Chinese Medical Journal 2008;121(18):1821-1829
Simvastatin attenuates bleomycin-induced pulmonary fibrosis in mice

OU Xue-mei,  FENG Yu-lin,  WEN Fu-qiang,  HUANG Xiang-yang,  XIAO Jun,  WANG Ke,  WANG Tao

OU Xue-mei (Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China; Department of Respiratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China)

FENG Yu-lin (Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China; Department of Respiratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China)

WEN Fu-qiang (Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China; Department of Respiratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China)

HUANG Xiang-yang (Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China; Department of Respiratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China)

XIAO Jun (Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China; Department of Respiratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China)

WANG Ke (Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China; Department of Respiratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China)

WANG Tao (Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China; Department of Respiratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China)

Correspondence to:WEN Fu-qiang,Department of Respiratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China (Tel: 86-28-85411561. Fax:86-28-85582944. E-mail:wenfuqiang@126.com)
Keywords
pulmonary fibrosis; simvastatin; transforming growth factor-β1; connective tissue growth factor
Abstract

Background  Bleomycin-induced fibrosis is extensively used to model aspects of the pathogenesis of interstitial pulmonary fibrosis. This study aimed to determine the benefic effects and mechanisms of simvastatin on bleomycin- induced pulmonary fibrosis in mice.
Methods  Bleomycin-induced pulmonary fibrosis mice were administered with simvastatin in different doses for 28 days. We measured inflammatory response, fibrogenic cytokines and profibrogenic markers in both bleomycin-stimulated and control lungs, and correlated these parameters with pulmonary fibrosis.
Results  Simvastatin attenuated the histopathological change of bleomycin-induced pulmonary fibrosis and prevented the increase of lung hydroxyproline content and collagen (I and III) mRNA expression induced by bleomycin. Moreover, simvastatin down-regulated the increased expression of transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF) induced by bleomycin at both gene and protein levels. Simultaneously, the accumulation of neutrophils and lymphocytes and the increased production of tumor necrosis factor-α (TNF-α) in bronchial alveolar lavage fluid were inhibited by simvastatin in early inflammatory phase after bleomycin infusion. The higher dose of simvastatin was associated with a more significant reduction in these inflammatory and fibrotic parameters. Furthermore, the inactivation of p38, RhoA and Smad2/3 signaling pathways was observed during simvastatin administration.
Conclusions
 Simvastatin attenuated bleomycin-induced pulmonary fibrosis, as indicated by decreases in Ashcroft score and lung collagen accumulation. The inhibitory effect of simvastatin on the progression of pulmonary fibrosis may be demonstrated by reducing inflammatory response and production of TGF-β1 and CTGF. These findings indicate that simvastatin may be used in the treatment of pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a consequence of lung injury and inflammation. The progressive interstitial lung disease has a high mortality rate and poor response to available medical therapy.1-3 Conventional treatment with corticosteroids and other immuno- suppressants has been disappointing, and the 5-year survival rate of patients with IPF is less than 50%.4,5 Therefore, novel therapeutic modalities are of particular interest.

IPF is characterized by inflammation, excessive proliferation of fibroblasts, and abnormal deposition of extracellular matrix (ECM) proteins. Bleomycin-induced fibrosis is extensively used to model aspects of the pathogenesis of interstitial pulmonary fibrosis. Intratracheal instillation of bleomycin develops an acute inflammatory response followed by a fibrotic interstitial reaction. There is growing evidence that both inflammation and fibrosis are mediated by cytokine activity, particularly tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β1.6,7 TGF-β1 links inflammation to fibrogenesis and is one of the key mediators in the fibrotic process.8-11 As a downstream mediator of TGF-β1, connective tissue growth factor (CTGF) plays a crucial role in TGF-β-induced connective tissue cell proliferation and ECM deposition.12 Moreover, enhanced expression of TGF-β1 and CTGF in the lung of IPF has been reported.13-17 These results suggest that the combined inhibition of inflammatory and fibrogenic cytokines might be a potential strategy of pulmonary fibrosis therapy.

