B - Gastrointestinal and Liver Physiology

Articles in PresS. Am J Physiol Gastrointest Liver Physiol (December 28, 2012). doi:10.1152/ajpgi.00391.2012
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Protective effects of branched-chain amino acids on hepatic ischemia/reperfusion-induced
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liver injury in rats: a direct attenuation of Kupffer cell activation.
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Tomomi Kitagawa, Yukihiro Yokoyama, Toshio Kokuryo, and Masato Nagino
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Division of Surgical Oncology, Department of Surgery,
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Nagoya University Graduate School of Medicine, Nagoya, Japan
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Running Head: Protective effects of BCAA
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Corresponding author:
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Yukihiro Yokoyama
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Division of Surgical Oncology, Department of Surgery
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Nagoya University Graduate School of Medicine
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65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan
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Tel: +81-52-744-2220
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Fax: +81-52-744-2230
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e-mail: yyoko@med.nagoya-u.ac.jp
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Copyright © 2012 by the American Physiological Society.
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ABSTRACT
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Objective: We determined whether there is a protective effect of branched-chain amino acid
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(BCAA) on hepatic ischemia/reperfusion (IR)-induced acute liver injury.
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Methods: Wister rats were divided into four groups: simple laparotomy with vehicle; simple
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laparotomy with BCAA (1 g/kg of body weight orally); IR (30 min clamp) with vehicle; and IR
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with BCAA. Serum liver function tests and the gene expression of adhesion molecules (ICAM
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and VCAM) and vasoconstrictor-related genes (endothelin-1) in the liver were examined. In the
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in vivo study, portal venous pressure, leukocyte adhesion, and hepatic microcirculation were
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evaluated. Furthermore, Kupffer cells were isolated and cultured with various concentrations of
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BCAA in the presence or absence of lipopolysaccharide (LPS).
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Results: Increased levels of liver function tests following IR were significantly attenuated by
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BCAA treatment. The increased expression of adhesion molecules and endothelin-1 were also
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significantly attenuated by BCAA treatment. Moreover, increased portal venous pressure,
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enhanced leukocyte adhesion, and deteriorated hepatic microcirculation following IR were all
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improved by BCAA treatment. In the experiment using isolated Kupffer cells, the expression of
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interleukin-6, interleukin-1β, and endothelin-1 in response to LPS stimulation was attenuated by
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BCAA in a dose-dependent fashion.
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Conclusions: These results indicate that perioperative oral administration of BCAA has excellent
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therapeutic potential to reduce IR-induced liver injury. These beneficial effects may result from
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the direct attenuation of Kupffer cell activation under stressful conditions.
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Keywords: inflammatory responses, microcirculatory failure, BCAA
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INTRODUCTION
During liver resection, ischemia/reperfusion (IR) is a mandatory procedure to minimize
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intraoperative blood loss. However, this procedure unfailingly induces liver injury and may
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sometimes lead to major postoperative complications such as liver failure (9). IR-induced liver
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injury is also a major problem in the process of liver transplantation (3; 6). Therefore, minimizing
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IR-induced liver injury is crucial to safely performing liver surgery.
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The major mechanism of hepatic IR-induced liver injury includes an enhanced
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inflammatory response (12) and microcirculatory failure (7). The upregulation of inflammatory
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cytokines in combination with an upregulation of adhesion molecules in the sinusoidal
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endothelial cells leads to the rolling, adhesion, and migration of leukocytes (14). The increased
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production of vasoconstrictors such as endothelin-1 (ET-1) leads to presinusoidal and sinusoidal
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vasoconstriction in the liver through an autocrine or paracrine mechanism (7). Moreover, the
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sensitivity of the hepatic microcirculatory system to vasoconstrictors is also enhanced after IR
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(36). These changes activate inflammatory processes and microcirculatory disturbances in the
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liver, which finally results in severe hepatic injury.
