Pirfenidone suppresses TGF‑β1‑induced human intestinal fibroblasts activities by regulating proliferation and apoptosis via the inhibition of the Smad and PI3K/AKT signaling pathway

Sun,Yanwu; Zhang,Yiyi; Chi,Pan

doi:10.3892/mmr.2018.9423

Pirfenidone suppresses TGF‑β1‑induced human intestinal fibroblasts activities by regulating proliferation and apoptosis via the inhibition of the Smad and PI3K/AKT signaling pathway

Authors:
- Yanwu Sun
- Yiyi Zhang
- Pan Chi
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Affiliations: Department of Colorectal Surgery, Fujian Medical University Union Hospital, Fuzhou, Fujian 350001, P.R. China
Published online on: August 22, 2018 https://doi.org/10.3892/mmr.2018.9423
Pages: 3907-3913
Copyright: © Sun et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Intestinal fibroblasts, the main effector cells of intestinal fibrosis, are considered to be a good target for anti‑fibrotic therapy. The aim of the present study was to examine the effects of pirfenidone (PFD) on human intestinal fibroblasts (HIFs) stimulated by transforming growth factor (TGF)‑β1 and to explore the potential mechanism. Prior to stimulation with TGF‑β1 (10 ng/ml), HIFs were treated with or without PFD (1 mg/ml). Cell proliferation was determined by Cell Counting Kit (CCK)‑8 and colony formation assays, and cell apoptosis was assessed using flow cytometry and a TUNEL assay. Reverse transcription‑quantitative polymerase chain reaction and western blotting were performed to evaluate the mRNA and protein expressions of α‑smooth muscle actin (α‑SMA), collagen I and fibronectin. The protein expression of TGF‑β1/mothers against decapentaplegic homolog (Smad) and phosphoinositide 3‑kinase (PI3K)/protein kinase B (AKT) signaling pathways was evaluated by western blotting. CCK‑8 and colony formation assays demonstrated that PFD significantly inhibited cell proliferation in HIFs stimulated with TGF‑β1. Flow cytometry and TUNEL assays revealed that PFD treatment significantly enhanced apoptosis in TGF‑β1‑stimulated HIFs. In addition, PFD markedly reduced TGF‑β1‑induced HIF activities, such as myofibroblast differentiation (α‑SMA), and collagen production (collagen I and fibronectin). These effects of PFD were mediated by the inhibition of the TGF‑β1/Smad and PI3K/AKT signaling pathways. Therefore, the present study demonstrated that PFD reduced TGF‑β1‑induced fibrogenic activities of HIFs, suggesting that PFD may be a potential therapeutic agent for intestinal fibrosis.

