A reduction in the levels of CYP2E1 protein and CYP2B1 and CYP2E1 mRNA expression was prevented by GdCl3.. The increased serum level of high mobility group box 1, hepatic level of Toll-l
Trang 1Role of Kupffer cells in pathogenesis of sepsis-induced
drug metabolizing dysfunction
Tae-Hoon Kim*, Sang-Ho Lee* and Sun-Mee Lee
School of Pharmacy, Sungkyunkwan University, Suwon, South Korea
Introduction
Sepsis, severe sepsis and septic shock are worldwide
problems and continue to be the most common causes
of death in surgical intensive care units [1] The
patho-genesis of sepsis has often been viewed to involve
excessive immune inflammation that can lead to lethal
multiple organ failure, suggesting that the
downregula-tion of immunity could be beneficial [2] As a result of
its major implications in essential metabolic functions
and host defense, the liver plays an important role in
the development of multiple organ failure [3]
Patients who are diagnosed with sepsis receive vari-ous therapeutic agents because of its complex patho-physiology and varied symptoms; the main clinical concern has been that patients on a stable drug regi-men would have increased exposure to an incidence of adverse drug events The cytochrome P450 (CYP) enzyme system constitutes one of the major aspects of hepatocyte function and contributes to the metabolism and elimination of exogenous and endogenous sub-stances [4] In various models and in clinical reports,
Keywords
CYP450; HMGB1; Kupffer cells; sepsis;
Toll-like receptor
Correspondence
S.-M Lee, School of Pharmacy,
Sungkyunkwan University, 300
Cheoncheon-dong, Jangan-gu, Suwon-si,
Gyeonggi-do 440-746, South Korea
Fax: +82 31 292 8800
Tel: +82 31 290 7712
E-mail: sunmee@skku.edu
*These authors contributed equally to this
work
(Received 2 December 2010, revised 19
April 2011, accepted 28 April 2011)
doi:10.1111/j.1742-4658.2011.08148.x
The present study aimed to determine the role of Kupffer cells (KCs) in cytochrome P450 (CYP) isozyme activity and the expression of its gene during polymicrobial sepsis For ablation of KCs, rats were pretreated with gadolinium chloride (GdCl3) at 48 and 24 h before cecal ligation and punc-ture (CLP) The depletion of KCs was confirmed by measuring the mRNA level of the KC marker gene CD163 Serum aminotransferase levels and lipid peroxidation showed an increase and hepatic glutathione content showed a decrease at 24 h after CLP These changes were prevented by GdCl3 pretreatment Catalytic activities of CYP1A1, 1A2 and 2E1 showed
a significant reduction at 24 h after CLP but were prevented by GdCl3
A reduction in the levels of CYP2E1 protein and CYP2B1 and CYP2E1 mRNA expression was prevented by GdCl3 Phosphorylation of CYP1A1⁄ 1A2 markedly increased 24 h after CLP, which was prevented by GdCl3 The increased serum level of high mobility group box 1, hepatic level of Toll-like receptors 2 and 4, and inducible nitric oxide synthase pro-tein expression were prevented by GdCl3 In addition, elevated serum con-centrations of tumor necrosis factor-a and interleukin-6, and increased hepatic mRNA levels of tumor necrosis factor-a and interleukin-6 were decreased by depletion of KCs Our findings suggest that ablation of KCs protects against hepatic drug-metabolizing dysfunction by modulation of the inflammatory response
Abbreviations
ALT, alanine aminotrasferase; AST, aspartate aminotrasferase; CLP, cecal ligation and puncture; CYP, cytochrome P450; GdCl3, gadolinium chloride; GSH, glutathione; GSSG, glutathione disulfide; HMGB1, high mobility group box 1; IL, interleukin; iNOS, inducible nitric oxide synthase; KCs, Kupffer cells; LPS, lipopolysaccharide; MDA, malondialdehyde; NO, nitric oxide; PAP, p-aminophenol;
RIPA, radioimmunoprecipitation assay; ROS, reactive oxygen species; TLR, Toll-like receptor; TNF, tumor necrosis factor.
