December 2003
Volume 44, Issue 12
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Cornea  |   December 2003
Inhibition by Triptolide of IL-1–Induced Collagen Degradation by Corneal Fibroblasts
Author Affiliations
  • Ying Lu
    From the Departments of Biomolecular Recognition and Ophthalmology and
  • Ken Fukuda
    Ocular Pathophysiology, Yamaguchi University School of Medicine, Yamaguchi, Japan.
  • Keisuke Seki
    Ocular Pathophysiology, Yamaguchi University School of Medicine, Yamaguchi, Japan.
  • Yoshikuni Nakamura
    From the Departments of Biomolecular Recognition and Ophthalmology and
  • Naoki Kumagai
    From the Departments of Biomolecular Recognition and Ophthalmology and
  • Teruo Nishida
    From the Departments of Biomolecular Recognition and Ophthalmology and
Investigative Ophthalmology & Visual Science December 2003, Vol.44, 5082-5088. doi:10.1167/iovs.03-0476
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      Ying Lu, Ken Fukuda, Keisuke Seki, Yoshikuni Nakamura, Naoki Kumagai, Teruo Nishida; Inhibition by Triptolide of IL-1–Induced Collagen Degradation by Corneal Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2003;44(12):5082-5088. doi: 10.1167/iovs.03-0476.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Extracts of the herb Tripterygium wilfordii hook f, the major component of which is triptolide, have been used in traditional Chinese medicine for the treatment of rheumatoid arthritis. Triptolide also exerts many other biological actions both in vitro and in vivo. The effect of this agent on collagen degradation by cultured corneal fibroblasts was examined.

methods. Rabbit corneal fibroblasts were cultured in three-dimensional gels of type I collagen and in the absence or presence of interleukin (IL)-1β or triptolide. The extent of collagen degradation was determined by measurement of the amount of hydroxyproline generated by acid–heat hydrolysis of the culture supernatants. The activities of matrix metalloproteinase (MMP)-1 and plasmin were measured with the specific substrates thiopeptolide and S-2251, respectively. The release of MMPs into the culture supernatant was assessed by immunoblot analysis and gelatin zymography, and the abundance of MMP mRNAs in the cells was determined by reverse transcription and real-time polymerase chain reaction.

results. Triptolide inhibited the IL-1β–induced degradation of collagen by corneal fibroblasts in a dose- and time-dependent manner. Neither the activity of purified recombinant MMP-1 nor that of plasmin in culture supernatants was affected by triptolide. The IL-1β–induced expression of MMP-1, -2, -3, and -9 by corneal fibroblasts was inhibited by triptolide at the protein or mRNA level.

conclusions. Triptolide inhibits collagen degradation by corneal fibroblasts by inducing downregulation of the production of MMPs, without directly affecting the collagenolytic activity of these enzymes.

