Anti-fibrotic effects of (S)-armepavine in HSC-T6
cells
T.-C. Weng 1,Y.-T. Huang 1*
1
Institute of Traditional Medicine, School of Medicine, National
Yang-Ming University, Taipei,
*Corresponding Author: Yi-Tsau Huang, Institute of Traditional
Medicine, School of Medicine, National Yang-Ming University,
No. 155, Li-Nong Street, Sec. 2, Taipei 112, Taiwan. Tel.
+886-2-28267179,
Fax
+886-2-28225044,
E-Mail
huangyt@ym.edu.
32
Background/Aims: Activation of hepatic stellate cells (HSCs) plays a
crucial role in liver fibrogenesis. (S)-armepavine (C19H23O3N), an active
compound from Nelumbo nucifera, has been shown to exert
immunosuppressive effects on T lymphoctytes and on lupus nephritic
mice. The aim of this study was to investigate whether armepavine could
inhibit the activation of HSCs.
Methods: A cell line of rat HSCs (HSC-T6) was stimulated with either
tumor necrosis factor- (TNF-, 10 ng/ml) or lipopolysaccharide (LPS, 1
g/ml). The inhibitory effects of armepavine on both inflammation- and
fibrosis-related markers were assessed.
Hypothesis of this study: Whether (S)-armepavine could exert
anti-hepatic fibrogenic effects via NFB and AP-1 and MAPK signaling
pathways both in vitro and in vivo or not.
Results: Both TNF- and LPS stimulated NFκB and AP-1 activities of
HSCs
in
the
reporter
gene
assays.
Armepavine
(1-10
M)
concentration-dependently inhibited both NFκB and AP-1 activities.
Moreover, armepavine inhibited the mitogen-activated protein kinases
(MAPK) and IB phosphorylation, nuclear translocation of NFκB and
mRNA expression of inducible nitric oxide synthase (iNOS) gene in
activated HSCs. Molecular fibrosis markers including protein levels of
-smooth muscle actin (-SMA) and collagen, and mRNA levels of
pro-collagen I and tissue inhibitor of metalloproteinase-1 (TIMP-1) genes
were also decreased by armepavine in TNF--stimulated HSCs.
Furthermore, armepavine inhibited the phosphorylation of p38, ERK 1/2
and JNK 1/2 in activated HSCs.
Conclusion:
Our
results
showed
that
(S)-armepavine
inhibited
33
TNF--induced activation and fibrogenesis of HSC-T6 cells, suggesting
the potential of armepavine as a therapeutic agent against hepatic fibrosis.
34
Introduction
Prolonged liver injury and inflammation result in hepatocyte damage,
which triggers activation of HSCs and recruitment of inflammatory cells
into the liver. Hepatic fibrosis is an outcome of many chronic liver
diseases, such as viral-infected (hepatitis B and C virus) and autoimmune
hepatitis, and of alcohol consumption and biliary obstruction and then
lead to liver cihhrosis and hepato cellular carcinoma finally. (Bataller and
Brenner, 2005; Kisseleva and Brenner, 2006)
TNF-α and LPS-induced hepatic fibrogenesis and inflammation response
in HSCs are triggered by NFκB and AP-1 signaling cascades. (Schwabe
and Brenner, 2006; Park et al., 2003; Schwabe et al., 2006) IKKα
superfamily is essential for rapid NFκB activation by proinflammatory
signaling cascades, such as those triggered by TNF-α or LPS. The
IKKα-dependent pathway leading to rapid degradation of IκBα is
commonly referred to as the NFκB signaling pathway. (Hacker and Karin,
2006) LPS-induced synthesis of NO, TNF-α and IL-6 in HSCs is
mediated by p38 and NFκB, with involvement of H2O2 in TNF-α
production. Recent evidence indicates LPS also up-regulates cell surface
expression of ICAM-1 and VCAM-1. (Park et al., 2003)
In response to injury proinflammatory cytokines are promptly increased
in wound areas, which induce matrix metalloproteinase (MMP)
expression by hepatic cells including HSC. The MMP secreted by HSCs
in the space of Disse degrade the normal extracellular matrix (ECM) such
35
as α-SMA, pro-collagen I and TIMP-1. TNF-α and LPS-induced ECM
degradation leads to activation of HSC. Consequently, a population of the
HSC undergo apoptosis while others trans-differentiate into
myofibroblasts that produce fibrillar ECM. (Han, 2006)
Nelumbo nucifera is a useful edible and medicinal plant for the treatment
of diarrhea, tissue inflammation, and hemostasis. (Liu et al., 2006)
(S)-armepavine, an active compound from Nelumbo nucifera, inhibited
cell proliferation and cytokines production, induces apoptosis in
CCRF-CEM leukemia cell line and as ligands for neuronal nicotinic
acetylcholine receptors. (Exley et al., 2005; Jow et al., 2004).
