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Oxidative stress, an important factor that induces liver fibrosis, represents a key feature of hepatitis induced by various
conditions, including anoxic/reoxygenation injury,
autoimmune hepatitis, viral hepatitis and alcoholic
hepatitis[1]. Less severe oxidative stress may sustain fibrosis progression by causing activation and morphological changes in hepatic stellate cells
(HSC), including promoting proliferative activity, synthesis and degradation/remodeling of the extracellular matrix (ECM),
chemotaxis, contractility, proinflammatory activity and retinoid
loss[1,2].
Carbon tetrachloride (CCl4) is a xenobiotic used extensively to induce oxidative stress. It is assumed to initiate free
radical-mediated lipid peroxidation, leading to the accumulation of lipid-derived oxidation products that cause liver injury and
excess collagen deposition in the liver, resulting in liver
fibrosis[3,4]. During hepatic fibrogenesis, there is an imbalance
between excess synthesis of ECM and/or its removal, with consequent fibrosis and
scarring[5,6]. The pathophysiology of
ECM formation during liver fibrosis is multifaceted and
complex[7,8]. It involves a change in the expression of ECM proteases
(matrix metalloproteinases; MMP) or their inhibitors (tissue inhibitors of metalloproteinases; TIMP) and an increase in the
synthesis of collagen and fibronectin driven by signaling pathways mediated by
pro-inflammatory cytokines such as transforming growth
factor-b1 (TGF-b1) and tumor necrosis factor-a
(TNF-a)[9-12].
Interleukin (IL)-10 is a cytokine that downregulates pro-inflammatory
responses[13]. Human IL-10 is a 160 amino acid
protein (molecular weight=18.5 kDa), and murine IL-10 is a 157 amino acid protein with 80% homology to the human
form[14]. Recombinant human IL-10 has been produced and tested in clinical trials. Studies suggest that IL-10 may be
effective against chronic hepatitis C and other liver
diseases[15]. Further, IL-10 gene therapy has been studied extensively in animal models for
autoimmune diabetes, thyroiditis, and
colitis[16-18]. Because the elimination half-life of recombinant IL-10 is relatively short
(=2 h)[19], it may be possible to utilize its therapeutic properties to develop a gene-based treatment regimen. In the present
study, we investigate whether IL-10 gene therapy is effective against
CCl4-induced liver fibrosis in mice.
Materials and methods
Subjects Male 6- to 8-week-old ICR mice were purchased from the National Science Council, Taiwan, China, and were
allowed to acclimatize for 5 d before experimentation. The mice were housed in Kaohsiung Chang Gung Memorial Hospital
Animal Facility under standard temperatures, and with a standard light and dark cycle. All procedures performed on the mice
were approved by the Kaohsiung Chang Gung Memorial Hospital Animal Care and Use Committee.
IL-10 expression plasmid preparation A human IL-10 expression plasmid (pCYIL-10 vector) was used in the present
study[20]. In brief, full-length human IL-10 cDNAs were subcloned into a pCY4B expression vector driven by a chicken
b-actin promoter with a cytomegalovirus immediate early enhancer. pCMV-Lacz was used as the vehicle control. These plasmids
were purified using the EndoFree Plasmid Giga Kit (Qiagen, Valencia, CA, USA).
Liver fibrosis induction and gene therapy
Based on the method used in a previous study, but with some modifica-tions,
the mice were administered CCl4 (1 mL/kg body
weight) dissolved in olive oil (1:1) twice a week for 10
weeks[21]. Sixteen mice were killed at the end of 6 weeks to confirm that liver fibrosis was established (group I). To evaluate the anti-fibrotic effect of
IL-10, gene therapy administration was started at the end of 6 and 8 weeks of
CCl4 treatment. Briefly, 30 µL bovine hyaluronidase
(0.4 IU/µL) (Sigma-Aldrich, St Louis, MO, USA) was injected into the anterior tibialis (AT) muscle of the mice 2 h before
electroporation. pCYIL-10 was injected into the bilateral AT muscles using a 27G needle (30 µL into each leg; 4 µg/µL; group
II, n=16). Electroporation was carried out using electrical pulses (8 pulses of 20 ms, 175 V/cm, and 1 s intervals) with
Tweezertrode electrode disks and an electrical pulse generator (T830; BTX, San Diego, CA,
USA)[22].
