Losartan Supports Liver Regrowth via Distinct Boost of Portal Vein Pressure in Rodents with 90 % Portal Branch Ligation

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  • ORIGINAL ARTICLE

    Losartan Supports Liver Regrowth via Distinct Boost of PortalVein Pressure in Rodents with 90 % Portal Branch Ligation

    Kezhou Li Xiaohong Qi Jiaying Yang

    Jianping Gong Chunlu Tan Qingjie Xia

    Jieran Long Zhongdin Wang

    Received: 27 February 2012 / Accepted: 27 March 2013 / Published online: 30 April 2013

    Springer Science+Business Media New York 2013

    Abstract

    Objectives The study used a model of 90 % portal branch

    ligation (PBL) in rats to study the effect of losartan on

    portal vein pressure (PVP) and liver regeneration in rats

    after PBL.

    Methods A total of 144 male SpragueDawley rats were

    arbitrarily designated into three treatment method groups: a

    sham operation group (Sham), a PBL treatment group

    (PBL), and a PBL plus losartan treatment group

    (PBL ? L). Losartan (2 mg/day) was intragastrically ga-

    vaged 3 days before the PBL or sham operation to time

    points of study.

    Results Both the PBL and PBL ? L groups showed an

    intense surge in PVP after PBL treatment, peaking at 12 h

    postsurgery, then lessening progressively afterwards. PVP

    was substantially greater in these two groups compared

    with the Sham group at 672 h postsurgery (p \ 0.01).Compared with the PBL group, the PBL ? L group

    showed a noticeable reduction in PVP 648 h postsurgery

    (p \ 0.05); the PBL group showed considerably raisedlevels of plasma ALT and AST 672 h postsurgery

    (p \ 0.01). Compared to the PBL group, the PBL ? Lgroup showed drastically reduced plasma ALT and AST

    levels 1272 h postsurgery (p \ 0.05).Conclusions Losartan supports liver regeneration in 90 %

    of rats that underwent PBL. The mechanism may be related

    to losartans ability to regulate PVP and increase serum

    hepatocyte growth factor levels.

    Keywords Liver regeneration Portal branch ligation Hepatocyte growth factor Losartan

    Introduction

    Extended hepatectomy is the recommended treatment for

    large liver malignancies, hilar cholangiocarcinoma (CCA),

    and colorectal liver metastasis [1]. Conversely, inadequate

    functional remnant liver (FRL) volume can cause postop-

    erative fatality by inducting hepatic complications, a

    component that often restricts the scientific application of

    this treatment [2, 3]. To decrease postoperative risk, some

    investigators recommend using portal vein embolization

    (PVE) as a preoperative measure to enhance the prognosis

    for extended hepatectomies, with reassuring outcomes.

    Functional remnant liver volume can be enhanced by

    means of compensatory hypertrophy induced by preoper-

    ative selective hepatic PVE. Subsequently, PVE may not

    only improve the surgical safety of radical liver resection,

    but also improve successful outcomes in some patients [4].

    Even with PVE, however, radical liver resection may not

    be suitable for 536 % of patients due to insufficient

    contralateral hepatic hypertrophy [5]. Liver regeneration is

    Kezhou Li and Xiaohong Qi contributed equally to this work.

    K. Li X. Qi J. Yang (&) C. Tan Z. WangDepartment of Hepatobiliopancreatic Surgery, West China

    Hospital, Sichuan University, Chengdu 610041, Sichuan

    Province, China

    e-mail: hepatobiliary@163.com

    J. Gong

    Hepatobiliary Surgery, Second Affiliated Hospital of Chongqing

    Medical University, Chongqing, China

    Q. Xia

    Advanced Medical Center, West China Hospital, Sichuan

    University, Chengdu, China

    J. Long

    Department of Oncology, West China Hospital, Sichuan

    University, Chengdu, China

    123

    Dig Dis Sci (2013) 58:22052211

    DOI 10.1007/s10620-013-2664-3

  • a sophisticated and stringently performed course of action,

    influenced by the complicated network of signaling path-

    ways that integrate extracellular signals, along with a

    cascade of intracellular molecular events. Numerous

    growth components and cytokines are involved during liver

    regeneration in animal models; similar components may

    also occur in human liver regeneration [6, 7]. Of the

    acknowledged components, hepatocyte growth factor

    (HGF) is the most essential proliferation revitalizing

    component, along with mitogen, which could mature liver

    cells. Liver regeneration can be promoted through esca-

    lating serum HGF levels in several animal models for liver

    regeneration [810].

