Pirfenidone Inhibits Cell Proliferation and Collagen I Production of Primary Human Intestinal Fibroblasts
Abstract
:1. Introduction
2. Materials and Methods
2.1. p-hIFs Isolation and Culture
2.2. Proliferation Assays
2.2.1. Real-Time Cell Analysis (RCTA)
2.2.2. BrdU Assay
2.2.3. p-hIFs Cell Counting
2.3. Real-Time Imaging of Cell Motility
2.4. Cytotoxicity and Cell Viability Assays
2.4.1. Sytox Green Assay
2.4.2. Caspase-3 Assay
2.4.3. WST-1 Assay
2.5. Quantitative Real–Time PCR (RT-qPCR)
2.6. Immunofluorescence Microscopy (IF)
2.7. Western Blotting
2.8. Statistical Analysis
3. Results
3.1. Pirfenidone Suppresses the Proliferation of p-hIFs, Which is Reversible
3.2. Pirfenidone Suppresses Extracellular Matrix Protein (ECM) Expression in p-hIFs
3.3. Pirfenidone Reduces TGF-β1-Induced COL1A1 mRNA Expression and Collagen I Synthesis
3.4. Pirfenidone Inhibits TGF-β1-Mediated Phosphorylation of TGF-β1/mTOR/p70S6K Signaling Pathway in p-hIFs
4. Discussion
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
- Baumgart, D.C.; Carding, S.R. Inflammatory bowel disease: Cause and immunobiology. Lancet 2007, 369, 1627–1640. [Google Scholar] [CrossRef]
- Kaplan, G.G. The global burden of IBD: From 2015 to 2025. Nat. Rev. Gastroenterol. Hepatol. 2015, 12, 720–727. [Google Scholar] [CrossRef] [PubMed]
- Latella, G.; Di Gregorio, J.; Flati, V.; Rieder, F.; Lawrance, I.C. Mechanisms of initiation and progression of intestinal fibrosis in IBD. Scand. J. Gastroenterol. 2015, 50, 53–65. [Google Scholar] [CrossRef] [PubMed]
- Bettenworth, D.; Rieder, F. Pathogenesis of Intestinal Fibrosis in Inflammatory Bowel Disease and Perspectives for Therapeutic Implication. Dig. Dis. 2017, 35, 25–31. [Google Scholar] [CrossRef] [PubMed]
- Lawrance, I.C.; Rogler, G.; Bamias, G.; Breynaert, C.; Florholmen, J.; Pellino, G.; Reif, S.; Speca, S.; Latella, G. Cellular and Molecular Mediators of Intestinal Fibrosis. J. Crohns Colitis 2017, 11, 1491–1503. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Rieder, F.; Fiocchi, C. Mechanisms of tissue remodeling in inflammatory bowel disease. Dig. Dis. 2013, 31, 186–193. [Google Scholar] [CrossRef] [PubMed]
- Allocca, M.; Fiorino, G.; Bonifacio, C.; Peyrin-Biroulet, L.; Danese, S. Noninvasive Multimodal Methods to Differentiate Inflamed vs Fibrotic Strictures in Patients With Crohn’s Disease. Clin. Gastroenterol. Hepatol. 2019, 12, 2397–2415. [Google Scholar] [CrossRef]
- Bettenworth, D.; Gustavsson, A.; Atreja, A.; Lopez, R.; Tysk, C.; van Assche, G.; Rieder, F. A Pooled Analysis of Efficacy, Safety, and Long-term Outcome of Endoscopic Balloon Dilation Therapy for Patients with Stricturing Crohn’s Disease. Inflamm. Bowel Dis. 2017, 23, 133–142. [Google Scholar] [CrossRef]
- van Loo, E.S.; Dijkstra, G.; Ploeg, R.J.; Nieuwenhuijs, V.B. Prevention of postoperative recurrence of Crohn’s disease. J. Crohns Colitis 2012, 6, 637–646. [Google Scholar] [CrossRef]
- Isaka, Y. Targeting TGF-beta Signaling in Kidney Fibrosis. Int. J. Mol. Sci. 2018, 19, 2532. [Google Scholar] [CrossRef] [Green Version]
- Laplante, M.; Sabatini, D.M. mTOR signaling at a glance. J. Cell Sci. 2009, 122, 3589–3594. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Lamouille, S.; Derynck, R. Cell size and invasion in TGF-beta-induced epithelial to mesenchymal transition is regulated by activation of the mTOR pathway. J. Cell Biol. 2007, 178, 437–451. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Richter, J.D.; Sonenberg, N. Regulation of cap-dependent translation by eIF4E inhibitory proteins. Nature 2005, 433, 477–480. [Google Scholar] [CrossRef] [PubMed]
