Effects of platelet-derived growth factor and interleukin-10 on Fas/Fas-ligand and Bcl-2/Bax mRNA expression in rat hepatic stellate cells in vitro.

Abstract

To investigate the effects of platelet-derived growth factor(PDGF) and interleukin-10 (IL-10) on Fas/Fas-ligand and Bcl-2/Bax mRNA expressions in rat hepatic stellate cells.
Rat hepatic stellate cells (HSCs) were isolated and purified from rat liver by in situ digestion of collagenase and pronase and single-step density Nycodenz gradient. After activated by culture in vitro, HSCs were divided into 4 groups and treated with nothing (group N), PDGF (group P), IL-10 (group I) and PDGF in combination with IL-10 (group C), respectively. Semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis was employed to compare the mRNA expression levels of Fas/FasL and Bcl-2/Bax in HSCs of each group.
The expression levels of Fas between the 4 groups had no significant differences (P>0.05). FasL mRNA level in normal culture-activated HSCs (group N) was very low. It increased obviously after HSCs were treated with IL-10 (group I) (0.091+/-0.007 vs 0.385+/-0.051, P<0.01), but remained the low level after treated with PDGF alone (group P) or PDGF in combination with IL-10 (group C). Contrast to the control group, after treated with PDGF and IL-10, either alone or in combination, Bcl-2 mRNA expression was down-regulated and Bax mRNA expression was up-regulated, both following the turn from group P, group I to group C. Expression of Bcl-2 mRNA in group C was significantly lower than that in group P (0.126+/-0.008 vs 0.210+/-0.024, P<0.01). But no significant difference was found between group C and group I, as well as between group I and group P (P>0.05). Similarly, the expression of Bax in group C was higher than that in group P (0.513+/-0.016 vs 0.400+/-0.022, P<0.01). No significant difference was found between group I and group P (P>0.05). But compared with group C, Bax expressions in group I tended to decrease (0.449+/-0.028 vs 0.513+/-0.016, P<0.05).
PDGF may promote proliferation of HSCs but is neutral with respect to HSC apoptosis. IL-10 may promote the apoptosis of HSCs by up-regulating the expressions of FasL and Bax and down-regulating the expression of Bcl-2, which may be involved in its antifibrosis mechanism.

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PO Box 2345, Beijing 100023, China World J Gastroenterol 2004;10(18):2706-2710
Fax: +86-10-85381893 World Journal of Gastroenterology
E-mail: wjg@wjgnet.com www.wjgnet.com Copyright © 2004 by The WJG Press ISSN 1007-9327
• BASIC RESEARCH •
Effects of platelet-derived growth factor and interleukin-10 on
Fas/Fas-ligand and Bcl-2/Bax mRNA expression in rat hepatic
stellate cells in vitro
Xiao-Zhong Wang, Sheng-Jun Zhang, Yun-Xin Chen, Zhi-Xin Chen, Yue-Hong Huang, Li-Juan Zhang
Xiao-Zhong Wang, Sheng-Jun Zhang, Yun-Xin Chen, Zhi-Xin Chen,
Yue-Hong Huang, Li-Juan Zhang, Department of Gastroenterology,
Union Hospital of Fujian Medical University, Fuzhou 350001, Fujian
Province, China
Supported by the Science and Technology Fundation of Fujian
Province, No. 2003D05
Correspondence to: Xiao-Zhong Wang, Department of Gastroenterology,
Union Hospital of Fujian Medical University, Fuzhou 350001, Fujian
Province, China. drwangxz@pub6.fz.fj.cn
Telephone: +86-591-3357896 Ext. 8482
Received: 2004-03-05 Accepted: 2004-03-12
Abstract
AIM: To investigate the effects of platelet-derived growth
factor(PDGF) and interleukin-10 (IL-10) on Fas/Fas-ligand
and Bcl-2/Bax mRNA expressions in rat hepatic stellate cells.
METHODS: Rat hepatic stellate cells (HSCs) were isolated
and purified from rat liver by in situ digestion of collagenase
and pronase and single-step density Nycodenz gradient.
