Inflamm. Res. (2010) 59:177–188 DOI 10.1007/s00011-009-0084-9
Inflammation Research
ORIGINAL RESEARCH PAPER
Anti-fibrotic effect of thalidomide through inhibiting TGF-b-induced ERK1/2 pathways in bleomycin-induced lung fibrosis in mice Jung-Yoon Choe Æ Hyun-Joo Jung Æ Ki-Yeun Park Æ Yoon-Seup Kum Æ Gwan Gyu Song Æ Dae-Sung Hyun Æ Sung-Hoon Park Æ Seong-Kyu Kim
Received: 8 May 2009 / Accepted: 18 August 2009 / Published online: 11 September 2009 Ó Birkha¨user Verlag, Basel/Switzerland 2009
Abstract Objectives This study is designed to confirm the antifibrotic effect of thalidomide on bleomycin-induced lung fibrosis in a mouse model and to identify whether this antifibrotic effect is associated with inhibition of the transforming growth factor-b (TGF-b)-induced extracellular signal-regulated kinase1/2 (ERK1/2). Methods and materials C57BL/6 female mice were administered blomycin sulfate. In cultured human lung fibroblasts, expressions of type I collagen, fibronectin, and either TGF-b or IL-6 were measured after thalidomide treatment by reverse transcription-polymerase chain reaction (RT–PCR). Expressions of ERK1/2, type I collagen, fibronectin, and TGF-b1 from lung tissues of blomycininduced mice and from mouse lung fibroblasts were evaluated using RT–PCR and western blotting.
Responsible Editor: M. Parnham. J.-Y. Choe D.-S. Hyun S.-H. Park S.-K. Kim (&) Department of Internal Medicine, Catholic University of Daegu School of Medicine, 3056-6 Daemyung 4-Dong, Namgu, Daegu 705-718, South Korea e-mail:
[email protected] J.-Y. Choe H.-J. Jung K.-Y. Park S.-H. Park S.-K. Kim Arthritis and Autoimmunity Research Center, Catholic University of Daegu School of Medicine, Daegu, South Korea Y.-S. Kum Department of Pathology, Catholic University of Daegu School of Medicine, Daegu, South Korea G. G. Song Division of Rheumatology, College of Medicine, Korea University, Seoul, South Korea
Results Thalidomide administration significantly inhibits TGF-b1 mRNA expression in a dose-dependant manner following administration of IL-6 and IL-6R. In the analysis of BAL fluids, total BAL inflammatory cell counts, TGF-b1, and IL-6 levels in thalidomide-treated mice were significantly reduced when compared with bleomycin-treated mice (p \ 0.01, p \ 0.01, and p \ 0.001, respectively). Thalidomide inhibited total ERK1/2 and phospho-ERK1/2 expression after TGF-b1 stimulation in the RT–PCR and western blotting. Conclusion The results of our study suggest that the antifibrotic effect of thalidomide on lung fibrosis may be related to suppression of the TGF-b1-induced ERK1/2 signaling pathway. Keywords Thalidomide Bleomycin Lung Fibrosis TGF-b ERK1/2
Introduction Pulmonary fibrosis is a progressive and chronic lung disease characterized by aberrant proliferation of fibroblasts and extracellular matrix (ECM) remodeling in pulmonary parenchyma [1]. Although the precise etiology of pulmonary fibrosis has not been clearly identified, a number of cytokines and other molecules including tumor necrosis factor-a (TNF-a) [2], interleukin-1 (IL-1) [2], transforming growth factor-b1 (TGF-b1) [3], and IL-6 [3] have been established to be related with the pathogenic mechanisms of pulmonary fibrosis, resulting in the proliferation and differentiation of fibroblasts in the lung tissue and then the subsequent development of pulmonary fibrosis. Among these pathogenic cytokines and growth factors, TGF-b is the prototype of a family of secreted growth
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factors involved with a diverse capacity of cell modulating processes including cell migration, apoptosis, differentiation and ECM production [4, 5]. It is well established that TGF-b is significantly involved during acute inflammatory phases with tissue damage followed by tissue repair and remodeling, and ultimately leading to human lung parenchymal fibrosis through increased expression of TGF-b in a variety of lung tissues and cellular sources including macrophage and epithelial cells [3, 6, 7]. TGF-b has two main signaling pathways: Smad and non-Smad pathways. The intracellular responses of TGF-b members are determined by complex interactions such as either phosphorylation or direct interaction with Smad between intracellular diverse molecules related to the TGF-b signaling downstream cascade from membrane to nucleus. Non-Smad signaling pathways include mitogenactivated protein kinases (MAPK), Rho-like GTPase and phosphatidylinositol-3-kinase/AKT pathways. Especially well-established non-Smad pathways of TGF-b signaling include extracellular