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Oral pirfenidone protects against fibrosis by inhibiting fibroblast proliferation and TGF-β signaling in a murine colitis model
Biochemical Pharmacology xxx (2016) xxx–xxx
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Oral pirfenidone protects against fibrosis by inhibiting fibroblast proliferation and TGF-b signaling in a murine colitis model Guanwei Li a, Jianan Ren a,⇑, Qiongyuan Hu a,b, Youming Deng a, Guopu Chen a, Kun Guo a, Ranran Li a, Yuan Li a, Lei Wu a, Gefei Wang a, Guosheng Gu a, Jieshou Li a a b
Department of Surgery, Jinling Hospital, Medical School of Nanjing University, Nanjing, China Medical School of Southeast University, No. 87 Dingjiaqiao Road, Nanjing 210009, China
a r t i c l e
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Article history: Received 14 July 2016 Accepted 2 August 2016 Available online xxxx Keywords: Pirfenidone Inflammatory bowel disease Intestinal fibrosis Antifibrotic therapy
a b s t r a c t Inflammatory bowel disease (IBD), particularly Crohn’s disease, frequently causes intestinal fibrosis that ultimately leads to formation of strictures requiring bowel resection. Currently there is no effective antifibrotic therapy available for this disease. Pirfenidone is a small compound that has a broad spectrum of antifibrogenic effect and has been used for the treatment of fibrotic diseases in various organs. The present study aimed to investigate the antifibrogenic effect of pirfenidone in a dextran sulfate sodium (DSS)induced murine colitis model. C57BL/6 mice were used and animals were randomly divided into groups receiving pirfenidone or vehicle by oral or transanal routes. Inflammation- and fibrosis-related indexes including body weight, colon length, disease activity, histological change, mRNA expression of proinflammatory and pro-fibrogenic cytokines were assessed. Furthermore, we performed in vitro analysis using CCD18-Co fibroblasts to evaluate cell proliferation, transdifferentiation, and viability after the cells were cultured with pirfenidone. It was found that oral administration of pirfenidone reduced deposition of collagen in colitis-associated fibrosis, and significantly suppressed the mRNA expression of col1a2, col3a1, and TGF-b. Moreover, pirfenidone inhibited the activation of TGF-b-related smad and MAPK pathways both in vitro and in vivo. Clinical and histological evaluation demonstrated that pirfenidone had no anti-inflammatory effect. The antifibrogenic effect was reduced when pirfenidone was administered in a delayed manner and was unobserved if given locally. Pirfenidone suppressed fibroblast proliferation and transdifferentiation without observed toxicity. Altogether, our results suggested that oral pirfenidone protects against fibrosis of DSS-induced colitis through inhibiting the proliferation of colonic fibroblasts and TGF-b signaling pathways. Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction Inflammatory bowel disease (IBD), particularly Crohn’s disease and ulcerative colitis, is featured by chronic inflammation of the gastrointestinal tract whose etiology remains unclear. Crohn’s disease is characterized by transmural inflammation that involves the entire thickness of the bowel leading to dysfunctional wound healing and abnormal collagen accumulation, which causes excessive fibrosis and formation of strictures [1]. Despite recent advances in exploring the pathogenesis and treatment of Crohn’s disease thus far, there is no effective medical therapy for prevention or cure of disease-associated fibrogenesis, making surgery the only option for symptomatic/complicated strictures. Approximately ⇑ Corresponding author at: Department of Surgery, Jinling Hospital, 305 East Zhongshan Road, Nanjing 210002, China. E-mail address: [email protected] (J. Ren).
75% of patients with Crohn’s disease eventually undergo at least one surgical procedure due to intestinal fibrosis [2]. Furthermore, 70–90% of patients suffer a relapse within one year after surgery, and more than half have recurrent strictures that could necessitate further bowel resections [3,4]. Intestinal fibrosis has therefore represented the main indication for surgery in patients with Crohn’s disease, and created substantial burden on healthcare resources and individuals. Therapies that are effective in attenuating mucosal inflammation do not prevent or reverse fibrosis in Crohn’s disease [5]. Given that fibrotic changes associated with diverse diseases in various organs share common pathogenic pathways such as the transforming growth factor beta (TGF-b) pathway [6], it is of great value to think ‘outside of the box’ and to learn from other fibrotic diseases in terms of pathogenesis and treatment. Pirfenidone, an active small molecule which consists of a modified phenylpyridone, has been used to treat various types of fibrotic diseases including
http://dx.doi.org/10.1016/j.bcp.2016.08.002 0006-2952/Ó 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: G. Li et al., Oral pirfenidone protects against fibrosis by inhibiting fibroblast proliferation and TGF-b signaling in a murine colitis model, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.08.002
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idiopathic pulmonary fibrosis, renal fibrosis, cardiac fibrosis, and liver cirrhosis with great efficacy and safety [7]. This compound exhibits remarkable antifibrotic effects in a variety of animal and cell-based models [8]. Kadir et al. reported that pirfenidone inhibited the proliferation and secretion of collagen, MMP-3 and TIMP-1 in fibroblasts isolated from patients of Crohn’s disease [9], reinforcing the notion that pirfenidone may have the potential to become a new avenue for therapeutic intervention of intestinal fibrosis. A recent study also showed promising antifibrotic effect of pirfenidone in a newly developed mouse model of intestinal fibrosis [10]. In this study, we examined whether administration of pirfenidone via oral or anal routes could prevent or attenuate colonic fibrosis in a dextran sulfate sodium (DSS)-induced colitis mouse model. Furthermore, we examined the potential effect of pirfenidone exerted on human colonic fibroblasts in vitro to explore its cellular mechanism of action. 2. Material and methods 2.1. Establishing a murine colitis model 6–8 week old female C57BL/6 mice weighing 18–20 g were purchased from the Cavens Lab (Changzhou, China), and maintained under specific pathogen-free conditions in the Animal Facility at Mergene Company (Nanjing, China). Chronic colonic fibrosis model was created by administering four cycles of 2.5% DSS (MP Biomedicals, USA) in drinking water. In brief, the mice received 2.5% DSS drinking water on days 1–5, 8–12, 15–19 and 22–26, and regular drinking water during the remaining days. Two pirfenidone treatment regimens were established. In the prophylactic setting, 300 mg/kg/d pirfenidone (TCI, Shanghai, China) was suspended in 0.5% carboxymethylcellulose solution (CMC, vehicle) and administered orally twice per day beginning from day 0. In the therapeutic setting, pirfenidone was administered beginning from day 14 of DSS induction of colitis. Mice were randomly divided into three groups: normal mice treated with CMC (Control group), DSS mice treated with CMC (DSS group) and DSS mice treated with pirfenidone (PFD group). The experimental dose of pirfenidone was selected with reference to published reports [11]. Additional groups of mice using the same experimental setting above received pirfenidone by a blunt needle via the transanal route to clarify the antifibrotic effects of local application. Each group consisted of 6–8 mice. Animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the Institute for Laboratory Animal Research and have received approval from Animal Investigation Ethics Committee of Jinling Hospital. 2.2. Disease activity index and histological scoring Disease activity index (DAI) at day 14 and 28 was the combined scores of weight loss, stool consistency and bleeding based on a previous scoring system [12]. All animals were sacrificed at day 29 except for a subgroup of mice serving as pre-treatment controls that were sacrificed at day 14 in the therapeutic setting. Colonic tissues were fixed in neutral buffered formalin and embedded in paraffin. Paraffin sections were cut and stained with hematoxylin-eosin. The sections were read and graded by two blinded investigators as previously described [13]. The amount and extent of inflammation, crypt damage as well as the percentage involved were evaluated to calculate the total histological scores. For evaluation of fibrosis, tissues were stained with Masson Trichrome Staining Kit, and mucosal and submucosal locations of fibrosis were scored using a 0–3 scale (0 = no fibrosis, 1 = mild fibrosis, 2 = moderate fibrosis, and 3 = severe fibrosis) [14].
