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From the Division of Pulmonary and Critical Care Medicine, Stanford University School of Medicine, Stanford, California
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A newer class of agents with antifibrotic properties is currently under
investigation. For example, pirfenidone,7
interferon-ß,8
and interferon-
9
are in
clinical trials. The exact mechanism by which these agents exert their
antifibrotic property is yet to be defined. Triptolide (PG-490) is
a diterpene triepoxide derived from the Chinese herb,
Tripterygium Wilfordii hook. It has potent antiproliferative
and immunosuppressive properties. Triptolide, for example, has been
used in traditional Chinese medicine in the treatment of rheumatoid
arthritis.10
Triptolide has also shown to possess
antileukemic activities and to inhibit proliferation of transformed
cell lines in vitro.11,12
An extract of
Tripterygium, PG 27, has been shown to prolong heart and kidney
allograft survival in rat transplantation models.11
PG27,
along with cyclosporine prolongs cardiac xenograft survival from a
hamster donor to a rat recipient by
inhibiting the production of serum anti-hamster IgM and IgG
antibodies.11
Wei and co-workers12
have shown
that PG27 suppresses the development of graft-versus-host
disease associated with allogenic bone marrow transplantation. Another
extract of Tripterygium, T2 (5% triptolide) has been shown to block
mitogen-induced early cytokine gene transcription in T
cells.13
Qiu and colleagues14
have shown that
PG490 (97% pure triptolide) blocks cytokine gene expression by
human peripheral blood lymphocytes and 16HBE human bronchial epithelial
cells by transcriptional inhibition of nuclear factor-
B (NF-B).
We have recently shown that PG490 promotes tumor necrosis factor-
mediated apoptosis of tumor cell lines by inhibiting
NF-B.15
Triptolide, therefore, exhibits diverse
immunosuppresive and antitumor effects.
In this study, we show that PG-490-88, a water-soluble succinate salt derivative of triptolide, blocks both inflammation and fibrosis in the bleomycin model of mouse lung fibrosis. PG490-88 at a dose of 0.25 mg/kg/day for 14 days, administered intraperitoneally, decreased inflammation and fibrosis after a single intratracheal instillation of bleomycin in C57BL/6 mice. PG490-88 blocked fibrosis and decreased inflammation when treatment was initiated both on the same day as bleomycin administration or if the treatment was initiated 5 days after bleomycin instillation. The bronchoalveolar lavage fluid (BALF) level of TGF-ß also showed a significant decrease in the PG490-88-treated group compared to the bleomycin group. Additionally, reverse transcriptase-polymerase chain reaction (RT-PCR) of normal human lung fibroblasts treated with bleomycin and PG490 (triptolide) showed an inhibition in TGF-ß gene expression in the PG-490-treated cells compared to bleomycin alone.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eight- to 10-week-old male C57BL/6 mice weighing between 22 to
26 g were obtained from the Stanford University (Palo Alto, CA)
animal facility. The animals were caged in groups of five and housed in
the Stanford animal care facility. They had free access to rodent chow
and water. All care was in accordance with the National Institutes of
Health Guide for Animal Welfare Act. Mice in each set of experiment
were randomly assigned into five groups of five each. Mice in groups 1
and 2 were treated intratracheally with 0.1 ml of sterile saline alone
or saline plus intraperitoneal PG490-88 (a gift from Pharmagenesis,
Palo Alto, CA). To induce pulmonary fibrosis, mice in groups 3, 4, and
5 were treated on day 1 with 0.1 ml of bleomycin (Blenoxane, Novaplus,
15 U/vial; VHA Inc., Nippon Kayaku Co., Tokyo, Japan) at a dosage of
0.1 U/mouse by intratracheal injection under anesthesia. The anesthetic
solution used is a mixture of 1 ml of ketamine (100 µg/ml) and 1 ml
of xylazine (100 µg/ml) and 4.6 ml of sterile bacteriostatic saline
(Stanford comparative medicine pharmacy) at a dose of 0.1 ml/30 g body
weight. Mice in groups 4 and 5 were treated daily with PG490-88 (0.25
mg/kg) starting the same day as bleomycin treatment or 5 days after
bleomycin installation. The animals were weighed every other day. All
of the animals that reached 17 g or below were euthanized and the
lungs were not used for analysis. Animals that died before day 15 were
also not included in the analysis. All of the animals were sacrificed
on day 15 after bleomycin or saline and PG490-88 treatment. The lungs
were removed and appropriately processed for light microscopy or were
frozen in liquid nitrogen for analysis of hydroxyproline content.