signal-regulated kinase1/2 (ERK1/2), c-jun N-terminal kinase (JNK), and p38 MAPK signaling pathways [8]. Although a number of therapies for lung fibrosis have been investigated, there are no reliably effective pharmacologic agents available. Currently corticosteroid and immunosuppressive agents, such as cyclophosphamide and azthioprine, are the mainstay of treatment for pulmonary fibrosis. Novel therapeutic trials have been introduced based on understandings of the pathogenic mechanisms of lung fibrosis [9]. In recent experimental studies, thalidomide has been demonstrated to have anti-fibrotic effects by suppressing the expression of IL-6, TGF-b, and angiogenesis-related growth factors that play a crucial role in the proliferation and differentiation of lung fibroblasts [10]. This study is designed to (1) identify whether thalidomide ameliorates pulmonary fibrosis by inhibiting fibrosis-related cytokines and ECM proteins such as IL-6, TGF-b, type I collagen, and fibronection, and (2) determine, using a bleomycin-induced lung fibrosis mouse model, if the antifibrotic effect of thalidomide is related to suppression of ERK1/2 in the TGF-b-stimulated MAPK signaling pathway.
Materials and methods Animal preparation and bleomycin administration C57BL/6 female mice 7 weeks old were purchased from Japan SLC (Shizuoka, Japan) and were maintained under pathogen-free conditions. Mice were quarantined for minimum of 1 week prior to use. Mice weighing approximately 20–25 g were used in this study. Lighting was artificial and maintained to a 12-h day–night cycle. The
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room temperature was kept constant at 22.3 ± 3°C, the relative humidity at 50 ± 10%. We created a bleomycin-induced lung fibrosis mouse model as previously published by Tabata et al. [10]. Lung fibrosis was created by injecting doses of 2.0 mg/mouse per day of bleomycin sulfate into C57BL/6 mice on days 1, 8, and 15 through the intraperitoneal route. Thalidomide used in this experiment was prepared by dissolving 4 mg/kg of thalidomide in 0.1 ml of 0.5% carboxymethylcellulose (CMC; Sigma–Aldrich, USA), and vortexing with sterile glass beads for 5 min. Control mice received 0.1 ml of the vehicle intraperitoneally. The drug was given 5 times per week from day 15. All drugs were prepared immediately before use. All animal procedures used in e thpresent study were reviewed and approved by the Ethical Review Board for Animal Study of Catholic University of Daegu. Methylthiazol tetrazolium (MTT) assay The effect of thalidomide on cell proliferation was determined by MTT assay in human lung fibroblasts. Thalidomide was treated for 96 h at variable dosages such as 10, 50, 100, 250, and 500 lg/ml. Preparation of bronchoalveolar lavage (BAL) fluids and blood samples BAL fluids and blood from mice were sampled at the end of 4 week after initiation of this experiment. We performed BAL to analyze the cytokine related with lung fibrosis such as TGF-b1 and IL-6 and to identify the differences of fractions of inflammatory cells including lymphocytes, neutrophils, and macrophages. A total of 1 mL BAL fluid in each experimental mouse was collected. A 22 gauge plastic needle was introduced into the trachea of the mouse, and we lavaged the lungs using two 0.5 mL aliquots of PBS. Total cell numbers were counted by trypan blue exclusion using a hemocytometer. After centrifugation, supernatants were stored -70°C until assay for cytokines. A pipette was introduced through the conjunctiva and into the orbital sinus by quickly rotating the tube to collect blood of experimental mice. After obtaining the required amount, blood was stored in hematocrit tubes. The blood clotted and left a clear serum above. This serum was slowly removed and collected in fresh Eppendorf tubes and centrifuged at 1,200 rpm for 10 min and the serum aspirated and stored at –70°C until assay. Measurements of cytokine levels using ELISA IL-6 and TGF-b1 in both BAL fluids and serum, and TGF-b1 in supernatants were assessed using IL-6 ELISA (eBioscience, USA) and TGF-b1 ELISA (R&D Systems,