The cornea is an avascular and transparent tissue. Corneal transparency is dependent on the fact that collagen fibrils are packed more densely and in a more organized manner in the cornea than in any other tissue of the body. Keratocytes, which are the major resident cells of the corneal stroma, play an important role in collagen metabolism. These cells thus both synthesize 1 and degrade collagen fibrils, the latter of which is achieved by the release of matrix-degrading enzymes such as matrix metalloproteinases (MMPs). 2 The functions of corneal fibroblasts (activated keratocytes in in vitro culture), including proliferation 3 and the synthesis of MMPs, 4 are also continuously modulated by interaction of the cells with extracellular collagen. We have shown that three-dimensional culture of these cells in collagen gels mimics the situation in vivo more closely than does monolayer culture. 3 5 We also have established an assay system with which to measure the collagenolytic activity of corneal fibroblasts in such three-dimensional cultures and have shown that interleukin (IL)-1, Pseudomonas aeruginosa elastase, and neutrophils each stimulate collagen degradation by corneal fibroblasts. 6 7 8 Excessive degradation of collagen in the corneal stroma, mediated in part by MMPs, results in corneal ulceration. 
The MMP family comprises at least 23 secreted or membrane-bound zinc-dependent endopeptidases, 9 including collagenases, gelatinases, stromelysins, and matrilysin. Substrates for these enzymes include all known proteins of the extracellular matrix. MMPs are synthesized and secreted as inactive proenzymes that are activated by serine proteinases, such as plasmin, in the extracellular space. Extracts of the Chinese herb Tripterygium wilfordii hook f, the major constituent of which is the diterpene triepoxide triptolide, have been used in traditional Chinese medicine for the treatment of rheumatoid arthritis. 10 Triptolide exhibits anti-inflammatory activity in immune cells such as T cells, B cells, and monocytes. 11 12 It also acts on tissue-resident cells such as epithelial cells 13 and fibroblasts, 14 and it inhibits the expression of MMPs in chondrocytes and synovial fibroblasts. 15 16  
With the use of our model system, we examined whether, through an effect on MMP production, triptolide inhibits collagen degradation by corneal fibroblasts in three-dimensional cultures. Specifically, we investigated the possible effect of triptolide on the expression of MMPs in corneal fibroblasts at both the protein and mRNA levels and its effect on the collagen degradation mediated by these cells in response to IL-1β. 
Materials and Methods
Eagle’s minimum essential medium (MEM), fetal bovine serum, trypsin-EDTA, and trypan blue were obtained from Invitrogen-Gibco (Osaka, Japan); 24- and 96-well culture plates from Corning Glass Co. (Corning, NY); native porcine type I collagen (acid solubilized) and 5× Dulbecco’s modified Eagle’s medium (DMEM) from Nitta Gelatin (Osaka, Japan); bovine plasminogen, collagenase, dispase, and dimethyl sulfoxide (DMSO) from Sigma-Aldrich (St. Louis, MO); recombinant human IL-1β from R&D Systems (Minneapolis, MN); triptolide from Alexis Biochemicals (Carlsbad, CA); filters (Ultrafree-MC) from Millipore (Bedford, MA); an MMP-1 colorimetric assay kit from Biomol (Plymouth Meeting, PA); synthetic substrate S-2251 for plasmin from Chromogenix (Milan, Italy); nitrocellulose membranes and a chemiluminescence (ECL) kit from Amersham Pharmacia Biotech (Uppsala, Sweden); kits for RNA purification (RNeasy Mini Kit) and polymerase chain reaction (QuantiTect SYBR Green PCR Kit) from Qiagen (Hilden, Germany); a reverse transcription system and a cytotoxicity assay (CytoTox 96 Non-Radioactive) from Promega (Madison, WI). Sheep antibodies to rabbit MMP-1 and -3 were kindly provided by Hideaki Nagase. All media and reagents used for cell culture were endotoxin minimized. 
Cell Isolation
Male Japanese albino rabbits (body weight, 2.0–2.5 kg) were obtained from Biotec (Saga, Japan). This study adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and was approved by the Animal Experimental Committee of Yamaguchi University School of Medicine. Rabbit corneal fibroblasts were isolated and maintained as described previously. 7 In brief, the endothelial layer of the cornea was removed mechanically, and the tissue was then incubated with dispase (2 mg/mL, in MEM) for 1 hour at 37°C. After mechanical removal of the epithelial sheet, the tissue was treated with collagenase (2 mg/mL, in MEM) at 37°C until a single-cell suspension of corneal fibroblasts was obtained. Isolated corneal fibroblasts were maintained in a humidified atmosphere containing 5% CO2 at 37°C in MEM supplemented with 10% fetal bovine serum. The cells were used for experiments after four to seven passages and were harvested at subconfluence, in the actively proliferating state. 
Three-Dimensional Culture
Collagen gels were prepared as described. 7 In brief, corneal fibroblasts were harvested by exposure to trypsin-EDTA, collected by centrifugation, and resuspended in serum-free MEM. Acid-solubilized type I collagen (3 mg/mL), 5× DMEM, reconstitution buffer (0.05 M NaOH, 0.26 M Na2CO3, and 0.2 M HEPES [pH 7.3]), and corneal fibroblast suspension (2.2 × 106 cells/mL in MEM) were mixed on ice at a ratio of 7:2:1:1. The resultant mixture (0.5 mL) was added to each well of a 24-well culture plate and allowed to solidify in an incubator under 5% CO2 at 37°C, after which 0.5 mL of serum-free MEM containing test agent and plasminogen (60 μg/mL) was overlaid and the cultures were returned to the incubator for the indicated times. Triptolide was dissolved and diluted in DMSO; the final DMSO concentration was 0.2% in all cultures containing this agent, and the same amount of vehicle was added to control cultures. 
Assay of Collagenolytic Activity
Measurement of degraded collagen in culture supernatants was performed as previously described. 7 8 In brief, the supernatants from collagen gel incubations were collected, and native collagen fibrils with a molecular size of more than 100 kDa were removed by ultrafiltration. The filtrate was then subjected to hydrolysis with 6 M HCl for 24 hours at 110°C. The amount of hydroxyproline in the hydrolysate was measured spectrophotometrically, and the amount of degraded collagen was expressed as micrograms of hydroxyproline per well. 
Measurement of MMP-1 Activity
The direct effect of triptolide on the protease activity of MMP-1 was evaluated with an assay kit. Corneal fibroblasts were cultured in collagen gels in the presence of plasminogen and IL-1β (0.1 ng/mL) for 48 hours, after which the culture supernatants were collected and centrifuged (4000g for 5 minutes) to remove cells and debris. The resultant supernatants were assayed immediately or stored at −80°C. The supernatants (20 μL) or recombinant human MMP-1 (rhMMP-1; 3 mU) were incubated for 30 minutes at 37°C in a 96-well plate with various concentrations of triptolide or of the specific MMP-1 inhibitor NNGH, after which the reaction was started by addition of the colorimetric substrate thiopeptolide. The absorbance of each well at 412 nm was measured continuously with a microplate reader between 5 and 60 minutes after substrate addition, and the reaction velocity was expressed as absorbance units per minute. 
