TNF-α neutralization improves experimental hepatopulmonary syndrome in rats

BAS IC STUDIES

TNF-a neutralization improves experimental hepatopulmonary syndromein ratsLi Liu1#, Nan Liu2#, Zhi Zhao1, Jiabao Liu2, Yingmei Feng1, Huiqing Jiang1 and Delan Han1

1 Department of Medicine, Hebei Key Laboratory of Gastroenterology, Second Hospital of Hebei Medical University, Shijiazhuang, China

2 The First Hospital of Shijiazhuang, Shijiazhuang, China

Keywords

cirrhosis – endotoxin – hepatopulmonary

syndrome – nitric oxide – tumour necrosis

factor

Abbreviations

CBDL, common bile duct ligation; HPS,

hepatopulmonary syndrome; iNOS, inducible

nitric oxide synthase; p-Akt, phospho-Akt;

PTX, pentoxifylline; TNF-a, tumuor necrosis

factor-a.

Correspondence

Li Liu, Department of Medicine, Second

Hospital of Hebei Medical Universaity, 215

Heping Road, Shijiazhuang 050000, China

Tel: + 86 13933177858

Fax: +86 31166002955

e-mail: [email protected]

or [email protected])

Received 23 February 2011

Accepted 16 April 2012

DOI:10.1111/j.1478-3231.2012.02821.x

Background/Aim: TNF-a is increased in hepatopulmonary syndrome (HPS).Pentoxifylline (PTX) mitigated experimental HPS through the inhibition ofTNF-a. However, PTX has pleiotropic effects besides the inhibition of TNF-a.This study is to neutralize TNF-a with specific monoclonal antibody toTNF-a (TNF-a McAb) to investigate the effect of TNF-a on HPS. Materialsand methods: Hepatopulmonary syndrome was induced by common bileduct ligation (CBDL); controls were sham operated. The endpoints were 1, 2,3, 4 and 5 weeks after surgery. 99mTechnetium-macroaggregated albumin(Tc-MAA) was to evaluate intrapulmonary arteriovenous shunts; Portalvenous pressure, cardiac output and mean blood pressure (MAP) were alsomeasured. Serum was for Alanine transaminase (ALT), endotoxin, TNF-aand nitric oxide (NO) measurements, liver for histology, lung for histologyand iNOS, PI3K/Akt expression assay. Results: Portal vein pressure was sig-nificantly elevated and MAP decreased in CBDL rats. Tc-MAA was mainlylocated in lung and very weak in brain in sham group and mainly in brain ofCBDL rats. TNF-a McAb significantly decreased the radioactivity in thebrain, reduced cardiac output, increased MAP and systemic vascular resis-tance (SVR) in CBDL animals. Serum ALT, endotoxin, TNF-a and NO weresignificantly increased. TNF-a McAb significantly decreased these serumindices in CBDL rats. TNF-a McAb significantly alleviated liver damage,decreased alveolar-arterial gradient and inhibited iNOS, PI3K/Akt and p-Aktexpression in lung tissue. Furthermore, TNF-a McAb significantly attenuatedthe inflammatory response in lung. Conclusion: TNF-a McAb improvesHPS in cirrhotic rats; this effect is likely mediated through the inhibition ofTNF-a PI3K/Akt-NO pathway.

The hepatopulmonary syndrome (HPS) is a clinicalthreesome composed of liver disease, intrapulmonaryvascular dilatation (IPVD) and arterial gas abnormali-ties. Ten to twenty-four percentage of patients with cir-rhosis have HPS. The presence of this syndrome is amarker of poor prognosis (1). Although this syndromehad been greatly investigated, its pathogenesis is not yetcompletely elucidated and there is yet no treatment thatoffers satisfactory results. Pathogenesis study is still thekey step in searching for the effective therapy. Xu et al.demonstrated that inhibition of TNFa significantlydecreased iNOS and LPS-induced pulmonary inflamma-tion. This effect was blocked by PI3K/Akt inhibitor (1).This implies that TNF-a-PI3Κ/Akt-NO pathway playsan important role in pulmonary diseases.