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Minneapolis, Minn., USA) methods according to manufacturer protocols. Assessment of histopathology and immunohistochemistry The mice were sacrificed at 4 weeks after the treatment. Lung tissues from sacrificed mice were fixed in 10% formaldehyde solution and embedded in paraffin. Sections of lung tissues were stained with hematoxylin and eosin (H&E) and Masson trichrome stain for evaluation the degree of lung fibrosis. The grading system of histologic severity for fibrosis was assessed by two investigators using a semi-quantitative method from grade 0–3 as follows: score of 0: normal lung, score of 1: alveolar inflammation without septal thickening, score of 2: extensive alveolar inflammation and septal thickening, and score of 3: obliteration of alveolar spaces or extensive thickening of the airway/vessel walls [11]. The sections were treated with xylene and ethanol to remove paraffin and for dehydration. The sections were boiled for 10 min with sodium citrate (pH 6.0), washed with secondary distilled water, and treated with normal serum at room temperature for 40 min to block non-specific reactions. Tissue sections were processed in 10 mM citrate buffer (pH 6.0) and heated to 100°C for 10 min for antigen retrieval. Sections then were incubated with antibodies against TGF-b1 (R&D Systems, Inc., Minneapolis, Minn., USA), fibronectin (Santa Cruz Biotechnology, Inc., CA, USA), and type I collagen (Santa Cruz Biotechnology, Inc., CA, USA) for 1 h at 37°C. The sections were then incubated in an EnVision system (EnVision kit, DAKO, CA, USA) at 37°C for 30 min with washing in PBS before incubation. DAB (3,30-diaminobenzidine tetrahydrochloride) was used as the color reagent, and hematoxylin was used as a counter stain. After washing, the sections were mounted with water-soluble mounting media and observed under light microscopy.
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starved for 24 h. After discarding the media-free serum media, cells were incubated with TGF-b1 (4 ng/ml) for 24 h in presence of the thalidomide for 48 h. RNA preparation and reverse transcription-polymerase chain reaction (RT–PCR) analysis Total RNA was isolated from lung tissues by the guanidinium isothiocyanate acidic phenol chloroform method using Trizol (Invitrogen Corp., Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA was reverse transcribed using the Access RT–PCR system (Promega, USA). Amplified PCR product was confirmed in a 1.2% agarose gel. After co-cultures of both IL-6 of 1 ng/ml and IL-6 receptor (IL-6R) of 20 ng/ml (R&D system, USA), which are known as mediators involve in the mechanism of lung fibrosis in human lung fibroblasts, thalidomide was applied dose-dependently for 96 h. RNA preparation and RT–PCR for expression of TGF-b1, and type I collagen were performed. Inhibitory effects of thalidomide on the fibrosis-related ability of TGF-b1 (R&D System, USA) was assessed in human lung fibroblasts. Human lung fibroblasts were cultured and treated with TGF-b1 (5 ng/ml) for 48 h, in the presence of the thalidomide at variable doses (1, 10, and 50 lg/ml) for 48 h. RNA preparation and RT–PCR were also performed. We tried to identify suppressive effects of thalidomide on the profibrotic mRNA expressions of type I collagen, fibronectin, and ERK1/2 in mouse lung fibroblasts treated with TGF-b1 of 4 ng/ml for 24 h in the presence of the thalidomide for 48 h. RNA was harvested, and RT–PCR was performed. In the lung tissues extracted from BLF, mRNA expressions of type I collagen, fibronectin, ERK1/2, and TGF-b1 were also assessed. Western blotting
Cell culture and treatment The human fetal lung fibroblast cell line, MRC-5 cells, was purchased from American Type Culture Collection (ATCC, USA) and maintained in MEM Alpha solution (Invitrogen, CA, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, USA), 100 U/ml penicillin and 100 lg/ml streptomysin. When cells were treated with Thalidomide (Phamicon, Ltd., USA) they were diluted in DMSO (Amresco, Solon, OH, USA). Mouse lung fibroblasts (MLFs), MLg cells, in alphaMEM were plated at 2 9 105 cells per 100 mm. Cells were plated and cultured at 37°C in 5% humidified air. For induction of fibrosis by recombinant protein TGF-b1, MLFs (4 9 105 cells) were seeded in 100 mm plates and
Proteins (50 lg) were separated on 10% SDS–polyacrylamide gels and then were transferred to nitrocellulose (NC) membranes (Bio-Rad Laboratories, CA, USA). NC membranes were incubated overnight at 4°C with one of the antibodies and then washed three times with TBS-T and incubated in horseradish peroxidase-conjugated secondary antibodies. After 1 h of gentle shaking, the membrane was washed and developed with an ECL detection system (Amersham, NJ, USA). Western analysis was used with antibodies that reacted selectively with type I collagen, fibronectin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phosphorylated ERK(p-ERK), and total ERK (Cell Signaling Technology, Inc., USA).