Separate experiments were conducted for each of the analysis. The
experiment was conducted four times (five animals per group) for
hydroxyproline analysis, two times (five animals per group) for light
microscopy with one lung for hematoxylin and eosin (H&E) and one lung
for trichrome staining. A single experiment was used for the analysis
of -smooth muscle actin expression by immunohistochemistry (five
animals per group) and a different experiment was used for the
enzyme-linked immunosorbent assay (ELISA) of TGF-ß from
bronchoalveolar lavage (three animals per group). The actual number of
animals used for analysis is given in Results and in the figures.
Histological Scoring of Fibrosis
The lung samples were fixed by inflation with buffered 10% formalin solution for 24 hours. After embedding in paraffin, the sections were prepared and stained by H&E or by trichrome stain. The severity of fibrosis was semiquantitatively assessed according to the method proposed by Ashcroft and co-workers.16 Briefly, the grade of lung fibrosis was scored on a scale from 0 to 8 by examining 44 randomly chosen fields per sample at a magnification of x100. Criteria for grading lung fibrosis were as follows: grade 0, normal lung; grade 1, minimal fibrous thickening of alveolar or bronchiolar walls; grade 3, moderate thickening of walls without obvious damage to lung architecture; grade 5, increased fibrosis with definite damage to lung structure and formation of fibrous bands or small fibrous masses; grade 7, severe distortion of structure and large fibrous areas; grade 8, total fibrous obliteration of fields. If there was difficulty in deciding between two odd-numbered categories, the field would be given the intervening even-numbered grade. The observations were then entered into a repeated measures analysis of variance. Treatment (three levels) was considered fixed and field (44 levels) was repeated with a compound symmetric structure. Scoring of the untreated and PG490-alone groups both gave values of zero so the data were not included in the analysis of variance because they violated the normality assumptions underlying the analysis.
Immunohistochemistry
Paraffin-embedded tissue from control and bleomycin-treated lungs
were processed for immunohistochemical localization of -smooth
muscle actin to identify myofibroblasts. Briefly, tissue sections were
dewaxed in xylene and rehydrated through graded concentrations of
ethanol. Slides were blocked with protein block serum-free (DAKO Corp.,
Carpinteria, CA) and overlaid with a 1:400 dilution of monoclonal mouse
anti--smooth muscle actin antibody overnight (Sigma-Aldrich Inc.,
St. Louis, MO) or phosphate-buffered saline (PBS) as negative control
at 4°C. Slides were then rinsed and overlaid with biotinylated
anti-mouse link immunoglobulins (Universal Dako LSAB Kit, Peroxidase;
DAKO) for 60 minutes. After washing with PBS, slides were then overlaid
with horseradish peroxidase conjugated to streptavidin (DAKO) and
incubated for 60 minutes in 3,3-diaminobenzidine (DAB) chromogen for
chromogenic localization of actin (liquid DAB substrate kit; ZYMED
Laboratories, Inc., San Francisco, CA). After optimal color
development, sections were immersed in sterile water, counterstained
with Harris alum hematoxylin, and coverslipped using an aqueous
mounting solution.
Hydroxproline Assay
To estimate the total amount of collagen deposited as an indicator
of pulmonary fibrosis, the hydroxyproline content of whole lung was
measured in each group according to the procedure described by
Woessner.17
A spectrophotometric assay was used to
quantify the lung hydroxyproline. Briefly, lungs were harvested and
homogenized in 5 ml of saline with a Polytron homogenizer
(IKA-Labortechnik, Staufen, Germany). Each sample (0.5 ml) was then
digested in 2 ml of 6 N HCl for 16 hours at 110°C. The samples were
neutralized with NaOH until pH 7. One ml of 0.5 mol/L Chloramine T
reagent was then added and the samples were left at room temperature
for 20 minutes. Next, 1 ml of 3.15 N perchloric acid and
P-dimethylaminobenzaldehyde were added to each sample, and
the samples were incubated for 20 minutes at 65°C. Samples were
cooled for 10 minutes and read at 557 nm on a DU-64 spectrophotometer
(Beckman Coulter, Inc., Hialeah, FL).
trans-4-hydroxy-L-proline (Sigma)
concentrations from 0 to 5 µg/ml were used to construct a standard
curve. The data were first log-transformed to remove the observed
correlation between the means and variances. The observations were then
entered into a mixed model analysis of variance, with experiment (four
levels) considered random and treatment (five levels) considered fixed.