Trang 2inflammation or infection is associated with a decrease
in hepatic expression and⁄ or activities of CYPs [5]
A previous study showed that hepatic CYP-mediated
drug metabolism is suppressed during polymicrobial
sepsis, particularly in the late phase [6]
The complex Toll-like receptor (TLR) and
associ-ated downstream regulators of immune cells play a
crucial role in the innate system as a first line of
defense against pathogens [7] TLR2 and TLR4
expres-sion in multi-organs, including the liver, lung, heart
and spleen, was significantly upregulated in
experimen-tal models of sepsis and in patients with sepsis [8]
Ghose et al [9] reported that the expression of hepatic
drug-metabolizing enzymes was regulated by a
TLR4-dependent mechanism in a lipopolysaccharide
(LPS)-induced inflammation model In addition, the TLR2
ligand, lipoteichoic acid, altered the expression of
hepatic genes involved in drug metabolism and
trans-port [10] Kupffer cells (KCs), the resident hepatic
macrophages, mainly mediate inflammatory responses
in the liver by presenting TLRs on their surface TLR4
associates with CD14 on the surface of KCs, mediating
LPS-induced signal transduction, and activates KCs to
produce several proinflammatory cytokines [11] In vivo
observation by immunoelectronmicroscopy shows the
accumulation of TLR2 to the membrane of KCs
dur-ing endotoxemia [12] A recent study reported that
KCs can release high mobility group box1 (HMGB1),
a critical late mediator of lethal sepsis, triggering the
production of proinflammatory cytokines and liver
injury [13]
Therefore, the present study aimed to elucidate the
role of KCs in the regulation of CYP isoform activities
and gene expression profiles, partly by investigating
the inflammatory signaling pathway
Results
Hepatic CD163 mRNA expression
Pretreatment with gadolinium chloride (GdCl3) alone
significantly decreased the hepatic mRNA level of
CD163 compared to that of sham group Twenty-four
hours after cecal ligation and puncture (CLP), the
hepatic mRNA level of CD163 was similar with that
of the sham group, which markedly decreased to
approximately 7.0% of that of the CLP group (Fig 1)
Serum aminotransferase activities and lipid
peroxidation
The serum level of alanine aminotransferase (ALT) in
sham-operated rats was 22.6 ± 1.6 UÆL)1 at 24 h after
CLP The serum ALT level in rats who underwent CLP was 1.8-fold that of sham-operated rats at 24 h after CLP, which was significantly attenuated by GdCl3 Similar to the ALT level, the serum aspartate aminotransferase (AST) level increased significantly at
24 h after CLP and this increase was attenuated by GdCl3 The malondialdehyde (MDA) level in CLP rats was 1.8-fold that of sham-operated rats The increase
in the MDA level at 24 h after CLP was significantly prevented by GdCl3(Table 1)
Hepatic glutathione (GSH) The hepatic GSH concentration showed a significant decrease at 24 h after CLP, and this decrease was pre-vented by depletion of KCs by GdCl3 Although the
CD163
β-actin Sham GdCl3 CLP GdCl3 + CLP
Fig 1 Effect of GdCl 3 on the hepatic CD163 mRNA expression levels 24 h after CLP Rats were pretreated intravenously with 7.5 mgÆkg)1GdCl3or saline alone 48 and 24 h before CLP The val-ues are represented as the mean ± SEM for eight to ten rats per group **P < 0.01, significantly different from sham. ++P < 0.01, significantly different from CLP.
Table 1 Effect of GdCl 3 on serum aminotransferase activites and lipid peroxidation after CLP Each value is presented as the mean ± SEM for eight to ten rats per group.
Groups ALT (UÆL)1) AST (UÆL)1)
MDA (nmolÆmg)1protein) Sham 22.6 ± 1.6 68.1 ± 3.4 0.98 ± 0.05 GdCl3 20.0 ± 1.9 82.6 ± 4.1 1.19 ± 0.07 CLP 41.4 ± 1.7** 143.6 ± 8.9** 1.72 ± 0.10** GdCl3+ CLP 24.7 ± 2.1 ++ 119.9 ± 5.0 + 1.18 ± 0.09 ++
**P < 0.01, significantly different from sham + P < 0.05,
++ P < 0.01, significantly different from CLP.