- Chung, J.; Kuo, C.J.; Crabtree, G.R.; Blenis, J. Rapamycin-FKBP specifically blocks growth-dependent activation of and signaling by the 70 kd S6 protein kinases. Cell 1992, 69, 1227–1236. [Google Scholar] [CrossRef]
- Li, C.; Han, R.; Kang, L.; Wang, J.; Gao, Y.; Li, Y.; He, J.; Tian, J. Pirfenidone controls the feedback loop of the AT1R/p38 MAPK/renin-angiotensin system axis by regulating liver X receptor-alpha in myocardial infarction-induced cardiac fibrosis. Sci. Rep. 2017, 7. [Google Scholar] [CrossRef] [Green Version]
- Armendariz-Borunda, J.; Islas-Carbajal, M.C.; Meza-Garcia, E.; Rincon, A.R.; Lucano, S.; Sandoval, A.S.; Salazar, A.; Berumen, J.; Alvarez, A.; Covarrubias, A.; et al. A pilot study in patients with established advanced liver fibrosis using pirfenidone. Gut 2006, 55, 1663–1665. [Google Scholar] [CrossRef] [Green Version]
- King, T.E., Jr.; Bradford, W.Z.; Castro-Bernardini, S.; Fagan, E.A.; Glaspole, I.; Glassberg, M.K.; Gorina, E.; Hopkins, P.M.; Kardatzke, D.; Lancaster, L.; et al. A phase 3 trial of pirfenidone in patients with idiopathic pulmonary fibrosis. N. Engl. J. Med. 2014, 370, 2083–2092. [Google Scholar] [CrossRef] [Green Version]
- Li, Z.; Liu, X.; Wang, B.; Nie, Y.; Wen, J.; Wang, Q.; Gu, C. Pirfenidone suppresses MAPK signalling pathway to reverse epithelial-mesenchymal transition and renal fibrosis. Nephrology 2017, 22, 589–597. [Google Scholar] [CrossRef]
- Herrera, M.; Islam, A.B.; Herrera, A.; Martin, P.; Garcia, V.; Silva, J.; Garcia, J.M.; Salas, C.; Casal, I.; de Herreros, A.G.; et al. Functional heterogeneity of cancer-associated fibroblasts from human colon tumors shows specific prognostic gene expression signature. Clin. Cancer Res. 2013, 19, 5914–5926. [Google Scholar] [CrossRef] [Green Version]
- Roshan Moniri, M.; Young, A.; Reinheimer, K.; Rayat, J.; Dai, L.J.; Warnock, G.L. Dynamic assessment of cell viability, proliferation and migration using real time cell analyzer system (RTCA). Cytotechnology 2015, 67, 379–386. [Google Scholar] [CrossRef] [Green Version]
- Conde de la Rosa, L.; Schoemaker, M.H.; Vrenken, T.E.; Buist-Homan, M.; Havinga, R.; Jansen, P.L.; Moshage, H. Superoxide anions and hydrogen peroxide induce hepatocyte death by different mechanisms: Involvement of JNK and ERK MAP kinases. J. Hepatol. 2006, 44, 918–929. [Google Scholar] [CrossRef] [PubMed]
- Schoemaker, M.H.; Conde de la Rosa, L.; Buist-Homan, M.; Vrenken, T.E.; Havinga, R.; Poelstra, K.; Haisma, H.J.; Jansen, P.L.; Moshage, H. Tauroursodeoxycholic acid protects rat hepatocytes from bile acid-induced apoptosis via activation of survival pathways. Hepatology 2004, 39, 1563–1573. [Google Scholar] [CrossRef] [PubMed]
- Blokzijl, H.; Vander Borght, S.; Bok, L.I.; Libbrecht, L.; Geuken, M.; van den Heuvel, F.A.; Dijkstra, G.; Roskams, T.A.; Moshage, H.; Jansen, P.L.; et al. Decreased P-glycoprotein (P-gp/MDR1) expression in inflamed human intestinal epithelium is independent of PXR protein levels. Inflamm. Bowel Dis. 2007, 13, 710–720. [Google Scholar] [CrossRef] [PubMed]
- Kurahara, L.H.; Hiraishi, K.; Hu, Y.; Koga, K.; Onitsuka, M.; Doi, M.; Aoyagi, K.; Takedatsu, H.; Kojima, D.; Fujihara, Y.; et al. Activation of Myofibroblast TRPA1 by Steroids and Pirfenidone Ameliorates Fibrosis in Experimental Crohn’s Disease. Cell. Mol. Gastroenterol. Hepatol. 2018, 5, 299–318. [Google Scholar] [CrossRef] [Green Version]
