Pirfenidone inhibits proliferation, arrests the cell cycle, and downregulates heat shock protein‑47 and collagen type I in rat hepatic stellate cells in vitro

Xiang,Xian‑Hong; Jiang,Tian‑Peng; Zhang,Shuai; Song,Jie; Li,Xing; Yang,Jian‑Yong; Zhou,Shi

doi:10.3892/mmr.2015.3403

Pirfenidone inhibits proliferation, arrests the cell cycle, and downregulates heat shock protein‑47 and collagen type I in rat hepatic stellate cells in vitro

Authors:
- Xian‑Hong Xiang
- Tian‑Peng Jiang
- Shuai Zhang
- Jie Song
- Xing Li
- Jian‑Yong Yang
- Shi Zhou
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Affiliations: Department of Interventional Radiology, The First Affiliated Hospital of Sun Yat‑Sen University, Guangzhou, Guangdong 510080, P.R. China, Department of Interventional Radiology, Affiliated Hospital of Guiyang Medical College, Guiyang, Guizhou 550000, P.R. China, Department of Interventional Radiology, Affiliated Cancer Hospital of Guiyang Medical College, Guiyang, Guizhou 550000, P.R. China
Published online on: March 3, 2015 https://doi.org/10.3892/mmr.2015.3403
Pages: 309-314

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Abstract

Pirfenidone (esbiret) is an established anti‑fibrotic and anti‑inflammatory drug used to treat idiopathic pulmonary fibrosis. In the present study, the dose‑dependent effects of pirfenidone on the cell cycle, proliferation and expression of heat shock protein (HSP)‑47 and collagen type I in a cultured rat hepatic stellate cell line (HSC‑T6) were investigated. Following pirfenidone treatment, cell proliferation was determined using the cell counting kit‑8 assay and the cell cycle was measured using flow cytometry. HSP‑47 expression was estimated using western blot analysis and collagen type I mRNA was assessed using reverse transcription quantitative polymerase chain reaction. Pirfenidone induced significant dose‑dependent inhibition of proliferation in HSC‑T6 cells. Cell viability was unaffected by treatment with pirfenidone (0, 10 or 100 µM) for 24 and 72 h. However, after 24 h, HSC‑T6 cells exhibited dose‑dependent decreases in HSP‑47 protein and collagen I mRNA levels. In conclusion, pirfenidone inhibited HSC‑T6 cell proliferation, arrested the cell cycle and reduced the expression of HSP‑47 and collagen type I, indicating that pirfenidone may be a promising drug in the treatment of liver fibrosis.

