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Introduction
Idiopathic pulmonary fibrosis is a chronic, progressive
form of interstitial lung disease, associated with an extremely
poor prognosis for survival in most patients. Most patients
die of progressive respiratory failure within 3_8 years of the
onset of symptoms. Considerable experimental evidence
implicated both increased collagen production and reduced
degradation, leading to an irreversible distortion of normal
tissue architecture and loss of
function[1_3]. However, the pathogenesis of idiopathic pulmonary fibrosis still remains
unknown; early lung inflammatory response and subsequent
fibrotic changes are well appreciated in the time course of
this disease, and until now, there has been no satisfactory
treatment for this disease. Bleomycin, a mixture of
glycopeptides derived from Streptomyces
verticillus, is a potent chemotherapeutic agent used for the treatment of
lympho-mas, head and neck cancers, and various tumors. Moreover,
bleomycin is also known to produce lung injury and fibrosis
in humans as well as in experimental
animals[4].
The Chinese traditional herb Tripterygium
wilfordii Hook F and its extracts have been widely used in the treatment of
autoimmune diseases, including rheumatoid arthritis and
systemic lupus erythematosus[5_8]. Triptolide is identified
as the most active component accounting for the
immunosuppressive effects of Tripterygium
wilfordii Hook F[9]. However, the strong toxicity of triptolide limits its
application to a great extent[10]. Recently,
(5R)-5-hydroxytriptolide (LLDT-8), a new compound derived from triptolide, was
synthesized and showed similar immunosuppressive activity to
that of triptolide, but its toxicity was greatly reduced
in vitro and in vivo. In
vitro, LLDT-8 significantly inhibited
mitogen-induced T and B cell proliferation, and mixed
lymphocyte reaction, inflammatory, and Th1 type cytokines
release[11]. In vivo, LLDT-8 suppressed the bovine type II
collagen-induced arthritis in DBA/1
mice[12] and adjuvant-induced arthritis in Wistar rat (our unpublished observations).
LLDT-8 prevented graft-versus-host disease and prolonged
allogeneic cardiac transplantation survival in
mice[13,14]. LLDT-8 attenuated the concanavalin A-induced liver
hepatitis[15] and prolonged mice survival in the MRL-lpr/lpr murine
model of systemic lupus erythematosus (our unpublished
observa-tions).
The purpose of the present study was to extend our
findings of LLDT-8 to an in vivo mouse lung fibrosis model for
further exploration of its anti-inflammatory and antifibrosis
action.
Materials and methods
Animals Female C57BL/6 mice (6_8 weeks old, 20_22 g)
were purchased from the Shanghai Experimental Animal
Center of the Chinese Academy of Sciences (Shanghai, China).
The animals were housed in specific pathogen-free
condi-tions. All mice were allowed to acclimatize in our facility for
1 week before any experiments were started. All experiments
were carried out according to the National Institutes of Health
Guide for the Care and Use of Laboratory Animals, and were
approved by the Bioethics Committee of the Shanghai
Institute of Materia Medica (Shanghai, China).
Test compound LLDT-8 was synthesized from triptolide
that was separated from Tripterygium
wilfordii Hook F. LLDT-8 is a white, amorphous powder with 99% purity by
reverse phase, high-performance liquid chromatography. The
stock solution of LLDT-8 (5 mg/mL) was prepared in 2-
methyl-1,3-propanediol, stored at 4 °C, and diluted to a
desired concentration with saline (10 mL/kg, ip). The final
concentration of 2-methyl-1,3-propanediol in the dosing
solution was 4%.
Experimental model of bleomycin-induced lung fibrosis
Lung fibrosis was induced as described by previous studies
with minor modifications[4,16]. Briefly, after the body weight
was recorded, the mice were anesthetized via intraperitoneal
injection of 30 mg/kg pentobarbital sodium. A midline
incision was made in the neck, and the trachea was exposed by
blunt dissection. Bleomycin hydrochloride (Nippon Kayaku,
Tokyo, Japan), was dissolved in 0.1 mL saline and injected
into the animals' lungs via the 0.25 mL syringes at a dose of
7.5 mg/kg body weight. The normal control received an equal
volume of sterile saline. After bleomycin or saline were
injected into the trachea, the animal operating plate was
erected and shaken to facilitate distribution of the solution
throughout the lungs. The day of bleomycin injection was
considered d 0 and the weight of the animals was recorded
every 3_4 d.