Hypothesis of this study:
Our recent result indicated that TNF- related signaling pathway in HSCs
has been proved to be a therapeutic target. (Chong et al., 2006; Hsu et al.,
2006) The aim of this study is that we try to investigate whether
(S)-armepavine inhibited TNF- and LPS-induced activation in HSCs in
inflammation signaling cascades including ERK1/2, p38 and JNK 1/2
phosphorylation and modulation of expressions of profibrogenic gene,
such as α-SMA, pro-collagen I and TIMP-1.
36
Materials and Methods
HSC-T6 Cell Line
The HSC-T6 cell line, a generous gift of Prof. S.L. Friedman, is
an immortalized rat HSCs which are transfected by the large T-antigen of
SV40 vector containing a Rous sarcoma virus promoter. (Vogel et al.,
2000) HSC-T6 cells were maintained in Waymouth’s medium (containing
10% FBS, pH 7.0) at 37 C in 5% CO2/95% air. 90% confluent
monolayer of HSCs were passaged by trypinization and HSCs were
plated in 75T culture flask at a number of 1  106 cells per flask in
Waymouth’s medium containing 10% FBS and incubated under 5% CO 2
in air at 37 C.
Evaluation of cytotoxicity of (S)-armepavine in HSCs
The assays of reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) were used to evaluate the potential
of cytotoxicity of (S)-armepavine. Cells were incubated in 24-well plates
containing Waymouth’s MB752/1 medium (FBS-free) with or without
(S)-armepavine at different concentrations for twenty four hrs at 37 ºC.
HSCs were incubated with minimum essential medium containing 0.1
mg/ml MTT in the last hour. After discarding medium, the formazan
particle was dissolved with DMSO. A540 absorption intensity was
measured by using enzyme-linked immunosorbent assay reader. (Hansen
et al. 1989) The optical density of the formazan formed in the control
cells was taken as 100% viability, and relative cell viability was
determined by the amount of MTT converted to the insoluble formazan
salt.
37
Luciferase assays in transiently transfected HSCs.
105 cells/well were seeded on 24-well plates the day before
transfection. Plasmid NFκB-Luc and AP-1-Luc (1μg/well) (Strategene,
La Jolla, CA) and pRL-SV40 (0.2 μg/well) (Promega, Madison. USA)
were transfected into cells by Fugene 6 (Roche, Indianapolis, IN, USA).
The pNFκB-Luc and the pAP-1-Luc consist of NFκB and AP-1 binding
region. Plasmid pRL-SV40 served as an internal control to normalize the
transfection efficiency. After treatment with TNF-α, LPS or drugs for
twenty four hours in 5% CO2 incubator at 37°C, cells were harvested and
lysed in 100μl of lysis reagent. 20μl of cell lysate was then mixed with
100μl of luciferin before luminescence detection. The intensity of
luciferase activity was measured with AutoLumat LB953 (Berthold
technologies, Bad Wildbad, Germany). The luciferase assay kits were
purchase from Promega (Madison. USA). (Chong et al., 2006)
Determiantion of MAPK, IκBα phosphorylation, NFB translocation
and -SMA
In brief, Cytoplasmic and nuclear extracts of HSCs were prepared
by washing ice-cold PBS twice. Then add lysis buffer A(10mM HEPES,
10mM KCl, 0.1mM EDTA, 1mM DTT, 0.5mM PMSF in distill water)
100μl in each dish with incubation on ice for 10 min. Collect cell lysates
in 1.5ml tubes and were centrifuged 1,2000 rpm,4℃,20min. Obtain
supernant (protein of cytoplasm) and lysate (protein of nucleus) add 50μl
lysis buffer B(20mM HEPES, 0.4M NaCl, 25% glycerol, 1mM EDTA,
38
1mM DTT, 0.5mM PMSF in distill water) in lysate of nucleus protein.