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Kinase inhibitor assay U0126 (ERK inhibitor) was purchased from Sigma– Aldrich (St. Louis, MO, USA) and dissolved in DMSO. MLF cells in 100 mm plates were treated with 10 lM inhibitors in serum-free medium for 24 h and in the presence of the thalidomide for 48 h. Statistical analysis Data were described as mean values ± standard deviation. Statistical analysis was performed using Student’s t test if appropriate. Data were analyzed by using the Statistical Package for the Social Sciences, version 13.0 (SPSS Inc., Chicago, IL, USA). A p value \ 0.05 was considered statistically significant.
Results Analysis for expression of fibrosis-related cytokines and ECMs in the cultured human lung fibroblasts The effects of thalidomide on cell proliferation were determined by MTT assay in lung fibroblasts. Thalidomide was applied for 96 h at variable doses of 10, 50, 100, 250, and 500 lg/ml. Thalidomide did not influence cell survival of mouse lung fibroblasts at dosages of 50 lg/ml or less. At thalidomide dosages of more than 100 lg/ml, cell viability was significantly reduced in a dose-dependent manner. Based on this finding, most assays in the cultured lung fibroblasts used a thalidomide dose of 50 lg/ml. We assessed whether thalidomide inhibited the expression of TGF-b1 by IL-6 stimulation in the cultured human lung fibroblasts. The TGF-b1 levels in the supernatants of cultured human lung fibroblasts following co-stimulation by IL-6 and IL-6R were significantly increased compared with normal cells without any stimulation (218.0 ± 23.4 ng/ml in IL-6 and IL-6R co-stimulated cells vs 175.8 ± 18.4 ng/ml in DMSO-treated cells, p \ 0.05) (Fig. 1a). TGF-b1 levels following treatment with thalidomide at doses 1, 10, and 50 lg/ml in the IL-6 and IL-6R co-stimulated lung fibroblasts were significantly reduced compared with nonthalidomide treated cells (134.3 ± 48.2 ng/ml with 1 lg/ml of thalidomide, 109.5 ± 7.2 ng/ml with 10 lg/ml, and 44.7 ± 37.2 ng/ml with 50 lg/ml vs 218.0 ± 23.4 ng/ml of non-thalidomide treated cells, p \ 0.05, p \ 0.001, and p \ 0.001, respectively). TGF-b1 mRNA expression following co-stimulation by IL-6 and IL-6R in cultured human lung fibroblasts was significantly reduced following thalidomide treatments in a dose-dependent manner at doses of 10 lg/ml and 50 lg/ml
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(Fig. 1b). Densitometric quantification also identified that both dosages of thalidomide suppressed the TGF-b1 expression (p \ 0.001 at 10 lg/ml of thalidomide, p \ 0.001 at 50 lg/ml of thalidomide). We assessed the changes of production of ECMs including fibronectin and type I collagen by thalidomide after stimulation of TGF-b1. Thalidomide, at doses of 10 and 50 lg/ml, significantly inhibited the mRNA expressions of type I collagen and fibronectin in human lung fibroblasts treated with 5 ng/ml of TGF-b1 for 48 h (Fig. 1c). Analysis of inflammatory cells and their fractions in BAL fluids of bleomycin-induced lung fibrosis mice BAL fluids were collected from individual experimental mice. Total and differential counts of inflammatory cells in the BAL fluids were quantified. In comparison of total cell counts, BAL fluid cell counts in bleomycin-treated mice were significantly increased compared to those in normal mice (47.0 ± 6.6/ll, 146.7 ± 20.2/ll, respectively, p \ 0.001). The levels of inflammatory cells in thalidomide-treated mice were significantly lower than those in bleomycin-treated mice (78.3 ± 17.6/ll, 146.7 ± 20.2/ll, respectively, p \ 0.01) (Fig. 2a). In analysis of lineages of inflammatory cells such as macrophages, neutrophils, and lymphocytes, the differences of proportions of these cells were not significant between