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1	Friedman SL: Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem. 275:2247–2250. 2000. View Article : Google Scholar : PubMed/NCBI
2	Milani S, Herbst H, Schuppan D, Surrenti C, Riecken EO and Stein H: Cellular localization of type I III and IV procollagen gene transcripts in normal and fibrotic human liver. Am J Pathol. 137:59–70. 1990.PubMed/NCBI
3	Bataller R and Brenner DA: Liver fibrosis. J Clin Invest. 115:209–218. 2005. View Article : Google Scholar : PubMed/NCBI
4	Taguchi T and Razzaque MS: The collagen-specific molecular chaperone HSP47: Is there a role in fibrosis. Trends Mol Med. 13:45–53. 2007. View Article : Google Scholar
5	Razzaque MS, Le VT and Taguchi T: Heat shock protein 47 and renal fibrogenesis. Contrib Nephrol. 148:57–69. 2005.PubMed/NCBI
6	Chen JJ, Jin PS, Zhao S, Cen Y, Liu Y, Xu XW, Duan WQ and Wang HS: Effect of heat shock protein 47 on collagen synthesis of keloid in vivo. ANZ J Surg. 81:425–430. 2011. View Article : Google Scholar
7	Brown KE, Broadhurst KA, Mathahs MM, Brunt EM and Schmidt WN: Expression of HSP47, a collagen-specific chaperone, in normal and diseased human liver. Lab Invest. 85:789–797. 2005. View Article : Google Scholar : PubMed/NCBI
8	Nakayama S, Mukae H, Sakamoto N, et al: Pirfenidone inhibits the expression of HSP47 in TGF-β1-stimulated human lung fibroblasts. Life Sci. 82:210–217. 2008. View Article : Google Scholar
9	Sato Y, Murase K, Kato J, et al: Resolution of liver cirrhosis using vitamin A-coupled liposomes to deliver siRNA against a collagen-specific chaperone. Nat Biotechnol. 26:431–442. 2008. View Article : Google Scholar : PubMed/NCBI
10	Kakugawa T, Mukae H, Hayashi T, Ishii H, Abe K, Fujii T, Oku H, Miyazaki M, Kadota J and Kohno S: Pirfenidone attenuates expression of HSP47 in murine bleomycin-induced pulmonary fibrosis. Eur Respir J. 24:57–65. 2004. View Article : Google Scholar : PubMed/NCBI
11	Zhao XY, Zeng X, Li XM, Wang TL and Wang BE: Pirfenidone inhibits carbon tetrachloride- and albumin complex-induced liver fibrosis in rodents by preventing activation of hepatic stellate cells. Clin Exp Pharmacol Physiol. 36:963–968. 2009. View Article : Google Scholar : PubMed/NCBI
12	Armendáriz-Borunda J, Rincón AR, Muñoz-Valle JF, et al: Fibrogenic polymorphisms (TGF-beta, PAI-1, AT) in Mexican patients with established liver fibrosis. Potential correlation with pirfenidone treatment. J Investig Med. 56:944–953. 2008.PubMed/NCBI
13	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.
14	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 rats. Br J Pharmacol. 133:687–694. 2001. View Article : Google Scholar : PubMed/NCBI
15	Sun G, Lin X, Zhong H, Yang Y, Qiu X, Ye C, Wu K and Yu M: Pharmacokinetics of pirfenidone after topical administration in rabbit eye. Mol Vis. 17:2191–2196. 2011.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	Di Sario A, Bendia E, Svegliati Baroni G, 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
18	Gurujeyalakshmi G, Hollinger MA and Giri SN: Pirfenidone inhibits PDGF isoforms in bleomycin hamster model of lung fibrosis at the translational level. Am J Physiol. 276:L311–L318. 1999.PubMed/NCBI
19	Grattendick KJ, Nakashima JM, Feng L, Giri SN and Margolin SB: Effects of three anti-TNF-alpha drugs: Etanercept, infliximab and pirfenidone on release of TNF-alpha in medium and TNF-alpha associated with the cell in vitro. Int Immunopharmacol. 8:679–687. 2008. View Article : Google Scholar : PubMed/NCBI
20	Oku H, Nakazato H, Horikawa T, Tsuruta Y and Suzuki R: Pirfenidone suppresses tumor necrosis factor-alpha, enhances interleukin-10 and protects mice from endotoxic shock. Eur J Pharmacol. 446:167–176. 2002. View Article : Google Scholar : PubMed/NCBI
21	Ginès P, Cárdenas A, Arroyo V and Rodés J: Management of cirrhosis and ascites. N Engl J Med. 350:1646–1654. 2004. View Article : Google Scholar : PubMed/NCBI
22	Rockey DC: Current and future anti-fibrotic therapies for chronic liver disease. Clin Liver Dis. 12:939–962. xi2008. View Article : Google Scholar : PubMed/NCBI