Common bile duct ligation (CBDL) in rats is an exper-imental HPS animal model, which mimics the pulmo-nary physiological abnormalities in human being (2, 3).It is now well documented that several factors, such asnitric oxide (NO), carbon monoxide (CO) and recently,TNF-a participated in the HPS formation in rats (4, 5).Bacterial translocation in cirrhotic rats(6) results in en-dotoxaemia which stimulates TNF-a and its downstreamproducts, NO and CO. These gas molecules cause vasodi-latation in cirrhotic pulmonary vasculature, (7) the typi-cal pathophysiological alteration in hepatopulmonarysyndrome. Zhang and colleagues demonstrated that pen-toxifylline improves HPS via the blockage of TNF-a.However, pentoxifylline, as a competitive nonselectivephospodiesterase inhibitor, has multifunction. Besidesthe inhibition of TNF-a, pentoxifylline raises intracellu-lar cAMP, activates protein kinase A (PKA) (8), inhibitsleukotriene and reduces inflammation and innateimmunity. Furthermore, pentoxifylline is a nonselective#Li Liu and Nan Liu contributed equally to this work.

Liver International (2012)© 2012 John Wiley & Sons A/S1018

Liver International ISSN 1478-3223

adenosine receptor antagonist, whereas adenosine is avasodilator (9). Hence, the distinctive effects of TNF-aon hepatopulmonary syndrome is still obscure.

This study was to investigate the effect of TNF-aon HPS. Specific monoclonal antibody to TNF-a(TNF-a McAb) was used to neutralize serumTNF-a. We assessed the consequences of TNF-aMcAb on the alveolar-arterial oxygen gradient P(A-a)O2, the intrapulmonary shunt fraction and lung histol-ogy. To explore the mechanism, serum endotoxin,TNF-a, nitric oxide, liver histology and ALT, pulmo-nary iNOS, PI3K, Akt and the phophorylated Akt(p-Akt) were evaluated.

Materials and methods

Animal model

Male Sprague-Dawley rats (250 ± 25 g) purchased fromthe animal resource centre of Hebei Medical University(certificate: 807145), were used in this study. The proto-col was approved by the Faculty of Medicine AnimalExperimentation Committee in accordance with guide-lines established by P.R. China administration of animalcare (GB14922-94). Cirrhosis was induced according tothe literature (10). Briefly, rats were anaesthetized byhalothane inhalation. An abdominal midline incisionwas used to expose the common bile duct, which wasdoubly ligated using 4-0 silk suture and cut between theligatures. The muscle and skin were sutured using3-0 silk separately. Surgery was carried out under sterileenvironment. Immediately after surgery, rats wereadministered 30 000 U penicillin G intramuscularly toprevent infection and allowed to recover in individualcages. Sham-operated control rats (n = 6) received thesame laparotomy except that the bile duct was notligated or sectioned. CBDL rats were divided into fivegroups and the endpoints were 1, 2, 3, 4 and 5 weekafter CBDL (n = 6 in each group). CBDL+TNF-a McAb(mouse anti-human TNFa antibody, immunology labo-ratory, the forth military medical university, half-life60 h, 0.1 g/kg/2d, n = 6 in each group), divided intofour groups: 2, 3, 4 and 5 week after CBDL. After CDBL1 week, TNF-a McAb was injected intraperitoneally.The endpoints of TNF-a McAb treated animals were 1,2, 3 and 4 weeks after TNF-a McAb administration.Same volume of mouse IgG was injected intraperitone-ally to CBDL and sham control rats.

For the haemodynamic study, the animals were an-aesthetized with 100 mg/kg ketamine intraperitoneally.The MAP was recorded from the femoral artery using apolygraph recorder(BL-420F biological experimentalsystem; Chengdu Technology & Market Co. Ltd.,Chengdu, China), calibrated at each use using a pressuremanometer. Cardiac output was measured by the ther-modilution technique using a Cardiomax II computer(Columbus Instruments, Columbus, OH, USA). Aknown volume (0.2 ml) of 19°C saline was injected in

the jugular vein catheter with the tip near the rightatrium, and changes in blood temperature were detectedusing a 1.5-Fr thermistor microprobe catheter (Colum-bus Instruments) positioned in the aortic arch advancedfrom the left carotid artery. Systemic vascular resistance(SVR) was calculated by dividing the mean systemicartery pressures by the cardiac index.