Statins, HMGCoA reductase inhibitors that have been used clinically as lipid lowering agents, have anti-inflammatory, antioxidant, and immunomodulatory actions in vitro and in vivo.18,19 Statins had been used successfully to decrease renal interstitial fibrosis in rats.20,21 Emerging data indicate that simvastatin inhibits fibrogenic cytokines expression and modulates profibrogenic markers in lung fibroblasts.22-24 Moreover, the inhibition of TGF-β1 and CTGF production in airway remodeling rat caused by simvastatin has been proved in our laboratory. Therefore we hypothesized that the anti-inflammatory and anti-fibrotic properties of simvastatin may counteract the harmful effects of bleomycin-induced lung inflammation and fibrosis. To demonstrate the effects of simvastatin on pulmonary fibrosis in the present study, we evaluated (1) the effects of simvastatin on fibrotic changes induced by bleomycin, (2) the effects on bleomycin-induced inflammatory injury in the lung and (3) the effects on the signal transduction pathway in responsible for fibrotic response. To our knowledge, this study is the first to show that simvastatin ameliorates bleomycin-induced pulmonary fibrosis in vivo, possibly by reducing inflammation and production of TGF-β1 and CTGF.

METHODS

Animal model preparation and treatment groups
This animal study was approved by the Panel on Laboratory Animal Care of West China School of Medicine, Sichuan University. To investigate whether simvastatin modulates bleomycin-induced pulmonary fibrosis, C57Bl/6 mice (6–8 weeks old) were randomly divided into six experimental groups (20 mice in each group): (A) control group (saline-only, CT), (B) simvastatin 5 mg/kg group, (C) simvastatin 20 mg/kg group, (D) bleomycin group (BLM), (E) bleomycin-plus- simvastatin 5 mg/kg group, and (F) bleomycin-plus- simvastatin 20 mg/kg group. To induce pulmonary fibrosis, mice were treated with intratracheal bleomycin (0.3 U/10 g. Sigma, St. Louis, MO, USA) on day 0. Control mice were subjected to the same protocol but received the same volume of intratracheal saline instead of bleomycin. After mice were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg), they were given an intratracheal injection of either bleomycin dissolved in 50 µl of 0.9% sterile saline or saline alone. Simvastatin (5 or 20 mg/kg. Merck Sharp & Dohme Ltd., UK) were administered by gavage in each simvastatin treatment groups from 3 days prior to the intratracheal instillation of bleomycin or saline up to the conclusion of the experiments. To study the effect of simvastatin on early inflammatory phase and late fibrotic phase, the mice were sacrificed on days 7 and 28 after bleomycin or saline instillation (randomly selected 10 mice/group each point).

Histopathological examination
Mouse lungs on day 28 after bleomycin infusion were fixed by perfusion with 4% paraformaldehyde-phosphate buffered solution (PBS) before routine processing and paraffin embedding. Sections (3 µm) were stained with hematoxylin and eosin for histological examination. The Ashcroft score was used for semi-quantitative assessment of fibrotic changes.25 The severity of fibrotic changes in each histological section of the lung was assessed as the mean score of severity from observed microscopic fields. Thirty fields in each section were analyzed. After examination of the whole fields of the section, the mean of the scores from all fields was considered the fibrotic score. To prevent bias of observation, grading was done in a blinded fashion by two observers.

Hydroxyproline assay
The total collagen content of the left lung was measured using a colorimetric assay26 to determine lung hydroxyproline content on day 28 after bleomycin infusion. In brief, the minced left lung lobes were homogenized in 6 mol/L HCl and hydrolyzed for 5 hours at 130°C. The pH was adjusted to 6.5–7.0 with NaOH, and the sample volume was adjusted to 30 ml with distilled water. The sample solution (1.0 ml) was mixed with 1.0 ml of chloramine T solution (0.05 mol/L), and then the mixture was incubated at room temperature for 20 minutes. When 1.0 ml of 20% dimethyl benzaldehyde solution was added, the mixture was incubated at 60°C for 20 minutes. The absorbance of each sample at 550 nm was measured. The results were calculated as μg hydroxyproline per mg wet lung weight using hydroxyproline standards (Sigma).