Measurement of Plasmin Activity
Plasmin activity was measured with the substrate S-2251, as described previously. 6 In brief, culture supernatants (100 μL) were incubated at 37°C in the wells of a 96-well plate with 20 μL of 50 mM Tris-HCl (pH 7.4) containing 0.01% Triton X-100 and with 100 μL of 0.6 mM S-2251. The release of p-nitroanilide during 30 minutes was monitored by measurement of absorbance at 405 nm with a microplate reader. 
Immunoblot Analysis
Immunoblot analysis of rabbit MMP-1 and MMP-3 was performed as described. 7 Culture supernatants were subjected to SDS-polyacrylamide gel electrophoresis on a 10% gel under reducing conditions, and the separated proteins were then transferred electrophoretically to a nitrocellulose membrane. After nonspecific sites were blocked, the membrane was incubated with sheep antibodies to rabbit MMP-1 or -3. Immune complexes were detected with the use of enhanced chemiluminescence reagents. 
Gelatin Zymography
Gelatin zymography of culture supernatants was performed as described previously. 7 In brief, culture supernatants (4 μL) were mixed with 2 μL of nonreducing SDS sample buffer(125 mM Tris-HCl [pH 6.8], 20% glycerol, 2% SDS, 0.002% bromphenol blue) and fractionated by SDS-polyacrylamide gel electrophoresis at 4°C on a 10% gel containing 0.1% gelatin. The gel was then washed in 2.5% Triton X-100 for 1 hour, to promote recovery of protease activity, before incubation for 18 hours at 37°C in a reaction buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM CaCl2, and 1% Triton X-100. The gel was finally stained with Coomassie brilliant blue. 
Quantitative Real-Time PCR Analysis
After culture for 12 hours, corneal fibroblasts were extracted from the collagen gel by incubation with 0.01% collagenase for 30 minutes at 37°C. Total RNA was then isolated from the cells and subjected to reverse transcription. The abundance of MMP-1, -2, -3, and -9, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs was quantified by real-time polymerase chain reaction (PCR) analysis with an automated PCR system (LightCycler; Roche Molecular Biochemicals, Indianapolis, IN), as described previously. 8 17 The sequences of the PCR primers for MMP-1, 8 -2, 18 -3, 8 and -9 19 and GAPDH 8 cDNAs were also as described previously. These primers yielded PCR products of the expected sizes of 649, 313, 306, 271, and 293 bp, respectively. 
Statistical Analysis
Data are expressed as the mean ± SEM. Statistical analysis was performed with Dunnett’s multiple comparison test or Student’s unpaired t-test. P < 0.05 was considered statistically significant. 
Results
Inhibitory Effect of Triptolide on Collagen Degradation by Rabbit Corneal Fibroblasts
We first examined the effect of IL-1β on collagen degradation in three-dimensional cultures of corneal fibroblasts. The cells were cultured in the presence of various concentrations of IL-1β for 48 hours. Consistent with our previous observations, 6 IL-1β increased collagen degradation by corneal fibroblasts in a dose-dependent manner. Its effect was statistically significant at a concentration of 0.001 ng/mL, and maximum effect was obtained at 0.1 ng/mL (Fig. 1)
To determine whether triptolide induces cytotoxic effects in corneal fibroblasts, we treated confluent monolayers of the cells with various concentrations of the agent (0.03–3.0 μM) for 48 hours. The cells were then stained with trypan blue, and the culture supernatants were assayed for the release of lactate dehydrogenase. Triptolide did not exhibit cytotoxicity at any of the concentrations examined (data not shown). 
We next examined the effect of triptolide on collagen degradation by corneal fibroblasts. The cells were incubated for 48 hours in the absence or presence of IL-1β (0.1 ng/mL) and in the presence of various concentrations of triptolide (0.03–3.0 μM). In the absence of IL-1β, triptolide had no significant effect on collagen degradation at any concentration examined. However, triptolide inhibited in a dose-dependent manner the collagen degradation induced by IL-1β. The inhibitory effect was statistically significant at a triptolide concentration of 0.3 μM (Fig. 2) . Time course analysis revealed that the inhibitory effect of triptolide (3 μM) on the collagen degradation induced by IL-1β (0.1 ng/mL) was significant at 24, 36, and 48 hours of culture (Fig. 3)
Effect of Triptolide on MMP-1 or Plasmin Activity
To investigate the mechanism by which triptolide inhibits collagen degradation by corneal fibroblasts, we first examined whether this agent exerts a direct effect on MMP activity. Whereas the specific MMP-1 inhibitor NNGH inhibited the activity of rhMMP-1 in a dose-dependent manner, triptolide (0.03–3.0 μM) did not affect the activity of this enzyme (Fig. 4) . Similarly, the MMP-1 activity present in the culture supernatants of corneal fibroblasts incubated in collagen gels for 48 hours with IL-1β (0.1 ng/mL) was inhibited by NNGH but not by triptolide. These results thus demonstrate that triptolide did not inhibit MMP-1 activity directly. 
We have shown that the addition of plasminogen is important for collagen degradation by corneal fibroblasts in our culture system. 6 Plasminogen activator mediates the conversion of plasminogen to plasmin, which then activates latent MMPs. We therefore investigated the possible effect of culture with triptolide on the activity of plasmin present in culture supernatants of corneal fibroblasts stimulated with IL-1β. The plasmin activity of the culture supernatants was not affected by the absence or presence of triptolide (Fig. 5)
Effects of Triptolide on the Expression of MMPs by Corneal Fibroblasts
We next examined the effects of triptolide on the expression of MMPs in corneal fibroblasts by immunoblot analysis. Analysis with antibodies to MMP-1 revealed that the culture supernatants of cells maintained in collagen gels for 48 hours in the absence of IL-1β and triptolide contained a relatively small amount of a 57-kDa immunoreactive protein corresponding to proMMP-1 as well as 49- and 45-kDa immunoreactive proteins corresponding to active MMP-1 (Fig. 6A) . Culture of cells in the presence of IL-1β (0.1 ng/mL) resulted in an increase in the intensity of the bands corresponding to proMMP-1 and active MMP-1. The additional presence of triptolide in the cultures induced a dose-dependent decrease in the abundance of both proMMP-1 and active MMP-1 in the culture supernatant. 
Immunoblot analysis with antibodies to MMP-3 revealed that, under basal conditions, corneal fibroblasts produced small amounts of a 57-kDa immunoreactive protein corresponding to proMMP-3 and a 45-kDa immunoreactive protein corresponding to active MMP-3 (Fig. 6A) . The abundance of both of these proteins was increased by culture of the cells in the presence of IL-1β (0.1 ng/mL), and this stimulatory effect of IL-1β was inhibited in a concentration-dependent manner by triptolide. 