The geometric least squares means were obtained by exponentiation from
the least squares means (see Figure 3
).
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The BALF was obtained 14 days after bleomycin treatment from three mice in each group. TGF-ß levels in the BALF were assayed using a commercially available TGF-ß ELISA kit (TGF-ß E max ImmunoAssay System; Promega Corp., Madison, WI). The kit contains a TGF-ß coat monoclonal antibody for a 96-well microtiter plate coating and immunomobilized mouse polyclonal antibody to TGF-ß with a reported sensitivity of 15.6 pg/ml. BALF was obtained from the mice under anesthesia using 1 ml of sterile isotonic saline. Lavage was performed four times in each mouse and the total volume collected separately. The volume of fluid collected in each mouse ranged from 3.0 to 3.5 ml. The lavage fluid was centrifuged at 1,500 rpm at 4°C for 15 minutes. To assay for total TGF-ß, the supernatant was first acidified to process TGF-ß from a latent form to the bioactive form. The active form is immunoreactive and detected by the anti-TGB-ß antibody. The representative standard curve was generated using the TGF-ß standard provided with the kit. The observations were entered into a mixed model analysis of variance, with experiment (two levels) considered random and treatment (four levels) considered fixed.
Culture of Normal Human Lung Fibroblasts (NHLFs)
The NHLF cell line was obtained from Clonetics-BioWhittaker (San Diego, CA). NHLFs were cultured in fibroblast growth medium (Clonetics) at 37°C in a 5% CO2 atmosphere. The cultures were incubated at various concentrations of bleomycin (0, 0.1, and 10 µg/ml; Blenoxane, Novaplus, 15 U/Vial) for 16 hours and the cells were then harvested for RNA extraction.
Analysis of TGF-ß mRNA Expression
RT-PCR for TGF-ß mRNA was done on NHLFs treated with varying doses of bleomycin (0, 0.1, and 10 U). TGF-ß mRNA was measured in the presence or absence of PG490 (20 ng/ml) that was added the same time as TGF-ß and samples were harvested after 24 hours. Total RNA from NHLFs was extracted using Total RNA/mRNA isolation reagent (RNA STAT-60; Tei-Test"B", Inc.). Four mg of total RNA was used for reverse-transcription into cDNA with a cDNA synthesis kit (Life Technologies, Inc., Rockville, MD) and then amplified for 35 cycles in MiniCycle PCR system (MJ Research, Inc.) with denaturation at 95°C for 30 seconds, primer annealing at 55°C for 30 seconds, and primer extension at 72°C for 2 minutes. TGF-ß and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense and antisense oligonucleotides were: TGF-ß sense, 5*-CGC ATA CAG TTA TCT CGG ACG A C-3*; antisense, 5*-TTT GTT GGC TGC TCT CAC GG-3*; GAPDH sense, 5*-GGA GCC AAA AGG GTC ATC TC-3*, antisense, 5*-AGT GGG TGT CGC TGT TGA AGT C-3*. PCR products were separated by electrophoresis on 2% agarose gel with ethidium bromide (EB) and were visualized with a electronic UV Transilluminator (Ultra-Lum, Inc.).