23	Zhao W, Li JJ, Cao DY, Li X, Zhang LY, He Y, Yue SQ, Wang DS and Dou KF: Intravenous injection of mesenchymal stem cells is effective in treating liver fibrosis. World J Gastroenterol. 18:1048–1058. 2012. View Article : Google Scholar : PubMed/NCBI
24	Behr J: Idiopathic pulmonary fibrosis: Modern guideline-concordant diagnostics and innovative treatment. Dtsch Med Wochenschr. 137:601–604. 2012.In German. PubMed/NCBI
25	Salazar-Montes A, Ruiz-Corro L, López-Reyes A, Castrejón-Gómez E and Armendáriz-Borunda J: Potent antioxidant role of pirfenidone in experimental cirrhosis. Eur J Pharmacol. 595:69–77. 2008. View Article : Google Scholar : PubMed/NCBI
26	Di Sario A, Bendia E, Macarri G, Candelaresi C, Taffetani S, Marzioni M, Omenetti A, De Minicis S, Trozzi L and Benedetti A: The anti-fibrotic effect of pirfenidone in rat liver fibrosis is mediated by downregulation of procollagen alpha1(I), TIMP-1 and MMP-2. Dig Liver Dis. 36:744–751. 2004. View Article : Google Scholar : PubMed/NCBI
27	Tada S, Nakamuta M, Enjoji M, Sugimoto R, Iwamoto H, Kato M, Nakashima Y and Nawata H: Pirfenidone inhibits dimethylnitrosamine-induced hepatic fibrosis in rats. Clin Exp Pharmacol Physiol. 28:522–527. 2001. View Article : Google Scholar : PubMed/NCBI
28	Navarro-Partida J, Martinez-Rizo AB, Gonzalez-Cuevas J, Arrevillaga-Boni G, Ortiz-Navarrete V and Armendariz-Borunda J: Pirfenidone restricts Th2 differentiation in vitro and limits Th2 response in experimental liver fibrosis. Eur J Pharmacol. 678:71–77. 2012. View Article : Google Scholar : PubMed/NCBI
29	Sauk JJ, Nikitakis N and Siavash H: Hsp47 a novel collagen binding serpin chaperone, autoantigen and therapeutic target. Front Biosci. 10:107–118. 2005. View Article : Google Scholar
30	Taguchi T, Nazneen A, Al-Shihri AA, Turkistani KA and Razzaque MS: Heat shock protein 47: A novel biomarker of phenotypically altered collagen-producing cells. Acta Histochem Cytochem. 44:35–41. 2011. View Article : Google Scholar : PubMed/NCBI
31	Nagata K: Therapeutic strategy for fibrotic diseases by regulating the expression of collagen-specific molecular chaperone HSP47. Nippon Yakurigaku Zasshi. 121:4–14. 2003.In Japanese. View Article : Google Scholar
32	Xia Z, Abe K, Furusu A, Miyazaki M, Obata Y, Tabata Y, Koji T and Kohno S: Suppression of renal tubulointerstitial fibrosis by small interfering RNA targeting heat shock protein 47. Am J Nephrol. 28:34–46. 2008. View Article : Google Scholar
33	Hagiwara S, Iwasaka H, Shingu C, Matumoto S, Hasegawa A and Noguchi T: Heat shock protein 47 (HSP47) antisense oligonucleotides reduce cardiac remodeling and improve cardiac function in a rat model of myocardial infarction. Thorac Cardiovasc Surg. 59:386–392. 2011. View Article : Google Scholar : PubMed/NCBI
34	Dams SD, de Liefde-van Beest M, Nuijs AM, Oomens CW and Baaijens FP: Heat shocks enhance procollagen type I and III expression in fibroblasts in ex vivo human skin. Skin Res Technol. 17:167–180. 2011. View Article : Google Scholar : PubMed/NCBI
35	Sauk JJ, Smith T, Norris K and Ferreira L: Hsp47 and the translation-translocation machinery cooperate in the production of alpha 1(I) chains of type I procollagen. J Biol Chem. 269:3941–3946. 1994.PubMed/NCBI
36	Sunamoto M, Kuze K, Tsuji H, Ohishi N, Yagi K, Nagata K, Kita T and Doi T: Antisense oligonucleotides against collagen-binding stress protein HSP47 suppress collagen accumulation in experimental glomerulonephritis. Lab Invest. 78:967–972. 1998.PubMed/NCBI
37	Hagiwara S, Nakamura K, Hamada H, et al: Inhibition of type I procollagen production by tRNAVal CTE-HSP47 ribozyme. J Gene Med. 5:784–794. 2003. View Article : Google Scholar : PubMed/NCBI
38	Nishino T, Miyazaki M, Abe K, Furusu A, Mishima Y, Harada T, Ozono Y, Koji T and Kohno S: Antisense oligonucleotides against collagen-binding stress protein HSP47 suppress peritoneal fibrosis in rats. Kidney Int. 64:887–896. 2003. View Article : Google Scholar : PubMed/NCBI