Portal venous pressure (PVP) was measured bydirectly puncturing the portal vein with PE-90 tube(Becton Dickinson and company, Sparks, MD, USA)with sharp end, which was connected to a pressuretransducer. Rectal temperature was maintained at 37°Cusing heating pads throughout this study.

Detection of intrapulmonary vascular dilatation

Technetium-macroaggregated albumin (200 lCi; meandiameter 20 lm, range 15–50 lm) were injectedthrough tail vein. Lung and brain radioactivity weremeasured using a gamma detector 30 min after theinjection, and the brain/lung radioactivity ratio was cal-culated. Six rats were studied in each group.

Arterial blood gas analysis

Arterial blood was drawn from the abdominal aorta.Arterial blood gas analysis was performed on an ABL520 radiometer (Radiometer America, Westlake, OH,USA) in the Clinical Laboratory, second hospital, HebeiMedical University. The alveolar-arterial oxygen gradi-ent was calculated as 150�(PaCO2/0.8)�PaO2. wherePaCO2 is the partial pressure of carbon dioxide and PaO2is the partial pressure of oxygen.

Blood endotoxin, TNF-a, nitric oxide and alaninetransaminase measurements

After pulmonary function evaluation [alveolar-arterialPO2 difference (P(A-a)O2)], samples were collected atdifferent time points, 1, 2, 3, 4 and 5 weeks after CBDL(Sham group was at 4 weeks). Serum was collected forendotoxin, ALT, NO and TNF-a measurement, liver forhistology, lung for histology, immunohistochemistryand Western blots.

Serum samples were serially diluted with endotoxin-free water and heat-treated to destroy inhibitors thatcan interfere with activation. Serum endotoxin wasassessed using tachypleus amebocyte lysate kit (BeijingZhongshan Biotechnological Company, Beijing, China),the end reactants were read within 24 h at OD405 nm.

Serum TNF-a levels were measured using a commer-cially available Iodine[125I] Tunmor Necrosis FactorRadioimmunoassay kit, according to the protocol sup-plied by the manufacturer (Beijing Zhongshan Biotech-nological Company, sensitivity 0.3 ng/ml).

Nitric oxide concentration: Serum was filtered usingmicropore filters (Ultrafree MC microcentrifuge device,UFC3; Millipore, Bedford, UK) to remove substances

Liver International (2012)© 2012 John Wiley & Sons A/S 1019

Liu et al. TNF-a antibody improves HPS

larger than 10 kDa (mainly haemoglobin). Filtrateswere analysed by an automated procedure based on theGriess reaction. Briefly, nitrate is converted to nitriteby the action of nitrate reductase from Apergih Niger(Sigma, St. Louis, MO, USA). The samples were dilutedwith nitrate/nitrite free water (1:1) and incubated withsubstrate buffer (l/2 volume of sample) and nitratereductase (14 mU/5Opl) at room temperature for45 min. The substrate buffer consists of imidazole0.1 mol/L, nicotinamide adenine dinucleotide phos-phate (reduced form, NAPDH) 210 mmol/L and flavinadenine dinucleotide (FAD) 3.8 mmol/L. The absor-bance at 540 nm was measured using plate reader, andnitrite concentrations were calculated from a sodiumnitrite standard curve. Because very little or no nitrite isfound in serum, we did not attempt to differentiatebetween the actual nitrate vs nitrite amounts.

Serum ALT was measured using automated chemis-try analyser (3600-020; Bayer, Leverkusen, Germany) inthe core lab in the second hospital, Hebei Medical Uni-versity.

PI3K and Akt protein content quantified using Westernblotting

Pulmonary tissue was lysed with RIPA lysis buffer (So-larbio Science & Technology Co. Ltd, Beijing, China)and homogenized by hands. After centrifuge, the pro-tein concentration of the supernant was determinedusing the Bio-Rad protein assay, using bovine serumalbumin as a standard. Equal amounts (150 lg) of thedenatured proteins per lane were loaded and separatedon 8% SDS-PAGE by electrophoresis. Proteins werethen transferred to nitrocellulose by wet electroblottingat 4°C for 2.5 h. Blots were blocked overnight at roomtemperature with 10% bovine serum albumin in 0.1%Tween Tris-buffered saline buffer (TBS-T) at pH7.5 containing 20 mM Tris base, 137 mM NaCl and0.1% Tween 20. The membranes were washed withTBS-T (three times, 10 min each) and then incubatedat room temperature for 90 min with highly specificrabbit polyclonal anti- PI3K, Akt antibody (all of theprimary antibodies were from Santa Cruz; Santa Cruz,CA, USA). The membranes were subsequently incu-bated with horseradish peroxidase-linked goat anti-rab-bit antibody (1:2000; Bo Ao Sen, Beijing, China). The