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Branched-chain amino acid (BCAA) is a group of essential amino acids comprised of
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valine, leucine, and isoleucine. In Japan, oral BCAA preparations containing valine, leucine, and
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isoleucine in a composition ratio of 1.2:2:1 have been used to correct protein and amino acids
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abnormalities in patients with chronic liver disease and hepatic encephalopathy (26). In addition,
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recent studies indicate that long-term administration of BCAA is effective in reducing the risk of
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cancer development in viral hepatitis (10; 39). However, the effect of BCAA administration on
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hepatic IR-induced liver injury has never been investigated. In this study, we hypothesized that
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oral administration of BCAA can attenuate excessive inflammatory responses, leukocyte
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adhesion, upregulation of vasoconstrictors, and microcirculatory liver failure and may also
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prevent liver damage following IR.
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MATERIALS AND METHODS
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Animals
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Male Wistar rats (Charles River Labs, Wilmington, MA) weighing 250 to 300 grams were
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purchased from Japan SLC (Nagoya, Japan) and housed in a temperature- and humidity-
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controlled room under a constant 12 h light/dark cycle. Animals were allowed free access to
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water and food at all times. All experiments were performed in compliance with the guidelines of
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the Institute for Laboratory Animal Research, Nagoya University Graduate School of Medicine.
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Surgical procedures
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Rats were anesthetized with diethyl ether. After an upper abdominal midline laparotomy,
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the hepatoduodenal ligament was clamped for 30 min with a vascular clip (BEAR Medic
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Corporation, Chiba, Japan) for the IR groups. Thereafter, the vascular clip was removed, and the
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liver was reperfused for 24 h. Although severe mesenteric congestion was observed during the
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clamping of hepatoduodenal ligament, no animal died within 24 h. In the Sham group, only
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laparotomy and mobilization of the hepatoduodenal ligament were performed. Twenty-four hours
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after the IR or Sham operation, blood samples were collected and liver tissue samples were
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removed for analysis (n=6 in each group).
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Administration of BCAA
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Oral BCAA preparations (containing valine, leucine, and isoleucine in a composition ratio
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of 1.2:2:1) were provided by Ajinomoto Pharm Co., Inc. (Tokyo, Japan). The protocol of BCAA
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administration was determined based on previously published studies with minor modification
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(20; 23). Briefly, BCAA was dissolved in 0.5% methylcellulose solution and administered orally
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(1 g/kg of body weight). The same volume of 0.5% methylcellulose solution was used as a
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vehicle. In the IR group, BCAA or vehicle was administered 0.5 h before ischemia and 7 h after
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reperfusion and 0.5 h before sampling. This administration protocol was established through
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multiple preliminary studies. We performed a protocol with only one time administration before
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IR, only one time administration after IR, or multiple administrations before and after IR.
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Although these protocols showed a similar trend, multiple administrations showed best results.
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BCAA or vehicle was also administered on the same time scale in the Sham operation group.
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Analysis of blood samples
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Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT),
hyaluronic acid (HA), and total bilirubin (T-Bil) were measured by standard laboratory methods
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(SRL, Tokyo, Japan). Serum concentrations of aromatic amino acids (AAA), BCAA, and
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methionine were also measured by standard laboratory methods (SRL, Tokyo, Japan). Fischer’s
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ratio was calculated by the ratio of serum BCAA to AAA concentrations.
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Histologic Evaluation
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The liver tissue samples were immersed immediately in 10% buffered formalin and stored
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overnight. The samples were then dehydrated in a graded ethanol series and embedded in paraffin.
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Six-micrometer-thick sections were mounted on glass slides and stained with hematoxylin and
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eosin. The number of necrotic focus was counted in paraffin-embedded liver samples by a third
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researcher who has no association with this study. The data was expressed as the average number
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of necrotic foci in high-power fields (HPF). Ten fields from one specimen were evaluated for
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three animals in each group. Other sections were subjected to immunohistochemistry to detect the
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expression of vascular cell adhesion molecule (VCAM) and intercellular adhesion molecule
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(ICAM). The automated slide preparation system Discovery XT (Ventana Medical Systems, Inc.,
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Tucson, AZ) was used. Before staining, paraffin sections were heated at 37°C for 30 min in a
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paraffin oven and were blocked with 5% nonfat milk. The staining procedure was carried out
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according to the manufacturer’s protocol (Ventana Medical Systems, Inc.). Anti-VCAM antibody
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(Abcam, Cambridge, UK) and anti-ICAM antibody (Abcam, Cambridge, UK) was diluted in
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Discovery Ab diluent (Ventana Medical Systems, Inc.).