After activated by culture in vitro, HSCs were divided into
4 groups and treated with nothing (group N), PDGF (group P),
IL-10 (group I) and PDGF in combination with IL-10 (group C),
respectively. Semi-quantitative reverse-transcriptase
polymerase chain reaction (RT-PCR) analysis was employed
to compare the mRNA expression levels of Fas/FasL and Bcl-
2/Bax in HSCs of each group.
RESULTS: The expression levels of Fas between the 4 groups
had no significant differences (P>0.05). FasL mRNA level
in normal culture-activated HSCs (group N) was very low.
It increased obviously after HSCs were treated with IL-10
(group I) (0.091±0.007 vs 0.385±0.051, P<0.01), but
remained the low level after treated with PDGF alone (group P)
or PDGF in combination with IL-10 (group C). Contrast to
the control group, after treated with PDGF and IL-10, either
alone or in combination, Bcl-2 mRNA expression was down-
regulated and Bax mRNA expression was up-regulated, both
following the turn from group P, group I to group C.
Expression of Bcl-2 mRNA in group C was significantly lower
than that in group P (0.126±0.008 vs 0.210±0.024, P<0.01).
But no significant difference was found between group C
and group I, as well as between group I and group P (P>0.05).
Similarly, the expression of Bax in group C was higher
than that in group P (0.513±0.016 vs 0.400±0.022, P<0.01).
No significant difference was found between group I and group
P (P>0.05). But compared with group C, Bax expressions in
group I tended to decrease (0.449±0.028 vs 0.513±0.016,
P<0.05).
CONCLUSION: PDGF may promote proliferation of HSCs
but is neutral with respect to HSC apoptosis. IL-10 may promote
the apoptosis of HSCs by up-regulating the expressions of FasL
and Bax and down-regulating the expression of Bcl-2, which
may be involved in its antifibrosis mechanism.
Wang XZ, Zhang SJ, Chen YX, Chen ZX, Huang YH, Zhang
LJ. Effects of platelet-derived growth factor and interleukin-
10 on Fas/Fas-ligand and Bcl-2/Bax mRNA expression in rat
hepatic stellate cells in vitro. World J Gastroenterol 2004; 10
(18): 2706-2710
http://www.wjgnet.com/1007-9327/10/2706.asp
INTRODUCTION
Liver fibrosis is a progressive pathological process involving
multi-cellular and molecular events that ultimately lead to
deposition of excess matrix proteins in the extracellular space. It
is generally accepted that hepatic stellate cells (HSCs) are central
to the process of fibrosis as the major source of extracellular
matrix (ECM) components[1-10]. Following acute or chronic liver
tissue injury, HSCs undergo a process of activation towards a
phenotype characterized by increasing proliferation, motility,
contractility and synthesis of ECM components. Cytokines play
an important role in the formation, development and reversibility
of fibrosis[9-14]. Activated HSCs secrete many important cytokines
through autocrine and paracrine, of which platelet-derived growth
factor (PDGF) can activate secretory cells and those quiescent
HSCs around[15,16] and promote the proliferation of HSCs[17].
IL-10 is a potent anti-inflammatory cytokine that inhibits the
synthesis of pro-inflammatory cytokines by T helper type 1 T
cells and mono/macrophages. Previous studies have shown that
endogenous IL-10 has the ability to inhibit the inflammation in
injured liver and block the advance of fibrosis[18-21]. Previous
works by our group have demonstrated that exogenous IL-10
has an anti-fibrogenic function[22]. But the underlying mechanism
remains obscure. In this study, in order to investigate the effects
of IL-10 and PDGF on the proliferation and apoptosis of rat HSCs,
culture-activated HSCs were treated with IL-10 and PDGF.
Fas/FasL and Bcl-2/Bax mRNA expressions in each group were
assayed by semiquantitative reverse-transcriptase polymerase
chain reaction (RT-PCR) analysis.
MATERIALS AND METHODS
M aterials
Male Wistar rats, weighing 450-500 g, were provided by
Shanghai Center for Laboratory Animals. Total RNA isolation
kit was obtained from Jingmei Biotechnology Company of
Shenzhen. Moloney murine leukemia virus (M-MLV) reverse
transcriptase was purchased from Promega. PCR reagent and
Dulbecco’s modified Eagle’s medium (DMEM) were respectively
provided by Shanghai Biotechnology Company and GibcoB. PCR
primers were synthesized by Shanghai Biotechnology Company.