Group assignment and drug administration
The mice were randomly assigned to 5 body weight-matched groups:
normal control, bleomycin, and the bleomycin with LLDT-8
treatment groups (LLDT-8 at 2 mg/kg, 1 mg/kg and 0.5
mg/kg). In most of the experiments, each group consisted of
8 mice, except the bronchoalveolar lavage assay, and 3
animals per group were analyzed. LLDT-8 was daily
administered to the mice by ip from d 1. The normal and bleomycin
control group were daily injected with vehicle solution by ip.
Bronchoalveolar lavage analysis On d 7 after bleomycin
treatment, the mice were sacrificed. The thorax was opened
by a median incision and the trachea was cannulated with a
plastic catheter attached to a 2 mL syringe. Bronchoalveolar
lavage was performed in 4 mL sterile saline with gentle
massaging of the lungs. The bronchoalveolar lavage fluid were
collected and centrifuged at 150×g for 10 min at 4 °C. The
total number of cells in the lavage fluid was counted with
trypan blue staining. The cell subsets were counted with
Giemsa staining by examining 200 cells per animal. The cell
numbers of macrophages, neutrophils, and lymphocytes in
the lavage fluid was calculated according to their respective
percentages in the total cells.
Lung tissue preparation and biochemical assay
One hour after LLDT-8 or vehicle administration on d 7 or 14, the
mice were sacrificed by bleeding. The lungs were removed
and weighed, washed twice with cold saline, and then each
lung was divided into 2 parts: the right one was fixed in 10%
formalin solution for histological examination and the left
one was prepared for biochemical assay and cytokine
detection.
The lung samples were prepared as 10% homogenate in
0.9% saline by homogenizer on ice according to their
respective weight. Then the homogenate was centrifuged, and the
supernatant was collected and diluted. The assay of
superoxide dismutase (SOD), malondialdehyde, and
hydroxyproline levels followed the manufacturer's instructions (Nanjing
Jiancheng Bioengineering Institute, Nanjing, China).
Histological examination of fibrosis The lung samples
were washed and fixed in buffered 10% formalin solution.
After embedded in paraffin, 5 µm sections were stained with
hematoxylin-eosin (HE), and examined by 2 pathologists who
were blinded to the experiment.
Measurement of tumor necrosis factor-α (TNF-α), interleukin-4 (IL-4), and transforming growth
factor-β (TGF-β) The lung homogenate (10%) was centrifuged at
10 600×g for 30 min at 4 °C to remove debris, and the
supernatants were assayed for TNF-α, IL-4, and
TGF-β concen-trations. The levels of TNF-α and IL-4 were determined
using sandwich ELISA kits from PharMingen (San Diego,
CA, USA) following the manufacturer's instructions. The
TGF-β level was determined using a Mv1Lu cell proliferation
assay[17]. Briefly, the Mv1Lu cells
(2×104/well) were cultured in the presence of diluted acidified samples or recombinant
TGF-β (R&D Systems, Minneapolis, MN, USA) in 96-well
plates for 24 h at 37 °C in an incubator with 5%
CO2. The cells were pulsed with 0.5 mCi of
[3H]-thymidine for 8 h and harvested onto glass fiber filters. The incorporated
radioactivity was then counted using a Beta Scintillation Counter
(MicroBeta Trilux, Perkin-Elmer Life Sciences, Boston, MA,
USA). The concentration of TGF-β in the lung homogenate
was calculated according to the rTGF-β inhibitory standard
curve. The anti-TGF-β 1,2,3 antibody (Genzyme, Framingham,
MA, USA) was used to confirm the specific inhibition by
TGF-β in the Mv1Lu cells. The sample was acidified to pH 2
with 1 mol/L HCl for 30 min on ice and then neutralized with
1 mol/L NaOH.