Sixteen mice received gene electro-transfer therapy using the same procedure as described above using pCMV-LacZ
(group III) as a vehicle control at the end of 6 and 8 weeks. All surviving mice (group II,
n=12; group III, n=7) were killed at
the end of the 10-week CCl4 treatment. Five mice were killed before
CCl4 intoxication as normal controls (group N).
Histopathology and immunohistochemistry For histopathology studies, mice were killed at 0, 6, and 10 weeks after
CCl4 administration. The liver was removed and fixed in 10% formalin solution. Five-micrometer sections were stained with 0.1%
Sirius red in picric acid (Sigma-Aldrich). Matrix density was quantified using a computerized image analysis system as
previously described[23]. For immunohistochemical studies, the sections were washed in phosphate-buffered saline (PBS),
and incubated in 3% normal goat serum with 0.3% Triton X-100 in PBS for 1 h. The sections were incubated free-floating at
4 ˇăC with IL-10 (specific for human origin; Santa Cruz Biotechnology, Santa Cruz, CA, USA), cyclooxygenase-2 (COX-2),
MMP-2, and TIMP-1 (Abcam, Cambridge, MA, USA) antibodies. Immunoreactivity was visualized using the Vectastain Elite
ABC Peroxidase method (Vector Laboratories, Burlingame, CA, USA) with diamino-benzidine (DAB) as the chromagen.
Soluble collagen measurement For soluble collagen analysis, the Sircol collagen assay (Biocolor, Belfast, UK) was
performed following the manufacturerˇŻs instructions as described in a previous
study[24]. Briefly, 50 mg of liver was
homogenized. Total acid pepsin-soluble collagens were
extracted overnight using 5 mg/mL pepsin in 500 µL of 0.5
mol/L acetic acid. One milliliter of Sircol dye reagent was added to
every 100 µL of each sample, in duplicate, and the mixture was incubated
at 25 ˇăC for 30 min. After centrifugation, the pellet was suspended in 1 mL of alkali reagent. The absorbance was read at 540
nm.
Immunoblotting The liver specimens were homogenized in a lysis buffer with complete protease inhibitor cocktail tablets
(Roche, Mannheim, Germany). For analysis of a-smooth muscle actin
(a-SMA) expression after CCl4 admini-stration, 20
mg of protein extracts were electrophoresed on a 10% acrylamide sodium
dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) gel and immunoblotted onto PVDF membranes. The membranes were blocked for 1 h at room temperature and
incubated overnight with a 1:1000 dilution of
a-SMA, and a-tubulin antibodies (Abcam). Antibody binding was detected
using horseradish peroxidase (HRP)-linked immunoglobulin G (IgG). Bands were visualized using an ECL detection system
(Amersham-Pharmacia Biotech, Little Chalfont, UK). Band intensities were quantified using an image analyzer (Densitograph
AE-6900M; Atto, Tokyo, Japan).
Reverse transcription-polymerase chain reaction
Livers were harvested at 0, 6, and 10 weeks after
CCl4 admini-stration. The expression levels of
TGF-b1, collagen a1, fibronectin, TNF-a, intercellular adhesion molecule-1
(ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), TIMP-1, and TIMP-2 mRNA were analyzed using reverse transcription-polymerase chain
reaction (RT-PCR) techniques. The total RNA was extracted and then reverse-transcribed into cDNA. PCR was performed at
a final concentration of 1ˇÁ PCR buffer, 1.0 µmol/L of each of the 3ˇŻ and 5ˇŻ primers, and 10 U of
Advan-Taq Plus DNA polymerase (Clontech, Palo Alto, CA, USA) in a total volume of 50 µL. The mixture was amplified for 32 cycles in a thermal
cycler (Stratagene, La Jolla, CA, USA). The b-actin was amplified to verify equal loading. The primer sequence and expected
product size were as previously described[25]
. The amplification products were separated by agarose gel electrophoresis and
visualized using ethidium bromide staining. The gel was scanned at a NucleoVision imaging workstation (NucleoTech, San
Mateo, CA, USA), and quantified using GelExpert release 3.5.
Gelatin zymography Gelatin zymography was carried out to explore MMP activity. Briefly, liver tissues were
homogenized in a protein extraction buffer. The supernatant of a centrifuged liver sample (20 µg of protein extract per line) was mixed
1:3 with a sample buffer and separated by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis in 8%
polyacrylamide gel copolymerized with 1 mg/mL gelatin (Sigma-Aldrich) as described
elsewhere[26]. Gels were incubated at 37 ˇăC
overnight in an MMP activation buffer. After Coomassie blue staining, the extent of gel digestion localized to bands of
active-MMP-2 (64-kDa) were quantified by densitometry.