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Real-time PCR for inflammatory cytokines and vasoconstrictor genes
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To validate the gene expression changes in the liver, quantitative real-time PCR analysis
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was performed with a Prism 7300 sequence detection system (Applied Biosystems Inc., Foster
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City, CA). Total RNA was isolated from whole liver or Kupffer cells using the RNeasy Mini kit
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(Qiagen, Hilden, Germany) according to the manufacturer’s protocol. cDNA was generated from
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total RNA samples by use of the High Capacity cDNA Reverse Transcription Kit (Applied
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Biosystems). Each reaction was performed in a 20 µl mixture with TaqMan universal PCR master
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mix according to the manufacturer’s instructions (Applied Biosystems). The expression of the
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genes encoding adhesion molecules [VCAM (assay identification number Rn00563627_m1) and
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ICAM (assay identification number Rn00564227_m1)], inflammatory cytokines [interleukin-1β
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(IL-1β; assay identification number Rn00580432_m1) and interleukin-6 (IL-6; assay
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identification number Rn99999011_m1)], and vasoconstrictor-related proteins (ET-1; assay
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identification number Rn00561129_m1) in the liver homogenate or the isolated Kupffer cells was
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determined by comparative quantitative real-time PCR using the Mx3000P Real-Time PCR
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System (Stratagene, La, Jolla, CA). 18S rRNA (assay identification number Hs99999901_s1)
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was used as an endogenous control. The reaction mixture was denatured for 1 cycle at 50 °C for 2
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min, 1 cycle at 95 °C for 10 min, 50 cycles at 95 °C for 15 sec, and 1 cycle at 60 °C for 1 min.
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All samples were tested in duplicate, and the average values were used for quantification. The
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analysis was performed using MxPro TM Software version 2.00 (Stratagene) according to the
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manufacturer’s instructions. The comparative cycle threshold (CT) method (ΔΔCT) was used for
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quantification of gene expression. The average of the Sham group with vehicle treatment was set
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as one fold induction, and the data were adjusted to that baseline.
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Measurement of portal venous pressure
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A total of 24 h after the IR or Sham operation, the animals were anesthetized with sodium
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pentobarbital (50 mg/kg body weight, intraperitoneally). Portal venous pressure (PVP) was
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measured by Power Lab AD Instruments (Castle Hill, Bella Vista, Australia) through a catheter
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(22G) inserted into the portal vein (n=6-8 in each group).
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In situ intravital microscopy
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Evaluation of leukocyte adhesion
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A total of 24 h after the IR or Sham operation, the animals were anesthetized with sodium
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pentobarbital (50 mg/kg body weight, intraperitoneally). Leukocytes were labeled by intravenous
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administration of rhodamine 6G (0.1437 mg/kg, Sigma-Aldrich, St. Louis, MO). As the liver
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preparation was stabilized onto a fluorescent microscope, the liver surface was epi-illuminated
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with a LED excitation system (Cool LED pE excitation system, UK) using 532-554 nm
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excitation and 573-613 nm emission band-pass filters to visualize fluorescent rhodamine 6G-
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labeled leukocytes in the sinusoids. In all experiments, four fields consisting of four to eight
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acini per field were recorded using a 20× objective (n=6 in each group). The number of adherent
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leukocytes was determined during playback of videotaped images.
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Evaluation of liver microcirculation
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Red blood cells (RBCs) were isolated and labeled using the PKH 67 Green Fluorescent Cell
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Linker Mini Kit for general cell membrane labeling (Sigma-Aldrich, St. Louis, MO) according to
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the manufacturer’s protocol. The liver surface was epi-illuminated with an LED excitation system
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(Cool LED pE excitation system) using 464.5-499.5 nm excitation and 516-556 nm emission
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band-pass filters. The images were recorded by a Digital CameraC10600 ORCA-R2 and
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processed using Aquacosmos software (Hamamatsu Photonics, Hamamatsu, Japan). Three acini
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were randomly recorded in each rat (n=4 in each group). Measurements of perfused sinusoidal
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diameters (Ds) was made directly from video playback. (5). For measurement of the RBC
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velocity (VRBC) in the sinusoids, 0.2 ml of fluorescent-labeled RBCs was injected into the carotid
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artery. The volumetric flow (VF) was calculated from the Ds and VRBC by the following equation
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as previously described: VF = VRBC×π×(Ds/2)2 (38).