Trang 3GSH disulfide (GSSG) concentration showed a marked
increase at 24 h after CLP, GdCl3 pretreatment did
not affect the GSSG concentration The GSH to
GSSG ratio showed a significant decline at 24 h after
CLP, which was attenuated by GdCl3 pretreatment
(Table 2)
Total hepatic CYP content and NADPH-CYP
reductase activity
The hepatic microsomal CYP content in the sham
group was 0.39 ± 0.03 nmolÆmg)1 protein At 24 h
after CLP, the hepatic microsomal CYP content
showed a significant decrease to 0.14 ± 0.01
nmolÆmg)1protein and this decrease was attenuated by
GdCl3 treatment Hepatic microsomal NADPH-CYP
reductase activity showed a significant decrease at 24 h
after CLP GdCl3 markedly attenuated this decrease
(Table 3)
Hepatic microsomal CYP isozyme activities
The results for the CYP isozyme activities are
summa-rized in Table 4 At 24 h after CLP, CYP1A1, 1A2
and 2E1 activities were reduced to levels approximately
46.2%, 45.8% and 34.3% of that observed in
micro-somes in sham-operated rats, respectively These
decreases were attenuated by GdCl3 pretreatment CYP2B1 activity remained unchanged across all experimental groups
CYP isozyme protein expression The amount of CYP1A1 and 1A2 protein expression in the microsome showed a significant decrease at 24 h after CLP GdCl3 pretreatment raised CYP1A1 and 1A2 protein expression levels without statistical signifi-cance No significant differences in CYP2B1 protein expression level were observed among any experimental groups The amount of 2E1 protein expression showed
a significant decrease at 24 h after CLP This decrease was prevented by GdCl3pretreatment (Fig 2)
CYP1A1⁄ 1A2 phosphorylation The phosphorylation of CYP1A1⁄ 1A2 significantly increased 24 h after CLP, which was attenuated by GdCl3pretreatment (Fig 3)
CYP isozyme mRNA expression
No differences were observed in CYP1A1 and 1A2 mRNA expression between the experimental groups The level of CYP2B1 mRNA expression showed a sig-nificant decrease at 24 h after CLP, and the decrease was prevented by GdCl3 Similar to CYP2B1, the level
of CYP2E1 mRNA expression showed a marked decline at 24 h after CLP, and the reduction was atten-uated by GdCl3(Fig 4)
Hepatic TLR2 and TLR4 protein expression The hepatic level of TLR2 and TLR4 protein expres-sion showed a marked increase at 24 h after CLP These increases were significantly attenuated by GdCl3 pretreatment (Fig 5)
Serum HMGB1 and hepatic inducible nitric oxide synthase (iNOS) protein expression
Serum levels of HMGB1 protein expression and hepa-tic iNOS protein expression showed a significant increase at 24 h after CLP These increases were mark-edly attenuated by pretreatment with GdCl3(Fig 6)
Serum tumor necrosis factor (TNF)-a and interleukin (IL)-6 levels
Compared to sham-operated rats, serum TNF-a and IL-6 levels showed a significant increase at 24 h
Table 2 Effect of gadolinium chloride on concentrations of GSH,
GSSG and GSH ⁄ GSSG ratio after CLP Each value is presented as
the mean ± SEM for eight to ten rats per group.
Groups
GSH
(nmolÆmg)1liver)
GSSG (nmolÆmg)1liver)
GSH ⁄ GSSG ratio Sham 4.02 ± 0.25 0.24 ± 0.02 17.61 ± 2.37
GdCl3 3.84 ± 0.14 0.24 ± 0.02 16.52 ± 1.86
CLP 3.01 ± 0.17** 0.33 ± 0.03* 9.32 ± 0.86**
GdCl 3 + CLP 3.66 ± 0.17+ 0.30 ± 0.01 12.41 ± 0.51+
*P < 0.05, **P < 0.01, significantly different from sham.+P < 0.05,
significantly different from CLP.
Table 3 Effect of gadolinium chloride on the total cytochrome
P450 content and NADPH-cytochrome P450 reductase activity after
CLP Each value is presented as the mean ± SEM for eight to ten
rats per group.