Isolation, culture and evaluation of HSCs
HSCs were isolated from normal male Wistar rats by in situ digestion
of collagenase and pronase and single-step density Nycodenz
gradient as Ramm GA[23] and Friedman SL[24] previously described,
and cultured in DMEM supplemented with 100 mL/L FBS.
Desmin immunocytochemistry was employed to determine the

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isolated HSCs’ purity. HSCs were subcultured 4 d after primary
culture. Alpha smooth muscle actin (α-SMA) immunocytochemistry
and electron microscope were employed to confirm that HSCs were
activated by culture in vitro and transformed into myofibroblasts.
Intervention and division of HSCs
The subcultured HSCs were diluted to a concentration of 5×104/mL
with DMEM containing 100 mL/L FBS and seeded onto the 24-
well plastic tissue culture plates. When HSCs spread the plate
fully, the culture medium was replaced with DMEM containing
10 mL/L FBS. After incubated for 24 h, HSCs were divided randomly
into 4 groups: one as control group cultured in 1 mL DMEM
containing 10 mL/L FBS, the other three were cultured in the same
medium and treated with 20 ng PDGF or 20 ng IL-10, either alone or
in combination, respectively. We named them group N, group P,
group I and group C, respectively. Each group included 5 wells.
RNA extraction
Total RNA was extracted from the above treated HSCs after
incubated for 24 h according to the RNA isolation kit instructions.
The content and purity of total RNA were determined by
spectrophotography. A260/A280 of total RNA was between 1.8-2.0.
RT-PCR for Fas/FasL and Bcl-2/Bax
For RT-PCR, total RNA was reverse-transcripted using M-MLV
reverse transcriptase and oligo (dT) at 37 °C for 60 min, followed
by at 70 °C for 10 min. Approximately 2 µg total RNA was used
in each reverse transcription reaction and the final volume was
25 µL. β-actin was used as internal control. The PCR reaction
volume was 50 ul, including 5 µL 10×PCR buffer, 2 mmol/L MgCl2,
1 µL 10 mmol/L dNTP, 1 µL 20 pmol/µL target gene sense and
anti-sense primers, 1 µL 20 pmol/µL β-actin primer pair, 2 µL RT
product, 1.5 U Tag DNA polymerase. The specific sets of primers
and the target gene amplification conditions are shown in Table 1.
Result determination
PCR products were run on 20 g/L agarose gel eletrophoresis and
visualized with ethidium bromide staining. Bio imagine system was
used to detect the densities of bands of the PCR products. The ratio
of target gene density to β-actin density was used to represent the
relative levels of Fas/FasL and Bcl-2/Bax mRNA expressions. The
semi-quantitative detection was analyzed 5 times repeatedly.
Statistical analysis
All data were expressed as mean±SE. The significance for the
difference between the groups was assessed with SPSS 10.0 by
one-way ANOVA. P<0.05 was considered statistically significant.
RESULTS
Evaluation of HSCs
Freshly isolated HSCs were round-shaped with many yellow droplets
in cytoplasm. After cultured for 5-6 d, the spread cells showed a
typical ‘star’-like configuration. Desmin immunocytochemistry
showed that the positive percentage was about 95% (Figure 1A),
indicating that 95% of the isolated cells were HSCs. α-SMA
immunocytochemistry showed that 98% of the cells were α-
SMA positive (Figure 1B), indicating that most of the cells were
activated. The myofilament could be seen in cytoplasm under the
electron microscope, confirming that HSCs were activated and
transformed into myofibroblasts after cultured in vitro (Figure 2).
Figure 1 Desmin and α-SMA immunocytochemistry (SP, origi-
nal magnification: ×100). A: Desmin immunocytochemistry
of HSCs 7 d after isolation; B: α-SMA immunocytochemistry
of HSCs 7 after isolation.