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Kupffer cell isolation
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The Kupffer cells were obtained by an in situ collagenase digestion method. The liver was
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perfused by oxygenized Hank’s balanced salt solution (HBSS; GIBCO-BRL, Gaithersburg, MD)
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for 10 min to wash out the blood and was perfused with 0.05% collagenase (Sigma-Aldrich, St.
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Louis, MO) for 5 min. After the digestion, the cell suspension was filtered through a sieve and
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centrifuged twice at 50×g for 2 min to separate the parenchymal and non-parenchymal cells.
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Non-parenchymal cells were collected and centrifuged at 2000×g for 15 min (4°C). Supernatants
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were discarded and pellets were suspended with RPMI 1640 (Invitrogen, Carlsbad, California).
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Cells were centrifuged at 800×g for 15 min through Percoll (GE Healthcare, Little Chalfont, UK)
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to remove contaminating non-parenchymal cells. Collected Kupffer cells were centrifuged at
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300×g for 15 min. The pellets were collected, and the cells were resuspended in RPMI 1640. The
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concentration of cells was adjusted to 1×105 cells/ml in RPMI, and 1 ml of cell suspension was
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added per well to a 12-well culture dish. After incubating for 3 h, the plates were washed with
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warm RPMI to remove non-adherent cells and 1 ml of RPMI was added. After 24 h, the adherent
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Kupffer cells were treated with vehicle or various concentrations of BCAA. After 30 min of
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vehicle or BCAA treatment, the Kupffer cells were stimulated with LPS (5 μg/ml) in the medium
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to induce inflammatory cytokine and endothelin-1 gene expression. Purity (>90%) was checked
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by the uptake of latex beads (Sigma-Aldrich, St. Louis, MO) in the Kupffer cells.
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Assessment of proinflammatory cytokine release
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IL-6 levels in the supernatant from the Kupffer cell culture were determined by sandwich
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enzyme linked immunosorbent assay (ELISA) methods. The assay was performed according to
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the manufacturer’s protocol (R&D, Minneapolis, MN).
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Statistical analysis
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Student’s t-test was used to compare the significant differences between two groups.
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Significant differences among multiple groups were analyzed by a one way ANOVA followed by
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the Dunnet test. When criteria for parametric testing were violated, the appropriate non-
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parametric (Mann–Whitney U-test) was used. A P-value of less than 0.05 was considered
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significant. All results are presented as the mean ± SE.
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RESULTS
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Blood tests
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The serum levels of AST, ALT, HA, and T-Bil significantly increased following IR with
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vehicle treatment (Figure 1A-D). However, all of these levels were significantly lower in the
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BCAA treatment group compared to the group with vehicle treatment. The serum levels of AAA
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were significantly higher following IR compared to those levels following the Sham operation
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(Figure 2A). However, in the IR with BCAA treatment group, the serum levels of AAA were
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significantly lower than in the IR with vehicle treatment group (Figure 2A). Fischer’s ratios
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(BCAA/AAA) were significantly lower following IR compared to those following the Sham
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operation in the group with vehicle treatment. However, with BCAA treatment, these ratios were
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substantially increased (Figure 2B). The serum levels of methionine, another amino acid that is
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related to liver injury (25), were significantly higher following IR compared to those following
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the Sham operation in the group with vehicle treatment. However, these levels were restored to
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the levels of the Sham group by BCAA treatment (Figure 2C).
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Histologic Evaluation
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Livers in the Sham group showed normal histology irrespective of BCAA administration,
indicating that a simple laparotomy did not damage the liver parenchyma (Figure 3A,
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Sham+vehicle; 3B, Sham+BCAA). In contrast, some necrotic foci were observed in the
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IR+vehicle group (Figure 3C). However, signs of necrosis were rarely observed in the IR+BCAA
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group (Figure 3D). The average number of necrotic foci in randomly selected high-power fields
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was significantly higher in the IR+vehicle group than that in the IR+BCAA group (Figure 3E).