normal mice and thalidomide-treated mice (73.3 ± 2.9 vs. 77.7 ± 3.1% of macrophages, p [ 0.05; 10.3 ± 2.5 vs. 7.0 ± 2.6% of neutrophils, p [ 0.05; 8.0 ± 2.6 vs. 10.0 ± 2.6% of lymphocytes, p [ 0.05, respectively) (Fig. 2b). However, the ratio of macrophages among inflammatory cells in BAL fluid of bleomycin-treated mice was significantly decreased compared to normal (73.3 ± 2.9 vs. 40.7 ± 4.0%, p \ 0.001), whereas proportions of neutrophils and lymphocytes were increased compared to normal (10.3 ± 2.5 vs. 18.3 ± 2.1% of neutrophils, p \ 0.05; 8.0 ± 2.6 vs. 21.0 ± 5.6% of lymphocytes, p \ 0.01). Macrophage, neutrophil, and lymphocyte fractions in BAL fluids after treatment of thalidomide were shown to be similar to the fractions in normal mice. Analysis of TGF-b1 and IL-6 level in BAL fluids and blood in bleomycin-induced lung fibrosis mice We assessed the TGF-b1 and IL-6 concentrations in BAL fluids from each experimental group. TGF-b levels from bleomycin-treated lung fibrosis mice were significantly increased compared to normal mice (221.0 ± 86.3 vs. 8.0 ± 10.3 ng/ml, p \ 0.001) (Fig. 3a). Thalidomide significantly reduced TGF-b1 levels compared with
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Fig. 1 Inhibitory effects of thalidomide on TGF-b-induced and IL-6-induced extracellular matrix production in cultured human fetal lung fibroblasts. a Co-stimulation with IL-6 (1 ng/ml) and IL-6R (20 ng/ml) induced enhancement of TGF-b1 expression. Thalidomide also dose-dependently reduced TGF-b1 expression in the supernatants
of human lung fibroblasts. b IL-6 and IL-6R enhanced TGF-b1 mRNA expression. Thalidomide inhibited these fibrosis-related molecules. c After stimulation of TGF-b1 (5 ng/ml), dosages of 10 lg/ml and 50 lg/ml of thalidomide suppressed mRNA expression of type I collagen and fibronectin in a dose-dependent manner
bleomycin-induced lung fibrosis (221.0 ± 86.3 vs. 14.4 ± 5.1 ng/ml, p \ 0.01). Bleomycin treatment increased production of IL-6 levels in BAL fluids, compared to normal mice (922.5 ± 26.8 vs. 109.8 ± 54.0 pg/ml, p \ 0.001) (Fig. 3b). Thalidomide significantly reduced IL-6 levels in bleomycin-treated mice (89.9 ± 36.7 vs. 922.5 ± 26.8 pg/ml, p \ 0.001). Serum analysis of TGF-b1 in normal, bleomycin-, or thalidomide-treated mice demonstrated that higher TGF-b1 expression in bleomycin-induced mice was noted than those of normal mice (272.1 ± 112.9 vs. 17.2 ± 4.3 ng/ml, p \ 0.01). And thalidomide markedly decreased TGF-b1 level in bleomycin-induced lung fibrosis (17.0 ± 2.9 vs. 272.1 ± 112.9 ng/ml, p \ 0.01) (Fig. 3c). Additionally, serum levels of IL-6 in thalidomide-treated mice were significantly reduced compared with those in bleomycintreated mice without thalidomide (1078.8 ± 288.9, 316.5 ± 230.9, respectively, p \ 0.001) (Fig. 3d).
Histopathological analysis for effect of thalidomide on the lung tissue of bleomycin-induced fibrosis mice Histopathological analysis using H&E and Masson’s trichrome staining were used to determine the degree of lung fibrosis. Lung fibrosis was demonstrated to be decreased by treatment with thalidomide (Fig. 4a). There were no specific changes in the degree of inflammation or fibrosis in either normal or CMC-treated mice. However, lung histology of bleomycin-treated mice demonstrated extensive peribronchial and interstitial infiltrations of inflammatory cells, predominantly lymphocytes, and multiple focal fibrotic lesions. However, less frequent infiltrations of inflammatory cells and attenuated fibrotic features were noted in thalidomide-treated mice. Fibrosis scores were estimated and demonstrated that thalidomide-treated mice had significantly lower fibrosis scores than bleomycintreated mice (1.8 ± 0.8 vs. 3.5 ± 0.5, p \ 0.001) (Fig. 4b).