Reverse transcription and real-time PCR revealed that culture of corneal fibroblasts in collagen gels for 12 hours with IL-1β (0.1 ng/mL) resulted in an 11.4-fold increase in the amount of MMP-1 mRNA compared with that present in cells cultured in the absence of cytokine (Fig. 6B) . Triptolide (3.0 μM) inhibited this effect of IL-1β by 62%. Similarly, IL-1β induced an 8.4-fold increase in the amount of MMP-3 mRNA in corneal fibroblasts, and triptolide inhibited this effect of IL-1β by 66%. 
Gelatin zymography of culture supernatants obtained after incubation of corneal fibroblasts in collagen gels under basal conditions for 48 hours revealed three major bands of 89, 65, and 57 kDa, corresponding to an intermediate form of MMP-9, proMMP-2, and active MMP-2, respectively (Fig. 7A) . Culture of cells in the additional presence of IL-1β (0.1 ng/mL) resulted in an increase in the intensity of the bands corresponding to proMMP-2 and active MMP-2, the disappearance of the band corresponding to the intermediate form of MMP-9, and the appearance of bands at 92 and 77 kDa corresponding to proMMP-9 and active MMP-9, respectively. Triptolide markedly inhibited the effects of IL-1β on the gelatinolytic bands corresponding to proMMP-2, active MMP-2, proMMP-9, and active MMP-9. 
Reverse transcription and real-time PCR revealed that culture of corneal fibroblasts in collagen gels for 12 hours with IL-1β (0.1 ng/mL) induced a 1.8-fold increase in the amount of MMP-2 mRNA (Fig. 7B) . This effect of IL-1β was not inhibited by triptolide (3.0 μM). IL-1β also induced a 5.2-fold increase in the abundance of MMP-9 mRNA, and triptolide inhibited this effect by 36%. 
Discussion
In the present study, triptolide inhibited IL-1β–induced collagen degradation by corneal fibroblasts. It did not inhibit MMP activity directly, nor did it inhibit the activity of plasmin present in culture supernatants, suggesting that its effect on collagen degradation is mediated by an action on corneal fibroblasts. Indeed, triptolide inhibited the production of MMPs by corneal fibroblasts at both the protein and mRNA levels. 
Many chemical agents, including EDTA, tetracycline, cysteine, acetylcysteine, a thiol peptide, and ilomastat, that inhibit MMP activity directly by binding to the active sites of these enzymes have been investigated for their potential to treat corneal ulceration. 20 21 22 Endogenous nonspecific inhibitors, such as α2-macroglobulin, as well as specific inhibitors such as tissue inhibitors of metalloproteinases (TIMPs) have also been shown to suppress corneal ulceration in animal models. 23 24 The inhibition of MMP activity by any of these various agents has not been sufficient, however, to promote full recovery from corneal ulceration. Our results now suggest that downregulation of the synthesis of MMPs in corneal fibroblasts by triptolide or similar agents may provide an alternative approach to the treatment of corneal ulceration. 
Cultured corneal fibroblasts produce MMP-1, -2, -3, and -9. 25 MMP-1 is the principal collagenolytic enzyme responsible for the degradation of type I collagen. Although MMP-3 does not degrade collagen type I, it participates in the activation of other MMPs. 26 The two gelatinases MMP-2 and -9 degrade type I collagen further after its initial cleavage by MMP-1 and subsequent denaturation of the three collagen chains. 2 In our study, that triptolide inhibited the IL-1β–induced synthesis of MMP-1, -2, -3, and -9 in corneal fibroblasts. These results are consistent with the previous observation that triptolide inhibited the IL-1β–induced synthesis of MMP-1 and -3 in human synovial fibroblasts. 16 T. wilfordii hook f extract, the major component of which is triptolide, also inhibited the synthesis of MMP-3 and -13 in human and bovine chondrocytes induced by tumor necrosis factor-α, IL-1, or IL-17. 15  
Many inflammatory cytokines, including tumor necrosis factor-α, IL-6, and, most prominently, IL-1, are implicated in corneal ulceration. 27 IL-1 stimulates both the synthesis of MMPs and collagen degradation by corneal fibroblasts, 6 and its concentration is increased in tear fluid of ulcerated eyes and in the ulcerated cornea. 28 29 Inhibition of IL-1 by an IL-1 receptor antagonist reduces the severity of mouse bacterial keratitis in vivo, indicating the importance of this cytokine in the pathogenesis of melting of the corneal stroma. 27 Our finding that triptolide inhibited IL-1β–induced collagen degradation by corneal fibroblasts in vitro thus suggests that this agent may ameliorate corneal ulceration in vivo. 
IL-1 also acts as an intermediary in the stimulation of MMP synthesis by other factors. Phorbol 12-myristate 13-acetate stimulates the synthesis and release of IL-1 in corneal fibroblasts, and the released cytokine then induces the synthesis of MMPs. 30 31 Integrin-mediated changes in cell shape also activate and sustain the IL-1 feedback loop and thereby stimulate MMP synthesis in synovial fibroblasts. 32 IL-1 induces activation of the MMP-1 gene promoter by nuclear factor (NF)-κB and activator protein (AP)-1 in corneal fibroblasts. 33 T. wilfordii hook f extract suppresses MMP gene expression by inhibiting the DNA binding activities of NF-κB and AP-1 in human and bovine chondrocytes. 15 Triptolide also inhibits NF-κB or AP-1 activity in several cell types, including T cells, human bronchial epithelial cells, and gastric cancer cells. 12 34 It is therefore possible that triptolide interferes with IL-1β signaling mediated by these transcription factors in corneal fibroblasts and thereby inhibits MMP expression in these cells. 
The plasminogen-plasmin system is implicated in the initiation and perpetuation of collagen degradation associated with corneal ulceration. 35 Human tear fluid contains both plasminogen activator and plasmin activities. 36 37 We have shown that exogenous plasminogen is important for collagen degradation by corneal fibroblasts in our assay system. Thus, in the absence of added plasminogen, IL-1 increases the synthesis of MMPs by corneal fibroblasts, but collagen degradation does not occur. 6 The plasminogen–plasmin system is thus required for the activation of proMMPs produced by the corneal fibroblasts. We also have shown that galardin inhibits collagen degradation by corneal fibroblasts by preventing the conversion of proMMPs to active MMPs. 6 Our present results, however, show that triptolide did not affect the plasmin activity in culture supernatants or the conversion of proMMPs to MMPs, but rather inhibited the synthesis of these proteases. 
An imbalance between MMPs and TIMPs has been implicated in the pathogenesis of corneal ulceration. 38 Triptolide promotes the expression of TIMP-1 and -2 in human synovial fibroblasts. 16 The possibility that triptolide also increases the expression of TIMPs in corneal fibroblasts remains to be examined. 
Corneal ulceration is a severe disorder that can cause blindness. We have now shown that triptolide inhibits collagen degradation by corneal fibroblasts by inducing downregulation of the production of MMPs, without directly affecting the collagenolytic activity of these enzymes. Triptolide may thus be an effective therapeutic agent for corneal ulceration. 
 