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Leukocyte adhesion
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The level of leukocyte adhesion observed by intravital microscopy was significantly
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greater in the IR group with vehicle treatment compared to that in the Sham group. However, the
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level was significantly lower in the IR group with BCAA treatment (Figure 4).
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Hepatic hemodynamics
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As previously reported (16; 22; 38), the levels of portal venous pressure in the IR group
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were significantly higher compared to the Sham group (Figure 5A). However, these levels in the
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IR group with BCAA treatment were significantly lower compared to those in the IR group with
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vehicle treatment. The average DS was significantly greater in the IR with BCAA treatment group
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compared with the IR with vehicle treatment group (Figure 5B). Although the VRBC was almost
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identical between the IR with vehicle treatment and BCAA treatment groups, the average VF of
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the sinusoids was significantly higher in the IR with BCAA treatment group compared to the
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vehicle treatment group (Figure 5C, D). The representative images of intravital microscopy in the
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IR with vehicle and BCAA treatment groups are shown in Figures 5E and 5F, respectively.
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Gene expression of adhesion molecules and vasoconstrictor genes
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The gene expression levels of VCAM and ICAM in the liver were significantly higher in
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the IR with vehicle treatment group compared to the Sham group (Figure 6A, B). However, these
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changes were significantly attenuated by BCAA treatment. The gene expression levels of ET-1, a
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potent vasoconstrictor in the liver, showed a similar trend (Figure 6C).
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Immunohistochemistry for VCAM and ICAM
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To confirm the expression of VCAM and ICAM in the liver tissue,
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immunohistochemistry was performed. After IR with vehicle treatment group, an increased
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expression of VCAM and ICAM in the sinusoidal endothelial cells was observed (Figure 7A, C).
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However, the expression was substantially suppressed in the IR with BCAA treatment group
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(Figure 7B, D).
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Study of isolated Kupffer cells
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The mechanisms of liver injury in response to various hepatic stresses, including IR (34),
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endotoxemia (exposure to the LPS) (33), and alcohol consumption (2; 21), are similar. Under
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these stressful conditions, the Kupffer cells are activated and produce excessive levels of
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inflammatory cytokines (i.e., IL-6 and IL-1β) (1) and vasoconstrictors (i.e., ET-1) (29). These
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changes lead to an enhanced inflammatory response and microcirculatory failure, thereby
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resulting in liver damage (37). Therefore, we next determined whether the BCAA directly
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modulated the excessive activation of Kupffer cells. In isolated Kupffer cells stimulated with LPS,
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the gene expression of inflammatory cytokines (such as IL-6 and IL-1β) and ET-1 was
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significantly upregulated (Figure 8A-C). However, the expression levels were attenuated by
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BCAA administration in the medium in a dose-dependent manner. We also determined the
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protein levels of IL-6 in the medium using sandwich ELISA technique. The levels of IL-6 after
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LPS treatment were substantially suppressed with 40 mM BCAA in the medium compared to the
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levels without BCAA administration (1135±612 pg/ml without BCAA vs. 78±39 mg/ml with
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BCAA).
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DISCUSSION
In this study, we found that the preoperative administration of BCAA significantly
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attenuated post-IR liver injury. Moreover, we demonstrated that the beneficial effects of BCAA
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on IR-induced liver injury are at least partly explained by the direct attenuation of Kupffer cell
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activation. These are novel findings, which have never been reported before and may have a
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substantial clinical impact on liver surgery. The results indicate that the perioperative oral
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administration of BCAA has excellent therapeutic potential for use in attenuating IR-induced
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liver injury.