- 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-beta signaling in a murine colitis model. Biochem. Pharmacol. 2016, 117, 57–67. [Google Scholar] [CrossRef]
- Sun, Y.W.; Zhang, Y.Y.; Ke, X.J.; Wu, X.J.; Chen, Z.F.; Chi, P. Pirfenidone prevents radiation-induced intestinal fibrosis in rats by inhibiting fibroblast proliferation and differentiation and suppressing the TGF-beta1/Smad/CTGF signaling pathway. Eur. J. Pharmacol. 2018, 822, 199–206. [Google Scholar] [CrossRef]
- Sun, Y.; Zhang, Y.; Chi, P. Pirfenidone suppresses TGFβ1induced human intestinal fibroblasts activities by regulating proliferation and apoptosis via the inhibition of the Smad and PI3K/AKT signaling pathway. Mol. Med. Rep. 2018, 18, 3907–3913. [Google Scholar] [CrossRef] [Green Version]
- Conte, E.; Gili, E.; Fagone, E.; Fruciano, M.; Iemmolo, M.; Vancheri, C. Effect of pirfenidone on proliferation, TGF-beta-induced myofibroblast differentiation and fibrogenic activity of primary human lung fibroblasts. Eur. J. Pharm. Sci. 2014, 58, 13–19. [Google Scholar] [CrossRef]
- Boehme, S.A.; Franz-Bacon, K.; DiTirro, D.N.; Ly, T.W.; Bacon, K.B. MAP3K19 Is a Novel Regulator of TGF-β Signaling That Impacts Bleomycin-Induced Lung Injury and Pulmonary Fibrosis. PLoS ONE 2016, 11. [Google Scholar] [CrossRef]
- Kurita, Y.; Araya, J.; Minagawa, S.; Hara, H.; Ichikawa, A.; Saito, N.; Kadota, T.; Tsubouchi, K.; Sato, N.; Yoshida, M.; et al. Pirfenidone inhibits myofibroblast differentiation and lung fibrosis development during insufficient mitophagy. Respir. Res. 2017, 18. [Google Scholar] [CrossRef] [Green Version]
- Woodcock, H.V.; Eley, J.D.; Guillotin, D.; Plate, M.; Nanthakumar, C.B.; Martufi, M.; Peace, S.; Joberty, G.; Poeckel, D.; Good, R.B.; et al. The mTORC1/4E-BP1 axis represents a critical signaling node during fibrogenesis. Nat. Commun. 2019, 10, 6. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Latella, G.; Rieder, F. Intestinal fibrosis: Ready to be reversed. Curr. Opin. Gastroenterol. 2017, 33, 239–245. [Google Scholar] [CrossRef] [PubMed]
- Rockey, D.C.; Bell, P.D.; Hill, J.A. Fibrosis- A common pathway to organ injury and failure. N. Engl. J. Med. 2015, 372, 1138–1149. [Google Scholar] [CrossRef] [PubMed]
- Kadir, S.I.; Wenzel Kragstrup, T.; Dige, A.; Kok Jensen, S.; Dahlerup, J.F.; Kelsen, J. Pirfenidone inhibits the proliferation of fibroblasts from patients with active Crohn’s disease. Scand. J. Gastroenterol. 2016, 51, 1321–1325. [Google Scholar] [CrossRef] [PubMed]
- Pan, C.; Kumar, C.; Bohl, S.; Klingmueller, U.; Mann, M. Comparative proteomic phenotyping of cell lines and primary cells to assess preservation of cell type-specific functions. Mol. Cell. Proteomics 2009, 8, 443–450. [Google Scholar] [CrossRef] [Green Version]
- Ji, X.; Naito, Y.; Weng, H.; Ma, X.; Endo, K.; Kito, N.; Yanagawa, N.; Yu, Y.; Li, J.; Iwai, N. Renoprotective mechanisms of pirfenidone in hypertension-induced renal injury: Through anti-fibrotic and anti-oxidative stress pathways. Biomed. Res. 2013, 34, 309–319. [Google Scholar] [CrossRef] [Green Version]
- Molina-Molina, M.; Machahua-Huamani, C.; Vicens-Zygmunt, V.; Llatjos, R.; Escobar, I.; Sala-Llinas, E.; Luburich-Hernaiz, P.; Dorca, J.; Montes-Worboys, A. Anti-fibrotic effects of pirfenidone and rapamycin in primary IPF fibroblasts and human alveolar epithelial cells. BMC Pulm. Med. 2018, 18. [Google Scholar] [CrossRef] [Green Version]