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tissues extracted from experimental mice. The results demonstrated that 4 mg/kg per mouse of thalidomide significantly inhibited mRNA expressions of TGF-b1, type I collagen, fibronectin, and ERK1/2 compared to those of bleomycin-treated mice (Fig. 5a). In the analysis of mouse lung fibroblasts treated with TGF-b1 (4 ng/ml), RT–PCR analysis showed that expressions of ERK1/2, type I collagen, and fibronectin were significantly decreased by treatment with thalidomide in a dose-dependent manner (Fig. 5b). Thalidomide, at dosages of both 4 and 8 lg/ml, significantly inhibited ERK1/2, type I collagen, and fibronectin mRNA expressions, as shown in Fig. 5b. We investigated whether the anti-fibrotic effect of thalidomide was related to suppression of ERK1/2 in TGF-bstimulated MARK pathways. U0126, an ERK inhibitor, inhibited expression of ERK1/2 and type I collagen expressions, but not of fibronectin (Fig. 5c). Immunoblotting assay demonstrated reduced p-ERK and total ERK expression in thalidomide-treated cells in a dose-dependent manner (Fig. 5d). Figure 5e shows synergic effects of thalidomide and ERK inhibitor reduced expressions of p-ERK, total ERK, type I collagen, and fibronectin.
Discussion
Fig. 2 Analysis of total inflammatory cell counts and their fractions in BAL fluids (n = 5 in each experiment group). a Total inflammatory cells were significantly reduced in mice treated with thalidomide. (*p \ 0.001 compared to normal mice, p \ 0.01 compared to bleomycin-induced mice). b The cellular differential fractions in the BAL fluids of mice treated with thalidomide were similar to those of normal controls. (*p \ 0.001 compared to normal mice, p \ 0.001 compared to bleomycin-induced mice in macrophages; *p \ 0.05 compared to normal mice, p \ 0.01 compared to bleomycin-induced mice in neutrophils; *p \ 0.01 compared to normal mice, p \ 0.05 compared to bleomycin-induced mice in lymphocytes)
Immunohistochemistry from lung tissues demonstrates that expressions of TGF-b1, type I collagen, and fibronectin antibodies were scanty in lung tissues of normal and CMC-treated mice (Fig. 4c). However, expressions of these antibodies were significantly increased in the bleomycin-treated lung tissue, and in thalidomide-treated lung tissues were markedly reduced compared to bleomycintreated tissue. Thalidomide inhibits TGF-b-related ERK1/2 signaling pathways in bleomycin-induced lung fibrosis To identify the effects of thalidomide on expression of TGF-b1, ERK1/2, type I collagen, and fibronectin, mRNA expressions of these molecules were assessed in lung
We verified that thalidomide significantly inhibits the TGF-b1-stimulated expression of type I collagen, fibronectin, and ERK1/2 in human and mouse lung fibroblasts. The release of fibrosis-related cytokines including TGF-b1 and IL-6 was reduced by thalidomide in the blood and BAL fluids analysis of bleomycin-induced lung fibrosis in mice. Histopathologic and immunohistochemical examination revealed that the severity of lung fibrosis induced by bleomycin could be attenuated by treatment with thalidomide. We also found that anti-fibrotic effects of thalidomide may be related to inhibition of TGF-b1-mediated ERK1/2 signaling pathways. The pathogenesis of pulmonary fibrosis has yet to be determined, despite a number of investigations of lung fibrosis for some decades. Present concepts for the pathogenesis of lung fibrosis on the basis of immunological evidence have been characterized yb failure of reepithelialization for alveolar epithelial cells after undefined injuries, aberrant recruitment and activation of fibroblasts/ myofibroblasts, and enhanced release or production of various fibrosis-related factors including TGF-b, IL-6, TNF-a, and platelet-derived growth factor [1]. However, no curative pharmacologic agents for halting or overcomjng these pathogenic processes have been elucidated, although many of therapeutic approaches for management of lung fibrosis have been tried. Recently, it was proposed that thalidomide might be considered as a novel therapeutic
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Fig. 3 Analysis of TGF-b1 and IL-6 levels in BAL fluids and blood (n = 5 in each experiment group). a and b Thalidomide inhibits TGF-b1 and IL-6 level in the BAL fluids, respectively. (*p \ 0.001 compared to normal mice, p \ 0.01 compared to bleomycin-induced mice in TGF-b1 levels; *p \ 0.001 compared to normal mice, p \ 0.001 compared to bleomycin-induced mice in IL-6 levels).