Statistical analysis All data (from at least 3 separate experiments) are presented as meanˇŔSEM.
Statistical analysis was performed using one-way ANOVA followed by the
t-test. P<0.05 was considered significant.
Results
Long-term IL-10 expression following electroporative gene transfer
There was only scanty staining of cells for human
IL-10 in the non-gene transfer groups (groups N, I, and III; Figure 1). Strong positive staining of cells for human IL-10 was
seen in the livers of the gene transfer group II and 4 weeks after electroporation (group II; Figure 1C).
IL-10 gene therapy reversed CCl4-induced liver fibrosis
There was no significant difference in food and water intake
throughout the study period between groups. After 6 weeks of
CCl4 administration, liver fibrosis was seen histo-pathologically.
Sirius red staining of liver sections revealed extensive fibrosis, portal-to-portal fibrous bridging, and nodular transformation
in groups I and III (Figure 2). Human IL-10 gene therapy (group II) significantly ameliorated hepatic fibrogenesis and reduced
matrix density (Figure 2C). These findings were further confirmed by measurements of liver collagen content (Table 1).
IL-10 gene therapy attenuated COX-2 increment after
CCl4 COX-2 was not detected immunohistochemically in the
normal group. COX-2 expression was upregulated after
CCl4 administration (groups I and III; Figure 3). IL-10 gene therapy
significantly diminished this COX-2 expression (Figure 3C).
IL-10 gene therapy suppressed hepatic stellate cell activation after
CCl4 a-SMA [activated hepatic stellate cell (HSC)
markers] are known to be activated after acute liver
injury[27,28]. In the present study, the expression of
a-SMA increased after chronic CCl4
administration as measured using immunoblotting (Figure 4). IL-10 gene therapy (group II) significantly reduced
this upregulation, indicating HSC inactivation
(P<0.01 vs group I; P<0.01
vs III). a-Tubulin was used as an internal control.
IL-10 gene therapy attenuated fibrogenic, proinflamma-tory, and cell adhesion molecule gene responses after
CCl4 treatment Expression of
TGF-b1, collagen a1, fibronectin, TNF-a, ICAM-1, and VCAM-1 mRNA were all upregulated
in the fibrotic liver as semi-quantified using RT-PCR (Figure
5). b-actin was amplified as an internal control. IL-10 gene
therapy (group II) significantly attenuated
these increase. In brief, IL-10 gene transfer suppressed the fibrogenic, pro-inflammatory, and cell
adhesion molecule gene responses after
CCl4 administration.
IL-10 gene therapy attenuated MMP-2 activation in the fibrotic
liver The expression of MMP after
CCl4 treatment was evaluated by using immunohistochemical and gelatin zymography methods. Immunohistochemical studies showed that RT-
when compared with normal livers, MMP-2 levels were significantly increased in the fibrotic livers (groups I and III;
Figure 6). IL-10 gene therapy attenuated this upregulation (group II; Figure 6C). The collagenolytic activity of MMP protein
in liver homogenates was examined by zymography (Figure 7). Gelatin zymography showed that the concentration of the 64
kDa active MMP-2 molecule increased in groups I and III after
CCl4 administration, and IL-10 gene therapy (group II)
abrogated this increase (P<0.01).
IL-10 gene therapy attenuated TIMP activation after
CCl4 treatment Expression of TIMP in the fibrotic livers was also
evaluated by RT-PCR and immunohistochemical methods.
PCR showed that TIMP-1 and TIMP-2 mRNA were significantly upregulated in the fibrotic liver. IL-10 gene therapy (group
II) significantly attenuated these increase
(P<0.01,Figure 5). Immunohistochemical studies revealed that the level of
TIMP-1 was increased after chronic CCl4
administration (Figure 8). IL-10 gene therapy (group II) significantly attenuated this
activation (Figure 8C).