Statistical analysis Data were expressed as mean±SEM
or mean±SD where indicated. Statistical differences were
analyzed according to the analysis of variance, followed by
post-hoc multiple comparison tests (LSD).
P<0.05 was considered to be significant.
Results
LLDT-8 attenuated bleomycin-induced lung injury
Compared with the normal control, the body weight in the
bleomycin-treated animals decreased gradually and reached
the lowest level at d 7 after bleomycin injection, and then
tended to recover. LLDT-8 displayed protective effects on
the loss of body weight (Figure 1). Moreover, LLDT-8 was
well tolerated in the bleomycin-treated mice, showing no
change in mobility, skin hair, and respiration throughout the
experiment.
In contrast to the loss of body weight, the weight of the
lungs increased obviously in the bleomycin-treated mice,
resulting in augmentation of the lung index (lung weight
versus body weight). LLDT-8 treatment dose-dependently
inhibited the increase of the lung index as compared with
that of the vehicle-treated bleomycin group on d 7 and 14
(Figure 2).
The lungs were examined histologically on d 14 after
bleomycin injection. Data are shown in Figure 3. The lung
architecture appeared intact in the normal control group. In
the bleomycin control group, there were multifocal diffuse
changes consisting of some combinations of thickened
alveolar septa, interstitial hyperplasia, intra-alveolar fibrosis
with myofibroblasts, occasional foci of dense fibrosis,
increased alveolar macrophages, and some infiltration of
inflammatory cells. However, in the LLDT-8-treated mice,
there was a marked decrease in inflammation and fibrosis.
No obvious fibrotic focal was observed in the 2 mg/kg
LLDT-8-treated group.
LLDT-8 suppressed lung inflammatory cell expansion
induced by bleomycin treatment The number of total cells
and the subsets including macrophages, lymphocytes, and
neutrophils in the bronchoalveolar lavage fluid were elevated
markedly in response to bleomycin on d 7 (Table 1). LLDT-8
administration dose-dependently reduced the numbers of
total cells, neutrophils, as well as lymphocytes, but weakly
affected the macrophages number.
LLDT-8 reduced hydroxyproline and malondialdehyde
production but enhanced SOD activity in bleomycin-treated
mice To assess the total collagen content and fibrotic
process, we determined the lung hydroxyproline level. Data
are presented in Table 2. In the lung homogenates from the
bleomycin control mice, the hydroxyproline level increased
on d 7 and 14. Administration of LLDT-8 reduced the
hydroxyproline production. On d 14, LLDT-8 at 2
mg/kg, 1 mg/kg, and 0.5 mg/kg decreased the hydroxyproline level by
30.4%, 23.9%, and 15.1%, respectively. Moreover, LLDT-8
at 2 mg/kg effectively reduced the hydroxyproline
production to the basal level as that in the normal control.
The deprivation of antioxidant enzyme SOD indirectly
reflects reactive oxygen species production in response to
bleomycin. As shown in Table 2, LLDT-8 significantly
prevented the decrease of SOD activity on d 7. On d 14, SOD
activity tended to recover in the bleomycin control group;
meanwhile, LLDT-8 still showed an enhancing effect on SOD
activity.
Malondialdehyde is a marker of lipid peroxidation. In
this study, malondialdehyde content in the lung tissue was
remarkably elevated after bleomycin treatment. The increased
percentages were 78.4% and 41.2% on d 7 and 14,
respec-tively. LLDT-8 inhibited bleomycin-induced
malondial-dehyde production in a dose-dependent manner. In the 2
mg/kg LLDT-8-treated group, its production was reduced to
the basal level as that in the normal control on d 7. On d 14,
the beneficial effect of LLDT-8 was still observed when given
at 2 mg/kg (Table 2).