Figure 1.
 
Stimulatory effect of IL-1β on collagen degradation by corneal fibroblasts. Cells were cultured in collagen gels in the presence of plasminogen and the indicated concentrations of IL-1β for 48 hours, after which the amount of degraded collagen was determined. Data are expressed as micrograms of hydroxyproline (HYP) per well and are the mean ± SEM of triplicate results from an experiment that was repeated three times with similar results. *P < 0.05, **P < 0.001 (Dunnett test) versus the corresponding value for cells cultured without IL-1β.
Figure 1.
 
Stimulatory effect of IL-1β on collagen degradation by corneal fibroblasts. Cells were cultured in collagen gels in the presence of plasminogen and the indicated concentrations of IL-1β for 48 hours, after which the amount of degraded collagen was determined. Data are expressed as micrograms of hydroxyproline (HYP) per well and are the mean ± SEM of triplicate results from an experiment that was repeated three times with similar results. *P < 0.05, **P < 0.001 (Dunnett test) versus the corresponding value for cells cultured without IL-1β.
Figure 2.
 
Dose-dependent inhibition by triptolide of IL-1β–induced collagen degradation by corneal fibroblasts. Cells were cultured in collagen gels in the presence of plasminogen, in the absence (○) or presence (•) of IL-1β (0.1 ng/mL), and in the presence of the indicated concentrations of triptolide for 48 hours, after which the amount of degraded collagen was determined. Data are the mean ± SEM of triplicate results from an experiment that was repeated three times with similar results. *P < 0.001 (Dunnett test) versus the corresponding value for cells cultured without triptolide.
Figure 2.
 
Dose-dependent inhibition by triptolide of IL-1β–induced collagen degradation by corneal fibroblasts. Cells were cultured in collagen gels in the presence of plasminogen, in the absence (○) or presence (•) of IL-1β (0.1 ng/mL), and in the presence of the indicated concentrations of triptolide for 48 hours, after which the amount of degraded collagen was determined. Data are the mean ± SEM of triplicate results from an experiment that was repeated three times with similar results. *P < 0.001 (Dunnett test) versus the corresponding value for cells cultured without triptolide.
Figure 3.
 
Time course of the inhibitory effect of triptolide on IL-1β–induced collagen degradation by corneal fibroblasts. Cells were cultured for the indicated times in collagen gels in the presence of plasminogen, in the absence (open symbols) or presence (closed symbols) of IL-1β (0.1 ng/mL), and in the absence (circles) or presence (squares) of 3.0 μM triptolide. The amount of degraded collagen was then determined. Data are the mean ± SEM of triplicate results from an experiment that was repeated three times with similar results. *P < 0.05, **P < 0.001 (Student’s t-test) versus the corresponding value for cells cultured with IL-1β and without triptolide.
Figure 3.
 
Time course of the inhibitory effect of triptolide on IL-1β–induced collagen degradation by corneal fibroblasts. Cells were cultured for the indicated times in collagen gels in the presence of plasminogen, in the absence (open symbols) or presence (closed symbols) of IL-1β (0.1 ng/mL), and in the absence (circles) or presence (squares) of 3.0 μM triptolide. The amount of degraded collagen was then determined. Data are the mean ± SEM of triplicate results from an experiment that was repeated three times with similar results. *P < 0.05, **P < 0.001 (Student’s t-test) versus the corresponding value for cells cultured with IL-1β and without triptolide.
Figure 4.
 
Lack of effect of triptolide effect on the activity of MMP-1. Either rhMMP-1 or culture supernatants from corneal fibroblasts (maintained in collagen gels for 48 hours in the presence of plasminogen and IL-1β [0.1 ng/mL]) were incubated for 30 minutes at 37°C in the presence of the indicated concentrations of triptolide or the specific MMP-1 inhibitor NNGH. The reaction was started by the addition of MMP-1 substrate. Data are expressed as a percentage of control (no NNGH or triptolide) and are the mean ± SEM of triplicate results from an experiment that was repeated three times with similar results. *P < 0.01, **P < 0.001 (Dunnett test) versus corresponding control.
Figure 4.
 