Groups
Cytochrome
P450 content
(nmolÆmg)1protein)
NADPH-cytochrome P450 reductase activity (nmolÆmg)1protein)
GdCl 3 + CLP 0.29 ± 0.03 + 68.9 ± 2.4 ++
**P < 0.01, significantly different from sham + P < 0.05,
++
P < 0.01, significantly different from CLP.
Trang 4after CLP (450.8 ± 22.6 pgÆmL)1 and 255.1 ± 40.8 pgÆmL)1, respectively) GdCl3 pretreatment atten-uated these increases (Fig 7)
Hepatic TNF-a and IL-6 mNRA expression
As shown in Fig 8, the hepatic level of TNF-a and IL-6 mRNA expression showed a significant increase
at 24 h after CLP, and this increase was attenuated by GdCl3
Discussion
Several studies have shown that interactions between KCs and endotoxin comprise the initiating event lead-ing to hepatotoxicity in liver injury, includlead-ing endotox-emia and ischendotox-emia⁄ reperfusion injury [14] In our studies, we employed GdCl3 to inactivate KCs based
on the findings of other investigators showing the destruction of KCs after the intravenous administra-tion of GdCl3 [15] Hardonk et al [15] demonstrated that large KCs were no longer present 24 h after GdCl3 treatment Splenic macrophages are less vulner-able to GdCl3because only some of the red pulp mac-rophages transiently disappear The white pulp
Sham GdCl3CLP GdCl3 + CLP
CYP1A1 β-actin
β-actin β-actin
β-actin
CYP2B1
CYP2E1 CYP1A2
Fig 2 Effects of KCs on hepatic CYP1A1, 1A2, 2B1 and 2E1 protein expression levels 24 h after CLP Rats were pretreated intravenously with 7.5 mgÆkg)1GdCl3or saline alone 48 and 24 h before CLP The values are represented as the mean ± SEM for eight to ten rats per group *P < 0.05, **P < 0.01, significantly different from sham +P < 0.05, significantly different from CLP.
Phospho-CYP1A1 CYP1A2 CYP1A1
IP : CYP1A1/1A2
Blot : phosphoserine/
threonine
Phospho-CYP1A2
GdCl3 – + – +
CLP
Fig 3 Effects of KCs on the phosphorylation of CYP1A1 ⁄ 1A2 24 h
after CLP Rats were pretreated intravenously with 7.5 mgÆkg)1
GdCl3or saline alone 48 and 24 h before CLP The livers samples
were subjected to immunoprecipitation (IP) using anti-CYP1A1 ⁄ 1A2
serum Immunoprecipitates were subjected to immunoblot analysis
using anti-phosphoserine ⁄ threonine serum The values are
repre-sented as the mean ± SEM for eight to ten rats per group.
*P < 0.05, **P < 0.01, significantly different from sham.+P < 0.05,
significantly different from CLP.
Table 4 Effect of gadolinium chloride on the hepatic microsomal cytochrome P450 isozyme activities after CLP Each value is represented
as the mean ± SEM for eight to ten rats per group.
Ethoxyresorufin O-deethylase (pmol resorufinÆmg)1protein) 69.9 ± 2.4 66.4 ± 1.7 32.3 ± 2.3** 59.2 ± 4.1 ++
Methoxyresorufin O-demethylase (pmol resorufinÆmg)1protein) 34.5 ± 0.7 25.8 ± 0.4 15.8 ± 1.4** 32.3 ± 1.9 ++
Penthoxyresorufin O-dealkylase (pmol resorufinÆmg)1protein) 18.6 ± 1.8 19.3 ± 2.5 16.9 ± 1.2 17.2 ± 1.0 Aniline p-hydroxylase (nmol PAPÆmg)1protein) 0.35 ± 0.01 0.36 ± 0.02 0.12 ± 0.01** 0.33 ± 0.02 ++