LLDT-8 inhibited pro-inflammatory and pro-fibrotic
cytokine production in lung homogenates To evaluate the
roles of key cytokines in lung fibrosis, we detected
TNF-α, IL-4, and TGF-β levels in lung homogenates. Data are
presented in Figure 4. Administration of LLDT-8 significantly
inhibited TNF-α production on d 7 in a dose-dependent
manner (Figure 4A). In addition, a remarkable increase of IL-4
levels was seen in the bleomycin control group on d 14 (Figure
4B), and LLDT-8 treatment effectively suppressed this
increase.
In the Mv1Lu cell proliferation assay, TGF-β
concentration in the lung tissue elevated from 580 pg/mL in the normal
control group to 880 pg/mL on d 7, and reached 1012 pg/mL
on d 14 after bleomycin injection (Figure 5). The increase of
the TGF-β level was suppressed after LLDT-8 treatment. In
addition, the anti-TGF-β1, 2, 3 antibody was taken to test the
specificity of proliferative inhibition by TGF-β. As expected,
the suppressed cell proliferation was almost completely
restored by this antibody (data not shown).
Discussion
Idiopathic pulmonary fibrosis is a progressive lung
disease with unknown pathogenesis. The role of the
inflammation and anti-inflammatory strategy still remain controversial.
It was suggested that in addition to inflammatory cells,
alveolar epithelial cells, mesenchymal precursor cells,
fibro-blasts, and myofibroblasts played an important role in the
diverse processes of fibrosis[2]. Thus, potential therapeutic
strategies can be developed at any stage which result in
idiopathic pulmonary fibrosis. These include agents that
interfere with the action of inflammatory mediators, agents
that prevent parenchymal cell damage, agents that prevent
the proliferation of fibroblasts and collagen synthesis, agents
that downregulate myofibroblast differentiation, and agents
that intervene with 1 or more key events in the pathogenesis
or signal transduction pathways of idiopathic pulmonary
fibrosis[1]. In addition, it is unlikely that any single treatment
can be sufficiently effective in the case of lung fibrosis.
Bleomycin-induced animal lung injury has been widely
used as a model of human lung fibrosis because some
biochemical and functional changes in the early stages in
animals resemble that in humans. Other studies from long-term
observations which reported the physiological and
histological changes at late stages (d 120) in rats were very
different from human disease[18], so that extrapolation of the data
from the animal model to humans needs to be taken with
caution. However some similarities exist between the
bleomycin animal model and human lung fibrosis, and this
model is informative for antifibrosis agent evaluation and
potential mechanism research.
The inflammatory response to bleomycin is orchestrated
partially by endogenous and migrating leukocytes, which is
also well demonstrated in our present study. These
activated leukocytes can synthesize and secrete various
cytokines, chemokines, reactive oxygen species, and
proteases that sustain the injury/repair processes. Neutrophils
isolated from the bronchoalveolar lavage had a greater
capacity to produce superoxide anion than those from the blood,
and resulted in lung damage[19]. Moreover, these leukocytes
together with lung epithelial and endothelial cells produced
a feedback circle where stimuli from injury responses could
activate alveolar and interstitial
macrophages[20]. LLDT-8 ameliorated exudation in lung tissue and reduced the
leukocytes number, which would result in a decrease in source of
free radicals.
Substantial data prove the cellular redox state and the
oxidant_antioxidant balance play a critical role in the
pathogenesis of lung fibrosis in animal models and possibly in
humans[21,22]. In addition to the inflammation mediator, the
high level of oxidants may increase TGF-β
release[23], activate protease, and enhance the fibrotic response; some
antioxidants including N-acetylcysteine, and SOD can decrease
collagen deposition and protect the lungs in a variety of
animal models or even in clinical
trials[24]. LLDT-8 served as a free-radical scavenger, enhanced SOD ability, and
inhibited lipid peroxidation, which helped to ameliorate
inflammatory reaction, meanwhile possibly contributing to decreased
TGF-β production and alleviating fibrosis change.
Cytokines are involved in the fibrosis process.