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1	Rieder F and Fiocchi C: Intestinal fibrosis in IBD-a dynamic, multifactorial process. Nat Rev Gastroenterol Hepatol. 6:228–235. 2009. View Article : Google Scholar : PubMed/NCBI
2	Stacey R and Green JT: Radiation-induced small bowel disease: Latest developments and clinical guidance. Ther Adv Chronic Dis. 5:15–29. 2014. View Article : Google Scholar : PubMed/NCBI
3	Hamama S, Delanian S, Monceau V and Vozenin MC: Therapeutic management of intestinal fibrosis induced by radiation therapy: From molecular profiling to new intervention strategies et vice et versa. Fibrogenesis Tissue Repair. 5 Suppl 1:S132012.PubMed/NCBI
4	Kendall RT and Feghali-Bostwick CA: Fibroblasts in fibrosis: Novel roles and mediators. Front Pharmacol. 5:1232014. View Article : Google Scholar : PubMed/NCBI
5	Zeisberg M and Kalluri R: Cellular mechanisms of tissue fibrosis. 1. Common and organ-specific mechanisms associated with tissue fibrosis. Am J Physiol Cell Physiol. 304:C216–C225. 2013. View Article : Google Scholar : PubMed/NCBI
6	Speca S, Giusti I, Rieder F and Latella G: Cellular and molecular mechanisms of intestinal fibrosis. World J Gastroenterol. 18:3635–3661. 2012. View Article : Google Scholar : PubMed/NCBI
7	Meng XM, Nikolic-Paterson DJ and Lan HY: TGF-β: The master regulator of fibrosis. Nat Rev Nephrol. 12:325–338. 2016. View Article : Google Scholar : PubMed/NCBI
8	Li Y, Liu H, Liang Y, Peng P, Ma X and Zhang X: DKK3 regulates cell proliferation, apoptosis and collagen synthesis in keloid fibroblasts via TGF-β1/Smad signaling pathway. Biomed Pharmacother. 91:174–180. 2017. View Article : Google Scholar : PubMed/NCBI
9	Conte E, Gili E, Fagone E, Fruciano M, Iemmolo M and Vancheri C: Effect of pirfenidone on proliferation, TGF-β-induced myofibroblast differentiation and fibrogenic activity of primary human lung fibroblasts. Eur J Pharm Sci. 58:13–19. 2014. View Article : Google Scholar : PubMed/NCBI
10	Abe Y, Murano M, Murano N, Morita E, Inoue T, Kawakami K, Ishida K, Kuramoto T, Kakimoto K, Okada T, et al: Simvastatin attenuates intestinal fibrosis independent of the anti-inflammatory effect by promoting fibroblast/myofibroblast apoptosis in the regeneration/healing process from TNBS-induced colitis. Dig Dis Sci. 57:335–344. 2012. View Article : Google Scholar : PubMed/NCBI
11	Azuma A, Nukiwa T, Tsuboi E, Suga M, Abe S, Nakata K, Taguchi Y, Nagai S, Itoh H, Ohi M, et al: Double-blind, placebo-controlled trial of pirfenidone in patients with idiopathic pulmonary fibrosis. Am J Respir Crit Care Med. 171:1040–1047. 2005. View Article : Google Scholar : PubMed/NCBI
12	Di Sario A, Bendia E, Svegliati Baroni G, Ridolfi F, Casini A, Ceni E, Saccomanno S, Marzioni M, Trozzi L, Sterpetti P, et al: Effect of pirfenidone on rat hepatic stellate cell proliferation and collagen production. J Hepatol. 37:584–591. 2002. View Article : Google Scholar : PubMed/NCBI
13	Miric G, Dallemagne C, Endre Z, Margolin S, Taylor SM and Brown L: Reversal of cardiac and renal fibrosis by pirfenidone and spironolactone in streptozotocin-diabetic rat. Br J Pharmacol. 133:687–694. 2001. View Article : Google Scholar : PubMed/NCBI
14	Shi Q, Liu X, Bai Y, Cui C, Li J, Li Y, Hu S and Wei Y: In vitro effects of pirfenidone on cardiac fibroblasts: Proliferation, myofibroblast differentiation, migration and cytokine secretion. PLoS One. 6:e281342011. View Article : Google Scholar : PubMed/NCBI
15	Duan L, Qi J, Huang T, Gu X, Xu D, Kong X and Qian XQ: Pirfenidone attenuates bladder fibrosis and mitigates deterioration of bladder function in a rat model of partial bladder outlet obstruction. Mol Med Rep. 12:3639–3647. 2015. View Article : Google Scholar : PubMed/NCBI