2.3. qRT-PCR Quantification of the gene expression of col1a2, col3a1, TGF-b, TNF-a, IL-1b, and IL-6 was performed by quantitative real-time PCR (qRT-PCR). Briefly, total RNAs were isolated from colon tissues by Trizol reagent (TAKARA) and reverse-transcribed into cDNAs using the reverse transcription kit (TAKARA, RR047, Takara Biomedical Technology, Beijing, China) according to the manufacturer’s instructions. Real-time PCR was subsequently performed in triplicate using the StepOnePlus PCR System (Applied Biosystems, Carlsbad, USA). All mRNA quantification data were normalized to GAPDH. 2.4. Cell cultures Human CCD-18Co colonic fibroblasts were cultured in minimal essential medium Eagle’s medium (WISENT, Nanjing, China) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. All experiments were performed after serum-free starvation for 24 h. Pirfenidone (0.5, 1.0 and 1.5 mg/ml) was dissolved in dimethylsulfoxide (DMSO). TGF-b (5 ng/ml) was added at the same time in some experiments to evaluate the effects of pirfenidone on myofibroblast trans-differentiation, collagen expression, and TGF-b related downstream signaling pathway molecules. 2.5. Assessment of cell proliferation CCK8 assay was used to measure the proliferative capability of CCD-18Co cells after pirfenidone treatment. Cells in the exponential growth phase were harvested, inoculated in 96-well plates, and incubated overnight. Pirfenidone mixed with MEME medium at the concentrations of 0.5, 1.0, 1 and 1.5 mg/ml was applied to CCD-18Co cells for 12, 24, 48, and 72 h following serum deprivation for 24 h. The CCK8 assay was performed with a commercially available kit (7sea biotech, Shanghai, China) according to the manufacturer’s instructions. The numbers of cells treated with different concentrations of pirfenidone were counted under a microscope. 2.6. Assessment of cell integrity The activity of lactate dehydrogenase (LDH) in CCD-18Co cells was examined using a commercialized kit (KeyGEN BioTECH, Nanjing, China). The trypan blue exclusion test was performed after treatment with pirfenidone (0.5, 1.0, 1.5 mg/ml) for 48 h. In LDH assay, the culture medium was collected and assayed according to the manufacturer’s instructions. LDH release following pirfenidone treatment was indicated as a percentage relative to the untreated control cultures. In trypan blue exclusion test, viable cells were counted in triplicate on a hemocytometer after trypan blue staining by a blinded investigator. The percentage of living cells was calculated and expressed as a ratio of viable cell count (unstained) to total cell count. 2.7. Western blot analysis Colonic CCD-18Co fibroblasts were incubated with or without pirfenidone (0.5, 1.0, 1.5 mg/ml) and TGF-b (5 ng/ml) for 24 h. The cells and the colon tissues from sacrificed mice were lysed with lysis buffer and the protein concentrations were determined with the BCA assay kit (Beyotime, Shanghai, China). Equal amounts of cell extracts were fractionated by 4–12% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separate proteins were then transferred to a Polyvinylidene Fluoride membrane (PVDF; MILLIPORE). Nonspecific binding was blocked in 5% nonfat milk or 5% BSA in TBST for 2 h, and then incu-
Please cite this article in press as: G. Li et al., Oral pirfenidone protects against fibrosis by inhibiting fibroblast proliferation and TGF-b signaling in a murine colitis model, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.08.002
G. Li et al. / Biochemical Pharmacology xxx (2016) xxx–xxx
bated overnight at 4 °C with primary antibodies against p-ERK(CST, 4511,), p-P38(CST, 4668), p-JNK(CST, 4370), p-smad2(CST, 3108), p-smad3(CST, 9520), (all from Cell Signaling Technology, Danvers), procol1A1 (Santa Cruz, sc-25973, Shanghai, China) and b-actin (zen bioscience, 200068-8F10, Chengdu, China). The membranes were then washed and incubated with horseradish peroxidaseconjugated secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized using chemiluminescent reagent and the signals were captured by a Tanon Imaging System (Tanon, Shanghai, China). The protein levels were quantified by densitometric analysis with Image J software and standardized by calculating the ratio of target to b-actin mean intensity. 2.8. Immunofluorescence staining Nuclear expression of ki67 reflecting the proliferative activity of cells was measured. In brief, colonic fibroblasts seeded on 24-well plates were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton following treatment with vehicle (DMSO) or pirfenidone at concentrations of 0.5, 1.0 and 1.5 mg/ml for 48 h. After blocking non-specific proteins with 10% goat serum, the cells were incubated with primary antibody against ki67 (1:500; Abcam, ab92353, Cambridge, UK) and then with Alexa Fluor 488conjugated secondary antibody. DAPI (4,6-diamidino-2phenylindole) staining was used to locate the nuclei. Transdifferentiation of fibroblasts into myofibroblasts was an important event involved in gut fibrogenesis. Therefore fibroblasts incubated with TGF-b in the absence or presence of pirfenidone (0.5, 1.0, 1.5 mg/ml) for 24 h were subjected to immunofluorescence staining to measure the expression of a-SMA, the marker of myofibroblasts. Primary antibody to a-SMA (1:100; Abcam, ab5694, Cambridge, UK) and Alexa Fluor 555-conjugated secondary antibody were used. For visualization of the collagen expression in fibroblasts in vivo, frozen colonic tissues were sectioned and fixed in acetone. Slides were incubated with primary antibodies including col1a2 (Abcam, ab96723, Cambridge, UK) and vimentin (Abcam, ab20346, Cambridge, UK) for 2 h at room temperature, and then incubated with Alexa Fluor 488- and 555-conjugated secondary antibodies. After rinsing in PBS, slides were stained with DAPI, observed and photographed under a confocal microscope (Zeiss, LSM 700). 2.9. Statistical analysis Results were expressed as mean ± SD and were analyzed using IBM SPSS software for Windows version 19.0. Comparison between two groups was performed by Fisher’s Exact test for categorical variables and Student’s t-test for continuous variables. For comparison between three groups, a One-way ANOVA was used and LSD post hoc method was used to analyze the intergroup differences. All P values were two-sided, and P < 0.05 was considered statistically significant.
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tored. It was found that the body weight of the DSS-treated mice was significantly lower than that of normal mice at days 14 and 28. Oral administration of pirfenidone showed no effect on the body weight loss whether it was started from day 0 or on day 14 (Fig. 1B). In both experimental settings, treatment with pirfenidone did not significantly alter the clinical status of DSS colitis (Fig. 1C). The length of colon in mice with chronic colitis induced by DSS was significantly shorter than that of normal mice (Fig. 1D). The length of the colon in the mice prophylactically receiving oral pirfenidone was significantly longer compared with the parallel CMC group (Fig. 1D). However pirfenidone used in a delay manner or by transanal route showed no significant effect (data not shown). Because shortening of the colon could result from the chronic inflammation and intestinal fibrosis in the DSS colitis model [15], we examined the histological change on the colonic tissues. Hematoxylin-eosin staining showed that the degree of colonic inflammation including inflammatory infiltrate and tissue damage caused by DSS remained unchanged after treatment with oral pirfenidone in both the prophylactic (Fig. 2A) and therapeutic settings (Fig. 2B). Similar results were observed when pirfenidone was used locally (intra-colon) (data not shown). Oral pirfenidone suppressed the expressions of mRNA of fibrosis-associated molecules including col1a2, col3a1 and TGF-b (Fig. 3A), but not the inflammationassociated cytokines including TNF-a, IL-1b and IL-6 (Fig. 3B). This result was consistent with the histological evaluation. These results collectively suggested that pirfenidone exerted no antiinflammatory effects on DSS-induced colitis model. 3.2. Oral administration of pirfenidone reduces colitis-associated intestinal fibrosis The antifibrotic effects of pirfenidone on DSS colitis-associated colonic fibrosis were next evaluated by Masson trichrome staining. Increased collagen deposition was observed in the colon mucosa and submucosa of mice with DSS-induced chronic colitis (Fig. 4). Oral administration of pirfenidone from day 0 (Fig. 4A) or day 14 (Fig. 4B) both inhibited the fibrosis, depicted by Masson trichrome staining as a lesser extent of collagen formation. The quantitative score of fibrosis was significantly reduced in DSS-treated mice that were fed pirfenidone prophylactically (Fig. 4A); the score did not reach statistical significance when pirfenidone treatment was postponed (Fig. 4B), suggesting that early use of pirfenidone is a necessity to prevent intestinal fibrosis. Immunofluorescence staining showed that collagen expression and vimentin-expressing fibroblasts were marked in the colons of DSS-treated mice, and significantly reduced after oral pirfenidone administration (Fig. 5). This result was consistent with the expression of mRNA of multiple types of collagen and profibrogenic cytokines determined by qRTPCR (Fig. 3A). However, the significant anti-fibrogenic effects of pirfenidone were not observed when pirfenidone was delivered intracolonically (data not shown). 3.3. Pirfenidone inhibits colonic fibroblast proliferation and transdifferentiation in vitro without cytotoxicity
3. Results 3.1. Pirfenidone exerts no anti-inflammatory effects on DSS-induced colitis in mice To investigate the roles of pirfenidone in murine colitis, mice were given four cycles of 2.5% DSS in drinking water on days 1– 5, 8–12, 15–19, and 22–26 to induce colitis. In the prophylactic setting, mice were orally administered pirfenidone twice per day from day 0 (Fig. 1A, left panel). In the therapeutic setting, mice were orally administered pirfenidone twice per day from day 14 (Fig. 1A, right panel). The body weight of these mice was moni-
To understand the cellular mechanism underlying the antifibrogenic effects of pirfenidone, we investigated the effect of pirfenidone on the proliferation of fibroblasts in vitro. Human colonic fibroblasts CCD-18Co cells were incubated with various concentrations of pirfenidone (0.5, 1.0, 1, and 1.5 mg/ml) or DMSO for 12, 24, 48, and 72 h following serum deprivation for 24 h. Cells were then subjected to the CCK8 assay. The assay showed that proliferation of colonic fibroblasts was suppressed by pirfenidone in a dose- and time-dependent manner (Fig. 6A). Furthermore, cells treated with different concentrations of pirfenidone or DMSO for 48 h and cell numbers were counted under a microscope. The
Please cite this article in press as: G. Li et al., Oral pirfenidone protects against fibrosis by inhibiting fibroblast proliferation and TGF-b signaling in a murine colitis model, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.08.002
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Fig. 1. Oral pirfenidone has no effect on colonic inflammation in mice with DSS-induced chronic colitis. (A) Pirfenidone treatment schematics. Mice received four cycles of 2.5% DSS in drinking water on days 1–5, 8–12, 15–19 and 22–26. In the prophylactic setting (left panel), pirfenidone was suspended in 0.5% CMC and administered orally twice per day from day 0. In the therapeutic setting (right panel), pirfenidone was administered after two weeks of DSS induction of colitis. (B) Percentage of body weight change of different groups at days 0, 14 and 28. (C) Disease activity index (DAI) at days 14 and 28 in CMC-treated and pirfenidone-fed groups in the DSS colitis model. The DAI in Normal + CMC group is 0. (D) The length of the colon after mice were sacrificed. n = 6–8 mice/group. *P < 0.05, **P < 0.01.