Bronchoalveolar lavage fluid (BALF)
Analysis of inflammatory cell profile of BALF
Bronchoalveolar lavage was performed through a tracheal cannula with PBS (pH 7.4) on days 7 and 28 after bleomycin instillation as previously described.27 This procedure was repeated five times. Cold sterile PBS (1 ml) was used to inflate the lung, and the lavage fluid was recovered with –80% of the original volume. The BALF was centrifuged (1000 × g, 10 minutes, 4°C) and the cell-free supernatant was used for the biochemical measurements. For each mouse, the cell pellet was then resuspended in PBS. The cells present in the lavage fluid were counted using a hemocytometer. Differential cells counts were performed on 200 cells stained with Wright-Giemsa.

Enzyme-linked immunosorbent assay (ELISA)
The supernatant of BALF was immediately stored at –70°C until the assay. We measured the TNF-α concentration in BALF with a mouse TNF-α ELISA kit (Pierce, Rockford, IL, USA) according to the manufacturers’ instructions.

Real-time RT-PCR
Lung tissues collagen (I and III), TGF-β1 and CTGF mRNA expression were detected by real-time RT-PCR on day 28 after bleomycin infusion. Total RNA was isolated from lung homogenates with Trizol (Gibco-BRL, Gaithersburg, MD, USA) and reverse transcribed. Each PCR was performed in a final volume of 25 µl (2 μl of cDNA, 0.2 μmol/L of both forward and reverse primers, 1.5 mmol/L MgCl2, 0.12 μmol/L TaqMan probe, and 180 μmol/L dNTP in 1×PCR-Gold buffer). The primers and Taqman probes were designed using primer express software (Applied Biosystems, USA). The sequences of primers used in the present study are as follows: type I collagen: forward: 5’-CAAGAGGAAGGCCAAGTCGAG-3’, reverse: 5’-TTGTCGCAGACGCAGATCC-3’; type III collagen: forward: 5’-CTGTGAATCATGCCCTACTGGTC-3’, reverse: 5’-TAGCCTGCGAGTCCTCCTACTG-3’; TGF-β1: forward: 5’-GCAACATGTGGAAC- TCTACCAGAA-3, reverse: 5’-GACGTCAAAAGACA- GCCACTCA-3’; CTGF: forward: 5’-CCCGCCAACC- GCAAGATT-3’, reverse: 5’-AGGCGGCTCTGCTTCTC- CA-3’; and GAPDH: forward: 5’-ACCCAGAAGACT- GTGGATGG-3’, reverse: 5’-TGTGAGGGAGATGCTC- AGTG-3’.


The fluorescence emitted by the reporter dye was detected in real time, and the threshold cycle (Ct) of each sample was recorded as a quantitative measure of the amount of PCR product in the sample. The Ct is the fractional cycle number at which the fluorescence generated by the reporter dye exceeds a fixed level above baseline. When indicated, the target signal was normalized against the relative quantity of GAPDH and expressed as ∆Ct = CtTarget–CtGAPDH. The changes in target signal relative to the total amount of genomic DNA were expressed as ∆∆Ct = ∆Cttreatment –∆Ctcontrol. Relative changes in metastasis were then calculated as 2-∆∆CT.28

Western blot
Protein concentration was measured using bicinchoninic acid protein assay (Pierce). Cytoplasmic protein samples (20 μg) were separated on precasted 15% SDS-polyacrylamide gels and semi-dry transferred to the polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Nonspecific binding to the membrane was blocked for 1 hour at room temperature with 5% bovine serum albumin (BSA) in TBS-T. Afterward, membranes were incubated with primary anti-TGF-β1 antibody (1:500. Santa Cruz Biotechnology), anti-CTGF antibody (2 μg/ml. Biovision), anti-pp38 antibody (1:1000, Cell Signaling Technology (CST), USA), anti-p38 antibody (1:1000. CST), anti-RhoA antibody (1:1000, CST), anti-pSmad2 (1:1000, CST), anti-pSmad3 (1:1000, CST), anti-Smad2 (1:1000, CST), anti-Smad3 (1:1000, CST) and anti-β-actin antibody (1:1000, CST) at 4°C overnight, followed by incubation for 1 hour at room temperature with a secondary horseradish peroxidase-conjugated IgG in 5% BSA-TBS-T (1:15 000). Immunodetected proteins were visualized using ECL assay kit (Minipore, Bedford) following the manufacturer’s recommended protocol.

Statistical analysis
All data were expressed as mean ± standard deviation (SD). Statistical analysis was performed with SPSS11.0 software. The data were analyzed by two-way analysis of variance with multiple comparisons to detect whether bleomycin causes fibrotic changes in lungs and whether simvastatin treatment had a protective effect on bleomycin stimulation. The data were considered statistically significant at P <0.05.