16	Lin X, Yu M, Wu K, Yuan H and Zhong H: Effects of pirfenidone on proliferation, migration, and collagen contraction of human Tenon's fibroblasts in vitro. Invest Ophthalmol Vis Sci. 50:3763–3770. 2009. View Article : Google Scholar : PubMed/NCBI
17	Hewitson TD, Kelynack KJ, Tait MG, Martic M, Jones CL, Margolin SB and Becker GJ: Pirfenidone reduces in vitro rat renal fibroblast activation and mitogenesis. J Nephrol. 14:453–460. 2001.PubMed/NCBI
18	Chan AL, Rafii R, Louie S and Albertson TE: Therapeutic update in idiopathic pulmonary fibrosis. Clin Rev Allergy Immunol. 44:65–74. 2013. View Article : Google Scholar : PubMed/NCBI
19	Cottin V: The role of pirfenidone in the treatment of idiopathic pulmonary fibrosis. Respir Res. 14 Suppl 1:S52013.PubMed/NCBI
20	Takeda Y, Tsujino K, Kijima T and Kumanogoh A: Efficacy and safety of pirfenidone for idiopathic pulmonary fibrosis. Patient Prefer Adherence. 8:361–370. 2014. View Article : Google Scholar : PubMed/NCBI
21	Meier R, Lutz C, Cosín-Roger J, Fagagnini S, Bollmann G, Hünerwadel A, Mamie C, Lang S, Tchouboukov A, Weber FE, et al: Decreased fibrogenesis after treatment with pirfenidone in a newly developed mouse model of intestinal fibrosis. Inflamm Bowel Dis. 22:569–582. 2016. View Article : Google Scholar : PubMed/NCBI
22	Li G, Ren J, Hu Q, Deng Y, Chen G, Guo K, Li R, Li Y, Wu L, Wang G, et al: Oral pirfenidone protects against fibrosis by inhibiting fibroblast proliferation and TGF-β signaling in a murine colitis model. Biochem Pharmacol. 117:57–67. 2016. View Article : Google Scholar : PubMed/NCBI
23	Sun YW, Zhang YY, Ke XJ, Wu XJ, Chen ZF and Chi P: Pirfenidone prevents radiation-induced intestinal fibrosis in rats by inhibiting fibroblast proliferation and differentiation and suppressing the TGF-β1/Smad/CTGF signaling pathway. Eur J Pharmacol. 822:199–206. 2018. View Article : Google Scholar : PubMed/NCBI
24	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
25	Ni BB, Li B, Yang YH, Chen JW, Chen K, Jiang SD and Jiang LS: The effect of transforming growth factor β1 on the crosstalk between autophagy and apoptosis in the annulus fibrosus cells under serum deprivation. Cytokine. 70:87–96. 2014. View Article : Google Scholar : PubMed/NCBI
26	Wu L, Zhang Q, Mo W, Feng J, Li S, Li J, Liu T, Xu S, Wang W, Lu X, et al: Quercetin prevents hepatic fibrosis by inhibiting hepatic stellate cell activation and reducing autophagy via the TGF-β1/Smads and PI3K/Akt pathways. Sci Rep. 7:92892017. View Article : Google Scholar : PubMed/NCBI
27	Takano M, Yamamoto C, Yamaguchi K, Kawami M and Yumoto R: Analysis of TGF-β1- and drug-induced epithelial-mesenchymal transition in cultured alveolar epithelial cell line RLE/Abca3. Drug Metab Pharmacokinet. 30:111–118. 2015. View Article : Google Scholar : PubMed/NCBI
28	Lee BS, Margolin SB and Nowak RA: Pirfenidone: A novel pharmacological agent that inhibits leiomyoma cell proliferation and collagen production. J Clin Endocrinol Metab. 83:219–223. 1998. View Article : Google Scholar : PubMed/NCBI
29	Luna J, Masamunt MC, Lawrance IC and Sans M: Mesenchymal cell proliferation and programmed cell death: Key players in fibrogenesis and new targets for therapeutic intervention. Am J Physiol Gastrointest Liver Physiol. 300:G703–G708. 2011. View Article : Google Scholar : PubMed/NCBI
30	Derynck R and Zhang YE: Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature. 425:577–584. 2003. View Article : Google Scholar : PubMed/NCBI
31	Choi K, Lee K, Ryu SW, Im M, Kook KH and Choi C: Pirfenidone inhibits transforming growth factor-β1-induced fibrogenesis by blocking nuclear translocation of Smads in human retinal pigment epithelial cell line ARPE-19. Mol Vis. 18:1010–1020. 2012.PubMed/NCBI
32	Downward J: PI 3-kinase, Akt and cell survival. Semin Cell Dev Biol. 15:177–182. 2004. View Article : Google Scholar : PubMed/NCBI