT (IUL)

Nog

o-B

(pg

mL)

r = 0062 P = 0285

(a)

1500

1200

900

600

300

08007006005004003002001000

AST (IUL)

Nog

o-B

(pg

mL)

r = 0112 P = 0052

(b)


900

800

700

600

500

400

300

200

100


Plas

ma N

ogo-

B le

vel (

pgm

L)

(a)

1500

1200

900

600

300

05004003002001000

PLT (109L)sect

Nog

o-B

(pg

mL)

r = minus024 P = 0739

(b)





6 Disease Markers




Acknowledgments


References




















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Disease Markers 3

HE F0 HE F2 HE F4








4 Discussion


4 Disease Markers

P lt 005


200

400

600

800

1000

1200

1400

1600

Rang

e

(a)

800

700

600

500

400

300

200

100

0

Hea

lthy

cont

rol

Hep

atiti

s B ci

rrho

sis

Alc

ohol

ic ci

rrho

sis

PBC

Plas

ma N

ogo-

B le

vel (

pgm

L)


lowast

lowastlowast

(b)


1000

900

800

700

600

500

400

300

200

100


Plas

ma N

ogo-

B le

vel (

pgm

L)




lowast

lowast

lowast

amp

(a)

1500

1200

900

600

300

04 6 8 10 12 14

Child-Pugh score

Nog

o-B

(pg

mL)

r = 0570 P lt 001

(b)





Disease Markers 5

1500

1200

900

600

300

0450400350300250200150100500

ALT (IUL)

Nog

o-B

(pg

mL)

r = 0062 P = 0285

(a)

1500

1200

900

600

300

08007006005004003002001000

AST (IUL)

Nog

o-B

(pg

mL)

r = 0112 P = 0052

(b)


900

800

700

600

500

400

300

200

100


Plas

ma N

ogo-

B le

vel (

pgm

L)

(a)

1500

1200

900

600

300

05004003002001000

PLT (109L)sect

Nog

o-B

(pg

mL)

r = minus024 P = 0739

(b)





6 Disease Markers




Acknowledgments


References




















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















4 Disease Markers

P lt 005


200

400

600

800

1000

1200

1400

1600

Rang

e

(a)

800

700

600

500

400

300

200

100

0

Hea

lthy

cont

rol

Hep

atiti

s B ci

rrho

sis

Alc

ohol

ic ci

rrho

sis

PBC

Plas

ma N

ogo-

B le

vel (

pgm

L)


lowast

lowastlowast

(b)


1000

900

800

700

600

500

400

300

200

100


Plas

ma N

ogo-

B le

vel (

pgm

L)




lowast

lowast

lowast

amp

(a)

1500

1200

900

600

300

04 6 8 10 12 14

Child-Pugh score

Nog

o-B

(pg

mL)

r = 0570 P lt 001

(b)





Disease Markers 5

1500

1200

900

600

300

0450400350300250200150100500

ALT (IUL)

Nog

o-B

(pg

mL)

r = 0062 P = 0285

(a)

1500

1200

900

600

300

08007006005004003002001000

AST (IUL)

Nog

o-B

(pg

mL)

r = 0112 P = 0052

(b)


900

800

700

600

500

400

300

200

100


Plas

ma N

ogo-

B le

vel (

pgm

L)

(a)

1500

1200

900

600

300

05004003002001000

PLT (109L)sect

Nog

o-B

(pg

mL)

r = minus024 P = 0739

(b)





6 Disease Markers




Acknowledgments


References




















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Disease Markers 5

1500

1200

900

600

300

0450400350300250200150100500

ALT (IUL)

Nog

o-B

(pg

mL)

r = 0062 P = 0285

(a)

1500

1200

900

600

300

08007006005004003002001000

AST (IUL)

Nog

o-B

(pg

mL)

r = 0112 P = 0052

(b)


900

800

700

600

500

400

300

200

100


Plas

ma N

ogo-

B le

vel (

pgm

L)

(a)

1500

1200

900

600

300

05004003002001000

PLT (109L)sect

Nog

o-B

(pg

mL)

r = minus024 P = 0739

(b)





6 Disease Markers




Acknowledgments


References




















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















6 Disease Markers




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 The Value of Circulating Nogo-B for ...

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