Table 1 Primer sequences for PCR and amplification conditions for each target gene
Primer (base) Sequence Amplification conditions
Fas 4145’-GA A TGCA A GGGA CTGA TA GC-3’
5’-TGGTTCGTGTGCAAGGCTC-3’
Denaturation at 94 °C for 45 s,
Annealing at 55 °C for 30 s and synthesizing
at 72 °C for 1 min for 25 cycles
Denaturation at 94 °C for 45 s,
Annealing at 55 °C for 30 s and synthesizing
at 72 °C for 1 min for 33 cycles
Denaturation at 94 °C for 45 s,
Annealing at 60 °C for 30 s and synthesizing
at 72 °C for 1 min for 33 cycles
Denaturation at 94 °C for 45 s,
Annealing at 60 °C for 30 s and synthesizing
at 72 °C for 1 min for 30 cycles
Changed according to different target genes
FasL 239 5’-GGA A TGGGA A GA CA CA TA TGGA A CTGC -3’
5’-CA TA TCTGGCCA GTA GTGCA GTA A TTC-3’
Bcl-2 525 5’-TA TGA TA A CCGGGA GA TCGTGA TC-3’
5’-GTGCA GA TGCCGGTTCA GGTA CTC-3’
Bax 3105’-GA CA CCTGA GCTGA CCTTGG-3’
5’-GA GGA A GTCCA GTGTCCA GC-3’
β-actin 6605’-CCA A CCGTGA A A A GA TGA CC-3’
5’-CA GGA GGA GCA A TGA TCTTG-3’
All initial denaturations were at 94 °C for 5 min. Finally an additional extension step at 72 °C for 7 min was done.
A
B
Wang XZ et al. PDGF and IL-10 on Fas/Fas-ligand and Bcl-2/Bax mRNA expression 2707

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Figure 2 A ctivated HSCs under the electron microscope.
The myofilament can be seen in the cytoplasm as the arrow
point shows.
Figure 3 Relative Fas/ FasL mRNA expression levels in
HSCs of different groups assessed by RT-PCR. A: Relative
Fas mRNA expression levels (P>0.05 between random two
groups.); B: Relative FasL mRNA expression levels (aP>0.05
vs group N, bP<0.01 vs group N, dP<0.01 vs group I.); group N:
Normal group as control; group P: PDGF treated group; group
I: IL-10 treated group; group C: Combined PDGF and IL-10
treatment group.
Figure 4 RT-PCR results of Fas/ FasL mRNA expression in
HSCs of different groups. A: RT-PCR results of Fas mRNA
expression; B: RT-PCR results of FasL mRNA expression; M:
100 bp DNA ladder (upper to lower: 1 000, 900, 800, 700, 600,
500, 400, 300, 200, and 100 bp); Lane 1: Normal group as control;
Lane 2: PDGF treatment group; Lane 3: IL-10 treatment group;
Lane 4: Combined PDGF and IL-10 treatment group.
Figure 5 Relative Bcl-2/ Bax mRNA expression levels in HSCs
of different groups assessed by RT-PCR. A: Relative Bcl-2
mRNA expression levels (bP<0.01 vs group P, group I and
group C, respectively; aP>0.05 vs group I, cP>0.05 vs group C,
dP<0.01 vs group P.). B: Relative Bax mRNA expression levels
(bP<0.01 vs group P, group I and group C, respectively; aP>0.05
vs group I, cP = 0.045<0.05 vs group C, dP<0.01 vs group P.).
Group N: Normal group as control; group P: PDGF treated
group; group I: IL-10 treated group; group C: Combined PDGF
and IL-10 treatment group.
Figure 6 RT-PCR results of Bcl-2/ Bax mRNA expression in
HSCs of different groups. A: Bcl-2 mRNA expression. B: Bax
mRNA expression. M: 100 bp DNA ladder (upper to lower:
1 000, 900, 800, 700, 600, 500, 400, 300, 200, and 100 bp); Lane 1:
Normal group as control; Lane 2: PDGF treatment group; Lane
3: IL-10 treatment group; Lane 4: Combined PDGF and IL-10
treatment group.
Effects of PDGF and IL-10 on Fas and FasL expressions in HSCs
Fas mRNA was expressed in HSCs of each group and the
expression levels had no significant difference among the 4
groups, as shown in Figures 3A, 4A, indicating that neither
PDGF nor IL-10 had effect on Fas mRNA expression in HSCs.