RESULTS

Simvastatin attenuates bleomycin-induced pulmonary fibrosis
To assess whether administration of simvastatin during bleomycin-induced pulmonary fibrosis would attenuate the fibrotic response, we measured Ashcroft score and collagen accumulation as an index of fibrosis on day 28 after bleomycin infusion.

Histopathology
Histological examination of lung specimens demonstrated that bleomycin stimulation induced fibrotic lesions in mouse lungs, whereas a well-alveolized normal histology was seen in mice treated with saline (Figure 1). Although the fibrotic lesions were observed in the simvastatin (5 and 20 mg/kg) treatment group, both its extent and intensity were less than those of the bleomycin group (BLM group). To confirm the effect of simvastatin on the histopathological change of bleomycin-induced pulmonary fibrosis, the overall grades of the fibrotic changes in the lungs were obtained by numerical score (Ashcroft score) (Table 1). The scores of the mice administered with simvastatin (5 and 20 mg/kg) were significantly suppressed compared to the BLM group on day 28 after bleomycin infusion (P <0.05). The higher dose of simvastatin was associated with a more significant reduction in Ashcroft score (P <0.05).


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Figure 1. Representative histological lung sections from each group. The lungs of the mice on day 28 after bleomycin infusion were fixed in 4% paraformaldehyde, routinely processed, and stained with hematoxylin-eosin (Original magnification: ×200). (A) Control group, (B) BLM group, (C) bleomycin-plus-simvastatin 5 mg/kg group, and (D) bleomycin-plus-simvastatin 20 mg/kg group (n=10).

Hydroxyproline content
Collagen deposition in the lung was assessed by measuring the hydroxyproline content on day 28 after bleomycin infusion. Compared with the control group, hydroxyproline content of the lung significantly increased in the BLM group. The increased hydroxyproline content was then significantly decreased in the lungs administered with simvastatin (5 and 20 mg/kg) than in those treated with bleomycin (P <0.05, Table 1). The higher dose of simvastatin was associated with a more significant reduction in hydroxyproline content (P <0.05). Simvastatin alone did not affect hydroxyproline content in the lung compared to the controls (P >0.05).


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Table 1. Grade of pulmonary fibrosis and marker of collagen accumulation

Collagen synthesis
To further evaluate the effect of simvastatin on collagen synthesis induced by bleomycin, the gene expression of type I collagen and type III collagen in the lung treated with either bleomycin or saline was measured on day 28 after bleomycin infusion by real-time PCR (Figure 2). Compared with the control group, the expression of type I and type III collagen mRNA in the lung were increased in the BLM group (P <0.05). The increased expression of type I and type III collagen mRNA was then significantly inhibited in the lungs administered with simvastatin (5 and 20 mg/kg) compared with the BLM group (P <0.05). The higher dose of simvastatin was associated with a more significant reduction in the expression of type I and type III collagen mRNA (P <0.05). However, simvastatin alone did not affect these gene expressions in the lung compared to the control group (P >0.05).
 

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Figure 2. Representative real-time PCR analysis of type I and III collagen mRNA expression in the lung. The expression of type I and III collagen mRNA was measured on day 28 after bleomycin infusion (n=10). All data presented as the fold-change over control in collagen gene expression. The expression of type I and III collagen mRNA induced by bleomycin was higher than that in the control mice. The increased expression of type I and III collagen mRNA was significantly attenuated by simvastatin (5 and 20 mg/kg). *P <0.05 compared with the control group, **P <0.05 compared with the BLM group, ***P <0.05 compared with the bleomycin-plus-simvastatin 5 mg/kg group.

Simvastatin inhibits profibrotic cytokines production in bleomycin-induced pulmonary fibrosis
A variety of fibrogenic cytokines are implicated in the development of bleomycin-induced pulmonary fibrosis, including TGF-β1 and CTGF. To assess the effects of simvastatin on TGF-β1 and CTGF production, their gene expression and protein in the lung were examined by real-time RT-PCR and Western blotting on day 28 after bleomycin infusion.