blots were detected using the enhanced chemilumines-cence method (ECL Western blot kit; Santa Cruz).GAPDH as internal control. The relative expression ofPI3K or Akt and GAPDH was quantified using com-puterized optical densitometric scanning system (Bio-rad; Benicia, CA, USA).

Immunohistochemistry

For immunohistochemical observation, the lungs wereimmediately fixed with ice-cold freshlymade 10% formalinand then embedded with paraffin. The slice cutting proce-dure was the same as regular cutting (4 lm). After hydra-tion, the slice was incubated with rabbit anti-iNOS (SantaCruz), rabbit anti- PI3K or Akt antibody (1:100), 4°Covernight. The incubation with the secondary (biotin)conjugated goat anti-rabbit antibody (1:2000), after Tween20-Tris-Buffered Saline wash, The sections were incubatedwith DAB and then stained with haematoxylin as back-ground. Image-Pro Plus5.0 (Media Cybernetics Inc.,Bethesda,MD,USA) was used to quantify the average opti-cal density (AOD).

Statistics

Data were expressed as mean ± standard deviation. Allstatistical analyses were performed using SPSS (SPSSInc., Chicago, IL, USA). The comparison among morethan two groups were analysed using one-way ANOVAand followed by Newman–Keuls correction. The signifi-cance level was set at P < 0.05.

Results

Haemodynamic studies

Haemodynamic studies were carried out at the timepoint of 4 weeks after surgery. Portal vein pressure wassignificantly increased in CBDL group compared withsham control (14.3 ± 1.3 vs 6.8 ± 0.5 mmHg, P <0.01). TNF-a McAb treatment did not significantlyreduce PVP in CBDL rats (13.2 ± 1.4 vs 14.3 ±1.3 mmHg, P > 0.05) and sham animals (6.5 ± 0.6mmHg vs 6.8 ± 0.5 mmHg, P > 0.05). Mean arterialpressure and peripheral vascular resistance were signifi-cantly decreased in CBDL rats compared with sham

Table 1. The effect of TNF-a McAb on haemodynamic circulation in cirrhotic rats

CBDL Sham CBDL + TNF-aMcAb Sham + TNF-aMcAb

MAP (mmHg) 95 ± 12** 115 ± 13 109 ± 11# 117 ± 13CO (ml/min/100 g) 45.4 ± 2.3** 38.2 ± 4.2 40.0 ± 2.7# 38.2 ± 4.4SVR (mmHg/min/ml/100 g) 2.1 ± 0.22** 3.01 ± 0.25 2.9 ± 0.27# 3.06 ± 0.26

MAP, mean arterial pressure; CO, cardiac output; SVR, systemic vascular resistance; CBDL, common bile duct ligation; TNF-a McAb, TNF-a monoclo-

nal antibody.

**P < 0.01 compared with sham control;

#P < 0.05 compared with CBDL, n = 6 in each group.


TNF-a antibody improves HPS Liu et al.

control; TNF-a McAb significantly reversed MAP andSVR in CBDL animals, but not in sham control rats.Cardiac output was significantly increased in CBDLgroup compared with sham control; TNF-a McAbsignificantly decreased cardiac output in CBDL rats, butnot in sham control animals (Table 1).

Intrapulmonary shunt and alveolar-arterial oxygendifference

Technetium-macroaggregated albumin showed that in-trapulmonary shunt fraction was significantly increasedin CBDL rats compared with sham control (19.7 ± 3.2%vs 6.5 ± 1.8%, P < 0.01), TNF-a McAb significantlydecreased intrapulmonary shunt (9.5 ± 2.3% vs 19.7 ±3.2%, P < 0.01). TNF-a McAb has no affect on shamanimals.