Then votex cell suspension for 10 seconds and were allowed to gently
agitate for 30 min at 4°C and then centrifuged at 15,000 g for 10 min at
4°C. Transfer the supernatant to a fresh 1.5ml tube, this supernatant
fraction is the nuclear extract for NFB translocation assay. 50g
proteins cytoplasmic fraction and nuclear fraction were separated on a
10% SDS-PAGE and transferred onto Immobilon-PVDF (Millipore,
Bedford, MA, USA) in a transfer buffer (6.2 mM boric acid, pH 8.0).
Blots were incubated initially with blocking buffer (5% BSA) for 1 hour
at room temperature, and then with specific primary antibodies against
mouse pIκBα, mouse -SMA, mouse -tubulin, mouse p65 and mouse
PCNA (Santa Cruz Biotechnology, Santa Cruz, California, CA, USA);
and determine levels of MAPK phosphorylation by specific primary
antibodies against rabbit p38, mouse ERK1/2 and rabbit JNK 1/2 (Cell
Signaling Inc, USA). Primary antibodies had been diluted (1:10000) with
Tris-buffered saline-Tween 20 (TBS-T) containing 1% non-fat milk. After
primary antibody incubation, the blots were washed with TBS-T for 1
hour and incubated with specific second antibody conjugated with
horseradish peroxidase (Becton Dickinson, Franklin Lakes, NJ, USA) for
1 hour at room temperature. After the washing of the secondary
antibodies (1:2000) with TBS-T, immunodection was performed, using an
enhanced chemiluminscence kit for western blot detection (Amersham
Pharmacia Biotech, Buckinghamshire, U.K.). Film exposure ranged from
a few seconds to 5 min.
Quantification of Collagen Deposition by Cultured HSCs
39
HSCs (in serum-free medium) were co-treated with TNF-α 10
ng/ml and (S)-armepavine for twenty four hours. Cells were washed and
collagen deposited in the wells was assayed using the Sircol collagen
assay kit (Biocolor, Belfast, Nothern Ireland) according to the
manufacturer’s instructions. (Chong et al., 2006) The sircol dye-collagen
complex disolved in 0.5% sodium hydroxide after wash twice with
ethanol. Collagen was quantitated by spectrophotometry at 540nm and
results were expressed as percentage of the untreated controls.
Hepato-fibrotic Animals (Thioacetamide (TAA) and BDL rats)
Hepatic fibrosis was induced by TAA administration in male
Sprague-Dawley rats (250~300 g) and we have recently documented
changes in molecular and cell biological parameters related to fibrosis in
these rats (Hsu et al., 2004). TAA (300 mg/kg) was injected
intraperitoneally for 3 consecutive days per week for 5 weeks, according
to the modified method of Gnainsky et al. (Gnainsky et al., 2004) TAA
was purchased from Sigma Chemical Co. (St. Louis, MO, USA) and
diluted 100 folds in saline with a final concentration of 10 mg/ml before
injection. Control rats were injected with saline alone. Rats were
maintained on a standard rat pellet diet and tap water ad libitum. Animal
studies were approved by the Institutional Animal Care and Use
Committee of the University and conducted humanely, in accordance
with the Guide for the Care and Use of Laboratory Animals [National
Academic Press, USA, 1996]. There were five groups of rats: (a) control
rats receiving 0.7% CMC, (b) TAA rats receiving 0.7% CMC, (c) TAA
40
rats receiving N-acethylcysteine (NAC) (50 mg/kg, mixed with 0.7%
CMC), (d) TAA rats receiving (S)-armepavine (3 mg/kg), and (e) TAA
rats receiving (S)-armepavine (10 mg/kg), each given by gavage twice
daily for 2 weeks starting after 3 week pre-TAA administration. Fiver
weeks after TAA or saline injection, the rats were examined for the
parameters listed below. Common bile duct ligation was performed in
male Sprague-Dawley rats (250~300 g) to induce hepatic fibrosis as
previous describe. (Huang et al., 2003) A double ligation of the bile duct
was performed in rats under anesthesia by a proximal ligature around the
bile duct in the hilus of the liver and by a distal ligature close to its entry
into the duodenum. A cut was then made between ligatures. On
sham-operated rats. The bile duct was mobilized but not ligated. Rats
were maintained on a standard rat pellet diet and tap water ad libitum.