As it could be informed from Figures 3B, 4B, FasL mRNA level
in normal culture-activated HSCs (group N) was very low. It
increased obviously after HSCs were treated with IL-10 (group
I) (0.091±0.007 vs 0.385±0.051, P<0.01), but remained the low
level after treated with PDGF alone (group P) or PDGF in
combination with IL-10 (group C) (0.085±0.006, 0.101±0.008,
respectively). The data suggested that IL-10 could improve FasL
mRNA expression in culture-activated HSCs and PDGF could
not. Furthermore, PDGF tended to abolish this effect of IL-10.
A
B
0.50
0.48
0.46
0.44
0.42
0.40
0.38
Group N Group P Group I Group C
Relative expression
levels of Fas mRNA
0.5
0.4
0.3
0.2
0.1
0
Group N Group P Group I Group C
Relative expression
levels of FasL mRNA
M 1 2 3 4
A
β-actin (660 bp)
B
Fas (414 bp)
M 1 2 3 4
β-actin (660 bp)
Fas (239 bp)
A
0.5
0.4
0.3
0.2
0.1
0
Group N Group P Group I Group C
Relative expression
levels of Bcl-2 mRNA
B
0.6
0.5
0.4
0.3
0.2
0.1
0
Group N Group P Group I Group C
Relative expression
levels of Bax mRNA
M 1 2 3 4
A
β-actin (660 bp)
Bcl-2 (525 bp)
M 1 2 3 4
B
β-actin (660 bp)
Bax (310 bp)
2708 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol September 15, 2004 Volume 10 Number 18
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b
d
a
b
c
d
a
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c
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Effects of PDGF and IL-10 on Bcl-2 and Bax expressions in HSCs
Bcl-2 and Bax mRNA were expressed in normal culture-activated
HSCs. Both of their expression levels were significantly changed
after treated with PDGF and IL-10, either alone or in combination.
Bcl-2 mRNA expression was down-regulated and Bax mRNA
expression was up-regulated, following the turn from group P,
group I to group C. The expression of Bcl-2 in group C was
significantly lower than that in group P (0.126±0.008 vs 0.210±0.024,
P<0.01). But no significant difference was found between group
C and group I, as well as between group I and group P (0.210±0.024
vs 0.166±0.017, 0.166±0.017 vs 0.126±0.008, P>0.05) (Figures 5A,
6A). Similarly, the expression of Bax in group C was higher
than that in group P (0.513±0.016 vs 0.400±0.022, P<0.01). No
significant difference was found between group I and group P
(0.400±0.022 vs 0.449±0.028, P>0.05). But compared with combined
treatment group, Bax expressions in group I tended to decrease
(0.449±0.028 vs 0.513±0.016, P = 0.045<0.05) (Figures 5B, 6B).
These results showed that both PDGF and IL-10 promoted the
Bax mRNA expression in HSCs and inhibited the Bcl-2
expression, but the differences of their effects were not
significant. Intervention with PDGF and IL-10 seemed to be
able to manifest effects on Bax expression than intervention
alone. IL-10 showed similar influences on culture-activated
HSCs and reactivated HSCs by PDGF.
DISCUSSION
It is generally accepted that hepatic stellate cells (HSCs) are
central to the process of hepatic fibrosis. They are the major
source of extracellular matrix and during fibrogenesis undergo
an activation process characterized by increased proliferation
and collagen synthesis[1-10,24]. So the activation, proliferation
and apoptosis of HSCs have close relationship with the
formation and development of liver fibrosis. To inhibit the
activation and proliferation of the HSCs and promote their
apoptosis has become the most important therapeutic approach
for liver fibrosis[7-10,14,25-28].
There is evidence that HSCs can be successfully isolated
by in situ digestion of collagenase and pronase and single-
step density Nycodenz gradient[23,24,29]. Desmin is a marker for
muscle cells and expressed by all muscle lineages including
HSCs (either quiescent or activated) in the liver. Alpha smooth
muscle actin (α-SMA) is an intermediate filament protein that
is expressed by activated HSCs and is widely accepted to be a
marker of activation. Both of them were used to identify and quantify
HSCs and their activation. The desmin immunocytochemistry result
showed that the purity of the isolated HSCs by this method was
satisfying (Figure 1A). The results of α-SMA immunocytochemistry
(Figure 1B) and electron microscope (Figure 2) confirmed that
HSCs were activated and transformed into myofibroblasts after
cultured in vitro.