Analysis of Cell Viability
Cell viability was measured by an of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Briefly, untreated cells or cells treated with PG490 in a 96-well plate at the dosages shown were harvested at 24 hours followed by the addition 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to the cells. Cells were then solubilized with 0.1 N acidified CH3Cl-HCl. The 96-well plate was read at a wavelength of 590 nm on an iEMS Labsystems plate reader.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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after sacrifice on day 15. The saline control group and the saline plus
PG490-88 (0.25 mg/kg/day) group showed normal lung architecture. The
bleomycin-alone group shows extensive inflammation and fibrosis. The
PG490-88 treatment groups showed a marked decrease in inflammation and
fibrosis when treatment is initiated on the same day as the bleomycin
insult or 5 days later. The animals treated with triptolide 5 days
after bleomycin were also sacrificed on day 15 so that they received
treatment with PG490-88 for 9 days. The extent of fibrosis was scored
by the method proposed by Ashcroft and colleagues.16
Forty-four randomly chosen fields from each animal (5 animals per
group) were used for analysis. Each sample was scored by a single
observer and numerical scores were assigned based on the degree of
fibrosis (see Materials and Methods for details). The mean scores are
as follows: saline control, 0; saline plus PG490, 0; bleomycin alone,
3.36; bleomycin plus PG490 treatment starting the same day, 1.07;
bleomycin plus PG490 treatment started 5 days later, 1.67 (Table 1)
. The degree of fibrosis was
significantly less (P < 0.001) in the treatment
group both when the treatment was started on the same day as bleomycin
or 5 days later. PG-490-88 was well tolerated with no change in
activity, skin color, and respiration. The PG490-88-treated mice also
showed an increase in body weight compared to the bleomycin group
(Table 1)
. There are no apparent adverse effects on long-term treatment
with PG490-88 at this dosage based on studies with up to 3 months of
dosing in a mouse xenograft tumor model (J. H. Fidler and G.
D. Rosen, unpublished results). However, dosages >0.75 to 1.0
mg/kg/day are associated with gastrointestinal toxicity manifested by
intestinal hemorrhage (J. H. Fidler and G. D. Rosen,
unpublished results).
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. The accumulation of
collagen is inhibited even when PG490-88 treatment is initiated 5 days
after bleomycin instillation.
We also immunostained the mouse lung tissue with -smooth muscle
actin that identifies myofibroblasts. Myofibroblast proliferation is
thought to be a hallmark of lung fibrosis and myofibroblasts in lung
fibrosis express -smooth muscle actin. There was a decrease in the
presence of myofibroblasts in the PG490-88-treated groups compared to
the bleomycin-alone group, again when given the same day or 5 days
after bleomycin (Figure 2)
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. The saline plus PG490 group had
hydroxyproline levels (0.519 µg/lung; n = 19) that
are almost identical to the control group (0.521 µg/lung;
n = 18) (Figure 3)
.
TGF-ß is known to play a central role in the pathogenesis of
pulmonary fibrosis.18-24
To examine the effect of
PG490-88 on TGF-ß, we assayed TGF-ß activity in BALF level by
ELISA: group 1: intratracheal saline plus PG490-88 (0.25 mg/kg); group
2: intratracheal bleomycin; group 3: bleomycin plus intraperitoneal
PG490-88 starting same day; and group 4: bleomycin plus intraperitoneal
PG490-88 (0.25 mg/kg) starting 5 days later. Bronchoalveolar lavage was
performed on day 15 in all of the groups under anesthesia and the
animals were then sacrificed. TGF-ß activity was significantly lower
in the treatment groups (P < 0.01): PG490-88
plus bleomycin (SD) (215.3 pg/ml) and PG490-88 plus bleomycin (5
days) (169.0 pg/ml) versus bleomycin alone (290.5 pg/ml)
(Figure 4)
.
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. It was possible that the
PG490-mediated suppression in TGF-ß transcription is a result of
antiproliferative or cytotoxic effects of PG490 on NHLFs. PG490 (20
ng/ml), however, only reduced NHLF viability by 15% after 24 hours of
treatment (Table 2)
. These data suggest,
therefore, that PG490 inhibits the bleomycin-mediated increase in
TGF-ß activity, at least in part, through inhibition of TGF-ß gene
expression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study was undertaken to demonstrate the antifibrotic properties of
PG490-88, a derivative of triptolide. Triptolide is an oxygenated
diterpine triepoxide isolated from Tripterygium wilfordii
hook.f (Celastraceae).26
The molecular structure of this
compound is shown in Figure 6
. Various
extracts from this plant have been used in traditional Chinese medicine
as a treatment for inflammatory conditions such as rheumatoid
arthritis.10
Triptolide has also been shown to have
antiproliferative, antileukemic, and immunosuppressive properties in
animal models and in transplant rejection models.15
For
example, we have recently shown that triptolide sensitizes tumor cells
to tumor necrosis factor--induced apoptosis by blocking tumor
necrosis factor--mediated activation of NF-B.15
Also, Qiu and colleagues showed that triptolide inhibits expression of
the proinflammatory cytokines interleukin-2 and interleukin-8 by
blocking activation of NF-B.14
Here we show that
PG490-88, a water-soluble derivative of triptolide inhibits
bleomycin-mediated inflammation and fibrosis. Triptolide may therefore
exert its action, in part, by blocking NF-B-mediated cytokine
production by immune cells in this model. Most agents that have been
studied using the bleomycin animal model show efficacy only when the
experimental drug is given simultaneously or before bleomycin. We show
here that PG490-88 inhibits pulmonary fibrosis even when administered 5
days after bleomycin.