c and d Serum TGF-b1 and IL-6 levels were significantly downregulated by thalidomide (*p \ 0.01 compared to normal mice, p \ 0.01 compared to bleomycin-induced mice in TGF-b1 levels; *p \ 0.001 compared to normal mice, p \ 0.001 compared to bleomycin-induced mice in IL-6 levels). CMC: carboxymethylcellulose
option for lung fibrosis [10]. The major focus of this study was to identify the mechanism of anti-fibrotic effects of thalidomide using bleomycin-induced lung fibrosis in mice. Thalidomide was firstly introduced as a sedative/tranquilizer in 1957. The drug was subsequently withdrawn from the world market following its identification as one of the major teratogens that results in various birth defects. However, since the Food and Drug Administration of the United States of America approved thalidomide as a therapeutic agent for erythema nodosum leprosum in 1998, a number of investigators have renewed interest in the clinical applications of thalidomide. Currently, thalidomide is prescribed for a wide spectrum of diseases including erythema nodosum leprosum, graft-versus-host disease, cutaneous lupus erythematosus, multiple myeloma, Behcet’s disease and aphthous oral ulceration. Although pharmacological mechanisms of thalidomide have often conflicted, thalidomide has been demonstrated to have immunomodulatory [12], anti-inflammatory [13], and antiangiogenic properties [13, 14]. Recent studies showed some evidence of anti-fibrotic activity of thalidomide in various fibrosis-related diseases such as lung fibrosis [10], liver fibrosis [15, 16], and remodeling after myocardial infarction [17]. One study reported that thalidomide exhibits potent anti-inflammatory or anti-fibrotic effects,
mediated by inhibiting the release of TNF-a, IL-12p40, and IL-18, in patients with interstitial lung diseases including idiopathic pulmonary fibrosis [18]. Results from these studies suggest that the therapeutic effects of thalidomide may be partially due to inhibition of TGF-b, IL-6, TNF-a, vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), and IL-18. Several fibrogenic cytokines and growth factors have been identified to be contributive to lung fibrosis and released from alveolar epithelial cells, including TNF-a [2], IL-1 [2], TGF-b1 [3], and IL-6 [3]. These molecules may induce proliferation and migration of lung fibroblasts and further differentiation of lung fibroblasts to activated phenotypes that are responsible for synthesis and remodeling of ECMs. Another characteristic of lung fibrosis is excessive deposition of ECM proteins such as type I collagen, and fibronectin. The present study also revealed markedly increased releases of TGF-b1 and IL-6 in both BAL fluids and blood after bleomycin treatment in mice, compatible with results from previous studies (Fig. 3). Increased releases of TGF-b1 and IL-6 by bleomycin treatment in BAL fluids and blood were significantly reduced by treatment with thalidomide in bleomycininduced lung fibrosis mice. In terms of ECM generation in lung fibrosis, we demonstrated here that stimulation with
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Fig. 4 Anti-fibrotic effects of thalidomide on bleomycin-induced lung fibrosis revealed by histopathological examination and immunohistochemistry. a Histopathology represents markedly decreased cellular proliferation and infiltration of connective tissues in mice treated with thalidomide compared with those treated with only bleomycin (9200). b Fibrosis scores in thalidomide-treated mice
were lower than bleomycin-induced mice (*p \ 0.001). c TGF-b1 positive cells and infiltration of type I collagen and fibronectin in connective tissues were increased in bleomycin-induced mice compared to thalidomide-treated mice. Thalidomide attenuates enhanced expressions of TGF-b1, type I collagen and fibronectin (9400)
TGF-b1 in human lung fibroblasts enhanced mRNA expressions of type I collagen and fibronectin, which was decreased by thalidomide treatment in a dose-dependent manner. The results of this study suggests that both fibrogenic factors, TGF-b1 and IL-6, may play an important role in the development of lung fibrosis and that thalidomide under stimulation of either TGF-b1 or IL-6 regulates negatively fibrosis-related processes. This finding is compatible with the results of a previous Japanese study [10]. The nature of the interaction between IL-6 and TGF-b1 in lung fibroblasts has been debated [10, 19]. TGF-b stimulates the production of IL-6 through regulation of its gene transcription in human lung fibroblast, strain CCL202 cells [19]. However, Tabata et al. reported no interaction between TGF-b and IL-6 in their experiment using IMR-90 human fetal lung fibroblasts. In contrast, we found that co-stimulation of IL-6 and IL-6R in the human lung fibroblast line, MRC-5 cells, increased TGF-b1 expression in culture supernatants (Fig. 1a) and IL-6 enhanced mRNA expression of TGF-b1 (Fig. 1b). In addition, IL-6 mRNA expression was augmented by addition of TGF-b1 (5 ng/ml) (Fig. 1c). We suggest that IL-6 and TGF-b may be involved in the pathogenesis of lung fibrosis, either independently or through reciprocal action.