PDGF, which is produced by HSCs, Kuffer cells and platelets,
is a major mitogen for connective tissues and certain other
cells. It was viewed as one of the most important growth factors
serving as the matrix-bound cytokines[11] and plays an important
role in the pathogenesis of liver fibrosis via promoting the activation
and proliferation of HSCs[12,15,25,30-32]. The best characterized
chemotactic factor for HSCs identified so far is the PDGF-BB[33-35]
which is also known as the most potent mitogen for HSCs over-
expressed during active hepatic fibrosis[36]. But there is also
evidence that PDGF is proapoptotic for fibroblasts in conditions
of low serum[37]. Saile B[38] reported that resting HSCs displayed
no sign of apoptosis and spontaneous apoptosis became
detectable in parallel with HSCs activation, suggesting that
apoptosis might represent an important mechanism terminating
proliferation of activated HSCs. He also found that Fas and
Fas-ligand in HSCs became increasingly expressed during the
course of activation. But our data demonstrated that PDGF
alone had no effect on the expression of Fas and FasL during
further activating the culture-activated HSCs, which was
supported by Issa R[39]. Bax and Bcl-2 are known as the
representatives of proapoptotic factor and contra-apoptotic
factor of Bcl-2 family, respectively[40,41]. In our study, evidences
showed that PDGF could promote Bax mRNA expression in HSCs
and inhibited Bcl-2 mRNA expression as well, resulting in the
apoptosis of HSCs[41]. All the above data demonstrated that
PDGF can accelerate the apoptosis of HSCs through Bcl-2/Bax
pathway in parallel with their proliferation[42]. In other words,
PDGF may promote proliferation but is neutral with respect to
HSCs apoptosis. But the proportion of apoptosis-inducing forces
and apoptosis-inhibiting forces would determine that PDGF-
activated HSCs tend to proliferate and increase[22].
Cytokine interleukin-10 (IL-10), produced by lymphocytes
and macrophages as well as cells within liver such as Kufffer
cells, hepatocytes and HSCs, has profound inhibitory actions
on macrophages and inflammation. The present studies showed
that IL-10 had additional effects on connective tissue cells,
such as HSCs and fibroblast. IL-10 could inhibit the activation
of HSCs by inflammatory cells[43], relieve the inflammation of
liver[18,19,44], suppress the function of NF-κB[45] and affect the
expression of collagen I and collagenase[20], thus exerting an
antifibrogenesis effect[46]. Failure for HSCs to sustain IL-10
expression might underlie pathologic progression to liver
cirrhosis[18,20] .Our previous studies also implied that IL-10 had
an antagonism on CCL4-induced rat hepatic fibrosis[22]. But the
underlying mechanism remains obscure. In this study, our results
showed that IL-10 could promote the expression of FasL and Bax
mRNA in culture-activated HSCs and meanwhile could inhibit Bcl-
2 mRNA expression, implying that IL-10 may induce the apoptosis
of HSCs through binding FasL to Fas on the cell membranes of
HSCs and increasing the proportion of Bax and Bcl-2. Saile B[38]
found that apoptosis could be fully blocked by Fas-blocking
antibodies in normal cells and HSCs already entering the apoptotic
cycle, implying that Fas/FasL system is the key pathway for the
apoptosis of HSCs. Our data, however, showed that Bax/Bcl-2
system was another important pathway involving in HSCs’
apoptosis[40,41]. In short, IL-10 could promote the apoptosis of
HSCs, which may be related to its mechanism of antifibrosis.
There is evidence that activated-HSCs could express IL-10
as well as its receptor[20,47]. In this study, PDGF had a similar
effect to IL-10 on Bax/Bcl-2 mRNA expression in HSCs. This
promotes us to hypothesize that PDGF may regulate the
expression of Bax and Bcl-2 mRNA by affecting the expression
of IL-10 in HSCs. But PDGF in combination with IL-10 did not
show a satisfying synergistic action, thus we can not exclude
the possibility that PDGF and IL-10 affect in different ways,
and further works are demanded.
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2710 ISSN 1007-9327 CN 14-1219/ R World J Gastroenterol September 15, 2004 Volume 10 Number 18