**P < 0.01, significantly different from sham ++ P < 0.01, significantly different from CLP.
Trang 5macrophages are not affected GdCl3pretreatment has
been demonstrated to have an effect on the prevention
of LPS-evoked release of reactive oxygen species
(ROS) and proinflammatory cytokines from KCs [16]
Our recent studies have shown that GdCl3 attenuated
the imbalanced vascular stress gene expression induced
by sepsis [17] In the present study, the depletion of
KCs was confirmed by dramatically reduced expression
of the KC marker gene CD163
In humans and animals, infections or inflammatory stimuli cause changes in the activities and expression levels of various forms of CYP in the liver In most cases, CYPs and their activities are suppressed; how-ever, some are unaffected or induced under these con-ditions [18] Our previous study reported on abnormalities in microsomal drug-metabolizing func-tion during the late phase of sepsis [6] However, the underlying mechanisms involved in hepatic dysfunction during sepsis remain elusive
Among various CYP isoforms, CYP1A1, 1A2, 2B1 and 2E1 are both present in hepatic microsome of human and normal rats The function and regulation
of these isozymes are highly conserved among mam-malian species [19]
CYP1A1 is not expressed in normal adult tissues but can be induced several fold by polycyclic or halo-genated hydrocarbons [20] CYP1A2, which is consti-tutively expressed in the liver, is primarily involved in the oxidative metabolism of xenobiotics and is capable
of the metabolic activation of numerous procarcino-gens, including aflatoxin B1 [21] In the present study, CYP1A1 and 1A2 activities were significantly decreased, with a concomitant decrease in their protein levels during the late phase of sepsis However, CYP1A1 and 1A2 mRNA expression was not altered Depletion of KCs restored CYP1A1 and 1A2 activities, whereas protein levels remained decreased There is evidence showing that oxidative stress contrib-utes to the inhibition of CYP activity observed in the absence of changes in protein expression in rabbit he-patocytes [22] ROS indirectly reduced the activity of selected isoforms of CYP by inducing phosphorylation
of the isoforms [23] Activated KCs cause oxidative stress on the surrounding tissue, releasing large amounts of ROS during sepsis [24] Interestingly, phos-phorylation of CYP1A1⁄ 1A2 occurred at 24 h after
Sham GdCl
3 CLP GdCl
3 + CLP
CYP1A1 β-actin
β-actin β-actin
β-actin
CYP2B1
CYP2E1 CYP1A2
Fig 4 Effects of KCs on hepatic CYP1A1, 1A2, 2B1 and 2E1 mRNA expression levels 24 h after CLP Rats were pretreated intravenously with 7.5 mgÆkg)1GdCl 3 or saline alone 48 and 24 h before CLP The values are represented as the mean ± SEM for eight to ten rats per group **P < 0.01, significantly different from sham + P < 0.05, ++ P < 0.01, significantly different from CLP.
Sham GdCl3 CLP GdCl3 + CLP
β-actin TLR4 TLR2
Fig 5 Effects of KCs on hepatic TLR2 and TLR4 protein
expres-sion levels 24 h after CLP Rats were pretreated intravenously with
7.5 mgÆkg)1GdCl 3 or saline alone 48 and 24 h before CLP The
val-ues are represented as the mean ± SEM for eight to ten rats per
group **P < 0.01, significantly different from sham ++ P < 0.01,
significantly different from CLP.
Trang 6CLP, and GdCl3 pretreatment attenuated this
phos-phorylation GdCl3 pretreatment also prevented lipid
peroxidation and a decrease in hepatic GSH⁄ GSSG
ratio during sepsis Thus, the results of the present
study suggest that ROS produced by KCs mediate the
sepsis-induced decrease in CYP1A1 and 1A2 activities
partly through a post-translational phosphorylation
Upregulation of CYP2E1 has been reported in
vari-able experimental pathological conditions, including
carbon tetachloride-induced hepatic fibrosis,
alcohol-induced liver diseases and hepatic ischemia⁄ reperfusion
injury, which were implicated in the activation of KCs
[25] The expression and activity of CYP2E1 were
downregulated in a rat hepatoma cell line after the
administration of proinflammatory cytokines, leading
to a loss of catalytic activity This downregulation was
at the level of transcription [26] In the present study,
the activity and protein and mRNA levels of CYP2E1
showed a significant decrease at 24 h after CLP These
decreases were attenuated by GdCl3 pretreatment Our results suggest that KCs are involved in the sepsis-induced downregulation of CYP2E1 at the transcrip-tional level
Depression of CYP-dependent hepatic drug metabo-lism in inflammatory reactions and infectious diseases has been attributed to the inflammatory events TLRs play a critical role in the immune system by providing
an early recognition of pathogen invasion and a facili-tation of the body’s subsequent immune responses [27] The stimulation of these receptors activates inflamma-tory responses characterized by the release of a wide range of proinflammatory cytokines, including IL-6
Sham GdCl3CLP GdCl3 + CLP
HMGB1
iNOS
β-actin
Fig 6 Effects of KCs on serum HMGB1 and hepatic iNOS protein expression levels 24 h after CLP Rats were pretreated intravenously with 7.5 mgÆkg)1GdCl 3 or saline alone 48 and 24 h before CLP The values are represented as the mean ± SEM for eight to ten rats per group **P < 0.01, significantly different from sham.++P < 0.01, significantly different from CLP.