Animal studies were approved by the Animal Experiment Committee of
the Unerversity and conducted humanly, in accordance with the Guide for
the Care and Use of Laboratory Animals (National Academic Press, USA,
1996.) Two weeks after bile duct ligation or sham operation, the rats were
examined for the parameters listed below. On the day of measurement,
venous blood was withdrawn from each rat under anesthesia, and
thereafter the rat was sacrificed by KCl injection to remove the liver for
homogenization and biochemical analysis.
Biochemical Analysis of Plasma
6 ml Blood samples of each rat were collected and immediately
centrifuged at 1300  g at 4 C, and plasma were kept at -80 C for liver
and renal function tests. Alanine transaminase (ALT), aspartate
41
transaminase (AST), and creatinine levels were measured using a
colorimetric analyzer (Dri-Chem 3000, Fuji Photo Film Co, Tokyo,
Japan), according to protocol of manufacturer. (Hsu et al., 2007)
Histological Examination
, The liver fragments were taken from the right lobe of each rat for
morphometric studies. Liver specimens were preserved in 4% buffered
paraformaldehyde and dehydrated in a graded alcohol series. Following
xylene treatment, the specimens were embedded in paraffin blocks, cut
into 5-μm thick sectons and placed on glass slides. The sections were then
stained with hematoxylin-eosin or Sirius red for collagen disturibution.
(Lotersztajn et al., 2005) A numerical scoring system for histologically
assessing the extent of fibrosis was adapted from the formula of Scheuer
with minor modification. (Scheuer et al., 1991) Briefly, fibrosis was
graded as: 0: no fibrosis; grade 1: enlarged, fibrous portal tracts; grade 2:
periportal or portal-portal septa, but intact architecture; grade 3: fibrosis
with architectural distortion; grade 4: probable or definite cirrhosis.
Fibrosis scores were given after the pathologist had examined throughout
three different areas in the tissue slide for each rat.
Collagen Determination
A portion of liver tissue was homogenized in acetic acid (0.5 M)
at 4 C using an ULTRA TURRAX homogenizer (Ika Labotechnik,
Staufen, Germany). The conversion of cross-linked collagen into soluble
gelatin was heated at 80 C for 60 min after acid extraction. The gelatin
contents of the acid extracts were assayed using the Sircol collagen assay
42
kit (Biocolor, Belfast, Nothern Ireland) according to the manufacturer’s
instructions. (Huang et al., 2003)
Analysis of transcripts of -SMA, TGF-β1, procollagen type I, ICAM-1
and iNOS genes
Total RNA was isolated and collected by partition with Trizol
reagent and chloroform (Sigma. For cDNA synthesis, 1 µg of total RNA
was reverse-transcribed in a 20 µl of reaction cocktail containing 10µM
dNTP mix, 500µg oligo(dT)12-18, 0.2µM DTT, 40 units of RNase
inhibitor, 200 units of M-MLV reverse transcriptase, and 5× buffer (1.5
mM MgCl2) (Invitrogen, Califonia, USA). The reaction cocktail was
incubated at 37 °C for 50 min and then denatured at 70°C for 15 min. For
quantitative real time PCR, specific primers and probe for -SMA,
procollagen type I, ICAM-1, iNOS and GAPDH were all purchased from
PE
Applied
Biosystems.