Lack of effect of triptolide effect on the activity of MMP-1. Either rhMMP-1 or culture supernatants from corneal fibroblasts (maintained in collagen gels for 48 hours in the presence of plasminogen and IL-1β [0.1 ng/mL]) were incubated for 30 minutes at 37°C in the presence of the indicated concentrations of triptolide or the specific MMP-1 inhibitor NNGH. The reaction was started by the addition of MMP-1 substrate. Data are expressed as a percentage of control (no NNGH or triptolide) and are the mean ± SEM of triplicate results from an experiment that was repeated three times with similar results. *P < 0.01, **P < 0.001 (Dunnett test) versus corresponding control.
Figure 5.
 
Lack of effect of triptolide on the activity of plasmin. Corneal fibroblasts were cultured in collagen gels in the presence of plasminogen, in the absence (○) or presence (•) of IL-1β (0.1 ng/mL) and in the presence of the indicated concentrations of triptolide for 48 hours, after which the activity of plasmin in the culture supernatants was determined. Data are expressed in absorbance units and are the mean ± SEM of triplicate results from an experiment that was repeated three times with similar results.
Figure 5.
 
Lack of effect of triptolide on the activity of plasmin. Corneal fibroblasts were cultured in collagen gels in the presence of plasminogen, in the absence (○) or presence (•) of IL-1β (0.1 ng/mL) and in the presence of the indicated concentrations of triptolide for 48 hours, after which the activity of plasmin in the culture supernatants was determined. Data are expressed in absorbance units and are the mean ± SEM of triplicate results from an experiment that was repeated three times with similar results.
Figure 6.
 
Effects of triptolide on the expression of MMP-1 and -3 by corneal fibroblasts. (A) Corneal fibroblasts were cultured in collagen gels for 48 hours in the presence of plasminogen, in the absence or presence of IL-1β (0.1 ng/mL), and in the presence of the indicated concentrations of triptolide. The culture supernatants were then subjected to immunoblot analysis with antibodies to either MMP-1 (top) or -3 (bottom). Data are representative of three independent experiments. The positions of the pro and active forms of MMP-1 and -3 are indicated on the right, and those of molecular size standards are shown on the left. (B) Corneal fibroblasts were cultured in collagen gels for 12 hours in the presence of plasminogen and in the absence or presence of IL-1β (0.1 ng/mL) or 3.0 μM triptolide, as indicated. The amounts of MMP-1 mRNA (left) and MMP-3 mRNA (right) in the cells were then determined by reverse transcription and real-time PCR analysis. Data are normalized on the basis of the abundance of GAPDH mRNA, are expressed in arbitrary units, and are the mean ± SEM of results in three experiments. *P < 0.001 (Student’s t-test) versus the corresponding value for cells cultured without IL-1β; #P < 0.01 (Student’s t-test) versus the corresponding value for cells cultured without triptolide.
Figure 6.
 
Effects of triptolide on the expression of MMP-1 and -3 by corneal fibroblasts. (A) Corneal fibroblasts were cultured in collagen gels for 48 hours in the presence of plasminogen, in the absence or presence of IL-1β (0.1 ng/mL), and in the presence of the indicated concentrations of triptolide. The culture supernatants were then subjected to immunoblot analysis with antibodies to either MMP-1 (top) or -3 (bottom). Data are representative of three independent experiments. The positions of the pro and active forms of MMP-1 and -3 are indicated on the right, and those of molecular size standards are shown on the left. (B) Corneal fibroblasts were cultured in collagen gels for 12 hours in the presence of plasminogen and in the absence or presence of IL-1β (0.1 ng/mL) or 3.0 μM triptolide, as indicated. The amounts of MMP-1 mRNA (left) and MMP-3 mRNA (right) in the cells were then determined by reverse transcription and real-time PCR analysis. Data are normalized on the basis of the abundance of GAPDH mRNA, are expressed in arbitrary units, and are the mean ± SEM of results in three experiments. *P < 0.001 (Student’s t-test) versus the corresponding value for cells cultured without IL-1β; #P < 0.01 (Student’s t-test) versus the corresponding value for cells cultured without triptolide.
Figure 7.
 
Effects of triptolide on the expression of MMP-2 and -9 by corneal fibroblasts. (A) Corneal fibroblasts were cultured in collagen gels for 48 hours in the presence of plasminogen, in the absence or presence of IL-1β (0.1 ng/mL), and in the presence of the indicated concentrations of triptolide. The culture supernatants were then subjected to gelatin zymography. Data are representative of three independent experiments. The positions of pro, intermediate, and active forms of MMP-2 or -9 are indicated on the right. (B) Corneal fibroblasts were cultured in collagen gels for 12 hours in the presence of plasminogen and in the absence or presence of IL-1β (0.1 ng/mL) or 3.0 μM triptolide, as indicated. The amounts of MMP-2 mRNA (left) and MMP-9 mRNA (right) in the cells were then determined by reverse transcription and real-time PCR analysis. Data are normalized on the basis of the abundance of GAPDH mRNA, are expressed in arbitrary units, and are the mean ± SEM of results in three experiments. *P < 0.05, **P < 0.001 (Student’s t-test) versus the corresponding value for cells cultured without IL-1β; #P < 0.01 (Student’s t-test) versus the corresponding value for cells cultured without triptolide.
Figure 7.
 