Fig 7 Effects of KCs on serum TNF-a and IL-6 levels 24 h after
CLP Rats were pretreated intravenously with 7.5 mgÆkg)1GdCl 3 or
saline alone 48 and 24 h before CLP The values are represented
as the mean ± SEM for eight to ten rats per group **P < 0.01,
significantly different from sham.++P < 0.01, significantly different
from CLP.
Sham GdCl3 CLP GdCl3 + CLP
IL-6
TNF-α β-actin β-actin
Fig 8 Effects of KCs on hepatic TNF-a and IL-6 mRNA expression levels 24 h after CLP Rats were pretreated intravenously with 7.5 mgÆkg)1GdCl 3 or saline alone 48 and 24 h before CLP The val-ues are represented as the mean ± SEM for eight to ten rats per group *P < 0.05, significantly different from sham + P < 0.05,
++ P < 0.01, significantly different from CLP.
Trang 7and TNF-a KCs strongly express all TLRs, except
TLR5 [28] TLR4 and TLR2 in hepatic and splenic
macrophages were significantly upregulated in mice
with experimental peritonitis induced by CLP [29] It
has been reported that the regulation of hepatic CYP
gene expression elicited by chemically-induced
inflam-matory bowel disease was entirely dependent on TLR4
[30] However, hepatic inflammation induced by
Cit-robacter rodentium infection was mainly
TLR4-inde-pendent because hepatic CYPs mRNA expression was
similarly downregulated and cytokine mRNAs were
similarly induced in both wild-type and TLR4-mutant
mice [31] Recently, the TLR2 ligand, lipoteichoic acid,
altered the expression of hepatic genes associated with
drug metabolism and transport [10] The results of the
present study show that inactivation of KCs by GdCl3
pretreatment attenuates any increases in hepatic TLR4
and TLR2 protein expression levels at 24 h after CLP
KCs mediated the specific downregulation of CYP2B1
via the release of TNF-a in a KCs⁄ hepatocyte
cocul-ture system [32] Moreover, proinflammatory cytokines
released from KC, although not the direct effects of
LPS, play an important role in downregulating hepatic
CYP1A2 expression in sepsis [33] In the present study,
increased serum levels of TNF-a and IL-6 and the
pro-tein expression of iNOS were markedly suppressed by
GdCl3 treatment This result suggests that septic insult
stimulates both TLR4 and TLR2 expression on KCs,
resulting in the release of proinflammatory mediators
and the downregulation of CYP enzymes in
hepato-cytes
HMGB1, a DNA-binding nuclear protein, is
released actively by monocytes⁄ macrophages and
pas-sively by cell death, and plays a critical role in the
mediation of immune responses in several
inflamma-tory disorders [34] The delayed secretion of HMGB1
was observed both in vitro and in vivo, and these
delayed secretions were crucial to the increased
mortal-ity in septic patients and experimental animals [35]
The findings reported in recent in vitro studies suggest
that some of the effects of HMGB1 result from its
interaction with TLR2 or TLR4, leading to the
media-tion of various cellular responses and the release of
proinflammatory cytokines [36] There is evidence that
LPS stimulation increases HMGB1 mRNA expression
in both cultured primary hepatocytes and KCs
How-ever, only KCs release HMGB1 protein into the
cul-ture media [13] In the present study, the inactivation
of KCs by GdCl3 treatment attenuated the increase in
serum HMGB1 and improved the survival rate at 24 h
after CLP (data not shown)
The full complexity of the regulatory mechanisms
underlying the alteration of CYP enzymes remains to
be elucidated; however, our results show that KCs dif-ferentially regulate the expression of each form of CYP among the various CYP subfamilies These dif-ferential regulations were attributed to the ability of KCs to develop exaggerated inflammatory responses through TLR overexpression, the release of HMGB1 and the upregulation of proinflammatory cytokines