TNF-α induces adhesion molecule expression by vascular
endothelial cells and intensifies the recruitment of inflammatory
cells into the lungs. Moreover, TNF-α is relevant to the
induction of fibrosis by augmenting synthesis of fibronectin,
prostaglandin, and TGF-β. Administration of the
anti-TNF-α antibody, soluble TNF-α is demonstrated to be beneficial
in suppressing bleomycin-induced lung
injury[25,26], and now a phase II clinical trial of soluble
TNF-α receptor (Etanercept) is under
way[1,27]. In our study, LLDT-8 markedly inhibited
TNF-α production, providing 1 possible mechanism of its
protective effect against lung injury.
The role of Th1 and Th2 cytokines in lung fibrosis
remains controversial[28_32]. Recent studies disclosed that IL-4
might play a selective anti-inflammatory role during initial
lung injury stages by limiting the early accumulation of T
cells, but IL-4 promoted fibroblast proliferation and collagen
deposition during the later stages of
fibrosis[33_36]. The IL-4 level in lung tissues was detected on d 7 and 14; the IL-4
increase on d 7 was not evident, and the inhibitory effect of
LLDT-8 was not significant (data not shown). However,
LLDT-8 suppressed IL-4 production on d 14 after bleomycin
injection, which possibly contributed to the blockade of lung
injury.
Moreover, we detected IFN-g and IL-10 levels in lung
homogenates on d 7 and 14 after bleomycin injection.
LLDT-8 did not affect these 2 cytokine production (data not shown).
However, LLDT-8 decreased IFN-g from concanavalin A or
Sac-stimulated murine spleen cells in
vitro[11]. The possible reason for this needs further investigation.
TGF-β is a pivotal mediator in lung fibrosis and has a
broad spectrum of activities in pulmonary inflammation,
tissue repair, and fibrosis. TGF-β can serve as a chemoattractant
for fibroblasts and monocytes/macrophages and
stimulate these cells to synthesize a number of pro-inflammatory and
fibrogenic cytokines such as TNF-α, IL-1b, and
TGF-β itself. At the same time, TGF-β is one of the most potent inducers
of extracellular matrix production. TGF-β reduces the
breakdown of collagen and other matrix proteins by inhibiting the
generation of plasminogen activators, matrix
metallopro-teinase, and elastase, as well as by enhancing the
expression of tissue inhibitors of metalloproteinases, plasminogen
activator inhibitor-1,2[1,37]. In our study,
TGF-β production in lung homogenates elevated gradually after
bleomycin treatment, while LLDT-8 potently suppressed
TGF-β production and retained it at basal level as that in the normal control.
Then, the reduced hydroxyproline production and lung
injury by LLDT-8 was at least partially attributed to its
inhibition of TGF-β production.
In conclusion, LLDT-8 demonstrated protective effects
against bleomycin-induced murine pulmonary fibrosis. The
beneficial effect of LLDT-8 might be closely associated with
its activities of anti-inflammation, antioxidant, and cytokine
inhibition.
Acknowledgement
We thank the research group of Dr Jin REN (Shanghai
Institute of Materia Medica ) for providing valuable
technical assistance with histology.
References
1 Gharaee-Kermani M, Phan SH. Molecular mechanisms of and
possible treatment strategies for idiopathic pulmonary fibrosis.
Curr Pharm Des 2005; 11: 3943_71.
2 Khalil N, O'Connor R. Idiopathic pulmonary fibrosis: current
understanding of the pathogenesis and the status of treatment.
Can Med Assoc J 2004; 171: 153_60.
3 Gross TJ, Hunninghake GW. Idiopathic pulmonary fibrosis. N
Engl J Med 2001; 345: 517_25.
4 Ozyurt H, Sogut S, Yildirim Z, Kart L, Iraz M, Armutcu F,
et al. Inhibitory effect of caffeic acid phenethyl ester on
bleomycin-induced lung fibrosis in rats. Clin Chim Acta 2004; 339: 65_75.
5 Asano K, Matsuishi J, Yu Y, Kasahara T, Hisamitsu T.
Suppressive effects of Tripterygium wilfordii
Hook f., a traditional Chinese medicine, on collagen arthritis in mice.