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Fischer’s ratio is important for assessing hepatic functional reserve in patients with liver
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cirrhosis (28). A low level of Fischer’s ratio indicates impaired physiological liver function,
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including protein metabolism and synthesis. The rationale of BCAA supplementation in these
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patients is to normalize amino acid profiles and nutritional status. In this study, we found
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significantly lower Fischer’s ratios following the IR group compared to those following the Sham
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group. Moreover, the levels of AAA and methionine were also significantly higher in the IR
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group than the Sham group. These results indicate a functional alteration in amino acid
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metabolism in the liver in response to IR stress. Although the precise mechanism was not
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elucidated in this study, supplementation of BCAA at least alleviated these alterations. It should
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be noted that the dose of BCAA used in this study seems pretty high (1 g/kg of body weight)
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compared to that used in humans. Metabolic pathway of BCAA in the liver may be different
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between rat and human (27). Therefore, it is difficult to directly extrapolate the results of animal
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study to human. Nevertheless, there are numerous reports showing a similar beneficial effect of
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BCAA administration both in the animal (11; 23) and human studies (15; 19). To determine the
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optimal dose of BCAA in protecting IR-induced liver damage in humans, a clinical study
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including patients who undergo hepatectomy while clamping hepatoduodenal ligament should be
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performed.
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One of the major mechanisms of hepatic injury following IR stress includes an excessive
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inflammatory response (18). Under the stress of IR, the adhesion, rolling, and migration of
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leukocytes is induced through the upregulation of adhesion molecules in both leukocytes and
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sinusoidal endothelial cells (30). The ICAM and VCAM molecules are representative adhesion
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molecules that are upregulated on endothelial cells following IR stress (8). As demonstrated in
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this study, BCAA treatment significantly attenuated the upregulation of ICAM and VCAM
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following IR. Decreased leukocyte adhesion on the sinusoidal endothelial cells was also observed
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by intravital microscopy. These effects may reduce excessive inflammatory responses and
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ameliorate liver injury following IR.
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Another important mechanism of hepatic injury following IR is microcirculatory failure (4;
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31). Under IR stress, ET-1, one of the most potent vasoconstrictors, is upregulated in the liver
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and acts on hepatic microvasculature through a paracrine and/or autocrine mechanism (17; 24).
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Moreover, the constrictive response of the portal vasculature system to ET-1 is also enhanced
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through the upregulation and remodeling of endothelin receptors (35; 36). Although the mRNA
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expression of ET-1 is robustly upregulated following IR, BCAA treatment significantly
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attenuated this change. In concordance with the upregulation of ET-1, hepatic microcirculatory
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failure is commonly observed following IR stress. Although the sinusoidal diameters become
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narrower compared to normal levels following IR, they were significantly greater in the group
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with the BCAA treatment compared to the vehicle treatment. We speculate that these differences
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are partly explained by the attenuated expression of ET-1 in the liver due to BCAA treatment.
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Under various hepatic stresses such as endotoxemia, IR, alcoholic consumption and
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biliary obstruction enhanced inflammatory response (13) and microcirculatory failure (32)
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commonly occur, and these changes finally lead to liver damage. Kupffer cells, which are hepatic
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resident macrophages, play a crucial role in these pathophysiological changes (13). Therefore, in
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the final experiment, we hypothesized that BCAA treatment directly modulates Kupffer cell
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activation. Kupffer cells were isolated from normal liver, and these cells were stimulated with
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LPS with/without BCAA pre-administration in the medium. As was expected, the upregulated
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mRNA expression of inflammatory cytokines and ET-1 by LPS stimulation were significantly
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attenuated by adding BCAA (30 min before LPS stimulation) to the medium in a dose-dependent
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fashion. Although the precise metabolic mechanisms on the molecular level remains unknown,
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we believe that the protective effect of BCAA on IR-induced liver injury is at least partly
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mediated by the direct modulation of the Kupffer cell function by BCAA. Further study is needed
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to elucidate this novel pharmacological effect by BCAA.
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During major hepatectomy, repeating IR is a necessary procedure to minimize
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intraoperative bleeding. However, this procedure paradoxically induces IR-related injury to the
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liver and may sometimes lead to severe postoperative complications. Based on our findings in
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this study, a preoperative administration of BCAA may have excellent therapeutic potential to
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reduce postoperative liver injury. This hypothesis should be investigated through a randomized
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controlled study.
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In summary, this study is the first to demonstrate a protective effect of BCAA treatment
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on hepatic IR-induced liver injury. The administration of BCAA prior to and following ischemia
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significantly attenuated IR-induced inflammatory responses and microcirculatory failure. The
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major mechanisms of these beneficial effects may include the direct attenuation of Kupffer cell
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activation in response to IR stress. These results strongly indicate the therapeutic potential of
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BCAA in preventing the hepatic IR-induced liver injury that inevitably occurs during liver
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surgery.