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-1 collagen (I) mRNA gene transcription and increases the
RNA stability of collagen and fibronectin.22
In addition,
in combination with platelet-derived growth factor and epidermal growth
factor, it is mitogenic for fibroblasts, increases the production of
fibronectin by macrophages, and stimulates fibroblast chemotaxis.
Moreover, TGF-ß acts in concert with other cytokines to inhibit
fibroblast degradation by inhibiting the expression of tissue
inhibitors of metalloproteinases and plasminogen activator/plasminogen
activator inhibitors.23
In animal models of
bleomycin-induced fibrosis, TGF-ß mRNA expression has been shown to
precede increases in collagen production that correlates with areas of
active fibrosis.24,28
We show that PG490-88 inhibits the bleomycin-mediated increase in
TGF-ß activity in vivo and that PG490 (triptolide)
inhibits the bleomycin-mediated increase in TGF-ß transcription in
normal human lung fibroblasts (Figures 4 and 5)
. TGF-ß activity in
BAL was significantly lower in PG490-88 treatment groups even when
administered 5 days after bleomycin. The mechanism of this suppression
may result, in part, from triptolide-mediated inactivation of other
transcription factors such as nuclear factor of activated T cells and
Sp1 (M. Gao and G. D. Rosen, unpublished results).14
We have also observed that triptolide is a potent antitumor agent
in vivo (J. J. Fidler and G. D. Rosen, unpublished
results). We are presently looking for intracellular targets of
triptolide in an effort to decipher its potent antifibrotic,
immunosuppresive, and antitumor effects. Our finding that PG490-88
inhibits bleomycin-mediated fibrosis even when administered 5 days
after bleomycin suggests a potential role in the treatment of patients
with pulmonary fibrosis.
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Acknowledgements |
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Footnotes |
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Supported by the Suha and Khalid Shoman Family fund.
G. K. and K. L. both contributed equally to preparation of the manuscript.
Accepted for publication November 17, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B transcriptional activation. J Biol Chem 1999, 274:13443-13450 to induce apoptosis in tumor cells. J Biol Chem 1999, 274:13451-13455-1 collagen promotor through intracellular and extra cellular signalling. J Biol Chem 1994, 269:13156-13161This article has been cited by other articles:
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  W. Wang, S. Yang, Y. Su, Z. Xiao, C. Wang, X. Li, L. Lin, B. M. Fenton, S. F. Paoni, I. Ding, et al. Enhanced Antitumor Effect of Combined Triptolide and Ionizing Radiation Clin. Cancer Res., August 15, 2007; 13(16): 4891 - 4899. [Abstract] [Full Text] [PDF]
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 Y. Lu, Y. Liu, K. Fukuda, Y. Nakamura, N. Kumagai, and T. Nishida Inhibition by triptolide of chemokine, proinflammatory cytokine, and adhesion molecule expression induced by lipopolysaccharide in corneal fibroblasts. Invest. Ophthalmol. Vis. Sci., September 1, 2006; 47(9): 3796 - 3800. [Abstract] [Full Text] [PDF]
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Y. Lu, K. Fukuda, Y. Nakamura, K. Kimura, N. Kumagai, and T. Nishida Inhibitory Effect of Triptolide on Chemokine Expression Induced by Proinflammatory Cytokines in Human Corneal Fibroblasts Invest. Ophthalmol. Vis. Sci., July 1, 2005; 46(7): 2346 - 2352. [Abstract] [Full Text] [PDF]
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