The
gene
glyceraldehyde-3-phosphate
dehydrogenase (G3PDH) was used as endogenous control to standardize
the amount of RNA in each reaction (PE Applied Biosystems).
Quantitative Realtime PCR was performed on the cDNA samples using
an ABI PRI(S)-armepavine® 7900HT Sequence Detection System (PE
Applied Biosystems). The Taqman® PCR Core reagent kit (PE Applied
Biosystems) was used according to the manufacturer's protocol. For each
sample tested, PCR reaction was carried out in a 50-µl volume containing
1 µl of cDNA reaction (equivalent to 50 ng of template RNA) and 2.5
units of AmpliTaq Gold. Oligonucleotide primers and fluorogenic probe
were added to a final concentration of 100 nM each. The amplification
step consisted of 60 cycles of 94°C for 45s, 58°C for 45s, and 65°C for 1
43
min.
Data analysis
Data are expressed as the mean  SEM. One-way analysis of
variance (ANOVA) was used for comparison of molecular parameters.
Statistical significance was accepted at p< 0.05.
44
Results
In Vitro Effects of (S)-armepavine on HSC-T6 Cells
Effect of (S)-armepavine on TNF-α-induced collagen deposition in HSCs
10uM of (S)-armepavine significantly inhibit collagen deposition
as HSCs co-treat with TNF-α (10ng/ml) for twenty-four hours and show
concentration-depend manners. TNF-α (10ng/ml)-stimulated collagen
deposition was 143 ± 9% of controls, and this ratio was significantly
reduced to 66 ± 23% by co-administration of (S)-armepavine (10 μM)
[figure 2].
The expressions of α-SMA and α-tubulin in HSCs
10uM of (S)-armepavine significantly inhibit -SMA expression as
HSCs co-treat with TNF- (10ng/ml) and LPS (1μg/ml) for twenty-four
hours and show concentration-dependent manners. TNF-α (10ng/ml) and
LPS (1μg/ml) stimulated α-SMA secretion and collagen deposition in
HSC-T6 cells [figure 3]. (S)-armepavine (1-10M) concentrationdependently attenuated TNF-and LPS (1μg/ml)-stimulated α-SMA
protein expression ratio, with higher concentrations of (S)-armepavine
(10 M) achieving significant reduction.
The gene expression level changes of iNOS, Procollagen type I , TIMP-1
andα-SMA in HSCs.
The gene expression levels of iNOS, Procollagen type I, TIMP-1
and α-SMA as HSCs co-treat with TNF-α (10ng/ml) for twenty-four hours
are significantly down-regulated by 10uM of (S)-armepavine, and show
concentration-depend manners. [Figure 8 and 9]
Effect on TNF-α-induced AP-1 and NFB luciferase reporter gene assay
45
in HSCs.
The luciferase levels of AP-1 and NFκB which co-treat with TNF-
(10ng/ml)
for
down-regulated
twenty-four
by
10uM
hours
of
in
HSCs
are
(S)-armepavine,
significantly
and
show
concentration-depend manners in cytoplasm of HSCs. We first transfected
the immortalized rat hepatic stellate cells (HSC-T6) with pAP-1-Luc
plasmid, which contains the AP-1-responsive region followed by the
firefly luciferase gene. Following TNF-α treatments, AP-1 presumably
will translocate into nucleus, bind to AP-1-binding sites on p AP-1-Luc
DNA, and trigger expression of luciferase gene. Namely, luciferase
activity corresponds to TNF-α and LPS-induced AP-1 activity. After
exogenously adding luciferin to cell lysates, the luciferase-luciferin
reactions generate luminescence with high sensitivity and can be
measured. The AP-1 responsive curve for different amount of TNF-α is
shown in [figure 1a]. TNF-α stimulated the luciferase activity in HSCs at
10 ng/ml and reached plateau (225 ± 18% of controls) at 10 ng/ml, and
LPS stimulated the luciferase activity in HSCs at 1 μg/ml and reached
significant increase (313 ± 35% of controls) at 1 μg/ml, which was taken
for further studies on (S)-armepavine.