P(A-a)O2, one of the three components in HPS (liverdisease, P(A-a)O2 and intrapulmonary vascular dilata-tion) was measured. We found that P(A-a)O2 was sig-nificantly increased in 2 weeks after CBDL (17.6 ± 2.67

vs 6.70 ± 3.20, P < 0.01) and kept at higher level.TNF-a McAb treatment significantly decreased theAlveolar-arterial gradient (Table 2).

Serum ALT, endotoxin, TNF-a, and NO concentration

Serum TNF-a was significantly increased one week laterafter CBDL, (1.60 ± 0.07 vs 0.75 ± 0.14 ng/ml,p < 0.01) and kept high level in the following weeks,TNF- McAb neutralized serum TNF-a (Fig. 1A) Oneweek after CBDL, serum ALT was significantly elevatedcompared with sham rats (177.00 ± 56.32 vs56.33 ± 9.29 U/L, P < 0.01), after treated by TNF-aMcAb, ALT was gradually decreased in the followingweeks after CBDL, but still significantly higher thansham animals. 2 weeks after TNF-a McAb treatment,ALT was significantly lower compared with the sametime point of CBDL only (Fig. 1B). Serum endotoxinwas significantly increased one week later afterCBDL compared with sham rats (22.68 ± 3.11 vs4.87 ± 1.16EU/L, P < 0.01). TNF-a McAb treatmentsignificantly decreased serum endotoxin levels in CBDLanimals (Fig. 1C). Serum nitric oxide started increasingin one week after CBDL , got a significant level in week2 (74.38 ± 2.67 vs 36.19 ± 1.59 umol/l, P < 0.01), andkept at a significant high level afterwards, TNF-a McAbsignificantly inhibited NO production in CBDL rats(Fig. 1D).

Liver histology

HE and Masson staining showed that 2 weeks later afterCBDL, there are massive ductile and connective tissue

Table 2. The level of P(A-a)O2 at various time points (mmHg;x � s, n = 6)

Groups Sham CBDL CBDL+TNF-aMcAb

1 week 6.70 ± 3.20 6.58 ± 3.10 -2 week 17.6 ± 2.67* 13.4 ± 2.56*3 week 17.5 ± 3.00* 13.8 ± 4.09*4 week 22.5 ± 4.24* 15.7 ± 2.03*,#5 week 51.8 ± 8.49* 10.5 ± 8.51#

*P < 0.05 vs Sham.

#P < 0.05 vs CBDL.

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(A) (B)

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Fig. 1. (A) Serum TNF-a was significantly increased 1 week later after CBDL and reached to peak value in 3 weeks. TNF-aMcAb significantlydecreased TNF-a level in CBDL rats. (B) ALT was significantly increased a week later after CBDL and then gradually decreased. Two weekslater after TNF-aMcAb administration, ALT was significantly decreased in CBDL rats. (C) serum endotoxin was significantly increased 1 weeklater after CBDL, 1 week later after TNF-a McAb administration, endotoxin was significantly decreased in CBDL rats. (D) Serum NO was sig-nificantly increased 2 weeks later after CBDL and reached to peak value in 3 weeks, TNF-a McAb significantly decreased NO level in CBDLrats (*significantly different compared with sham; #significantly different compared with CBDL only).



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Fig. 3. Immunohistochemical staining for lung iNOS (original magnification 9400). (A) sham; (B) 4 weeks later after CBDL. (C) TNF-aMcAbtreated. iNOS positive cells were mainly phagocytes. iNOS expression was significantly increased in CBDL lungs compared with those of shamcontrols, TNF-aMcAb significantly inhibited iNOS expression. (*significantly different compared with sham; #significantly different comparedwith CBDL only).