    Scientific studies propose that angiotensin II is actually a

    highly effective inhibitor of HGF. Implementing angio-

    tensin II receptor 1 (AT1) antagonist also has been proven

    to minimize lesions of the cardiovascular and pulmonary

    systems by increasing serum HGF levels in animal models

    of diabetes and pulmonary fibrosis [11, 12]. However, the

    results of AT1 antagonist on serum HGF levels and liver

    regeneration in 90 % of PBL rats have not been docu-

    mented. Research has shown that, as a possible AT1

    antagonist, losartan can minimize portal vein pressure

    (PVP) in patients with portal hypertension in liver cirrho-

    sis. The consequence of losartan on PVP, however, as a

    result of stages after PVE has not yet been shown. For this

    reason, our research addresses the consequences of losartan

    on PVP, serum HGF levels, and liver regeneration in rats

    after 90 % PBL.

    Materials and Methods

    Animal Experiments

    A total of 144 male SpragueDawley rats (8 weeks old),

    weighing 180220 g, obtained from the Laboratory Animal

    Center, West China Center of Medical Science, were used in

    our study. The rats consumed conventional water and food

    under controlled temperature and moisture and a 12-h light/

    dark cycle. All rats in the study were observed for 1 week,

    then randomized into three groups: the sham operation group

    (Sham group); the model group (PBL only), and the treat-

    ment group (PBL ? losartan 2 mg/kg 9 day). The rats were

    anesthetized by injection of 1.5 % sodium pentobarbital

    (40 mg/kg) into the abdominal cavity. Under the micro-

    scopic lense, portal vein branches, which provide to the

    median, left, and right lateral lobes of the liver, were sepa-

    rated and ligated carefully. Only portal vein branch supply to

    the caudate lobe was preserved. During this process, proper

    care was taken to not damage the hepatic artery and bile

    ducts. The ligated liver lobes comprised 90 % of the liver

    weight. All PBL surgeries were performed between 9:00 and

    12:00 a.m. In the sham operation group, the portal vein

    branches were dissociated but not ligated, and the abdominal

    cavity was then closed immediately. Daily for 3 days before

    surgery until the end of the experiment, the sham operation

    group and the PBL group received physiological saline,

    whereas the PBL ? L group was given losartan.

    Portal Vein Pressure, Blood Samples, and Liver

    Samples

    To measure PVP, the abdomen was opened along the ori-

    ginal incision under anesthetization at 6, 12, 24, 48, 72,

    120, or 168 h after surgery for each of the groups. An

    electronic manometer (Nihon Kohden, Tokyo, Japan) with

    a heparinized 22-gauge needle was inserted directly into

    the portal vein to measure PVP. The zero graduation line

    was defined by the anatomical position of portal vein, and

    results were indicated in millimeters of mercury. All blood

    samples were taken directly from the heart immediately

    after liver extraction and centrifuged. The levels of ALT

    and AST in the sera were detected with a standard clinical

    automatic analyzer (Arkray, Kyoto, Japan). The complete

    livers were surgically removed, blood was blotted with

    filter paper, and the tissue was observed for color and

    texture. The unligated liver lobes were maintained in 20 %

    buffered formalin. The mass of unligated liver lobes and

    total liver were weighed (accurate to 0.01 g), and LW/TW

    index was calculated.