The levels of TGF-β1 mRNA expression and protein induced by bleomycin were higher than those in the control mice (P <0.05, Figure 3). The increased levels of TGF-β1 mRNA expression and protein were significantly inhibited by administration of simvastatin (5 and 20 mg/kg), as compared to the BLM group (P <0.05). The higher dose of simvastatin was associated with a more significant reduction in the level of TGF-β1 mRNA expression and protein (P <0.05). However, simvastatin alone did not affect the level of the gene and protien in the lung compared to the controls (P >0.05).


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Figure 3. The level of TGF-β1 mRNA expression and protein in the lung. (A) TGF-β1 mRNA expression was measured by real-time PCR on day 28 after bleomycin infusion (n=10). All data presented as the fold-change over control in TGF-β1 gene expression. (B) TGF-β1 protein in the lung was measured by Western blot on day 28 after bleomycin infusion (n=10). The levels of TGF-β1 mRNA expression and protein induced by bleomycin were higher than those in the control mice. The increased levels of TGF-β1 mRNA expression and protein were significantly inhibited by administration of simvastatin (5 and 20 mg/kg). *P <0.05 compared with the control group, **P <0.05 compared with the BLM group, ***P <0.05 compared with the bleomycin-plus-simvastatin 5 mg/kg group.

The levels of CTGF mRNA expression and protein induced by bleomycin were higher than those in the control mice (P <0.05, Figure 4). The increased levels of CTGF mRNA expression and protein were significantly inhibited by administration of simvastatin (5 and 20 mg/kg) compared to the BLM group (P <0.05). The higher dose of simvastatin was associated with a more significant reduction in the level of CTGF mRNA expression and protein (P <0.05). However, simvastatin alone did not affect the level of the gene and protien in the lung compared to the controls (P >0.05).


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Figure 4. The level of CTGF mRNA expression and protein in the lung. (A) CTGF mRNA expression was measured by real-time PCR on day 28 after bleomycin infusion (n=10). All data presented as the fold-change over control in CTGF gene expression. (B) CTGF protein in the lung was measured by Western blot on day 28 after bleomycin infusion (n=10). The levels of CTGF mRNA expression and protein induced by bleomycin were higher than those in the control mice. The increased levels of CTGF mRNA expression and protein were significantly inhibited by administration of simvastatin (5 and 20 mg/kg). *P <0.05 compared with the control group, **P <0.05compared with the BLM group, ***P <0.05compared with the bleomycin-plus-simvastatin 5 mg/kg group.

Simvastatin attenuates inflammatory response in bleomycin-induced pulmonary fibrosis
Total and differential cell count in BALF
All BALFs were obtained on days 7 and 28 after intratracheal instillation of bleomycin. The number of total cells and the number of macrophages, neutrophils and lymphocytes were increased on day 7 after bleomycin infusion (P <0.05, Table 2). Simvastatin (5 and 20 mg/kg) administration significantly decreased the numbers of total cells, neutrophils and lymphocytes in bleomycin-stimulated mice (P <0.05), but did not affect the number of macrophages in BALF (P >0.05). The higher dose of simvastatin was associated with a more significant reduction in the numbers of total cells, neutrophils and lymphocytes (P <0.05). Similar changes were observed on day 28 after intratracheal instillation of bleomycin (Table 3).


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Table 2. Inflammatory cells and cytokines in BALF on day 7 after bleomycin infusion (n=10)

Inflammatory cytokine in BALF
Intratracheal instillation of bleomycin induces the production of inflammatory cytokines that contribute to the pathogenesis of pulmonary fibrosis. Hence, we investigated the effect of simvastatin pretreatment on TNF-α production on days 7 and 28 after bleomycin infusion. The level of TNF-α concentration significantly increased on day 7 after bleomycin infusion (P <0.05, Table 2). The increased expression of TNF-α concentration was then inhibited by simvastatin (5 and 20 mg/kg) administration (P <0.05). The higher dose of simvastatin was associated with a more significant reduction in TNF-α concentration (P <0.05). Similar changes were observed on day 28 after intratracheal instillation of bleomycin (Table 3).
 