Materials and methods
Animals
Male Sprague-Dawley rats, weighing 280–320 g, were sup-plied by the Jeil Animal Breeding Company (Deajeon, Korea) The animals were housed in cages located in
photocycle, and received water and food ad libitum for at least 1 week All animal procedures were approved by the Sungkyunkwan University Animal Care Committee and were performed in accordance with the guidelines of the National Institutes of Health
Treatment with GdCl3and experimental groups
injected via the tail vein at 48 and 24 h before the perfor-mance of CLP or sham operation, based on the findings of other investigators who showed the destruction of KCs
[15] In vehicle-treated rats, physiological saline solution was injected with the same volume and in the same manner
CLP
Polymicrobial sepsis was induced by CLP in accordance with the method previously described by Chaudry et al [37] After anesthetization with ether, a 2 cm ventral mid-line incision was performed The cecum was then carefully exposed, ligated just distal to the ileocecal valve to avoid
18-gauge needle The punctured cecum was squeezed to expel a small amount of fecal material and returned to the abdominal cavity, and the abdominal incision was closed in two layers Sham-operated animals underwent the same surgical procedure, except that the cecum was neither ligated, nor punctured All animals received normal saline
after surgery (i.e fluid resuscitation) At 24 h (i.e late phase of sepsis) after CLP, blood was obtained from the
Trang 8abdominal aorta The left and median lobes of the liver
assayed
Isolation of hepatic microsomal fraction
The excised liver was minced and then homogenized in four
volumes of ice-cold 1.15% KCl for 1 g of liver, and
centri-fuged at 9000 g for 60 min The supernatant was collected
and centrifuged at 105 000 g for 60 min, and the
precipi-tates (microsomal fractions) were resuspended with four
volumes of 0.1 m phosphate buffer at pH 7.4, for 1 g of
Analytical procedures
Serum ALT and AST activities were determined by
stan-dard spectrophotometric procedures using a diagnostic kit
(Sigma Chemical Co., St Louis, MO, USA) Lipid peroxide
was assayed by the method of Buege and Aust [38], and
1,1,3,3-tetraethoxypropane (MDA tetraethyl acetal) was
used as the standard Total GSH was determined in liver
homogenates after precipitation with 1% picric acid, using
yeast GSH reductase, 5,5¢-dithio-bis(2-nitrobenzoic acid)
and NADPH at 340 nm GSSG was determined by the
same method in the presence of 2-vinylpyridine and reduced
GSH was calculated from the difference between total
glu-tathione and GSSG [39] CYP content was calculated using
the molar extinction coefficient for the absorbance
differ-ence between 450 and 480 nm, as measured with a
differen-tial spectrophotometer [40] The activity of NADPH-CYP
reductase was indirectly determined by its
NADPH-cyto-chome c reductase activity [41] The catalytic activity of
CYP1A1, 1A2 and 2B1 in liver microsomal fractions was
measured as 7-ethoxyresourfin O-deethylase,
methoxyre-sourfin O-demethylase and pentoxyremethoxyre-sourfin O-dealkylase
described by Burke et al [42] Microsomal CYP2E1 activity
was determined by measurement of 4-hydroxylation of
aniline to p-aminophenol (PAP) [43]
ELISA
Serum concentrations of TNF-a and IL-6 were determined
using ELISA kits in accordance with the manufacturer’s
instructions (BD Biosciences, San Diego, CA, USA)
Western blot immunoassay
Protein samples (10–20 lg per well) from liver tissue and
transferred to nitrocellulose membranes using a semi-dry
transfer process Bands were immunologically detected using
polyclonal antibodies against rat CYP1A1, 1A2, 2B1 and