    Histology and Immunohistochemistry

    The unligated liver lobes taken from each time point were

    fixed with 10 % formaldehyde, embedded in paraffin, and

    sliced into 4-lm sections. Hepatic tissue sections wereroutinely dewaxed and stained with hematoxylin and

    eosin (H&E) to observe the pathological changes in liver

    structures. Proliferating cell nuclear antigen (PCNA) in

    unligated liver lobes was assayed immunohistochemically

    using the SP method, then incubated at 1:100 dilution

    for 60 min with polyclonal antibody against mouse PCNA

    (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After

    washing with Tris-buffered saline, sections were incu-

    bated with the secondary antibody and immunostained by

    the EnVision (Dako A/S, Grostup, Denmark) method

    according to instructions. The criterion for immunohisto-

    chemical-positive cells in terms of PCNA index was

    brown-yellow stained nucleus with a distinct boundary.

    Each section was counted for PCNA-positive nuclei in at

    least five random areas, at 2009 magnification ([300cells), and the percentages of labeled nuclei were

    calculated.

    2206 Dig Dis Sci (2013) 58:22052211

    123

  • Assays of HGF in Plasma

    The plasma concentration of HGF was detected using the

    ELISA method (HGF Assay Kit, Institute of Immunology,

    Tokyo, Japan). The value of A492 was determined by

    WellscanMK-3 type. Based on different concentrations of

    standard serum A value, the specification curve and HGF

    concentrations were attained.

    Statistical Analysis

    Data were expressed as mean SEM and analyzed by

    t test or Pearson correlative analysis with SPSS II.5 soft-

    ware. p values B0.05 were considered statistically

    significant.

    Results

    Losartan Inhibits Rise of PVP

    The normal value of PVP in rats is approximately

    10.56 0.75 mmHg. Compared with that in the sham

    operation group, the PVP of rats in the PBL group and the

    PBL ? L group increased sharply after PBL. For these two

    groups, postsurgery PVP peaked at 12 h and then gradually

    decreased to normal levels approximately 120 h after sur-

    gery. The average PVP of the PBL group was significantly

    higher than that of the sham operation group between 6 and

    72 h (p \ 0.01), whereas the PVP of the PBL ? L groupdecreased significantly at 648 h after surgery (p \ 0.05)(Fig. 1).

    Losartan Inhibits the Increase of ALT and AST Levels

    The typical ALT level in rats is approximately

    40.33 7.09. The ALT levels in the PBL group increased

    rapidly after PBL, peaking (488.83 41.80) at 24 h after

    surgery, then decreased gradually. Compared with ALT

    levels in the sham operation group, ALT levels in the PBL

    group increased dramatically between 6 and 72 h

    (p \ 0.01). The ALT levels in the PBL ? L group weresignificantly higher than those in the sham operation group,

    yet significantly lower than those in the PBL group

    between 12 and 72 h postsurgery (p \ 0.01 or p \ 0.05)(Fig. 2).

    The AST levels in rats are typically approximately

    75.00 17.09. After PBL, AST levels in the PBL group

    peaked (806.16 59.56) at 12 h, and then dropped grad-

    ually. The AST levels in the PBL group were significantly

    higher than those in the sham operation group at 672 h

    postsurgery (p \ 0.01). In comparison with the PBL group,AST levels in the PBL ? L group decreased gradually

    after 12 h and reached a normal level at 72 h. Between 6

    and 72 h, AST levels in the PBL ? L group were signifi-

    cantly lower than those in the PBL group (p \ 0.01 orp \ 0.05) (Fig. 3).