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Table 3. Inflammatory cells and cytokines in BALF on day 28 after bleomycin infusion (n=10)

Simvastatin inhibits signal molecules activation in bleomycin-induced pulmonary fibrosis
Expression of p38 MAPK in the lung
The total expression and phosphorylation of p38 protein in the lung were analyzed by Western blotting on day 7 after intratracheal instillation of bleomycin (Figure 5A). The total expression and phosphorylation of p38 protein increased significantly in bleomycin-induced pulmonary fibrosis (P <0.05). The increased level of p38 phosphorylation was inhibited by simvastatin (5 and 20 mg/kg) administration (P <0.05). However, the total expression of p38 protein was not affected by simvastatin treatment (P >0.05).

Expression of RhoA in the lung
RhoA protein in the lung was analyzed by Western blot on day 28 after intratracheal instillation of bleomycin (Figure 5B). The expression of RhoA protein significantly increased in bleomycin-induced pulmonary fibrosis (P <0.05). The increased expression of RhoA was inhibited by simvastatin (5 and 20 mg/kg) administration compared to the BLM group (P <0.05).

Expression of Smad2 and Smad3 in the lung
The total expression and phosphorylation of Smad2 and Smad3 in the lung were analyzed on day 28 after intratracheal instillation of bleomycin (Figure 5C and 5D). The total expression and phosphorylation of Smad2 and Smad3 increased significantly in bleomycin-induced pulmonary fibrosis (P <0.05). The increased levels of Smad2 and Smad3 phosphorylation were inhibited by simvastatin (5 and 20 mg/kg) administration compared to the BLM group (P <0.05). However, the total expression of Smad2 and Smad3 was not affected by simvastatin treatment (P >0.05).


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Figure 5. The signaling pathway detected by Western blotting. (A) Expression of p38 MAPK in the lung was analyzed on day 7 after intratracheal instillation of bleomycin (n=10). (B) RhoA expression was analyzed on day 28 after bleomycin infusion (n=10). (C and D) The expression of Smad2 and Smad3 in the lung was analyzed on day 28 after bleomycin infusion (n=10). *P <0.05 compared with the control group, **P <0.05 compared with the BLM group, ***P <0.05 compared with the bleomycin-plus-simvastatin 5 mg/kg group.

DISCUSSION

This study examined whether treatment with simvastatin might attenuate pulmonary fibrosis that followed bleomycin infusion. Because inflammation and fibrogenesis are the two determinants of the progression of pulmonary fibrosis, potential benefit effects of simvastatin on bleomycin-induced pulmonary fibrosis were measured in the early inflammatory phase and late fibrotic phase. In this study, simvastatin had the following effects in bleomycin-induced pulmonary fibrosis: (1) attenuating the increase of Ashcroft score, hydroxyproline content and collagen (I and III) mRNA expression in the lung induced by bleomycin; (2) attenuating the production of TGF-β1 and CTGF in the lung; (3) decreasing inflammatory cells infiltration, TNF-α production and p38 MAPK activation in the early inflammatory phase after bleomycin infusion; and (4) inhibiting RhoA expression and Smad2/3 phosphorylation in bleomycin-induced pulmonary fibrosis.

Intratracheal administration of bleomycin induces acute alveolitis and interstitial inflammation, characterized by the sequential recruitment of leukocytes in the early inflammatory phase.29,30 Sustained and augmented expressions of some inflammatory cytokines in the lung are relevant to recruitment of inflammatory cells and accumulation of extracellular matrix components. There are compelling reasons to believe that TNF-α plays an important role in the pathogenesis of pulmonary fibrosis, which mediates pulmonary fibrosis via TGF-β1 production.31,32 In this study, simvastatin significantly reduced the numbers of neutrophils and lymphocytes, and the concentration of TNF-α in BALF that followed bleomycin instillation. The decrease of TNF-α concentration associated with the reduction of TGF-β1 and collagen expression in the lung. We therefore assume that the effect of simvastatin on bleomycin-induced pulmonary fiborsis may be partly due to the suppression of inflammatory cells infiltration and TNF-α production in the early inflammatory phase.

Although there are many reports on the anti-inflammatory potential of simvastatin, the underlying mechanisms that simvastatin regulates airway inflammation have not been elucidated. The p38 MAPK signal pathway is an important mediator for proinflammatory cytokines production (such as IL-1β and TNF-α) and TNF-α-induced inflammatory damage.31-33 It was proved that interruption of the p38 MAPK signal pathway could ameliorate the formation of bleomycin-induced pulmonary fibrosis.34 We have shown that the increased level of p38 phosphorylation after bleomycin stimulation is inhibited by simvastatin administration. Moreover, the decrease of p38 phosphorylation is correlated to the reduction of TNF-α concentration in BALF. These findings suggest that simvastatin may inhibit p38 MAPK activation, thus reducing proinflammatory cytokines production in bleomycin-induced pulmonary inflammation.