(I)(H)(G)

(F)

(C)(B)

(D)

(A)

(E)

Fig. 2. Representative liver and lung histological changes: HE staining (original magnification 9100 and 9400 for liver) and Massion stain-ing(9400). (A, D) sham-operated liver, (B, E) 5 weeks later after CBDL, there was severe fibrotic proliferation and pseudolobuli formation.(C, F) TNF-aMcAb treatment mitigated the liver structural change and prevented the pseudolobuli formation. (D–F) Lung. HE staining (origi-nal magnification 9200). (G) Sham-operated rat. (H) Five weeks later after CBDL, there were massive inflammatory cell filtration, the capil-lary density was significantly increased; alveolar space was shrunken down in some area and there were some macrophages. The Alveolarsepta were wider compared with sham control. (I) TNF-aMcAb alleviated these pathological changes.



proliferation and cholangiectasis in portal area.Five weeks later after CBDL, there was severe fibroticproliferation and pseudolobuli formation, liver tissuewas detoured. TNF-a McAb treatment mitigated theliver structural change and prevented the pseudolobuliformation (Fig. 2).

Lung histology

HE stain showed that there is no obvious pulmonaryoedema in all groups. In CBDL group, there werespread ecchymoses, inflammation was obvious in pul-monary interstitium, the capillary density was signifi-cantly increased. Alveolar space was shrunken down insome area and there were some macrophage in it. TheAlveolar septa were wider compared with sham control.TNF-a McAb alleviated these pathological changes(Fig. 2).

Lung iNOS and PI3K expression byimmunohistochemistry

Immunohistochemistry showed that iNOS expressionwas mainly in the plasma of phagocytes and significantlyincreased in CBDL groups and reached at 4 weeks(0.39 ± 0.11 vs 0.084 ± 0.09, P < 0.01, n = 6), TNF-aMcAb significantly decreased iNOS density in CBDLlungs (0.253 ± 0.108, P < 0.05 vs CBDL, n = 6), TNF-aMcAb did not affect iNOS expression in sham animals(Fig. 3).

PI3K positive cells were mainly in macrophages.PI3K expression was significantly increased in CBDLlungs compared with those of sham controls, TNF-aMcAb significantly inhibited PI3K expression (Fig. 4).

Lung PI3K protein content quantified using Western blots

PI3K was significantly increased 2 weeks later afterCBDL compared with sham controls (2.15 ± 0.10 vs0.35 ± 0.12, P < 0.01) and kept at a high level. TNF-a

*0.4CBDL CBDL + TNF-a McAb

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Fig. 4. Immunohistochemical staining for lung PI3K (original magnification 9400). (A) sham; (B) 5 weeks later after CBDL. (C) TNF-a McAbtreated. PI3K positive cells were mainly macrophages. PI3K expression was significantly increased in CBDL lungs compared with those ofsham controls, TNF-a McAb significantly inhibited PI3K expression(*significantly different compared with sham; #significantly different com-pared with CBDL only).

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Fig. 5. Lung PI3K protein content quantified using Western blots.PI3Kwas significantly increased 2 weeks later after CBDL comparedwith sham controls and kept at a high level. TNF-aMcAb treatmentsignificantly decreased PI3K content in CBDL rats (lane 1: sham; 2:CCBDL 5 week; 3: CCBDL 5 week +TNF-a McAb 4 week; 4:CCBDL 4 week; 5: CCBDL 4 week+TNF-a McAb 3 week; 6: CCBDL3 week; 7: CCBDL3 week +TNF-aMcAb 2week; 8: CCBDL 2 week;9: CCBDL 2 week +TNF-a McAb 1 week; 10: CCBDL 1 week.*significantly different compared with sham; #significantly differentcompared with CBDL only).



McAb treatment significantly decreased PI3K content inCBDL rats (Fig 5).

Lung Akt density by immunohistochemistry

Immunohistochemistry showed that Akt positive cellswere mainly macrophages. Akt expression was

significantly increased 2 weeks later after CBDL com-pared with that of sham controls and reached to peakvalue in 4 weeks (0.31 ± 0.03 vs 0.09 ± 0.03, P < 0.01).TNF-a McAb significantly decreased the content of Aktin CBDL rats (Fig. 6).

Lung phosphorylated Akt level quantified by Westernblots

p-Akt was significantly increased 3 weeks later afterCBDL compared with that in sham operation rats(2.45 ± 0.23 vs 1.08 ± 0.20, P < 0.01). One week laterafter TNF-a McAb administration, p-Akt was signifi-cantly decreased compared with CBDL only at the sametime point (Fig 7).