2E1 (Gentest, Woburn, MA, USA); iNOS (Transduction Laboratories, San Jose, CA, USA); TLR4 and TLR2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and HMGB1 (Abcam, Cambridge, MA, USA) Binding of all of the anti-bodies was detected using an ECL detection system (iNtRON Biotechnology, Seoul, Korea) in accordance with the manu-facturer’s instructions The intensity of the immunoreactive bands was determined using densitometric analysis software (image gauge, version 3.12; Fujifilm, Tokyo, Japan)
Immunoprecipitation
Liver tissues were homogenized with ice-cold radioimmuno-precipitation assay (RIPA) buffer (150 mm NaCl, 50 mm Tris, 1% Triton X-100, 1% deoxycholic acid, 0.1% SDS,
pH 7.4) containing protease and phosphatase inhibitor cocktail set (Calbiochem, La Jolla, CA, USA) Aliquots of
Biotechnology) for 30 min and then incubated overnight at
was then added, and the samples were incubated for a
com-plexes were washed three times in a RIPA buffer for 30 s After the third wash, the immunoprecipitants were resus-pended in Laemmli sample buffer The samples were then analyzed by western blotting using the polyclonal
anti-body Binding of all of the antibodies was detected using an ECL detection system (iNtRON Biotechnology) in accor-dance with the manufacturer’s instructions The intensity of the immunoreactive bands was determined using densito-metric analysis software (image gauge, version 3.12)
Total RNA extraction and RT-PCR
Isolation of total RNA was carried out in accordance with the method previously described by Chomczynski and Sacchi [44] Reverse transcription of total RNA was performed for
(Tech-Line; Invitrogen Carlsbad, CA, USA) The PCR reaction was performed with a diluted cDNA sample and amplified in each 20 lL reaction volume The final reaction concentrations were: primers (Table 5), 10 pmol; dNTP mix,
250 lL; 10· PCR buffer; and Ex Taq DNA polymerase, 0.5 U per reaction All PCR reactions had an initial
for 5 min using the GeneAmp 2700 thermocycler (Applied Biosystems, Foster City, CA, USA) PCR amplification
Trang 930 s, 60C for 30 s, 72 C for 60 s, 23 cycles for CYP2E1;
and 25 cycles for TNF-a, IL-6 and b-actin, respectively
After RT-PCR, 10 lL samples of the amplified products
were resolved by electrophoresis in 1.5% agarose gel, and
stained with ethidium bromide The intensity of each PCR
product was semi-quantitatively evaluated using a digital
camera (DC120; Eastman Kodak, New Haven, CT, USA)
and densitometric scanning analysis software (1d main;
Advanced American Biotechnology, Fullerton, CA, USA)
Statistical analysis
All results are presented as the mean ± SEM Overall
sig-nificance was tested by one-way analysis of variance
differ-ences between groups at specific time points, with the
appropriate Bonferroni correction being made for multiple comparisons
Acknowledgements
This research was supported by Basic Science Research Program through the National Research Foundation
of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0028646)
References
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Gene
(accession
number)
Primer sequences (5¢- to 3¢)
Product length (bp) CD163
(XM_053094.2)
Sense:
AGCTGGGCTGTGCAGACAACG Antisense:
TGAATGACCCCCGAGGATTTCAGC
736
CYP1A1
(X00469)
Sense:
CTGGTTCTGGATACCCAGCTG Antisense:
CCTAGGGTTGGTTACCAGG
331
CYP1A2
(X01031)
Sense:
CAGTCACAACAGCCATCTTC Antisense:
CCACTGCTTCTCATCATGGT
302
CYP2B1
(XM_342078)
Sense:
TTGTTTGGTGCTGGGACAGAG Antisense:
GGCTAGGCCCTCTCCTGCACA
443
CYP2E1
(M20131)
Sense:
AAACTTCATGAAGAAATTGAC Antisense:
TCTCCAACACACACACGCTTTCC
311
TNF-a
(X66539)
Sense:
GTAGCCCACGTCGTAGCAAA Antisense:
CCCTTCTCCAGCTGGAAGAC
346
IL-6
(NM_012589)
Sense:
GAAAGTCAACTCCATCTGCC Antisense:
CATAGCACACTAGGTTTGCC
678
b-actin
(BC063166)
Sense:
TTGTAACCAACTGGGACGATATGG Antisense:
GATCTTGATCTTCATGGTGCTAG
764
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