    Fig. 1 PVP values at the various time points after surgical treatment(millimeters of mercury s). PVP values are clearly higher in both

    the PBL and PBL ? losartan groups compared with the sham-

    operation group; however, PVP in PBL ? losartan group is lower

    than that in the PBL group. *p \ 0.01 versus sham-operation group;**p \ 0.01 or p \ 0.05 versus the PBL group

    Fig. 2 Serum ALT value after 90 % PBL at different time points(U/L, s). ALT values were clearly higher in both the PBL and the

    PBL ? losartan groups compared with the sham-operation group;

    however, ALT in the PBL ? losartan group was lower than that in the

    PBL group. *p \ 0.01 versus sham operation group; **p \ 0.01 orp \ 0.05 versus PBL group

    Dig Dis Sci (2013) 58:22052211 2207

    123

  • Losartan Increases the Plasma Concentration of HGF

    Portal branch ligation resulted in a rise in plasma HGF

    concentrations in both the PBL group and the PBL ? L

    group. After PBL, HGF levels in the PBL group reached

    seven times their initial preoperative values. HGF

    concentrations then decreased gradually, finally maintain-

    ing a level of approximately 2.0 ng/ml after 168 h. Com-

    pared with HGF levels in the PBL group, HGF levels in the

    PBL ? L group were notably higher between 6 and 168 h

    postsurgery and remained significantly higher at 168 h

    postsurgery (p \ 0.01) (Fig. 4).

    Losartan Increases the PCNA Index

    The PCNA index was used to evaluate cell proliferation

    in vivo in the regenerating liver after PBL. The basic value

    of the PCNA index is 5.55 % in rat liver tissue. In the PBL

    group, the PCNA index was significantly elevated by 12 h

    after PBL, and peaked at 120 h. Compared with that in the

    sham operation group, the PCNA index was significantly

    higher between 12 and 168 h (p \ 0.01). For the PBL ? Lgroup, the PCNA index peaked at 72 h. Compared with

    that in the PBL group, the PCNA index for the PBL ? L

    group was significantly elevated at 12168 h postsurgery

    (p \ 0.01 or p \ 0.05) (Figs. 5, 6ac).

    Losartans Effects on the LW/TW Ratio

    After PBL, the ligated liver lobes atrophied and darkened,

    whereas the unligated liver lobes underwent hypertrophy.

    In the sham operation group, the caudate lobe accounted

    for 10 % of the liver weight. Compared with the LW/TW

    ratio in the sham operation group, the LW/TW ratio in the

    PBL group increased significantly from 24 h (p \ 0.01),increasing gradually, reaching 27.83 % by 168 h

    Fig. 3 Serum AST value after 90 % PBL at different time points (U/l, s). AST values were obviously higher in both the PBL and

    PBL ? losartan groups compared with the sham operation group;

    however, AST in the PBL ? losartan group is lower than that in the

    PBL group. *p \ 0.01 versus sham operation group; **p \ 0.01versus PBL group

    Fig. 4 HGF levels after PBL at different time points (ng/ml, s).HGF values are higher in both the PBL and PBL ? losartan groups

    compared with the sham operation group; however, HGF in the

    PBL ? losartan group is higher than that in the PBL group. *p \ 0.01versus sham operation group; **p \ 0.01 or p \ 0.05 versus PBLgroup

    Fig. 5 PCNA indexes after PBL at different time points. *p \ 0.01versus sham operation group. PCNA values clearly higher in both the

    PBL and the PBL ? losartan groups compared with the sham

    operation group; however, PCNA in the PBL ? losartan group is

    higher than that in the PBL group. **p \ 0.01 versus PBL group

    2208 Dig Dis Sci (2013) 58:22052211

    123

  • postsurgery. The LW/TW ratio for the PBL ? L group

    reached 31.15 % by 168 h postsurgery. In the PBL ? L

    group, the LW/TW ratio was higher than that in the PBL

    group between 24 and 168 h postsurgery (p \ 0.01)(Fig. 7).

    Pathologic Changes in Hepatic Tissue

    In the sham operation group, the hepatic cells arrayed

    radially around the central vein. After PBL, cells of unli-

    gated liver lobes showed slight edema and hepatic sinu-

    soids dilated significantly by 24 h. Hepatic lobules were

    severely distorted by 72 h. Thereafter, the hepatic tissue

    gradually returned to normal. In the PBL ? L group,

    hepatocyte edema and dilation of hepatic sinusoids were

    less severe than in the PBL group, and took less time to

    r...

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