TGF-b members are pleiotropic cytokines critical for growth and differentiation in many organ systems [4, 5]. However, the enhanced expression of TGF-b from lung injury caused by noxious stimuli leads to inflammatory changes as well as tissue remodeling and repair, and ultimately to lung fibrosis [3, 6, 7]. The pathogenic roles of TGF-b members in lung fibrosis may reflect influence on the chemotaxis of fibroblasts and macrophage, increased ECM protein production by fibroblasts, and proliferation of fibroblasts. Blocking TGF-b1 mRNA over-expression in an animal model results in reduced production of type I
Fig. 5 Anti-fibrotic effects of thalidomide on the production of c ECMs through suppression of ERK1/2 signal pathway. a Thalidomide reduced mRNA expression of TGF-b1, ERK1/2, type I collagen, and fibronectin in the lung tissues (N normal mice, BC bleomycin-treated mice, BT4 thalidomide (4 mg/kg per mouse)-treated mice, CMC carboxymethylcellulose-treated mice). b Thalidomide also decreased mRNA expression of ERK1/2, type I collagen, and fibronectin after TGF-b1-stimulated mouse lung fibroblasts. c ERK inhibitor, U0126, effectively suppressed ERK1/2 and type I collagen in mouse lung fibroblasts. d p-ERK and total ERK were inhibited by thalidomide in a dose-dependent manner. e Synergic effects of thalidomide and U0126 on blocking p-ERK, total ERK, type I collagen, and fibronectin are shown
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Anti-fibrotic effect of thalidomide on lung fibrosis
collagen [10, 20]. Administration of some anti-fibrotic agents, such as CD36 synthetic peptide [21] and proteoglycans decorin [22], reduced the biologic activity of TGF-b and ultimately decreased or prevented lung fibrosis [21, 22]. In the present study, thalidomide was demonstrated to inhibit TGF-b-induced expression of type I collagen and fibronectin expressions in human lung fibroblasts. Mechanism of TGF-b signal is mainly responsible for both Smad and non-Smad pathways, of which the latter consists of MAPK, Rho-like GTPase, phosphatidylinositol3-kinase/AKT pathways in the pathogenesis of lung fibrosis [8]. Three well established signaling molecules of MARKs pathways with regard to TGF-b signaling are ERK1/2, JNK, and p38 MAPK [8]. MAPKs are serine/threonine-specific protein kinases that have been implicated in the regulation of intracellular processes including cell survival, apoptosis, differentiation and gene expression through binding to extracellular mitogens such as growth factors, stress, and inflammatory cytokines [23]. Signaling mediated by TGF-b ligands including TGF-b1, TGF-b2, TGF-b3, Activin, and Nodals occurs through binding to heterodimeric type I and type II TGF-b receptors containing an intracellular serine/ threonine kinase domain. A study using MRC-5 cells demonstrated that TGF-b1 induced expression of ERK1/2 and JNK but not of p38 [24]. In the current study, we investigated whether thalidomide regulated TGF-b1-induced ERK1/2 activation in MCR-5 cells. After obtaining lung tissues from sacrificed experimental fibrosis mice, mRNA expressions of TGF-b1, type I collagen, and fibronectin were significantly different in bleomycin-induced lung fibrosis with and without addition of thalidomide. Thalidomide inhibits expression of ERK1/2, type I collagen, and fibronectin mRNA in lung fibroblast treated with TGF-b1 in a dosedependent manner. Furthermore, ERK1/2 phosphorylation was markedly reduced by thalidomide administration. In summary, we demonstrated that thalidomide improved histopathologic changes for lung fibrosis in the lung tissues extracted from bleomycin-induced mice and inhibited the production of TGF-b1, IL-6, and ECMs, including type I collagen and fibronectin, through inhibition of ERK1/2 signaling. Our results indicate that thalidomide may have an application as a novel therapeutic approach for the treatment of lung fibrosis. Acknowledgments This work was supported by the grant of Research Institute of Medical Science, Catholic University of Daegu (2008).
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