Discussion

To our knowledge, this is the first study to investigatethe direct relationship between TNFa and hepatopul-monary syndrome. We found that TNF-a McAbdecreased blood endotoxin concentration, alleviatedlung inflammation, attenuated nitric oxide productionthrough the inhibition of PI3K/Akt expression in cir-rhotic rats, induced by CBDL and finally, significantlymitigated hyperdynamic circulation in cirrhotic rats,decreased intrapulmonary shunt and improved pulmo-nary function. Another interesting finding was thatTNF-a McAb had a protective effect on liver injuryinduced by cholestasis.

The HPS is a severe complication of cirrhosis, 10–24% of Cirrhotic patients have HPS (11, 12), itspresence indicates the poor prognosis. The mortalityassociated with hepatopulmonary syndrome is high,

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Fig. 6. Immunohistochemical staining for lung Akt (original magnification 9400). (A) sham; (B) 5 weeks of CBDL. Akt positive cells weremainly macrophages and significantly increased. (C) TNF-aMcAb significantly decreased the amount of Akt positive cells(*significantly differ-ent compared with sham; #significantly different compared with CBDL only).

p-Akt60 KD

Sham CBDL CBDL+TNF-α McAb

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Fig. 7. Lung phosphorylated p-Akt level quantified using Westernblots. p-Akt was significantly increased in CBDL rats, TNF-a McAbadministration significantly decreased p-Akt compared with CBDLonly at the same time point((lane 1: sham; 2: CCBDL 5 week; 3:CBDL 5 week +TNF-a McAb 4 week; 4: CBDL 4 week; 5: CBDL4 week+TNF-aMcAb 3 week; 6: CBDL 3 week; 7: CBDL3 week+TNF-aMcAb 2week; 8: CBDL 2 week; 9: CBDL 2 week +TNF-aMcAb 1 week; 10: CBDL 1 week. *significantly different comparedwith sham; #significantly different compared with CBDL only).



with a median survival of 10.6 months. The onlydefinitive treatment is liver transplantation, for which a5-year survival rate of 76% has been reported (13).However, owing to the shortage of liver donor, it is veryimportant to find an effective therapeutic way toimprove patients’ life quality and to prolong life. Methy-lene blue infusion, a dye that inhibits the effect of NOon soluble guanylate cyclase, has shown a transientimprovement in oxygenation (14), but there is no soliddata showing the long-term benefits.

Although there is a tense study on HPS in the last twodecades, its mechanism still remains unclear. There is hy-perdynamic circulation in the patients with liver diseaseand this shortens blood transit time through the lung vas-culature. Bacterial translocation in cirrhosis leads toincreased TNF-a, which leads to increased macrophageadherence to pulmonarymicrovasculature with increasedinducible NO-synthase-derivedNOproduction.

Pulmonary production of nitric oxide (NO) has beenimplicated in the development of intrapulmonary vaso-dilatation in patients with HPS (15). Acute inhibition ofNO production or its action with administering NG-nitro-L-arginine methyl ester (L-NAME) or methyleneblue, respectively, transiently improves HPS (16).

In addition, compared with time-matched sham rats,MAP and SVR were significantly lower in the BDL rats,whereas cardiac output was significantly increased, indi-cating the development of a hyperdynamic state. Fur-thermore, PVP was significantly increased in the BDLrats, indicating the existence of portal hypertension. In-trapulmonary shunt and P(A-a)O2 were significantlyincreased in CBDL rats. All of these findings confirmedthe establishment of experimental HPS in rats.

TNF-a is a pleiotropic cytokine and participates in avariety of pathogenesis, such as inflammation, immuneresponse, cancer etc. (12, 17). Serum TNF-a is increasedin cirrhosis because of endotoxaemia caused by bacterialtranslocation (18). TNF-a enhances nitric oxide synthe-sis via PI3K/Akt pathway (19). However, the effects ofTNF-a on HPS still remains unclear. Using pentoxifyl-line (PTX) as an inhibitor, Sztrymf and colleagues foundthat PTX prevents the development of both hepatopul-monary syndrome and hyperdynamic circulation. Theybelieved that TNF-a plays a central role in the genesis ofhepatopulmonary syndrome (5). Another study, usinganti- TNF-a polyclonal antibodies, demonstrated thatneutralization of TNF-a significantly decreased nitricoxide and prostacyclin production and thereforeincreased systemic vascular resistance (SVR) and finally,reversed the hyperdynamic circulation induced by por-tal vein stenosis (20). Using the same inhibitor (PTX),Zhang and coworkers found that PTX only partiallyreversed the increase in circulating TNF-a levels. Theeffect of PTX on hepatopulmonary improvement ismainly via the inhibition of pulmonary microvascularendothelin B ET(B) receptor, Akt and eNOS expression.TNF-a had no effects on endothelial ET(B) and eNOSalterations (4). Besides the inhibition of Akt and eNOS

