Elsevier

European Journal of Pharmacology

Volume 822, 5 March 2018, Pages 199-206
European Journal of Pharmacology

Full length article
Pirfenidone prevents radiation-induced intestinal fibrosis in rats by inhibiting fibroblast proliferation and differentiation and suppressing the TGF-β1/Smad/CTGF signaling pathway

https://doi.org/10.1016/j.ejphar.2018.01.027Get rights and content

Abstract

Radiation-induced intestinal fibrosis (RIF) is a chronic toxicity following radiation, and can be very difficult to treat. Pirfenidone is a promising anti-fibrotic agent that inhibits fibrosis progression in various clinical and experimental studies. This study was aimed to explore whether pirfenidone could protect against RIF, and to evaluate the underlying mechanism. An animal model of RIF was induced by exposure of a single dose of 20 Gy to the pelvis. Rats were orally administered with pirfenidone (200, 400 md/kg/d) for 12 weeks. Primary rat intestinal fibroblasts were cultured to determine the effects of pirfenidone on TGF-β1-induced (5 ng/ml) proliferation and transdifferentiation of fibroblasts. The expression of collagen I, α-SMA, and TGF-β1/Smad/CTGF pathway proteins were analyzed by qRT-PCR and/or western blot analysis. The cell proliferation rate was determined by CCK-8 assay. The results indicated that pirfenidone significantly attenuated fibrotic lesion in irradiated intestines and reduced collagen deposition by inhibiting TGF-β1/Smad/CTGF pathway in rat models. Moreover, in primary rat intestinal fibroblasts, pirfenidone decreased the up-regulation of TGF-β1-induced collagen I and α-SMA by suppressing TGF-β1/Smad/CTGF signaling pathway. Altogether, our findings suggested that pirfenidone attenuated RIF by inhibiting the proliferation and differentiation of intestinal fibroblasts and suppressing the TGF-β1/Smad/CTGF signaling pathway.

Introduction

Radiation-induced intestinal fibrosis (RIF) is a chronic toxicity of pelvic radiation. It is characterized by transmural accumulation of extracellular matrix within intestinal layers, which induces loss of intestinal compliance, and might leads to strictures formation requiring surgical resection (Larsen et al., 2007). Currently, there is no reliable clinical or laboratory marker available for RIF; progressive fibrosis and consequent intestinal strictures are not often diagnosed until irreversible. Moreover, current drug therapies have limited effect on fibrotic development, making surgery the only practical option for severe symptomatic intestinal fibrosis (Hamama et al., 2012, Henson et al., 2012). Bear in mind that excessive synthesis and deposition of collagens are the main pathological process of fibrotic diseases and the pathogenesis of fibrosis often shares common signaling pathways, it is of great value to learn from other fibrotic diseases in terms of pathogenesis and treatment (Urban et al., 2015).

Pirfenidone (5-methyl-N-phenyl-2- (1H)-pyridone), a promising anti-fibrotic agent, has exhibited remarkable antifibrotic properties in various clinical and animal-based experimental studies (Azuma et al., 2005, Di Sario et al., 2002, Duan et al., 2015, Hewitson et al., 2001, Rodríguez-Castellanos et al., 2015, Shi et al., 2011). In vitro studies have shown that pirfenidone inhibits proliferation and/or activation of a variety of fibroblasts responsible for the pathogenesis of fibrosis (Hewitson et al., 2001, Lin et al., 2009, Shi et al., 2011). Due to its great efficacy and safety, pirfenidone has been approved for the treatment of idiopathic pulmonary fibrosis in Europe and Japan (Cottin, 2013, Takeda et al., 2014). Recently, pirfenidone has been reported to inhibit the proliferation and secretory activities of fibroblasts isolated from patients with Crohn's disease (Kadir et al., 2016). In addition, this agent also showed promising antifibrotic effects in a newly developed mouse model of intestinal fibrosis, suggesting that pirfenidone might be used as an antifibrotic agent for intestinal fibrosis (Meier et al., 2016).

In the present study, we explored whether pirfenidone could protect against RIF in a rat model. Additionally, primary rat intestinal fibroblasts were incubated with transforming growth factor-β1 (TGF-β1) to possibly mimic in vivo situation, and we investigated the inhibitory effects of pirfenidone on critical fibrotic process, such as fibroblast proliferation as well as TGF-β1-induced myofibroblasts differentiation and fibrogenic activities. We also evaluated pirfenidone effects on the TGF-β1/Smad/CTGF signaling pathways underlying these antifibrotic activities both in vivo and in vitro.

Section snippets

Animal experiments

Six-week-old male Sprague–Dawley (SD) rats weighing 180–200 g were purchased from the Laboratory Animal Center, Fujian Medical University (Fuzhou, China). Rats were maintained in a controlled environment (23 ± 3 °C and 12 h light/dark illumination cycle) and fed with a standard chow diet and water. Experimental protocols were approved by Institutional Animal Care and Use Committee of Fujian Medical University.

After acclimatization for one week, rats were randomly divided into four groups (n =

Pirfenidone attenuates radiation-induced intestinal fibrosis in rats

The first symptom of RIF in rats is body weight loss. A gradual body weight loss was observed in the radiation group compared with the control group, but the extent of weight loss was decreased after treatment with pirfenidone (P< 0.01, Fig. 1A). One rat in the radiation group died from acute radiation injury at week 3, and four rats developed remarkable chronic bowel obstruction due to stenosis of the irradiated intestines (Fig. 1B). The remaining 5 rats also developed intestinal strictures of

Discussion

Radiation-induced intestinal fibrosis is a chronic toxicity after pelvic radiation, which is considered progressive and irreversible the majority of clinicians (Barcellos-Hoff and Costes, 2006). Currently, effective drug therapy for RIF is still lacking. In the present study, we explored the role of pirfenidone, a safe and well-tolerated drug, in the prevention or treatment of RIF. We found that pirfenidone attenuated RIF both in vivo and in vitro by inhibiting TGF-β1/Smad/CTGF signaling

Conclusion

The present study demonstrated that pirfenidone protected against the progression of RIF both in vivo and in vitro by inhibiting the proliferation and differentiation of intestinal fibroblasts and suppressing TGF-β1/Smad/CTGF signaling pathways. Further studies are needed to translate pirfenidone into the treatment of intestinal fibrosis in patients following pelvic radiation.

Acknowledgement

This study was supported by National Clinical Key Specialty Construction Project (General Surgery) of China (Grant no. 2012-649), guiding key project of social development by the Fujian Provincial Science and Technology Department (Grant no. 2015Y0058), and National Natural Science Fund for Young Scholars of China (Grant no. 81602798). We thank Dr. Ben-Hua Xu and Wen-Yao Li at the radiotherapy department of the Fujian Medical University Union Hospital for pelvic irradiation of rat models.

Conflict of interest

The authors declare that they have no conflict of interest.

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