result of cell number counting (Fig. 6B) was consistent with that of the CCK8 assay. Cell integrity was investigated by LDH release assay (Fig. 6C) and trypan blue exclusion test (Fig. 6D) after treating the cells with pirfenidone (0.5, 1.0, 1.5 mg/ml) for 48 h. It was found that pirfenidone had no cellular toxicity and was safe to use at the tested concentration and time duration. The colonic fibroblasts after treatment with DMSO or pirfenidone at concentrations of 0.5, 1.0, and 1.5 mg/ml for 48 h were subjected to Ki67 immunofluoresence staining, another indicator of cell proliferation. It was found that Ki67 positive cells significantly decreased dose-dependently (Fig. 7). This result further confirmed that pirfenidone inhibited fibroblast cell proliferation. In addition to fibroblast proliferation, transdifferentiation of fibroblasts toward myofibroblast phenotype is another key event involved in the fibrogenic process of chronic colitis. TGF-b induces the transdifferentiation of CCD-18Co fibroblasts toward myofi-
broblast phenotype. When fibroblasts were incubated with TGF-b in the absence or presence of pirfenidone (0.5, 1.0, 1.5 mg/ml) for 24 h and subjected to immunofluorescence staining, we found that pirfenidone inhibits the TGF-b-induced transdifferentiation of CCD-18Co fibroblasts toward myofibroblasts indicated by a-SMA expression (Fig. 8). This result demonstrated that pirfenidone inhibited TGF-b-induced transdifferentiation of myofibroblasts from fibroblasts. 3.4. Pirfenidone inhibits TGF-b-related Smad and MAPK pathways in vitro and in vivo TGF-b is a key profibrotic factor and a central mediator of fibrogenesis that signals both in a classical smad pathway and in an atypical MAPK pathway [16]. We examined CCD-18Co cells treated with TGF-b (5 ng/ml) for 24 h in the absence or presence
Please cite this article in press as: G. Li et al., Oral pirfenidone protects against fibrosis by inhibiting fibroblast proliferation and TGF-b signaling in a murine colitis model, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.08.002
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Fig. 2. Oral pirfenidone has no effect on histological inflammation in mice with DSS-induced chronic colitis. Representative hematoxylin-eosin stained image of colonic tissue sections from the prophylactic (A) and therapeutic (B) experimental settings. Histology score was based on hematoxylin-eosin staining images of colonic tissues, and evaluated by the amount and extent of inflammation, crypt damage as well as the percentage involvement. n = 6–8 mice/group. *P < 0.05, **P < 0.01.
of various concentrations of pirfenidone (0.5, 1.0, 1.5 mg/ml). We found that pirfenidone significantly reduced TGF-b-stimulated procollagen expression in a dose-dependent manner (Fig. 9A, C). We also investigated by Western blot the change of activated states of several molecules that initiate and modulate these pathways, that is, the phosphorylation of smad2, smad3,
ERK, p38, and JNK. The activation of these molecules in fibroblasts was all inhibited by pirfenidone treatment (Fig. 9A, C), with the highest effect obtained at the maximum concentration (1.5 mg/ml). Significant activation of smad and MAPK pathways observed in colitis-associated fibrosis was also inhibited in mice treated with pirfenidone (Fig. 9B, D).
Please cite this article in press as: G. Li et al., Oral pirfenidone protects against fibrosis by inhibiting fibroblast proliferation and TGF-b signaling in a murine colitis model, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.08.002
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Fig. 3. Oral pirfenidone suppresses the increased expressions of profibrotic but not proinflammatory mediators in mice with DSS-induced chronic colitis. Pirfenidone was administered from day 0. Mice were sacrificed at day 29 and total RNA was isolated from colon tissues for qRT-PCR assay. (A) The mRNA expression levels of fibrosisassociated factors including col1a2, col3a1 and TGF-b. (B) The mRNA expression levels of inflammation-associated cytokines including TNF-a, IL-1b and IL-6. n = 6 mice/group. **P < 0.01.
4. Discussion Long term tissue damage and dysregulated wound healing accompanying Crohn’s disease lead to abnormal in situ deposition of collagen and progressive luminal narrowing. Despite advances in the treatment of Crohn’s disease, specific intestinal antifibrotic therapy is still lacking [17]. The incidence of stricture formation and rate of surgery in Crohn’s disease has not decreased [17]. In this study, we investigated the roles of pirfenidone, a compound that has shown great potential in attenuating the fibrogenic process in various organ systems, in the prevention or cure of intestinal fibrosis using a murine DSS-colitis model. We found that pirfenidone administered by oral route significantly protected the colons from fibrogenic development without obvious effects on the inflammatory condition. Double immunofluorescence staining indicated less number of vimentin-expressing fibroblasts and reduced accumulation of collagen in the injured intestinal tissue of pirfenidone-treated mice. One of the key events leading to intestinal fibrosis is the exposure of mesenchymal cells to a variety of mediators that induce a profibrotic phenotype, among which TGF-b is the central regulator that activates smad and MAPK pathways for controlling collagen deposition and fibrogenesis. To address the mechanisms underlying the antifibrotic ability of pirfenidone in the context of the gut, human CCD-18Co colonic fibroblasts were cultured with or without TGF-b in the absence or presence of pirfenidone. We showed that pirfenidone inhibited the proliferation but not viability of intestinal fibroblasts, hampered the TGF-b-induced myofibroblast differentiation, and reduced the cellular secretion of procollagen. Our results also indicated that the downregulated expression of collagen in response to pirfenidone was mediated by the inhibition of smad and MAPK pathways in vitro and in vivo.
Pirfenidone was the first approved drug for idiopathic pulmonary fibrosis (IPF), a chronic, progressive, and fibrogenic lung disease characterized by abnormal extracellular matrix deposition and irreversible loss of lung function. IPF was the most studied disease with the most established curative efficacy of pirfenidone for its antifibrotic activities. A phase 3 trial published in the New England Journal of Medicine showed results of IPF patients who received pirfenidone therapy had significantly improved lung function, exercise tolerance, and progression-free survival compared to placebo [18]. A variety of profibrogenic factors including growth factors and cytokines such as TGF-b, platelet-derived growth factor (PDGF), CTGF, IL4, and IL-13 function in a similar way in various organ systems [19]. This fact supports the notion that results stemmed from IPF can be transferable to other fibrogenic diseases, and rationalizes the clinical application of pirfenidone in the treatment of renal fibrosis, cardiac fibrosis, and liver cirrhosis [7]. Pirfenidone has also been shown to have antifibrotic effects in a wide range of clinical and animal-based studies involving multiple sclerosis [20], scleroderma [21], bladder fibrosis [22], radiation-induced fibrosis [23], and others. In the context of the gastrointestinal tract, a previous study found that pirfenidone protected against stricture formation in an animal model of caustic esophageal burn when used in the form of gel by gavage [24], indicating that pirfenidone might also play a protective role in strictures caused by other gastrointestinal disease such as Crohn’s disease. DSS-induced colitis is driven by a Th1 mediated cytokine immune response that mimics IBD [25], and is commonly used as a model of chronic intestinal inflammation and fibrosis [26,27]. In this study, pirfenidone administered either by gavage or transanal route showed no significant effect on the inflammatory condition of the murine colitis, demonstrated by the histolog-
Please cite this article in press as: G. Li et al., Oral pirfenidone protects against fibrosis by inhibiting fibroblast proliferation and TGF-b signaling in a murine colitis model, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.08.002
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Fig. 4. Oral pirfenidone decreases the amount of intestinal fibrosis in mice with DSS-induced chronic colitis. Representative image of Masson Trichrome staining for collagen from the prophylactic (A) and therapeutic (B) experimental settings. Collagen deposited in the mucosal and submucosal layer of DSS-treated mice was assessed by fibrosis score. n = 6–8 mice/group. *P < 0.05, **P < 0.01.
ical evaluation of the injured colons and the mRNA expression levels of multiple inflammatory cytokines. Most of the previous studies focused on the protective role of pirfenidone in fibrogenic process of various organs [8], and there were only several reports with respect to inflammation [28–31]. Hirano et al. reported that subcutaneous injections of pirfenidone attenuated allergic airway inflammation along with inhibited secretion of IL-4, IL-5, and
IL-13 after chronic allergen challenge [29]. Oral administration of pirfenidone reduced tissue level of multiple cytokines including TNF-a, IFN-c, IL-1b, IL-6, IL-12p40, and IL-18 in bleomycininduced murine pulmonary fibrosis [28]. Whether pirfenidone possesses anti-inflammatory activity in colitis remains inconclusive. Drugs targeting fibrosis such as pirfenidone seemed not to affect inflammatory status in our study, therefore pirfenidone should
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Fig. 5. Oral pirfenidone reduces collagen expression in colonic fibroblasts in vivo. Pirfenidone was administered from day 0 and mice in the normal, CMC-treated and pirfenidone-fed groups were sacrificed at day 29. Immunofluorescence staining showed col1a2 (green), fibroblast marker vimentin (red), and nuclei DAPI (blue). Merging images resulted in a yellowish color that indicated the colocalization of collagen expression in colonic fibroblasts. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 6. Pirfenidone inhibits proliferation of colonic CCD-18Co fibroblasts with no cytotoxicity effects. (A) CCK8 assay. Various concentrations of pirfenidone (0.5, 1.0, 1 and 1.5 mg/ml) or DMSO were applied to CCD-18Co cells for 12, 24, 48 and 72 h following serum deprivation for 24 h. Cells were then subjected to the CCK8 assay. (B) Cell count. Cells treated with different concentrations of pirfenidone or DMSO for 48 h, and were resuspended and counted under a microscope. Cytotoxicity effects were assessed after treatment with pirfenidone (0.5, 1.0, 1.5 mg/ml) for 48 h by measuring LDH activity (C) and by the trypan blue exclusion test (D). *P < 0.05, **P < 0.01, ***P < 0.001.