Effects of triptolide on the expression of MMP-2 and -9 by corneal fibroblasts. (A) Corneal fibroblasts were cultured in collagen gels for 48 hours in the presence of plasminogen, in the absence or presence of IL-1β (0.1 ng/mL), and in the presence of the indicated concentrations of triptolide. The culture supernatants were then subjected to gelatin zymography. Data are representative of three independent experiments. The positions of pro, intermediate, and active forms of MMP-2 or -9 are indicated on the right. (B) Corneal fibroblasts were cultured in collagen gels for 12 hours in the presence of plasminogen and in the absence or presence of IL-1β (0.1 ng/mL) or 3.0 μM triptolide, as indicated. The amounts of MMP-2 mRNA (left) and MMP-9 mRNA (right) in the cells were then determined by reverse transcription and real-time PCR analysis. Data are normalized on the basis of the abundance of GAPDH mRNA, are expressed in arbitrary units, and are the mean ± SEM of results in three experiments. *P < 0.05, **P < 0.001 (Student’s t-test) versus the corresponding value for cells cultured without IL-1β; #P < 0.01 (Student’s t-test) versus the corresponding value for cells cultured without triptolide.
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Figure 1.
 
Stimulatory effect of IL-1β on collagen degradation by corneal fibroblasts. Cells were cultured in collagen gels in the presence of plasminogen and the indicated concentrations of IL-1β for 48 hours, after which the amount of degraded collagen was determined. Data are expressed as micrograms of hydroxyproline (HYP) per well and are the mean ± SEM of triplicate results from an experiment that was repeated three times with similar results. *P < 0.05, **P < 0.001 (Dunnett test) versus the corresponding value for cells cultured without IL-1β.
Figure 1.
 
Stimulatory effect of IL-1β on collagen degradation by corneal fibroblasts. Cells were cultured in collagen gels in the presence of plasminogen and the indicated concentrations of IL-1β for 48 hours, after which the amount of degraded collagen was determined. Data are expressed as micrograms of hydroxyproline (HYP) per well and are the mean ± SEM of triplicate results from an experiment that was repeated three times with similar results. *P < 0.05, **P < 0.001 (Dunnett test) versus the corresponding value for cells cultured without IL-1β.
Figure 2.
 
Dose-dependent inhibition by triptolide of IL-1β–induced collagen degradation by corneal fibroblasts. Cells were cultured in collagen gels in the presence of plasminogen, in the absence (○) or presence (•) of IL-1β (0.1 ng/mL), and in the presence of the indicated concentrations of triptolide for 48 hours, after which the amount of degraded collagen was determined. Data are the mean ± SEM of triplicate results from an experiment that was repeated three times with similar results. *P < 0.001 (Dunnett test) versus the corresponding value for cells cultured without triptolide.
Figure 2.
 
Dose-dependent inhibition by triptolide of IL-1β–induced collagen degradation by corneal fibroblasts. Cells were cultured in collagen gels in the presence of plasminogen, in the absence (○) or presence (•) of IL-1β (0.1 ng/mL), and in the presence of the indicated concentrations of triptolide for 48 hours, after which the amount of degraded collagen was determined. Data are the mean ± SEM of triplicate results from an experiment that was repeated three times with similar results. *P < 0.001 (Dunnett test) versus the corresponding value for cells cultured without triptolide.
Figure 3.
 
Time course of the inhibitory effect of triptolide on IL-1β–induced collagen degradation by corneal fibroblasts. Cells were cultured for the indicated times in collagen gels in the presence of plasminogen, in the absence (open symbols) or presence (closed symbols) of IL-1β (0.1 ng/mL), and in the absence (circles) or presence (squares) of 3.0 μM triptolide. The amount of degraded collagen was then determined. Data are the mean ± SEM of triplicate results from an experiment that was repeated three times with similar results. *P < 0.05, **P < 0.001 (Student’s t-test) versus the corresponding value for cells cultured with IL-1β and without triptolide.
Figure 3.
 
Time course of the inhibitory effect of triptolide on IL-1β–induced collagen degradation by corneal fibroblasts. Cells were cultured for the indicated times in collagen gels in the presence of plasminogen, in the absence (open symbols) or presence (closed symbols) of IL-1β (0.1 ng/mL), and in the absence (circles) or presence (squares) of 3.0 μM triptolide. The amount of degraded collagen was then determined. Data are the mean ± SEM of triplicate results from an experiment that was repeated three times with similar results. *P < 0.05, **P < 0.001 (Student’s t-test) versus the corresponding value for cells cultured with IL-1β and without triptolide.
Figure 4.
 
Lack of effect of triptolide effect on the activity of MMP-1. Either rhMMP-1 or culture supernatants from corneal fibroblasts (maintained in collagen gels for 48 hours in the presence of plasminogen and IL-1β [0.1 ng/mL]) were incubated for 30 minutes at 37°C in the presence of the indicated concentrations of triptolide or the specific MMP-1 inhibitor NNGH. The reaction was started by the addition of MMP-1 substrate. Data are expressed as a percentage of control (no NNGH or triptolide) and are the mean ± SEM of triplicate results from an experiment that was repeated three times with similar results. *P < 0.01, **P < 0.001 (Dunnett test) versus corresponding control.
Figure 4.
 
Lack of effect of triptolide effect on the activity of MMP-1. Either rhMMP-1 or culture supernatants from corneal fibroblasts (maintained in collagen gels for 48 hours in the presence of plasminogen and IL-1β [0.1 ng/mL]) were incubated for 30 minutes at 37°C in the presence of the indicated concentrations of triptolide or the specific MMP-1 inhibitor NNGH. The reaction was started by the addition of MMP-1 substrate. Data are expressed as a percentage of control (no NNGH or triptolide) and are the mean ± SEM of triplicate results from an experiment that was repeated three times with similar results. *P < 0.01, **P < 0.001 (Dunnett test) versus corresponding control.
Figure 5.
 