It is generally assumed that leukocytes infiltrating in the lung are involved in the evolution of pulmonary fibrosis by secreting reactive oxygen species, fibrogenic cytokines, and growth factors. Bleomycin up-regulates the production of proinflammatory and fibrogenic cytokines, which stimulate excessive collagen accumulation in the lung parenchyma. TGF-β1, as a link between inflammation and fibrosis, appears to play an integral role in promoting lung architecture changes secondary to the underlying inflammatory process.8,35,36 The upregulation of TGF-β1 and CTGF play critical roles in the pathogenesis of bleomycin-induced pulmonary fibrosis.10,37-39 It was proved that simvastatin inhibited TGF-β1 and CTGF gene expression in lung fibroblasts.23 We found that simvastatin treatment attenuated the protein and gene expression of TGF-β1 and CTGF in the lung of bleomycin-stimulated mice. The reduction of TGF-β1 and CTGF production paralleled to the decrease of type I collagen and type III collagen synthesis in the lung. These findings suggest that simvastatin may downregulate fibrogenic cytokines production, thus modulating the collagen synthesis in bleomycin-induced pulmonary fibrosis.

RhoA plays an important role in inflammatory and fibrotic response40,41 and acts as upstream of MAPK in the inflammatory process.42 Recent studies highlighted the effectiveness of simvastatin in inhibiting growth factors and profibrogenic markers expression via a Rho signaling mechanism in human lung fibroblasts.23,40,41,43 In the present study, simvastatin attenuated RhoA protein expression in bleomycin-stimulated lungs. The reduction of RhoA expression paralleled to the decrease of fibrogenic cytokines in the lung. Therefore, we assumed that the effect of simvastatin on bleomycin-induced pulmonary fibrosis may be partly due to the suppression of the Rho-signaling pathway.

In addition, we observed the activation of the Smad signal pathway including the total expression and phosphorylation of Smad2 and Smad3 in bleomycin- induced pulmonary fibrosis. The increased phosphorylation of Smad2 and Smad3 was attenuated by simvastain, but the total expression of Smad2 and Smad3 was not affected. To date there is no evidence that simvastatin has direct impact on the activation of the Smads signal pathway. As we know that the phosphorylation of Smad2 and Smad3 by the activated TGF-β1 receptor I is a major step in the initiation of TGF-β1 signals transduction. Many studies have tried to link Smad signaling with RhoA activity. Kamaraju and Roberts44 revealed that the Rho/ROCK pathway affects the linker region phosphorylation of Smad2/3 in human breast carcinoma cells. Recently, Chen et al45 reported that RhoA modulates the phosphorylation of Smad2 and Smad3 during TGF-β-induced smooth muscle differentiation. Therefore, we supposed that the inactivation of the Smad signaling pathway may be caused by inhibition of inflammatory mediators or interaction with other signal molecules. The detailed mechanisms need further investigation.

In summary, simvastatin attenuates bleomycin-induced pulmonary fibrosis, as indicated by decreases in Ashcroft score and lung collagen accumulation. Simvastatin generates significant impact on inflammatory cells infiltration and proinflammatory cytokines production in the early inflammatory phase and fibrogenic cytokines production in the late fibrotic phase, which may be responsible for the beneficial effect of simvastatin on bleomycin-induced pulmonary fibrosis. Although the inhibition of the p38 MAPK, RhoA and Smad signal transduction has been observed during the simvastatin administration, the detailed mechanisms by which simvastatin acts to prevent bleomycin-induced pulmonary fibrosis remain to be determined in light of the pleiotropic effects of simvastatin and the complex pathogenesis of pulmonary fibrosis. Current results suggest that simvastatin may have potential benefitial effects on pulmonary fibrosis.

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  1. China Medical Board of New York to Dr. WEN Fu-qiang,No. 00-722 and 06-834;National Natural Science Foundation of China,No. 3042500, 30370627, 30670921;