Please cite this article in press as: G. Li et al., Oral pirfenidone protects against fibrosis by inhibiting fibroblast proliferation and TGF-b signaling in a murine colitis model, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.08.002
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Fig. 7. Pirfenidone treatment inhibits nuclear expression of ki67 in CCD-18Co fibroblasts. Representative image of ki67 staining in colonic fibroblasts was shown after treatment with DMSO or pirfenidone at concentrations of 0.5, 1.0 and 1.5 mg/ml for 48 h.
be used in association with other compounds that exert an antiinflammatory effect in the intestine. Previous studies supported the possibility that local administration of pirfenidone may be protective against tissue fibrogenesis in skin lesions [21,32]. To test the efficacy of topical pirfenidone treatment in DSS-inducing colitis, additional groups of mice received pirfenidone via the transanal route. The mice developed fibrosis as the parallel groups treated by vehicle indicating that the topical effect of pirfenidone may be tissuespecific. Besides, the effects of transanal administration of pirfenidone can be influenced by other factors such as mucous barrier and intraluminal feces, and there was also the possibility that the enema given by a blunt needle could be partly excreted from the anal. We did not rule out the potential of pirfenidone to be used transanally. Pirfenidone is able to move through cell membranes without requiring a receptor, and it is rapidly absorbed after oral administration and reaches its maximum levels in blood after about one hour [33]. The findings in this study suggested that orally administered pirfenidone significantly suppress the fibrogenic process when given prior to the fibrotic phase of DSS model. As for the therapeutic regimen, the
antifibrotic efficacy of pirfenidone was reduced when the drug was given after the occurrence of colitis, and it seemed that pirfenidone posed no effect on the existed collagen deposition at day 14. The results derived from this preliminary study should be treated cautiously and required further investigation. In vitro studies using colonic fibroblast cell line CCD-18Co demonstrated that preventive effect of pirfenidone against progression of intestinal fibrosis may involve several key events of the fibrogenic process, including the proliferation of fibroblasts, transdifferentiation into myofibroblasts, and synthesis of extracellular matrix. The proliferative activity of colonic fibroblasts cultured with pirfenidone were remarkably reduced while cell viability remained unaffected, consistent with a number of previous studies based on fibroblasts isolated from patients with Crohn’s disease [9] and various cell lines of fibroblasts [34–36]. In vivo study demonstrated a decrease in vimentin-expressing fibroblasts in the colonic tissues of pirfenidone-treated mice. However, the CCK-8 assay by Meier et al. using primary murine colonic fibroblasts yielded divergent results [10]. In their study, commercial EsbrietÒ tablets dissolved in methanol were used instead of a pure compound of pirfenidone.
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Fig. 8. Immunofluoresent staining of a-SMA. Pirfenidone inhibits the TGF-b-induced transdifferentiation of CCD-18Co fibroblasts toward myofibroblast phenotype indicated by a-SMA expression. Fibroblasts incubated without TGF-b (A), with TGF-b alone (B), with TGF-b in the presence of DMSO (C) or 0.5 mg/ml (D), 1.0 mg/ml (E) and 1.5 mg/ml (F) pirfenidone for 24 h were subjected to immunofluorescence staining for a-SMA (yellow) and nuclei DAPI (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 9. Pirfenidone inhibits activation of smad and MAPK pathways in vitro and in vivo. CCD-18Co cells were treated with TGF-b (5 ng/ml) for 24 h in the absence or presence of various concentrations of pirfenidone (0.5, 1.0, 1.5 mg/ml) (A, C). Pirfenidone was administered from day 0 and mice in the normal, CMC-treated and pirfenidone-fed groups were sacrificed at day 29 (B, D). Expression of procol1A1 and phosphorylation of smad2, smad3, p38, ERK and JNK were assessed by Western blot analysis. Representative blots of one out of three separated experiments were shown (A, B). Graphs with means ± SD of target/b-actin gene ratios were obtained by densitometric analysis (C, D). *P < 0.05, **P < 0.01 compared with TGF-b-treated cells/CMC-treated colitis mice.
TGF-b is one of the most potent profibrotic factors that trigger the conversion of fibroblasts into myofibroblasts and secretion of collagen through the activation of smad and MAPK pathways. In the present study, these processes were downregulated and collectively contributed to the reduced expression of collagen in vitro
and in vivo following pirfenidone treatment. The findings derived from colonic fibroblasts were also in accordance with the results based on human lung fibroblasts [34] and orbital fibroblasts [37]. The universal inhibition on TGF-b pathways may explain the broad antifibrogenic spectrum of pirfenidone against fibrosis in various
Please cite this article in press as: G. Li et al., Oral pirfenidone protects against fibrosis by inhibiting fibroblast proliferation and TGF-b signaling in a murine colitis model, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.08.002
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organs. Increased tyrosine phosphorylation of p125 FAK is a central regulator of cell migration, which may also be involved as demonstrated by Meier et al. using a heterotopic transplantation model [10]. In conclusion, we demonstrated that oral pirfenidone protected against the progression of DSS colitis-related fibrosis without affecting the inflammatory status of colonic tissues. The mechanisms involved may be related to the downregulation of profibrotic TGF-b and inhibition of the TGF-b downstream smad and noncanonical MAPK pathways, as well as the antiproliferative activity against collagen-produced colonic fibroblasts. Antifibrotic therapy should be considered independently from antiinflammatory drugs in the treatment regimen, especially for those patients with obstructive phenotype, in order to prevent strictures. Pirfenidone has been efficaciously used in treating fibrosis in various organs with a sufficient safety profile and could be a promising alternative for the prevention and treatment of colitis-related fibrosis. Conflict of interest All authors declare no conflicts of interest. Acknowledgements This work was supported by grants from National Natural Science Foundation of China [81270478, 81571881]. References [1] J. Cosnes, S. Cattan, A. Blain, L. Beaugerie, F. Carbonnel, R. Parc, J.P. Gendre, Long-term evolution of disease behavior of Crohn’s disease, Inflamm. Bowel Dis. 8 (4) (2002) 244–250. [2] A. Spinelli, C. Correale, H. Szabo, M. Montorsi, Intestinal fibrosis in Crohn’s disease: medical treatment or surgery? Curr. Drug Targets 11 (2) (2010) 242– 248. [3] G. Olaison, K. Smedh, R. Sjodahl, Natural course of Crohn’s disease after ileocolic resection: endoscopically visualised ileal ulcers preceding symptoms, Gut 33 (3) (1992) 331–335. [4] E. Louis, A. Collard, A.F. Oger, E. Degroote, F.A. Aboul Nasr El Yafi, J. Belaiche, Behaviour of Crohn’s disease according to the Vienna classification: changing pattern over the course of the disease, Gut 49 (6) (2001) 777–782. [5] F. Zorzi, E. Calabrese, G. Monteleone, Pathogenic aspects and therapeutic avenues of intestinal fibrosis in Crohn’s disease, Clin. Sci. (Lond.) 129 (12) (2015) 1107–1113. [6] D.C. Rockey, P.D. Bell, J.A. Hill, Fibrosis–a common pathway to organ injury and failure, N. Engl. J. Med. 372 (12) (2015) 1138–1149. [7] D. Bettenworth, F. Rieder, Medical therapy of stricturing Crohn’s disease: what the gut can learn from other organs – a systematic review, Fibrogenesis Tissue Repair 7 (1) (2014) 5. [8] C.J. Schaefer, D.W. Ruhrmund, L. Pan, S.D. Seiwert, K. Kossen, Antifibrotic activities of pirfenidone in animal models, Eur. Respir. Rev. 20 (120) (2011) 85–97. [9] S.I. Kadir, T. Wenzel Kragstrup, A. Dige, S. Kok Jensen, J.F. Dahlerup, J. Kelsen, Pirfenidone inhibits the proliferation of fibroblasts from patients with active Crohn’s disease, Scand. J. Gastroenterol. (2016) 1–5. [10] R. Meier, C. Lutz, J. Cosin-Roger, S. Fagagnini, G. Bollmann, A. Hunerwadel, C. Mamie, S. Lang, A. Tchouboukov, F.E. Weber, A. Weber, G. Rogler, M. Hausmann, Decreased fibrogenesis after treatment with pirfenidone in a newly developed mouse model of intestinal fibrosis, Inflamm. Bowel Dis. 22 (3) (2016) 569–582. [11] M. Inomata, K. Kamio, A. Azuma, K. Matsuda, N. Kokuho, Y. Miura, H. Hayashi, T. Nei, K. Fujita, Y. Saito, A. Gemma, Pirfenidone inhibits fibrocyte accumulation in the lungs in bleomycin-induced murine pulmonary fibrosis, Respir. Res. 15 (2014) 16. [12] H.S. Cooper, S.N. Murthy, R.S. Shah, D.J. Sedergran, Clinicopathologic study of dextran sulfate sodium experimental murine colitis, Lab. Invest. 69 (2) (1993) 238–249. [13] L.W. Maines, L.R. Fitzpatrick, K.J. French, Y. Zhuang, Z. Xia, S.N. Keller, J.J. Upson, C.D. Smith, Suppression of ulcerative colitis in mice by orally available inhibitors of sphingosine kinase, Dig. Dis. Sci. 53 (4) (2008) 997–1012. [14] J.H. Yoo, S. Ho, D.H. Tran, M. Cheng, K. Bakirtzi, Y. Kukota, R. Ichikawa, B. Su, D. H. Tran, T.C. Hing, I. Chang, D.Q. Shih, R.E. Issacson, R.L. Gallo, C. Fiocchi, C. Pothoulakis, H.W. Koon, Anti-fibrogenic effects of the anti-microbial peptide cathelicidin in murine colitis-associated fibrosis, Cell. Mol. Gastroenterol. Hepatol. 1 (1) (2015) 55–74 e1.