Discussion
Animal models of hepatic fibrosis provide a means to study the cellular and molecular mediators of fibrosis in a serial
manner during both progression and recovery. Several approaches to the induction of fibrosis have been described. Of
these, CCl4 intoxication in rats and mice is probably the most widely
studied[29]. In addition, the CCl4
model is the best characterized with respect to histological, biochemical, cellular, and molecular changes associated with the development of
fibrosis[30,31]. CCl4 can be given intraperitoneally or by oral gavage; it induces hepatocyte necrosis and apoptosis with
associated HSC activation and tissue fibrosis. With ongoing treatment
CCl4 can be used to induce bridging hepatic fibrosis
(4 weeks of twice-weekly treatment), cirrhosis (8 weeks of twice-weekly treatment) and advanced
micronodular cirrhosis (12 weeks of twice-weekly
treatment)[31].
IL-10 is a potent anti-inflammatory cytokine that inhibits the synthesis of pro-inflammatory
cytokines[32]. IL-10 has been shown to downregulate the synthesis of collagen type I and TIMP in previous
investigations[33,34]. It also plays an
anti-fibrogenic role by decreasing the levels of pro-fibrogenic
cytokines, including TGF-b1 and
TNF-a[33]. In the present study, we demonstrated that electroporative IL-10 gene therapy provided an effective expression method for long-term use. This
treatment reversed established liver fibrosis and reduced collagen synthesis in mice. IL-10 gene therapy also inhibited HSC
activation after CCl4 administration. The fibrogenic gene
(TGF-b1 and TNF-a) response attenuation may be responsible for
the hepatoprotective effect of IL-10.
COX-2 is a key executor of uncontrolled
inflammation[35]. Overexpression of COX-2 has been demonstrated in
CCl4-induced liver fibrosis and post-viral human
cirrhosis[36,37]. Further, COX-2 can contribute to hepatic carcinogenesis by
increasing necroinflammatory activity, promoting
proliferation, and enhancing
angiogenesis[38,39]. Selective COX-2 blockers
are known to reduce CCl4-induced liver
fibrosis[36]. Hence, COX-2 may be a new therapeutic target for treatments for liver
cirrhosis. IL-10 is known as the central regulator of
COX-2[40]. Therefore, IL-10 gene therapy might have exerted its
anti-hepatic fibrogenesis effect through COX-2 inactiva-tion.
Cell adhesion molecules are known as prognostic markers of liver
fibrosis[41]. Expression of ICAM-1 and VCAM-1
modulated by TNF-a are upregulated in alcoholic hepatitis,
CCl4-induced liver injury, and nutritional
fibrosis[42-44]. A previous study showed that ICAM-1 and VCAM were upregu-lated in IL-10 knockout-colitis in
mice[45]. In addition, IL-10 can attenuate ICAM-1 activation in cisplatin
nephrotoxicity[46]. Therefore, cell adhesion molecule regulation may be involved in the
anti-fibrotic effect of IL-10.
The imbalance between MMP and TIMP in the ECM contributes to the pathogenesis of liver fibrosis.
Matrix metalloproteinases are a family of zinc-dependent proteases capable of degrading hepatic ECM, thereby playing a central role
in tissue remodeling and repair after
injury[47]; however, persistent overexpression of MMP may contribute to the pathogenesis
of liver diseases. Inhibition of MMP-2 produced by activated stellate cells blocks
lethal hepatitis and apoptosis induced by
TNF-a[48]. Furthermore, MMP-2-deficient mice have decreased hepatocyte apoptosis and necrosis, and enhancsed survival
in this model. Recent studies have also revealed a strong correlation between MMP-2 activity and severity of human liver
disease[49]. MMP activity is regulated by the TIMP, which binds in a substrate- and tissue-specific manner to MMP, blocking
their proteolytic activity[50]. Antibodies and antisense oligonucleotides directed at TIMP-1 attenuate rat liver
fibrosis[50,51]. IL-10 is known to suppress MMP-2 and TIMP-1 expression in HSC during liver
fibrosis[52]. In the present study, we
demonstrated that IL-10 gene therapy attenuated MMP-2 and TIMP activation in the fibrotic liver. Therefore,
its collagenolytic effect might be attributed to MMP and TIMP modulation.
In the present study, we demonstrated the anti-hepatic fibrogenic effect of IL-10 in mice. IL-10 gene therapy reversed
established CCl4-induced liver fibrosis in mice through fibrogenic gene response attenuation. In conclusion, IL-10 gene
therapy may be a new therapeutic modality for liver cirrhosis with potential clinical use.
Acknowledgement
We would like to thank Dr Xian-min MENG of Thomas Jefferson University, Philadelphia, PA, USA, for providing the
human IL-10 expression plasmid (pCYIL-10 vector) used in this study.
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