Secondly, we used N-acetylcysteine (NAC) as positive inhibitors in
this assay. We observed that NAC (5 mM) reduced the AP-1 activity
induced by TNF-α (10 ng/ml) in HSCs [figure 6]. Moreover,
(S)-armepavine (1- 10 μM ) was shown to reduce the NFκB activity
induced by TNF-α [figure 4]. TNF-α-stimulated luciferase activity was
273 ± 28% of controls, and this ratio was reduced to 98 ± 34% by
co-administration of (S)-armepavine (5). The inhibitory effect of
46
NAC, and (S)-armepavine was not due to the cytotoxicity [figure 1]. The
luciferase levels of NFB which co-treat with TNF- (10ng/ml) and LPS
(1ug/ml) for twenty-four hours in HSCs are significantly down-regulated
by 10uM of (S)-armepavine, and show concentration-depend manners in
cytoplasm of HSCs.
The translocation of nucleus p65, IκB and MAPK phosphorylation
in
HSCs.
The translocation levels of p65 and IκB phosphorylation as HSCs
co-treat with TNF- (10ng/ml) or LPS(1μg/ml) for twenty-four hours are
significantly down-regulated p65 translocation into nucleus and inhibit
IκB phosphorylation by 10uM of (S)-armepavine [figure 5], and show
concentration-depend manners. Following TNF-α and LPS treatment,
MAPK phosphorylation showed induced by TNF-α and LPS treatment.
We also observed that the ERK1/2, p38 and JNK 1/2 phosphorylation
was attenuated by (S)-armepavine treatment [figure 7]. The amounts of
the NFκB (p65) protein in nuclear extracts of cells were also
concentration-dependly reduced by (S)-armepavine treatment [figure 6].
In Vivo Effects of (S)-armepavine on TAA and BDL Rats
(A) General Features
TAA rats showed decrease in liver weight (  vs.  g, p<
0.01) as compared with control rats. Neither (S)-armepavine nor
silymarin treatment changed the liver weight in TAA rats. The body
weight of TAA rats was significantly lower than that of control rats ( 
47
vs.  g, p< 0.01). TAA rats also displayed a sickening appearance. The
body weight of TAA rats was significantly improved by treatment of
either low or high dose of (S)-armepavine (  and  g, respectively,
vs.  g). BDL rats showed increase in liver weight (  vs.  g, p<
0.01) as compared with control rats. Neither (S)-armepavine nor
silymarin treatment changed the liver weight in BDL rats (table 1). The
body weight of TAA rats was significantly lower than that of control rats
( 
vs.

g, p< 0.01). BDL rats also displayed a sickening
appearance. The body weight of BDL rats was significantly improved by
treatment of either low or high dose of (S)-armepavine (  and  g,
respectively, vs. 
g).
(B) Plasma Biochemistry
TAA rats showed significantly higher plasma ALT (  vs. 
U/ml, p< 0.01) and AST (  vs.  U/ml, p< 0.01) levels compared
with control rats, indicating hepatic injury (Table 1). Levels of either ALT
or AST in TAA rats were significantly decreased by high-dose (  vs.

U/ml, p< 0.05 and

vs.

U/ml, p< 0.05, respectively) or
low-dose (  vs.  U/ml, p< 0.05 and  vs.  U/ml, p> 0.05,
respectively) (S)-armepavine and silymarin (  vs.  U/ml, p> 0.05
and

vs.

U/ml, p< 0.05, respectively), suggesting that
(S)-armepavine and silymarin ameliorated hepatic injury in TAA rats
(table 1). There were no difference in plasma creatinine levels between
each group of TAA rats and control rats, suggesting no manifest renal
48
impairment in TAA rats.
BDL rats showed significantly higher plasma ALT (
 vs. 
U/ml, p< 0.01) and AST (  vs.  U/ml, p< 0.01) levels compared
with control rats, indicating hepatic injury (Table 1). Levels of either ALT
or AST in BDL rats were significantly decreased by high-dose (  vs.

U/ml, p< 0.05 and

vs.