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Oral pirfenidone protects against fibrosis by inhibiting fibroblast proliferation and TGF-b signaling in a murine colitis model Guanwei Li a, Jianan Ren a,⇑, Qiongyuan Hu a,b, Youming Deng a, Guopu Chen a, Kun Guo a, Ranran Li a, Yuan Li a, Lei Wu a, Gefei Wang a, Guosheng Gu a, Jieshou Li a a b
Department of Surgery, Jinling Hospital, Medical School of Nanjing University, Nanjing, China Medical School of Southeast University, No. 87 Dingjiaqiao Road, Nanjing 210009, China
a r t i c l e
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Article history: Received 14 July 2016 Accepted 2 August 2016 Available online xxxx Keywords: Pirfenidone Inflammatory bowel disease Intestinal fibrosis Antifibrotic therapy
a b s t r a c t Inflammatory bowel disease (IBD), particularly Crohn’s disease, frequently causes intestinal fibrosis that ultimately leads to formation of strictures requiring bowel resection. Currently there is no effective antifibrotic therapy available for this disease. Pirfenidone is a small compound that has a broad spectrum of antifibrogenic effect and has been used for the treatment of fibrotic diseases in various organs. The present study aimed to investigate the antifibrogenic effect of pirfenidone in a dextran sulfate sodium (DSS)induced murine colitis model. C57BL/6 mice were used and animals were randomly divided into groups receiving pirfenidone or vehicle by oral or transanal routes. Inflammation- and fibrosis-related indexes including body weight, colon length, disease activity, histological change, mRNA expression of proinflammatory and pro-fibrogenic cytokines were assessed. Furthermore, we performed in vitro analysis using CCD18-Co fibroblasts to evaluate cell proliferation, transdifferentiation, and viability after the cells were cultured with pirfenidone. It was found that oral administration of pirfenidone reduced deposition of collagen in colitis-associated fibrosis, and significantly suppressed the mRNA expression of col1a2, col3a1, and TGF-b. Moreover, pirfenidone inhibited the activation of TGF-b-related smad and MAPK pathways both in vitro and in vivo. Clinical and histological evaluation demonstrated that pirfenidone had no anti-inflammatory effect. The antifibrogenic effect was reduced when pirfenidone was administered in a delayed manner and was unobserved if given locally. Pirfenidone suppressed fibroblast proliferation and transdifferentiation without observed toxicity. Altogether, our results suggested that oral pirfenidone protects against fibrosis of DSS-induced colitis through inhibiting the proliferation of colonic fibroblasts and TGF-b signaling pathways. Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction Inflammatory bowel disease (IBD), particularly Crohn’s disease and ulcerative colitis, is featured by chronic inflammation of the gastrointestinal tract whose etiology remains unclear. Crohn’s disease is characterized by transmural inflammation that involves the entire thickness of the bowel leading to dysfunctional wound healing and abnormal collagen accumulation, which causes excessive fibrosis and formation of strictures [1]. Despite recent advances in exploring the pathogenesis and treatment of Crohn’s disease thus far, there is no effective medical therapy for prevention or cure of disease-associated fibrogenesis, making surgery the only option for symptomatic/complicated strictures. Approximately ⇑ Corresponding author at: Department of Surgery, Jinling Hospital, 305 East Zhongshan Road, Nanjing 210002, China. E-mail address: [email protected] (J. Ren).
75% of patients with Crohn’s disease eventually undergo at least one surgical procedure due to intestinal fibrosis [2]. Furthermore, 70–90% of patients suffer a relapse within one year after surgery, and more than half have recurrent strictures that could necessitate further bowel resections [3,4]. Intestinal fibrosis has therefore represented the main indication for surgery in patients with Crohn’s disease, and created substantial burden on healthcare resources and individuals. Therapies that are effective in attenuating mucosal inflammation do not prevent or reverse fibrosis in Crohn’s disease [5]. Given that fibrotic changes associated with diverse diseases in various organs share common pathogenic pathways such as the transforming growth factor beta (TGF-b) pathway [6], it is of great value to think ‘outside of the box’ and to learn from other fibrotic diseases in terms of pathogenesis and treatment. Pirfenidone, an active small molecule which consists of a modified phenylpyridone, has been used to treat various types of fibrotic diseases including
http://dx.doi.org/10.1016/j.bcp.2016.08.002 0006-2952/Ó 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: G. Li et al., Oral pirfenidone protects against fibrosis by inhibiting fibroblast proliferation and TGF-b signaling in a murine colitis model, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.08.002
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idiopathic pulmonary fibrosis, renal fibrosis, cardiac fibrosis, and liver cirrhosis with great efficacy and safety [7]. This compound exhibits remarkable antifibrotic effects in a variety of animal and cell-based models [8]. Kadir et al. reported that pirfenidone inhibited the proliferation and secretion of collagen, MMP-3 and TIMP-1 in fibroblasts isolated from patients of Crohn’s disease [9], reinforcing the notion that pirfenidone may have the potential to become a new avenue for therapeutic intervention of intestinal fibrosis. A recent study also showed promising antifibrotic effect of pirfenidone in a newly developed mouse model of intestinal fibrosis [10]. In this study, we examined whether administration of pirfenidone via oral or anal routes could prevent or attenuate colonic fibrosis in a dextran sulfate sodium (DSS)-induced colitis mouse model. Furthermore, we examined the potential effect of pirfenidone exerted on human colonic fibroblasts in vitro to explore its cellular mechanism of action. 2. Material and methods 2.1. Establishing a murine colitis model 6–8 week old female C57BL/6 mice weighing 18–20 g were purchased from the Cavens Lab (Changzhou, China), and maintained under specific pathogen-free conditions in the Animal Facility at Mergene Company (Nanjing, China). Chronic colonic fibrosis model was created by administering four cycles of 2.5% DSS (MP Biomedicals, USA) in drinking water. In brief, the mice received 2.5% DSS drinking water on days 1–5, 8–12, 15–19 and 22–26, and regular drinking water during the remaining days. Two pirfenidone treatment regimens were established. In the prophylactic setting, 300 mg/kg/d pirfenidone (TCI, Shanghai, China) was suspended in 0.5% carboxymethylcellulose solution (CMC, vehicle) and administered orally twice per day beginning from day 0. In the therapeutic setting, pirfenidone was administered beginning from day 14 of DSS induction of colitis. Mice were randomly divided into three groups: normal mice treated with CMC (Control group), DSS mice treated with CMC (DSS group) and DSS mice treated with pirfenidone (PFD group). The experimental dose of pirfenidone was selected with reference to published reports [11]. Additional groups of mice using the same experimental setting above received pirfenidone by a blunt needle via the transanal route to clarify the antifibrotic effects of local application. Each group consisted of 6–8 mice. Animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the Institute for Laboratory Animal Research and have received approval from Animal Investigation Ethics Committee of Jinling Hospital. 2.2. Disease activity index and histological scoring Disease activity index (DAI) at day 14 and 28 was the combined scores of weight loss, stool consistency and bleeding based on a previous scoring system [12]. All animals were sacrificed at day 29 except for a subgroup of mice serving as pre-treatment controls that were sacrificed at day 14 in the therapeutic setting. Colonic tissues were fixed in neutral buffered formalin and embedded in paraffin. Paraffin sections were cut and stained with hematoxylin-eosin. The sections were read and graded by two blinded investigators as previously described [13]. The amount and extent of inflammation, crypt damage as well as the percentage involved were evaluated to calculate the total histological scores. For evaluation of fibrosis, tissues were stained with Masson Trichrome Staining Kit, and mucosal and submucosal locations of fibrosis were scored using a 0–3 scale (0 = no fibrosis, 1 = mild fibrosis, 2 = moderate fibrosis, and 3 = severe fibrosis) [14].