expression, the same group later also demonstrated thatPTX improved HPS via the inhibition of angiogenesis(21). This might be another mechanism for the TNFa toimprove HPS because like PTX, TNFa also inhibit Akt.

What a role TNF-a plays exactly in HPS? There areseveral ways to demonstrate this, one is gene knockoutmodel. One is to block on its expression level (siRNA).Another reliable approach is to use antibody directly toneutralize TNF-a. This study showed that TNF-a McAbcompletely neutralized the increased serum TNF-a inCBDL rats, paralleling with TNF-a, PI3K/Akt was con-sequently attenuated in TNF-a McAb treated CBDLgroups. As per the definition of HPS, intrapulmonaryvascular dilatation is the pivotal pathogenesis of HPS.After TNF-a neutralization, nitric oxide was signifi-cantly decreased. This result is consistent with Munozand colleagues, (20) they demonstrated that diminishedTNF-a improves vasodilatation in both systemic andpulmonary territories and this effect is via the inhibitionof nitric oxide. Our study showed that TNF-a neutral-ization also alleviated intrapulmonary shunt and P(A-a)O2 in cirrhotic rats.

Besides the improvement of hepatopulmonary syn-drome, liver damage was also significantly milder inTNF-a McAb treated CBDL animals compared withCBDL rats. As TNF-a is a mediator of hepatotoxicity inseveral animal models, the previous study by Gabele andcoworkers found that after CBDL, the survival rate inTNF-a knockout mice was higher than those of wild-typewith the same surgery, serum alanine transaminases(ALT) levels were significantly higher in wild-type com-pared with knockout mice. More than these, the collagenexpression and liver fibrosis were lower in knockout miceafter CBDL compared with those in wild-type animals.TNF-a is required for cholestasis-induced liver fibrosisin mouse (22). Theoretically, it is the bacterial transloca-tion in cirrhotic rats that causes endotoxaemia, the laterstimulates the mononuclear cells to produce TNF-a (18,23). Our results showed the opposite, neutralization ofTNF-a results in the mitigation of endotoxaemia. Howto explain this? As TNF-a McAb protects the liver fromdamage, the liver function was preserved and thereforethe capability of catalyzing endotoxin was conserved.Another explanation is, milder disarrangement of liveralleviates portosystemic shunting, which is anotherimportant factor of endotoxaemia in cirrhosis. Someonemight argue that TNF-a McAb inhibits NO through theprotection of liver damage and decrease endotoxin con-centration. However, using acute animal model, Chenand coworkers demonstrated that blockage of TNF-aimmediately diminished NO synthesis (24), we thereforeconclude that TNF-a McAb improves pulmonary func-tion through both the protection of liver and lung fromdamage and direct inhibition of nitric oxide.

Antibody to TNF-a (Infliximab) has approved tohave therapeutic effects on crohn’s disease and rheuma-toid arthritis. However, clinical study in alcoholic hepa-titis did not show any beneficiary effects (25). The



studies on TNF-a are not consistent between animaland humans. In animals, antibody to TNF-a has thera-peutic effects on both ischaemic heart disease and alco-holic hepatitis, these animal data did not apply tohuman being. This study demonstrated that antibody toTNF-a improved HPS; whether this study is applicableto human beings need further investigation.

In conclusion, endotoxin-TNF-a -PI3K/Akt-NOpathway was activated in hepatopulmonary syndrome.TNF-a McAb decreased PI3K/Akt expression, attenu-ated nitric oxide production and finally, significantlyimproved intrapulmonary shunt and pulmonary func-tion in HPS model animals.

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TNF-α neutralization improves experimental hepatopulmonary syndrome in rats