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FIGURE LEGENDS
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Figure 1
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The serum aspartate aminotransferase (AST) (A), alanine aminotransferase (ALT) (B),
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Hyaluronic acid (HA) (C), and total bilirubin (T-Bil) (D) were measured by standard laboratory
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methods.
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† P<0.05 versus Sham+vehicle; ∗ P<0.05 versus IR+vehicle
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Figure 2
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The serum levels of aromatic amino acids (AAA) (A), Fisher’s ratios (BCAA/AAA) (B), and
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methionine (C) were measured by standard laboratory methods.
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† P<0.05 versus Sham+vehicle; ∗ P<0.05 versus IR+vehicle
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Figure 3
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Histology of the livers in the Sham+vehicle (A), Sham+BCAA (B), IR+vehicle (C), and
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IR+BCAA (D) groups. The micrographs depict representative hematoxylin and eosin staining of
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paraffin-embedded liver slides, recorded using a 4× objective. Several necrotic foci were
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observed in the IR+vehicle group (arrows). The average number of necrotic foci in high-power
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fields (HPF) (E). Ten fields from one specimen were evaluated for three animals in each group.
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∗ P<0.05 versus IR+vehicle
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Figure 4
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The level of leukocyte adhesion in the liver evaluated by intravital microscopy in the
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Sham+vehicle group (A), Sham+BCAA group (B), IR+vehicle group (C), and IR+BCAA group
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(D). The level of leukocyte adhesion shown in a high power field (HPF) (E). Four fields
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consisting of four to eight acini per field were recorded using a 20× objective. The number of
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adherent leukocytes was determined during playback of videotaped images.
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† P<0.05 versus Sham+vehicle; ∗ P<0.05 versus IR+vehicle
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Figure 5
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Portal venous pressure (PVP) (A) was measured by Power Lab AD Instruments through a
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catheter (22G) inserted into the portal vein. Measurements of sinusoidal diameters (Ds) (B) was
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made directly from video playback. For measurement of the red blood cell velocity (VRBC) (C) in
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the sinusoids, 0.2 ml of fluorescent-labeled RBCs was injected into the carotid artery. The
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volumetric flow (VF) was calculated from the Ds and VRBC by the following equation as
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previously described: VF = VRBC×π×(Ds/2)2 (pl/sec) (D).
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† P<0.05 versus Sham+vehicle; ∗ P<0.05 versus IR+vehicle
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Figure 6
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The expression levels of the genes encoding adhesion molecules [VCAM (A) and ICAM (B)] and
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vasoconstrictor-related proteins (ET-1) (C) in the liver, detected by real-time RT-PCR. Total
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RNA was extracted from the frozen liver tissue and was subjected to comparative quantitative
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real-time RT-PCR using specific primers. 18S rRNA was used as an internal control for each
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sample. The average expression level of the Sham+vehicle group’s data was set to 1.0, and other
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data were adjusted to this baseline.
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† P<0.05 versus Sham+vehicle; ∗ P<0.05 versus IR+vehicle
382
Figure 7
383
Immunohistochemistry for VCAM protein in the IR+vehicle (A) and IR+BCAA (B) groups, and
384
ICAM protein in the IR+vehicle (C) and IR+BCAA (D) groups. The images were recorded using
385
a 10× objective.
386
Figure 8
387
Kupffer cells were isolated and incubated for 20 h. The adherent Kupffer cells were treated with
388
vehicle or various concentrations of BCAA. After 30 min of vehicle or BCAA treatment, the
389
Kupffer cells were stimulated with LPS (5 μg/ml) in the medium. After 4h, the expression levels
390
of the genes encoding IL-6 (A), IL-1β (B), and ET-1 (C), were detected by real-time RT-PCR.