- Lipton, J.O.; Sahin, M. The neurology of mTOR. Neuron 2014, 84, 275–291. [Google Scholar] [CrossRef] [Green Version]
- Saxton, R.A.; Sabatini, D.M. mTOR Signaling in Growth, Metabolism, and Disease. Cell 2017, 168, 960–976. [Google Scholar] [CrossRef] [Green Version]
- Li, J.; Kim, S.G.; Blenis, J. Rapamycin: One drug, many effects. Cell Metab. 2014, 19, 373–379. [Google Scholar] [CrossRef] [Green Version]
- Lopez-de la Mora, D.A.; Sanchez-Roque, C.; Montoya-Buelna, M.; Sanchez-Enriquez, S.; Lucano-Landeros, S.; Macias-Barragan, J.; Armendariz-Borunda, J. Role and New Insights of Pirfenidone in Fibrotic Diseases. Int. J. Med. Sci. 2015, 12, 840–847. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Fois, A.G.; Posadino, A.M.; Giordo, R.; Cossu, A.; Agouni, A.; Rizk, N.M.; Pirina, P.; Carru, C.; Zinellu, A.; Pintus, G. Antioxidant Activity Mediates Pirfenidone Antifibrotic Effects in Human Pulmonary Vascular Smooth Muscle Cells Exposed to Sera of Idiopathic Pulmonary Fibrosis Patients. Oxid. Med. Cell. Longev. 2018, 2018. [Google Scholar] [CrossRef] [PubMed]
- Noble, P.W.; Albera, C.; Bradford, W.Z.; Costabel, U.; Glassberg, M.K.; Kardatzke, D.; King, T.E., Jr.; Lancaster, L.; Sahn, S.A.; Szwarcberg, J.; et al. Pirfenidone in patients with idiopathic pulmonary fibrosis (CAPACITY): Two randomised trials. Lancet 2011, 377, 1760–1769. [Google Scholar] [CrossRef]
- Noble, P.W.; Albera, C.; Bradford, W.Z.; Costabel, U.; du Bois, R.M.; Fagan, E.A.; Fishman, R.S.; Glaspole, I.; Glassberg, M.K.; Lancaster, L.; et al. Pirfenidone for idiopathic pulmonary fibrosis: Analysis of pooled data from three multinational phase 3 trials. Eur. Respir. J. 2016, 47, 243–253. [Google Scholar] [CrossRef] [PubMed]
- Gareb, B.; Dijkstra, G.; Kosterink, J.G.W.; Frijlink, H.W. Development of novel zero-order release budesonide tablets for the treatment of ileo-colonic inflammatory bowel disease and comparison with formulations currently used in clinical practice. Int. J. Pharm. 2019, 554, 366–375. [Google Scholar] [CrossRef] [Green Version]
- Khanna, D.; Albera, C.; Fischer, A.; Khalidi, N.; Raghu, G.; Chung, L.; Chen, D.; Schiopu, E.; Tagliaferri, M.; Seibold, J.R.; et al. An Open-label, Phase II Study of the Safety and Tolerability of Pirfenidone in Patients with Scleroderma-associated Interstitial Lung Disease: The LOTUSS Trial. J. Rheumatol. 2016, 43, 1672–1679. [Google Scholar] [CrossRef] [Green Version]
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Cui, Y.; Zhang, M.; Leng, C.; Blokzijl, T.; Jansen, B.H.; Dijkstra, G.; Faber, K.N. Pirfenidone Inhibits Cell Proliferation and Collagen I Production of Primary Human Intestinal Fibroblasts. Cells 2020, 9, 775. https://doi.org/10.3390/cells9030775
Cui Y, Zhang M, Leng C, Blokzijl T, Jansen BH, Dijkstra G, Faber KN. Pirfenidone Inhibits Cell Proliferation and Collagen I Production of Primary Human Intestinal Fibroblasts. Cells. 2020; 9(3):775. https://doi.org/10.3390/cells9030775
Chicago/Turabian StyleCui, Yingying, Mengfan Zhang, Changsen Leng, Tjasso Blokzijl, Bernadien H. Jansen, Gerard Dijkstra, and Klaas Nico Faber. 2020. "Pirfenidone Inhibits Cell Proliferation and Collagen I Production of Primary Human Intestinal Fibroblasts" Cells 9, no. 3: 775. https://doi.org/10.3390/cells9030775