U/ml, p< 0.05, respectively) or
low-dose (  vs.  U/ml, p< 0.05 and  vs.  U/ml, p> 0.05,
respectively) (S)-armepavine and silymarin (  vs.  U/ml, p> 0.05
and

vs.

U/ml, p< 0.05, respectively), suggesting that
(S)-armepavine and silymarin ameliorated hepatic injury in TAA rats
(table 1). There were no difference in plasma creatinine levels between
each group of BDL rats and control rats, suggesting no manifest renal
impairment in BDL rats.
(C) Histological Examination
Histological examination of livers from TAA rats revealed the
following changes: progressive increase and expansion of fibrous septa
and loss of hepatocytes, compared with control rats. Collagen fibers, as
stained by Sirius-red, were distinctly deposited in the liver of TAA rats
(fig. ). Fibrosis scores of livers from TAA rats (  ) were significantly
reduced in TAA rats treated with either low (  , P< 0.01) or high (  , P<
0.01) dose of (S)-armepavine, and silymarin (  , P< 0.01).
Histological examination of livers from BDL rats revealed the
following changes: progressive increase and expansion of fibrous septa
and loss of hepatocytes, compared with control rats. Collagen fibers, as
49
stained by Sirius-red, were distinctly deposited in the liver of BDL rats
(fig. 3). Fibrosis scores of livers from BDL rats (
significantly reduced in BDL rats treated with either low (


) were
, P< 0.01)
or high (  , P< 0.01) dose of (S)-armepavine, and silymarin (
 ,
P< 0.01).
(D) Hepatic Collagen Content
Hepatic collagen levels were significantly increased in TAA rats
compared with control rats ( 
vs.

mg/g liver weight, p<),
suggesting abundant accumulation of collagen in the liver of TAA rats.
Hepatic collagen levels were significantly decreased by low-dose ( 
mg/g, p<) or high-dose (  mg/g, P<) (S)-armepavine, and silymarin
( 
mg/g, p=), suggesting that (S)-armepavine and silymarin
ameliorated hepatic collagen deposition in TAA rats
Hepatic collagen levels were significantly increased in BDL rats
compared with control rats (  vs.  mg/g liver weight, p< 0.01),
suggesting abundant accumulation of collagen in the liver of BDL rats.
Hepatic collagen levels were significantly decreased by low-dose ( 
mg/g, p< 0.01) or high-dose (  mg/g, P< 0.01) (S)-armepavine, and
silymarin (

mg/g, p= 0.02), suggesting that (S)-armepavine and
silymarin ameliorated hepatic collagen deposition in TAA rats
(E) Analysis of Transcripts of α-SMA, Procollagen, ICAM-1and iNOS
Genes
There were significant increases in hepatic mRNA expression of α-SMA,
50
TGF-1, CTGF, TIMP-1, pro-collagen I, iNOS, ICAM-1, IL-6 and
metallothionein genes relative to G3PDH in TAA or BDL rats compared
with control rats (figs. 4 and 5). The mRNA expression levels of
profibrogenic genes in TAA and BDL rats were all attenuated in
(S)-armepavine- and silymarin-treated groups (figs. 4 and 5), suggesting
that fibrosis-related gene transcripts were attenuated by (S)-armepavine
or silymarin treatment.
51
Discussion
In the present study, we observed that (S)-armepavine exerted
inhibitory effects on NFB activation in HSC-T6 cells, including (a)
(S)-armepavine significantly inhibited TNF-α and LPS-induced AP-1 and
NFB activity, IBα phosphotylation and NFB translocation in HSC-T6
cells. (b)(S)-armepavine also reduced the mRNA expression of NFB
responsive
gene
ICAM-1,
induced
by
TNF-α
and
LPS.
(c)
(S)-armepavine concentration-dependently attenuated TNF-α-stimulated
α-SMA protein expression ratio and collagen deposition by HSC-T6 cells
Taken together, these results suggest that (S)-armepavine exerted
anti-fibrotic effects in vitro which of the mechanism of actions were
associated
with
that
(S)-armepavine
could
attenuated
the
pro-inflammatory and fibrogenic pathways in HSC-T6 cells stimulated by
TNF-α and LPS.