391
∗ P<0.05 versus BCAA 0 mM
392
393
394
395
21
396
397
398
399
400
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25
510
26
AST
A
ALT
B
†
700
600
∗
400
300
lU/l
400
500
lU/l
†
500
300
∗
200
200
100
100
0
0
Sham
+vehicle
Sham
+BCAA
IR
+vehicle
IR
+BCAA
Sham
+vehicle
HA
Sham
+BCAA
IR
+vehicle
IR
+BCAA
T-Bil
C
D
†
70
60
mg/dl
40
30
0.05
∗
0.04
mg/dl
∗
50
†
0.06
0.03
0.02
20
0.01
10
0
Sham
+vehicle
Sham
+BCAA
IR
+vehicle
IR
+BCAA
0
Sham
+vehicle
Sham
+BCAA
IR
+vehicle
IR
+BCAA
Figure 1
AAA
A
†
nmol/m
ml
140
∗
120
100
80
60
40
20
0
Sham
+vehicle
Sham
IR
+BCAA +vehicle
IR
+BCAA
Fischer’s ratio
B
35
∗
30
nmol/ml
25
5
†
4
3
2
1
0
Sham
+vehicle
IR
Sham
+BCAA +vehicle
IR
+BCAA
Methionine
C
†
80
∗
70
nmol/ml
60
50
40
30
20
10
0
Sham
+vehicle
Sham
IR
+BCAA +vehicle
IR
+BCAA
Figure 2
A
B
Sham+vehicle
Sham+BCAA
D
C
IR+BCAA
IR+vehicle
E
Number off necrotic
foci/H
HPF
1.4
1.2
1
0.8
0.6
∗
0.4
0.2
0
IR
+vehicle
IR
+BCAA
Figure 3
A
B
Sham+vehicle
Sham+BCAA
CC
D
IR+BCAA
IR+vehicle
E
Number of leukocyte
on/HPF
adhesio
25
†
20
15
10
∗
5
0
Sham
+vehicle
Sham
IR
+BCAA +vehicle
IR
+BCAA
Figure 4
10
∗
8
mmHg
D
†
12
6
4
B
600
∗
500
400
†
300
100
Sham
+vehicle
Sham
+BCAA
IR
+vehicle
0
IR
+BCAA
Sham
+vehicle
Sham
+BCAA
IR
+vehicle
IR
+BCAA
IR+vehicle
DS
E
35
30
∗
25
μm
m
800
200
2
0
VF
00
700
VF(pl/Sec)
A
PVP
†
20
15
10
5
0
Sham
+vehicle
Sham
+BCAA
IR
+vehicle
IR
+BCAA
IR+BCAA
C
VRBC
F
1400
1200
μ
μm/Sec
1000
800
600
400
200
0
Sham
+vehicle
Sham
+BCAA
IR
+vehicle
IR
+BCAA
Figure 5
VCAM
A
†
Arbitrary unit
2.5
2.0
∗
1.5
1.0
0.5
0.0
Sham
+vehicle
Sham
IR
+BCAA +vehicle
IR
+BCAA
ICAM
B
†
ary unit
Arbitra
2.0
∗
1.5
1.0
0.5
0.0
Sham
+vehicle
hi l
Sham
+BCAA
BCAA
IR
+vehicle
hi l
IR
+BCAA
BCAA
ET-1
C
†
4.0
3.5
ary unit
Arbitra
3.0
∗
2.5
2.0
1.5
1.0
0.5
0.0
Sham
+vehicle
Sham
+BCAA
IR
+vehicle
IR
+BCAA
Figure 6
VCAM
A
B
IR+vehicle
IR+BCAA
ICAM
D
C
IR+vehicle
IR+BCAA
Figure 7
Arbiitrary unit
A
9.0
8.0
7.0
6.0
5
0
5.0
4.0
3.0
2.0
1.0
0.0
IL-6
LPS (-)
LPS (+)
∗
∗
0
10
20
∗
40
BCAA (mM)
IL-1β
LPS ((-))
LPS (+)
14.0
12.0
Arb
bitrary unit
B
∗
10.0
∗
8.0
∗
6.0
4.0
2.0
0.0
0
10
20
40
BCAA (mM)
ET-1
LPS (-)
LPS (+)
2.5
Arbitrary unit
A
C
2.0
2
0
∗
1.5
∗
∗
1.0
0.5
0.0
0
10
20
BCAA (mM)
40
Figure 8