2.3. qRT-PCR Quantification of the gene expression of col1a2, col3a1, TGF-b, TNF-a, IL-1b, and IL-6 was performed by quantitative real-time PCR (qRT-PCR). Briefly, total RNAs were isolated from colon tissues by Trizol reagent (TAKARA) and reverse-transcribed into cDNAs using the reverse transcription kit (TAKARA, RR047, Takara Biomedical Technology, Beijing, China) according to the manufacturer’s instructions. Real-time PCR was subsequently performed in triplicate using the StepOnePlus PCR System (Applied Biosystems, Carlsbad, USA). All mRNA quantification data were normalized to GAPDH. 2.4. Cell cultures Human CCD-18Co colonic fibroblasts were cultured in minimal essential medium Eagle’s medium (WISENT, Nanjing, China) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. All experiments were performed after serum-free starvation for 24 h. Pirfenidone (0.5, 1.0 and 1.5 mg/ml) was dissolved in dimethylsulfoxide (DMSO). TGF-b (5 ng/ml) was added at the same time in some experiments to evaluate the effects of pirfenidone on myofibroblast trans-differentiation, collagen expression, and TGF-b related downstream signaling pathway molecules. 2.5. Assessment of cell proliferation CCK8 assay was used to measure the proliferative capability of CCD-18Co cells after pirfenidone treatment. Cells in the exponential growth phase were harvested, inoculated in 96-well plates, and incubated overnight. Pirfenidone mixed with MEME medium at the concentrations of 0.5, 1.0, 1 and 1.5 mg/ml was applied to CCD-18Co cells for 12, 24, 48, and 72 h following serum deprivation for 24 h. The CCK8 assay was performed with a commercially available kit (7sea biotech, Shanghai, China) according to the manufacturer’s instructions. The numbers of cells treated with different concentrations of pirfenidone were counted under a microscope. 2.6. Assessment of cell integrity The activity of lactate dehydrogenase (LDH) in CCD-18Co cells was examined using a commercialized kit (KeyGEN BioTECH, Nanjing, China). The trypan blue exclusion test was performed after treatment with pirfenidone (0.5, 1.0, 1.5 mg/ml) for 48 h. In LDH assay, the culture medium was collected and assayed according to the manufacturer’s instructions. LDH release following pirfenidone treatment was indicated as a percentage relative to the untreated control cultures. In trypan blue exclusion test, viable cells were counted in triplicate on a hemocytometer after trypan blue staining by a blinded investigator. The percentage of living cells was calculated and expressed as a ratio of viable cell count (unstained) to total cell count. 2.7. Western blot analysis Colonic CCD-18Co fibroblasts were incubated with or without pirfenidone (0.5, 1.0, 1.5 mg/ml) and TGF-b (5 ng/ml) for 24 h. The cells and the colon tissues from sacrificed mice were lysed with lysis buffer and the protein concentrations were determined with the BCA assay kit (Beyotime, Shanghai, China). Equal amounts of cell extracts were fractionated by 4–12% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separate proteins were then transferred to a Polyvinylidene Fluoride membrane (PVDF; MILLIPORE). Nonspecific binding was blocked in 5% nonfat milk or 5% BSA in TBST for 2 h, and then incu-
Please cite this article in press as: G. Li et al., Oral pirfenidone protects against fibrosis by inhibiting fibroblast proliferation and TGF-b signaling in a murine colitis model, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.08.002
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bated overnight at 4 °C with primary antibodies against p-ERK(CST, 4511,), p-P38(CST, 4668), p-JNK(CST, 4370), p-smad2(CST, 3108), p-smad3(CST, 9520), (all from Cell Signaling Technology, Danvers), procol1A1 (Santa Cruz, sc-25973, Shanghai, China) and b-actin (zen bioscience, 200068-8F10, Chengdu, China). The membranes were then washed and incubated with horseradish peroxidaseconjugated secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized using chemiluminescent reagent and the signals were captured by a Tanon Imaging System (Tanon, Shanghai, China). The protein levels were quantified by densitometric analysis with Image J software and standardized by calculating the ratio of target to b-actin mean intensity. 2.8. Immunofluorescence staining Nuclear expression of ki67 reflecting the proliferative activity of cells was measured. In brief, colonic fibroblasts seeded on 24-well plates were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton following treatment with vehicle (DMSO) or pirfenidone at concentrations of 0.5, 1.0 and 1.5 mg/ml for 48 h. After blocking non-specific proteins with 10% goat serum, the cells were incubated with primary antibody against ki67 (1:500; Abcam, ab92353, Cambridge, UK) and then with Alexa Fluor 488conjugated secondary antibody. DAPI (4,6-diamidino-2phenylindole) staining was used to locate the nuclei. Transdifferentiation of fibroblasts into myofibroblasts was an important event involved in gut fibrogenesis. Therefore fibroblasts incubated with TGF-b in the absence or presence of pirfenidone (0.5, 1.0, 1.5 mg/ml) for 24 h were subjected to immunofluorescence staining to measure the expression of a-SMA, the marker of myofibroblasts. Primary antibody to a-SMA (1:100; Abcam, ab5694, Cambridge, UK) and Alexa Fluor 555-conjugated secondary antibody were used. For visualization of the collagen expression in fibroblasts in vivo, frozen colonic tissues were sectioned and fixed in acetone. Slides were incubated with primary antibodies including col1a2 (Abcam, ab96723, Cambridge, UK) and vimentin (Abcam, ab20346, Cambridge, UK) for 2 h at room temperature, and then incubated with Alexa Fluor 488- and 555-conjugated secondary antibodies. After rinsing in PBS, slides were stained with DAPI, observed and photographed under a confocal microscope (Zeiss, LSM 700). 2.9. Statistical analysis Results were expressed as mean ± SD and were analyzed using IBM SPSS software for Windows version 19.0. Comparison between two groups was performed by Fisher’s Exact test for categorical variables and Student’s t-test for continuous variables. For comparison between three groups, a One-way ANOVA was used and LSD post hoc method was used to analyze the intergroup differences. All P values were two-sided, and P < 0.05 was considered statistically significant.
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tored. It was found that the body weight of the DSS-treated mice was significantly lower than that of normal mice at days 14 and 28. Oral administration of pirfenidone showed no effect on the body weight loss whether it was started from day 0 or on day 14 (Fig. 1B). In both experimental settings, treatment with pirfenidone did not significantly alter the clinical status of DSS colitis (Fig. 1C). The length of colon in mice with chronic colitis induced by DSS was significantly shorter than that of normal mice (Fig. 1D). The length of the colon in the mice prophylactically receiving oral pirfenidone was significantly longer compared with the parallel CMC group (Fig. 1D). However pirfenidone used in a delay manner or by transanal route showed no significant effect (data not shown). Because shortening of the colon could result from the chronic inflammation and intestinal fibrosis in the DSS colitis model [15], we examined the histological change on the colonic tissues. Hematoxylin-eosin staining showed that the degree of colonic inflammation including inflammatory infiltrate and tissue damage caused by DSS remained unchanged after treatment with oral pirfenidone in both the prophylactic (Fig. 2A) and therapeutic settings (Fig. 2B). Similar results were observed when pirfenidone was used locally (intra-colon) (data not shown). Oral pirfenidone suppressed the expressions of mRNA of fibrosis-associated molecules including col1a2, col3a1 and TGF-b (Fig. 3A), but not the inflammationassociated cytokines including TNF-a, IL-1b and IL-6 (Fig. 3B). This result was consistent with the histological evaluation. These results collectively suggested that pirfenidone exerted no antiinflammatory effects on DSS-induced colitis model. 3.2. Oral administration of pirfenidone reduces colitis-associated intestinal fibrosis The antifibrotic effects of pirfenidone on DSS colitis-associated colonic fibrosis were next evaluated by Masson trichrome staining. Increased collagen deposition was observed in the colon mucosa and submucosa of mice with DSS-induced chronic colitis (Fig. 4). Oral administration of pirfenidone from day 0 (Fig. 4A) or day 14 (Fig. 4B) both inhibited the fibrosis, depicted by Masson trichrome staining as a lesser extent of collagen formation. The quantitative score of fibrosis was significantly reduced in DSS-treated mice that were fed pirfenidone prophylactically (Fig. 4A); the score did not reach statistical significance when pirfenidone treatment was postponed (Fig. 4B), suggesting that early use of pirfenidone is a necessity to prevent intestinal fibrosis. Immunofluorescence staining showed that collagen expression and vimentin-expressing fibroblasts were marked in the colons of DSS-treated mice, and significantly reduced after oral pirfenidone administration (Fig. 5). This result was consistent with the expression of mRNA of multiple types of collagen and profibrogenic cytokines determined by qRTPCR (Fig. 3A). However, the significant anti-fibrogenic effects of pirfenidone were not observed when pirfenidone was delivered intracolonically (data not shown). 3.3. Pirfenidone inhibits colonic fibroblast proliferation and transdifferentiation in vitro without cytotoxicity
3. Results 3.1. Pirfenidone exerts no anti-inflammatory effects on DSS-induced colitis in mice To investigate the roles of pirfenidone in murine colitis, mice were given four cycles of 2.5% DSS in drinking water on days 1– 5, 8–12, 15–19, and 22–26 to induce colitis. In the prophylactic setting, mice were orally administered pirfenidone twice per day from day 0 (Fig. 1A, left panel). In the therapeutic setting, mice were orally administered pirfenidone twice per day from day 14 (Fig. 1A, right panel). The body weight of these mice was moni-
To understand the cellular mechanism underlying the antifibrogenic effects of pirfenidone, we investigated the effect of pirfenidone on the proliferation of fibroblasts in vitro. Human colonic fibroblasts CCD-18Co cells were incubated with various concentrations of pirfenidone (0.5, 1.0, 1, and 1.5 mg/ml) or DMSO for 12, 24, 48, and 72 h following serum deprivation for 24 h. Cells were then subjected to the CCK8 assay. The assay showed that proliferation of colonic fibroblasts was suppressed by pirfenidone in a dose- and time-dependent manner (Fig. 6A). Furthermore, cells treated with different concentrations of pirfenidone or DMSO for 48 h and cell numbers were counted under a microscope. The
Please cite this article in press as: G. Li et al., Oral pirfenidone protects against fibrosis by inhibiting fibroblast proliferation and TGF-b signaling in a murine colitis model, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.08.002
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Fig. 1. Oral pirfenidone has no effect on colonic inflammation in mice with DSS-induced chronic colitis. (A) Pirfenidone treatment schematics. Mice received four cycles of 2.5% DSS in drinking water on days 1–5, 8–12, 15–19 and 22–26. In the prophylactic setting (left panel), pirfenidone was suspended in 0.5% CMC and administered orally twice per day from day 0. In the therapeutic setting (right panel), pirfenidone was administered after two weeks of DSS induction of colitis. (B) Percentage of body weight change of different groups at days 0, 14 and 28. (C) Disease activity index (DAI) at days 14 and 28 in CMC-treated and pirfenidone-fed groups in the DSS colitis model. The DAI in Normal + CMC group is 0. (D) The length of the colon after mice were sacrificed. n = 6–8 mice/group. *P < 0.05, **P < 0.01.