In conclusion, our results showed that (S)-armepavine also inhibited
fibrogenic responses of HSC-T6 cells to LPS and proinflammatory events
of HSC-T6 cells to both TNF-α and LPS.
52
ACKNOWLEDGMENTS
We gratefully acknowledge the kind provision of HSC-T6 cells by
Dr. Scott L. Friedman, Division of Liver Diseases, The Mount Sinai
School of Medicine, New York, NY, USA. We also obtain two pressure
gifts-armepavine oxalate which are prepared by semisynthesis from Dr.
Chien-Chang Shen (National Research Institute of Chinese Medicine,
Taiwan, ROC), and NAC from Dr. Yun-Lian Lin (National Research
Institute of Chinese Medicine, Taiwan, ROC).
53
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Figure 1. Effects of (S)-armepavine on cell viability of HSC-T6 cells at
twenty four hours after treatments (n=3).
Figure 2. Effects of (S)-armepavine on collagen deposition by HSC-T6
cells after TNF-α stimulation for twenty four hours. Collagen deposition
by HSC-T6 cells was quantified by sircol collagen assay. *,p < 0.05 vs.
Control; #,p< 0.05 vs. TNF-α alone, (n=3).
Figure 3 (S)-armepavine reduced the protein expression of α-SMA
induced by TNF-α (10 ng/ml) in HSC-T6 cells for twenty four hours.
Representative results from three independent experiments are shown
here. *,p< 0.05 vs. Control; #,p < 0.05 vs. TNF-α alone.
Figure 4. Effect of (S)-armepavine on TNF-α induced NFkB luciferase
reporter gene assay in HSC-T6 cells for 24h,NAC as a positive control.
Representative results from three independent experiments are shown
here. *,p< 0.05 vs. Control; #,p < 0.05 vs. TNF-α alone.
Figure 5. The expressions of nucleus p65 and pIκB in HSC-T6 cells
treated with TNF-α and (S)-armepavine. HSC-T6 cells treat with TNF-α
for twenty four hours. Representative results from three independent
experiments are shown here. PCNA and α-tubulin as internal control.
Figure 6. Effect of (S)-armepavine on TNF-α induced AP-1 luciferase
reporter gene assay in HSC-T6 cells for 24h. Representative results
from three independent experiments are shown here. *,p< 0.05 vs.
Control; #,p < 0.05 vs. TNF-α alone.
Figure 7. The levels of ERK1/2, p38 and JNK 1/2 phosphorylation in
HSC-T6 cells treated with TNF-α and (S)-armepavine. HSC-T6 cells treat
with TNF-α for 0-120 minutes. Representative results from duplicate are
shown here.
Figure 8. The expressions of TIMP-1 and α-SMA in HSC-T6 cells
treated with TNF-α and drugs. HSC-T6 cells treat with TNF-α for
twenty four hours. Representative results from duplicate are shown here.
Figure 9. The expressions of iNOS and Procollagen type I in HSC-T6
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cells treated with TNF-α and drugs. HSC-T6 cells treat with TNF-α for
twenty four hours. Representative results from duplicate are shown here.
Figure 10 (S)-armepavine reduced the protein expression of α-SMA
induced by LPS(1 μg/ml) in HSC-T6 cells for twenty four hours.
Representative results from three independent experiments are shown
here. *,p< 0.05 vs. Control; #,p < 0.05 vs. TNF-α alone.
Figure 11. Effect of (S)-armepavine on LPS(1 μg/ml)-induced AP-1
luciferase reporter gene assay in HSC-T6 cells for 24h. Representative
results from three independent experiments are shown here. *,p< 0.05 vs.
Control; #,p < 0.05 vs. TNF-α alone.
Figure 12 The levels of ERK1/2, p38 and JNK 1/2 phosphorylation in
HSC-T6 cells treated with LPS and (S)-armepavine. HSC-T6 cells treat
with TNF-α for 0-120 minutes. Representative results from duplicate are
shown here.
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