Immunopharma-cology 1998; 39: 117_26.
6 Gu WZ, Banerjee S, Rauch J, Brandwein SR. Suppression of renal
disease and arthritis, and prolongation of survival in MRL-lpr
mice treated with an extract of Tripterygium
wilfordii Hook f. Arthritis Rheum 1992; 35: 1381_6.
7 Gu WZ, Brandwein SR. Inhibition of type II collagen-induced
arthritis in rats by triptolide. Int J Immunopharmacol 1998; 20:
389_400.
8 Tao X, Davis LS, Hashimoto K, Lipsky PE. The Chinese herbal
remedy, T2, inhibits mitogen-induced cytokine gene
transcription by T cells, but not initial signal transduction. J Pharmacol
Exp Ther 1996; 276: 316_25.
9 Chen BJ. Triptolide, a novel immunosuppressive and anti-
inflammatory agent purified from a Chinese herb
Tripterygium wilfordii Hook F. Leuk Lymphoma 2001; 42: 253_65.
10 Huynh PN, Hikim AP, Wang C, Stefonovic K, Lue YH, Leung A,
et al. Long-term effects of triptolide on spermatogenesis,
epididymal sperm function, and fertility in male rats. J Androl 2000;
21: 689_99.
11 Zhou R, Zhang F, He PL, Zhou WL, Wu QL, Xu JY,
et al. (5R)-5-hydroxytriptolide (LLDT-8), a novel triptolide analog
mediates immunosuppressive effects in vitro and
in vivo. Int Immuno-pharmacol 2005; 5: 1895_903.
12 Zhou R, Tang W, Ren YX, He PL, Zhang F, Shi LP,
et al. (5R)-5-hydroxytriptolide attenuated collagen-induced arthritis in
DBA/1 mice via suppressing interferon-gamma production and its
related signaling. J Pharmacol Exp Ther 2006b; 318: 35_44.
13 Tang W, Yang Y, Zhang F, Li YC, Zhou R, Wang JX,
et al. Prevention of graft-versus-host disease by a novel
immuno-suppressant, (5R)-5-hydroxytriptolide (LLDT-8), through
expansion of regulatory T cells. Int Immunopharmacol 2005; 5:
1904_13.
14 Tang W, Zhou R, Yang Y, Li YC, Yang YF, Zuo JP. Suppression
of (5R)-5-hydroxytriptolide (LLDT-8) on allograft rejection in
full MHC-mismatched mouse cardiac transplantation.
Transplantation 2006; 81: 927_33.
15 Zhou R, Tang W, Ren YX, He PL, Yang YF, Li YC,
et al. Preventive effects of (5R)-5-hydroxytriptolide on concanavalin
A-induced hepatitis. Eur J Pharmacol 2006a; 537: 181_9.
16 Kremer S, Brever R, Lossos IS. Effect of immunomodulators on
bleomycin-induced lung injury. Respiration 1999; 66: 455_62.
17 Li XF, Takiuchi H, Zou JP, Katagiri T, Yamamoto N, Nagata T,
et al. Transforming growth factor-β (TGF-β)-mediated
immunosuppression in the tumor-bearing state: enhanced production
of TGF-β and a progressive increase in TGF-β susceptibility of
anti-tumor CD4+ T cell function. Jpn J Cancer Res 1993; 84:
315_25.
18 Borzone G, Moreno R, Urrea R, Meneses M, Oyarzun M.
Bleomycin-induced chronic lung damage does not resemble
human idiopathic pulmonary fibrosis. Am J Respir Crit Care Med
2001; 163: 1648_53.
19 Tarnell EB, Oliver BL, Johnson GM, Watts FL, Thrall, RS.
Superoxide anion production by rat neutrophils at various stages of
bleomycin-induced lung injury. Lung 1992; 170: 41_50.
20 Oury TD, Thakker K, Menache M, Chang LY, Crapo JD, Day BJ.
Attenuation of bleomycin-induced pulmonary fibrosis by a
catalytic antioxidant metalloporphyrin. Am J Respir Crit Care Med
2001; 164: 164_9.