result of cell number counting (Fig. 6B) was consistent with that of the CCK8 assay. Cell integrity was investigated by LDH release assay (Fig. 6C) and trypan blue exclusion test (Fig. 6D) after treating the cells with pirfenidone (0.5, 1.0, 1.5 mg/ml) for 48 h. It was found that pirfenidone had no cellular toxicity and was safe to use at the tested concentration and time duration. The colonic fibroblasts after treatment with DMSO or pirfenidone at concentrations of 0.5, 1.0, and 1.5 mg/ml for 48 h were subjected to Ki67 immunofluoresence staining, another indicator of cell proliferation. It was found that Ki67 positive cells significantly decreased dose-dependently (Fig. 7). This result further confirmed that pirfenidone inhibited fibroblast cell proliferation. In addition to fibroblast proliferation, transdifferentiation of fibroblasts toward myofibroblast phenotype is another key event involved in the fibrogenic process of chronic colitis. TGF-b induces the transdifferentiation of CCD-18Co fibroblasts toward myofi-
broblast phenotype. When fibroblasts were incubated with TGF-b in the absence or presence of pirfenidone (0.5, 1.0, 1.5 mg/ml) for 24 h and subjected to immunofluorescence staining, we found that pirfenidone inhibits the TGF-b-induced transdifferentiation of CCD-18Co fibroblasts toward myofibroblasts indicated by a-SMA expression (Fig. 8). This result demonstrated that pirfenidone inhibited TGF-b-induced transdifferentiation of myofibroblasts from fibroblasts. 3.4. Pirfenidone inhibits TGF-b-related Smad and MAPK pathways in vitro and in vivo TGF-b is a key profibrotic factor and a central mediator of fibrogenesis that signals both in a classical smad pathway and in an atypical MAPK pathway [16]. We examined CCD-18Co cells treated with TGF-b (5 ng/ml) for 24 h in the absence or presence
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Fig. 2. Oral pirfenidone has no effect on histological inflammation in mice with DSS-induced chronic colitis. Representative hematoxylin-eosin stained image of colonic tissue sections from the prophylactic (A) and therapeutic (B) experimental settings. Histology score was based on hematoxylin-eosin staining images of colonic tissues, and evaluated by the amount and extent of inflammation, crypt damage as well as the percentage involvement. n = 6–8 mice/group. *P < 0.05, **P < 0.01.
of various concentrations of pirfenidone (0.5, 1.0, 1.5 mg/ml). We found that pirfenidone significantly reduced TGF-b-stimulated procollagen expression in a dose-dependent manner (Fig. 9A, C). We also investigated by Western blot the change of activated states of several molecules that initiate and modulate these pathways, that is, the phosphorylation of smad2, smad3,
ERK, p38, and JNK. The activation of these molecules in fibroblasts was all inhibited by pirfenidone treatment (Fig. 9A, C), with the highest effect obtained at the maximum concentration (1.5 mg/ml). Significant activation of smad and MAPK pathways observed in colitis-associated fibrosis was also inhibited in mice treated with pirfenidone (Fig. 9B, D).
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Fig. 3. Oral pirfenidone suppresses the increased expressions of profibrotic but not proinflammatory mediators in mice with DSS-induced chronic colitis. Pirfenidone was administered from day 0. Mice were sacrificed at day 29 and total RNA was isolated from colon tissues for qRT-PCR assay. (A) The mRNA expression levels of fibrosisassociated factors including col1a2, col3a1 and TGF-b. (B) The mRNA expression levels of inflammation-associated cytokines including TNF-a, IL-1b and IL-6. n = 6 mice/group. **P < 0.01.
4. Discussion Long term tissue damage and dysregulated wound healing accompanying Crohn’s disease lead to abnormal in situ deposition of collagen and progressive luminal narrowing. Despite advances in the treatment of Crohn’s disease, specific intestinal antifibrotic therapy is still lacking [17]. The incidence of stricture formation and rate of surgery in Crohn’s disease has not decreased [17]. In this study, we investigated the roles of pirfenidone, a compound that has shown great potential in attenuating the fibrogenic process in various organ systems, in the prevention or cure of intestinal fibrosis using a murine DSS-colitis model. We found that pirfenidone administered by oral route significantly protected the colons from fibrogenic development without obvious effects on the inflammatory condition. Double immunofluorescence staining indicated less number of vimentin-expressing fibroblasts and reduced accumulation of collagen in the injured intestinal tissue of pirfenidone-treated mice. One of the key events leading to intestinal fibrosis is the exposure of mesenchymal cells to a variety of mediators that induce a profibrotic phenotype, among which TGF-b is the central regulator that activates smad and MAPK pathways for controlling collagen deposition and fibrogenesis. To address the mechanisms underlying the antifibrotic ability of pirfenidone in the context of the gut, human CCD-18Co colonic fibroblasts were cultured with or without TGF-b in the absence or presence of pirfenidone. We showed that pirfenidone inhibited the proliferation but not viability of intestinal fibroblasts, hampered the TGF-b-induced myofibroblast differentiation, and reduced the cellular secretion of procollagen. Our results also indicated that the downregulated expression of collagen in response to pirfenidone was mediated by the inhibition of smad and MAPK pathways in vitro and in vivo.
Pirfenidone was the first approved drug for idiopathic pulmonary fibrosis (IPF), a chronic, progressive, and fibrogenic lung disease characterized by abnormal extracellular matrix deposition and irreversible loss of lung function. IPF was the most studied disease with the most established curative efficacy of pirfenidone for its antifibrotic activities. A phase 3 trial published in the New England Journal of Medicine showed results of IPF patients who received pirfenidone therapy had significantly improved lung function, exercise tolerance, and progression-free survival compared to placebo [18]. A variety of profibrogenic factors including growth factors and cytokines such as TGF-b, platelet-derived growth factor (PDGF), CTGF, IL4, and IL-13 function in a similar way in various organ systems [19]. This fact supports the notion that results stemmed from IPF can be transferable to other fibrogenic diseases, and rationalizes the clinical application of pirfenidone in the treatment of renal fibrosis, cardiac fibrosis, and liver cirrhosis [7]. Pirfenidone has also been shown to have antifibrotic effects in a wide range of clinical and animal-based studies involving multiple sclerosis [20], scleroderma [21], bladder fibrosis [22], radiation-induced fibrosis [23], and others. In the context of the gastrointestinal tract, a previous study found that pirfenidone protected against stricture formation in an animal model of caustic esophageal burn when used in the form of gel by gavage [24], indicating that pirfenidone might also play a protective role in strictures caused by other gastrointestinal disease such as Crohn’s disease. DSS-induced colitis is driven by a Th1 mediated cytokine immune response that mimics IBD [25], and is commonly used as a model of chronic intestinal inflammation and fibrosis [26,27]. In this study, pirfenidone administered either by gavage or transanal route showed no significant effect on the inflammatory condition of the murine colitis, demonstrated by the histolog-
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Fig. 4. Oral pirfenidone decreases the amount of intestinal fibrosis in mice with DSS-induced chronic colitis. Representative image of Masson Trichrome staining for collagen from the prophylactic (A) and therapeutic (B) experimental settings. Collagen deposited in the mucosal and submucosal layer of DSS-treated mice was assessed by fibrosis score. n = 6–8 mice/group. *P < 0.05, **P < 0.01.
ical evaluation of the injured colons and the mRNA expression levels of multiple inflammatory cytokines. Most of the previous studies focused on the protective role of pirfenidone in fibrogenic process of various organs [8], and there were only several reports with respect to inflammation [28–31]. Hirano et al. reported that subcutaneous injections of pirfenidone attenuated allergic airway inflammation along with inhibited secretion of IL-4, IL-5, and
IL-13 after chronic allergen challenge [29]. Oral administration of pirfenidone reduced tissue level of multiple cytokines including TNF-a, IFN-c, IL-1b, IL-6, IL-12p40, and IL-18 in bleomycininduced murine pulmonary fibrosis [28]. Whether pirfenidone possesses anti-inflammatory activity in colitis remains inconclusive. Drugs targeting fibrosis such as pirfenidone seemed not to affect inflammatory status in our study, therefore pirfenidone should
Please cite this article in press as: G. Li et al., Oral pirfenidone protects against fibrosis by inhibiting fibroblast proliferation and TGF-b signaling in a murine colitis model, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.08.002
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Fig. 5. Oral pirfenidone reduces collagen expression in colonic fibroblasts in vivo. Pirfenidone was administered from day 0 and mice in the normal, CMC-treated and pirfenidone-fed groups were sacrificed at day 29. Immunofluorescence staining showed col1a2 (green), fibroblast marker vimentin (red), and nuclei DAPI (blue). Merging images resulted in a yellowish color that indicated the colocalization of collagen expression in colonic fibroblasts. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 6. Pirfenidone inhibits proliferation of colonic CCD-18Co fibroblasts with no cytotoxicity effects. (A) CCK8 assay. Various concentrations of pirfenidone (0.5, 1.0, 1 and 1.5 mg/ml) or DMSO were applied to CCD-18Co cells for 12, 24, 48 and 72 h following serum deprivation for 24 h. Cells were then subjected to the CCK8 assay. (B) Cell count. Cells treated with different concentrations of pirfenidone or DMSO for 48 h, and were resuspended and counted under a microscope. Cytotoxicity effects were assessed after treatment with pirfenidone (0.5, 1.0, 1.5 mg/ml) for 48 h by measuring LDH activity (C) and by the trypan blue exclusion test (D). *P < 0.05, **P < 0.01, ***P < 0.001.