Lack of effect of triptolide on the activity of plasmin. Corneal fibroblasts were cultured in collagen gels in the presence of plasminogen, in the absence (○) or presence (•) of IL-1β (0.1 ng/mL) and in the presence of the indicated concentrations of triptolide for 48 hours, after which the activity of plasmin in the culture supernatants was determined. Data are expressed in absorbance units and are the mean ± SEM of triplicate results from an experiment that was repeated three times with similar results.
Figure 5.
 
Lack of effect of triptolide on the activity of plasmin. Corneal fibroblasts were cultured in collagen gels in the presence of plasminogen, in the absence (○) or presence (•) of IL-1β (0.1 ng/mL) and in the presence of the indicated concentrations of triptolide for 48 hours, after which the activity of plasmin in the culture supernatants was determined. Data are expressed in absorbance units and are the mean ± SEM of triplicate results from an experiment that was repeated three times with similar results.
Figure 6.
 
Effects of triptolide on the expression of MMP-1 and -3 by corneal fibroblasts. (A) Corneal fibroblasts were cultured in collagen gels for 48 hours in the presence of plasminogen, in the absence or presence of IL-1β (0.1 ng/mL), and in the presence of the indicated concentrations of triptolide. The culture supernatants were then subjected to immunoblot analysis with antibodies to either MMP-1 (top) or -3 (bottom). Data are representative of three independent experiments. The positions of the pro and active forms of MMP-1 and -3 are indicated on the right, and those of molecular size standards are shown on the left. (B) Corneal fibroblasts were cultured in collagen gels for 12 hours in the presence of plasminogen and in the absence or presence of IL-1β (0.1 ng/mL) or 3.0 μM triptolide, as indicated. The amounts of MMP-1 mRNA (left) and MMP-3 mRNA (right) in the cells were then determined by reverse transcription and real-time PCR analysis. Data are normalized on the basis of the abundance of GAPDH mRNA, are expressed in arbitrary units, and are the mean ± SEM of results in three experiments. *P < 0.001 (Student’s t-test) versus the corresponding value for cells cultured without IL-1β; #P < 0.01 (Student’s t-test) versus the corresponding value for cells cultured without triptolide.
Figure 6.
 
Effects of triptolide on the expression of MMP-1 and -3 by corneal fibroblasts. (A) Corneal fibroblasts were cultured in collagen gels for 48 hours in the presence of plasminogen, in the absence or presence of IL-1β (0.1 ng/mL), and in the presence of the indicated concentrations of triptolide. The culture supernatants were then subjected to immunoblot analysis with antibodies to either MMP-1 (top) or -3 (bottom). Data are representative of three independent experiments. The positions of the pro and active forms of MMP-1 and -3 are indicated on the right, and those of molecular size standards are shown on the left. (B) Corneal fibroblasts were cultured in collagen gels for 12 hours in the presence of plasminogen and in the absence or presence of IL-1β (0.1 ng/mL) or 3.0 μM triptolide, as indicated. The amounts of MMP-1 mRNA (left) and MMP-3 mRNA (right) in the cells were then determined by reverse transcription and real-time PCR analysis. Data are normalized on the basis of the abundance of GAPDH mRNA, are expressed in arbitrary units, and are the mean ± SEM of results in three experiments. *P < 0.001 (Student’s t-test) versus the corresponding value for cells cultured without IL-1β; #P < 0.01 (Student’s t-test) versus the corresponding value for cells cultured without triptolide.
Figure 7.
 
Effects of triptolide on the expression of MMP-2 and -9 by corneal fibroblasts. (A) Corneal fibroblasts were cultured in collagen gels for 48 hours in the presence of plasminogen, in the absence or presence of IL-1β (0.1 ng/mL), and in the presence of the indicated concentrations of triptolide. The culture supernatants were then subjected to gelatin zymography. Data are representative of three independent experiments. The positions of pro, intermediate, and active forms of MMP-2 or -9 are indicated on the right. (B) Corneal fibroblasts were cultured in collagen gels for 12 hours in the presence of plasminogen and in the absence or presence of IL-1β (0.1 ng/mL) or 3.0 μM triptolide, as indicated. The amounts of MMP-2 mRNA (left) and MMP-9 mRNA (right) in the cells were then determined by reverse transcription and real-time PCR analysis. Data are normalized on the basis of the abundance of GAPDH mRNA, are expressed in arbitrary units, and are the mean ± SEM of results in three experiments. *P < 0.05, **P < 0.001 (Student’s t-test) versus the corresponding value for cells cultured without IL-1β; #P < 0.01 (Student’s t-test) versus the corresponding value for cells cultured without triptolide.
Figure 7.
 
Effects of triptolide on the expression of MMP-2 and -9 by corneal fibroblasts. (A) Corneal fibroblasts were cultured in collagen gels for 48 hours in the presence of plasminogen, in the absence or presence of IL-1β (0.1 ng/mL), and in the presence of the indicated concentrations of triptolide. The culture supernatants were then subjected to gelatin zymography. Data are representative of three independent experiments. The positions of pro, intermediate, and active forms of MMP-2 or -9 are indicated on the right. (B) Corneal fibroblasts were cultured in collagen gels for 12 hours in the presence of plasminogen and in the absence or presence of IL-1β (0.1 ng/mL) or 3.0 μM triptolide, as indicated. The amounts of MMP-2 mRNA (left) and MMP-9 mRNA (right) in the cells were then determined by reverse transcription and real-time PCR analysis. Data are normalized on the basis of the abundance of GAPDH mRNA, are expressed in arbitrary units, and are the mean ± SEM of results in three experiments. *P < 0.05, **P < 0.001 (Student’s t-test) versus the corresponding value for cells cultured without IL-1β; #P < 0.01 (Student’s t-test) versus the corresponding value for cells cultured without triptolide.
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