21 Kinnula VL, Fattman CL, Tan RJ, Oury TD. Oxidative stress in
pulmonary fibrosis. Am J Respir Crit Care Med 2005; 172:
417_22.
22 Kuwanl K, Nakashima N, Inoshima I, Hagimoto N, Fujita M,
Yoshimi M, et al. Oxidative stress in lung epithelial cells from
patients with idiopathic interstitial pneumonias. Lab Invest 2002;
82: 1695_706.
23 Bellocq A, Azoulay C, Marullo S, Flahault A, Fouqueray B, Philippe
C, et al. Reactive oxygen and nitrogen intermediates increase
transforming growth factor-beta 1 release from human epithelial
alveolar cells through two different mechanisms. Am J Respir
Cell Mol Biol 1999; 21: 128_36.
24 Salvemini D, Riley DP, Cuzzocrea S. SOD mimetics are coming
of age. Nat Rev Drug Discov 2002; 1: 367_74.
25 Piguet PF, Ribaux C, Karpuz V, Grau GE, Kapanci Y. Expression
and localization of tumor necrosis factor-alpha and its mRNA in
idiopathic pulmonary fibrosis. Am J Pathol 1993; 143: 651_5.
26 Yara S, Kawakami K, Kudeken N, Tohyama M, Teruya K, Chinen
T, et al. FTS reduced bleomycin-induced cytokine and chemokine
production and inhibits pulmonary fibrosis in mice. Clin Exp
Immunol 2001; 124: 77_85.
27 Walter N, Collard HR, King TE Jr. Current perspectives on the
treatment of idiopathic pulmonar fibrosis. Proc Am Thorac Soc
2006; 3: 330_8.
28 Izbicki G, Segel MJ, Christensen TG, Conner MW, Breuer R.
Time course of bleomycin-induced lung fibrosis. Int J Exp Path
2002; 83: 111_9.
29 Gharaee-Kermani M, Phan SH. Role of cytokines and cytokine
therapy in wound healing and fibrotic diseases. Curr Pharm Des
2001; 11: 1083_103.
30 Lossos IS, Or R, Goldstein RH, Conner MW, Breuer R.
Amelioration of bleomycin-induced pulmonary injury by cyclosporin A.
Exp Lung Res 1996; 22: 337_49.
31 Zhu J, Cohen DA, Goud SN, Kaplan AM. Contribution of T
lymphocytes to the development of bleomycin-induced
pulmonary fibrosis. Ann NY Acad Sci 1996; 796: 194_202.
32 Sharma SK, MacLean JA, Pinto C, Kradin RL. The effect of an
anti-CD3 monoclonal antibody on bleomycin-induced
lymphokine production and lung injury. Am J Respir Crit Care Med 1996;
154: 193_200.
33 Huaux F, Liu T, McGarry B, Ullenbruch M, Phan SH. Dual roles
of IL-4 in lung injury and fibrosis. J Immunol 2003; 170: 2083_92.
34 Arai T, Abe K, Matsuoka H, Yoshida M, Mori M, Goya S,
et al. Introduction of the interleukin-10 gene into mice inhibited
bleomycin-induced lung injury in vivo. Am J Physiol Lung Cell
Mol Physiol 2000; 278: L914_22.
35 Sakamoto H, Zhao LH, Jain F, Kradin R. IL-12p40(-/-) mice
treated with intratracheal bleomycin exhibit decreased
pulmonary inflammation and increased fibrosis. Exp Mol Pathol 2002;
72: 1_9.
36 Gharaee-Kermani M, Nozaki Y, Hatano K, Phan SH. Lung
interleukin-4 gene expression in a murine model of
bleomycin-induced pulmonary fibrosis. Cytokine 2001; 15: 138_47.
37 Krishna G, Liu K, Shigemitsu H, Gao M, Raffin TA, Rosen GD.
PG490-88, a derivative of triptolide, blocks bleomycin-induced
lung fibrosis. Am J Pathol 2001; 158: 997_1004.
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