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Fig. 7. Pirfenidone treatment inhibits nuclear expression of ki67 in CCD-18Co fibroblasts. Representative image of ki67 staining in colonic fibroblasts was shown after treatment with DMSO or pirfenidone at concentrations of 0.5, 1.0 and 1.5 mg/ml for 48 h.
be used in association with other compounds that exert an antiinflammatory effect in the intestine. Previous studies supported the possibility that local administration of pirfenidone may be protective against tissue fibrogenesis in skin lesions [21,32]. To test the efficacy of topical pirfenidone treatment in DSS-inducing colitis, additional groups of mice received pirfenidone via the transanal route. The mice developed fibrosis as the parallel groups treated by vehicle indicating that the topical effect of pirfenidone may be tissuespecific. Besides, the effects of transanal administration of pirfenidone can be influenced by other factors such as mucous barrier and intraluminal feces, and there was also the possibility that the enema given by a blunt needle could be partly excreted from the anal. We did not rule out the potential of pirfenidone to be used transanally. Pirfenidone is able to move through cell membranes without requiring a receptor, and it is rapidly absorbed after oral administration and reaches its maximum levels in blood after about one hour [33]. The findings in this study suggested that orally administered pirfenidone significantly suppress the fibrogenic process when given prior to the fibrotic phase of DSS model. As for the therapeutic regimen, the
antifibrotic efficacy of pirfenidone was reduced when the drug was given after the occurrence of colitis, and it seemed that pirfenidone posed no effect on the existed collagen deposition at day 14. The results derived from this preliminary study should be treated cautiously and required further investigation. In vitro studies using colonic fibroblast cell line CCD-18Co demonstrated that preventive effect of pirfenidone against progression of intestinal fibrosis may involve several key events of the fibrogenic process, including the proliferation of fibroblasts, transdifferentiation into myofibroblasts, and synthesis of extracellular matrix. The proliferative activity of colonic fibroblasts cultured with pirfenidone were remarkably reduced while cell viability remained unaffected, consistent with a number of previous studies based on fibroblasts isolated from patients with Crohn’s disease [9] and various cell lines of fibroblasts [34–36]. In vivo study demonstrated a decrease in vimentin-expressing fibroblasts in the colonic tissues of pirfenidone-treated mice. However, the CCK-8 assay by Meier et al. using primary murine colonic fibroblasts yielded divergent results [10]. In their study, commercial EsbrietÒ tablets dissolved in methanol were used instead of a pure compound of pirfenidone.
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Fig. 8. Immunofluoresent staining of a-SMA. Pirfenidone inhibits the TGF-b-induced transdifferentiation of CCD-18Co fibroblasts toward myofibroblast phenotype indicated by a-SMA expression. Fibroblasts incubated without TGF-b (A), with TGF-b alone (B), with TGF-b in the presence of DMSO (C) or 0.5 mg/ml (D), 1.0 mg/ml (E) and 1.5 mg/ml (F) pirfenidone for 24 h were subjected to immunofluorescence staining for a-SMA (yellow) and nuclei DAPI (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 9. Pirfenidone inhibits activation of smad and MAPK pathways in vitro and in vivo. CCD-18Co cells were treated with TGF-b (5 ng/ml) for 24 h in the absence or presence of various concentrations of pirfenidone (0.5, 1.0, 1.5 mg/ml) (A, C). Pirfenidone was administered from day 0 and mice in the normal, CMC-treated and pirfenidone-fed groups were sacrificed at day 29 (B, D). Expression of procol1A1 and phosphorylation of smad2, smad3, p38, ERK and JNK were assessed by Western blot analysis. Representative blots of one out of three separated experiments were shown (A, B). Graphs with means ± SD of target/b-actin gene ratios were obtained by densitometric analysis (C, D). *P < 0.05, **P < 0.01 compared with TGF-b-treated cells/CMC-treated colitis mice.
TGF-b is one of the most potent profibrotic factors that trigger the conversion of fibroblasts into myofibroblasts and secretion of collagen through the activation of smad and MAPK pathways. In the present study, these processes were downregulated and collectively contributed to the reduced expression of collagen in vitro
and in vivo following pirfenidone treatment. The findings derived from colonic fibroblasts were also in accordance with the results based on human lung fibroblasts [34] and orbital fibroblasts [37]. The universal inhibition on TGF-b pathways may explain the broad antifibrogenic spectrum of pirfenidone against fibrosis in various
Please cite this article in press as: G. Li et al., Oral pirfenidone protects against fibrosis by inhibiting fibroblast proliferation and TGF-b signaling in a murine colitis model, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.08.002
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organs. Increased tyrosine phosphorylation of p125 FAK is a central regulator of cell migration, which may also be involved as demonstrated by Meier et al. using a heterotopic transplantation model [10]. In conclusion, we demonstrated that oral pirfenidone protected against the progression of DSS colitis-related fibrosis without affecting the inflammatory status of colonic tissues. The mechanisms involved may be related to the downregulation of profibrotic TGF-b and inhibition of the TGF-b downstream smad and noncanonical MAPK pathways, as well as the antiproliferative activity against collagen-produced colonic fibroblasts. Antifibrotic therapy should be considered independently from antiinflammatory drugs in the treatment regimen, especially for those patients with obstructive phenotype, in order to prevent strictures. Pirfenidone has been efficaciously used in treating fibrosis in various organs with a sufficient safety profile and could be a promising alternative for the prevention and treatment of colitis-related fibrosis. Conflict of interest All authors declare no conflicts of interest. Acknowledgements This work was supported by grants from National Natural Science Foundation of China [81270478, 81571881]. References [1] J. Cosnes, S. Cattan, A. Blain, L. Beaugerie, F. Carbonnel, R. Parc, J.P. Gendre, Long-term evolution of disease behavior of Crohn’s disease, Inflamm. Bowel Dis. 8 (4) (2002) 244–250. [2] A. Spinelli, C. Correale, H. Szabo, M. Montorsi, Intestinal fibrosis in Crohn’s disease: medical treatment or surgery? Curr. Drug Targets 11 (2) (2010) 242– 248. [3] G. Olaison, K. Smedh, R. Sjodahl, Natural course of Crohn’s disease after ileocolic resection: endoscopically visualised ileal ulcers preceding symptoms, Gut 33 (3) (1992) 331–335. [4] E. Louis, A. Collard, A.F. Oger, E. Degroote, F.A. Aboul Nasr El Yafi, J. Belaiche, Behaviour of Crohn’s disease according to the Vienna classification: changing pattern over the course of the disease, Gut 49 (6) (2001) 777–782. [5] F. Zorzi, E. Calabrese, G. Monteleone, Pathogenic aspects and therapeutic avenues of intestinal fibrosis in Crohn’s disease, Clin. Sci. (Lond.) 129 (12) (2015) 1107–1113. [6] D.C. Rockey, P.D. Bell, J.A. Hill, Fibrosis–a common pathway to organ injury and failure, N. Engl. J. Med. 372 (12) (2015) 1138–1149. [7] D. Bettenworth, F. Rieder, Medical therapy of stricturing Crohn’s disease: what the gut can learn from other organs – a systematic review, Fibrogenesis Tissue Repair 7 (1) (2014) 5. [8] C.J. Schaefer, D.W. Ruhrmund, L. Pan, S.D. Seiwert, K. Kossen, Antifibrotic activities of pirfenidone in animal models, Eur. Respir. Rev. 20 (120) (2011) 85–97. [9] S.I. Kadir, T. Wenzel Kragstrup, A. Dige, S. Kok Jensen, J.F. Dahlerup, J. Kelsen, Pirfenidone inhibits the proliferation of fibroblasts from patients with active Crohn’s disease, Scand. J. Gastroenterol. (2016) 1–5. [10] R. Meier, C. Lutz, J. Cosin-Roger, S. Fagagnini, G. Bollmann, A. Hunerwadel, C. Mamie, S. Lang, A. Tchouboukov, F.E. Weber, A. Weber, G. Rogler, M. Hausmann, Decreased fibrogenesis after treatment with pirfenidone in a newly developed mouse model of intestinal fibrosis, Inflamm. Bowel Dis. 22 (3) (2016) 569–582. [11] M. Inomata, K. Kamio, A. Azuma, K. Matsuda, N. Kokuho, Y. Miura, H. Hayashi, T. Nei, K. Fujita, Y. Saito, A. Gemma, Pirfenidone inhibits fibrocyte accumulation in the lungs in bleomycin-induced murine pulmonary fibrosis, Respir. Res. 15 (2014) 16. [12] H.S. Cooper, S.N. Murthy, R.S. Shah, D.J. Sedergran, Clinicopathologic study of dextran sulfate sodium experimental murine colitis, Lab. Invest. 69 (2) (1993) 238–249. [13] L.W. Maines, L.R. Fitzpatrick, K.J. French, Y. Zhuang, Z. Xia, S.N. Keller, J.J. Upson, C.D. Smith, Suppression of ulcerative colitis in mice by orally available inhibitors of sphingosine kinase, Dig. Dis. Sci. 53 (4) (2008) 997–1012. [14] J.H. Yoo, S. Ho, D.H. Tran, M. Cheng, K. Bakirtzi, Y. Kukota, R. Ichikawa, B. Su, D. H. Tran, T.C. Hing, I. Chang, D.Q. Shih, R.E. Issacson, R.L. Gallo, C. Fiocchi, C. Pothoulakis, H.W. Koon, Anti-fibrogenic effects of the anti-microbial peptide cathelicidin in murine colitis-associated fibrosis, Cell. Mol. Gastroenterol. Hepatol. 1 (1) (2015) 55–74 e1.
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Please cite this article in press as: G. Li et al., Oral pirfenidone protects against fibrosis by inhibiting fibroblast proliferation and TGF-b signaling in a murine colitis model, Biochem. Pharmacol. (2016), http://dx.doi.org/10.1016/j.bcp.2016.08.002