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Cited by 50TGF-β signaling in diseasesInflammatory Bowel DiseaseInterstitial Lung Diseases and Idiopathic Pulmonary Fibrosis

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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

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Sun, Yanwu, Zhang, Yiyi, Chi, Pan·2018·Molecular Medicine Reports
DOI 10.3892/mmr.2018.9423PMC PMC6131636
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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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Figure 1. PFD inhibits cell proliferation in HIFs following TGF-β1 stimulation. (A) HIFs were pretreated with DMSO and different concentrations of PFD (0, 0.01, 0.05, 0.1, 0.5 and 1 mg/ml) for 24 h. Percentage of cytotoxicity was evaluated by a lactate dehydrogenase cytotoxicity assay. (B) HIFs were pretreated with PFD for 1 h prior to stimulation with TGF-β1 (10 ng/ml) for 1, 2, 3, 4 and 5 days. Cell Counting Kit-8 assays revealed a robust proliferative response to TGF-β1 when compared with normal cells, and PFD pretreatment abrogated TGF-β1-induced proliferation in HIFs, restraining cell numbers to basal levels. (C and D) HIFs were pretreated with PFD for 1 h prior to stimulation with TGF-β1 (10 ng/ml) for 14 days. Colony formation assays demonstrated the mean colony number was significantly increased following TGF-β1 stimulation in the colony formation assay, and PFD administration markedly reduced colony growth. Data are presented as the mean ± standard deviation of triplicate experiments. *P<0.05 vs. control group; #P<0.05 vs. TGF-β1 group. PFD, pirfenidone; HIFs, human intestinal fibroblasts; TGF, transforming growth factor; PFD, pirfenidone; OD, optical density.
Figure 1. PFD inhibits cell proliferation in HIFs following TGF-β1 stimulation. (A) HIFs were pretreated with DMSO and different concentrations of PFD (0, 0.01, 0.05, 0.1, 0.5 and 1 mg/ml) for 24 h. Percentage of cytotoxicity was evaluated by a lactate dehydrogenase cytotoxicity assay. (B) HIFs were pretreated with PFD for 1 h prior to stimulation with TGF-β1 (10 ng/ml) for 1, 2, 3, 4 and 5 days. Cell Counting Kit-8 assays revealed a robust proliferative response to TGF-β1 when compared with normal cells, and PFD pretreatment abrogated TGF-β1-induced proliferation in HIFs, restraining cell numbers to basal levels. (C and D) HIFs were pretreated with PFD for 1 h prior to stimulation with TGF-β1 (10 ng/ml) for 14 days. Colony formation assays demonstrated the mean colony number was significantly increased following TGF-β1 stimulation in the colony formation assay, and PFD administration markedly reduced colony growth. Data are presented as the mean ± standard deviation of triplicate experiments. *P<0.05 vs. control group; #P<0.05 vs. TGF-β1 group. PFD, pirfenidone; HIFs, human intestinal fibroblasts; TGF, transforming growth factor; PFD, pirfenidone; OD, optical density.CC BY-NC-ND · PubMed Central
Figure 2. PFD induces apoptosis in HIFs stimulated with TGF-β1. HIFs were pretreated with PFD for 1 h prior to TGF-β1 stimulation for 48 h. (A and B) Flow cytometry assay indicated that TGF-β1 treatment induced a reduction in cell apoptosis, and pretreatment with PFD reduced the TGF-β1-induced reduction in apoptosis. (C) Representative images of TUNEL assays (n=3 per group). Magnification, ×400. (D) Western blotting and (E) quantitative analysis of Bcl-2 and Bad protein expression. Data are presented as the mean ± standard deviation of triplicate experiments. *P<0.05 vs. control group; #P<0.05 vs. TGF-β1 group. PFD, pirfenidone; HIFs, human intestinal fibroblasts; TGF, transforming growth factor; Bcl-2, B-cell lymphoma 2; Bad, Bcl-2 associated agonist of cell death.
Figure 2. PFD induces apoptosis in HIFs stimulated with TGF-β1. HIFs were pretreated with PFD for 1 h prior to TGF-β1 stimulation for 48 h. (A and B) Flow cytometry assay indicated that TGF-β1 treatment induced a reduction in cell apoptosis, and pretreatment with PFD reduced the TGF-β1-induced reduction in apoptosis. (C) Representative images of TUNEL assays (n=3 per group). Magnification, ×400. (D) Western blotting and (E) quantitative analysis of Bcl-2 and Bad protein expression. Data are presented as the mean ± standard deviation of triplicate experiments. *P<0.05 vs. control group; #P<0.05 vs. TGF-β1 group. PFD, pirfenidone; HIFs, human intestinal fibroblasts; TGF, transforming growth factor; Bcl-2, B-cell lymphoma 2; Bad, Bcl-2 associated agonist of cell death.CC BY-NC-ND · PubMed Central
Figure 3. PFD inhibits HIFs differentiation and the collagen deposition induced by TGF-β1. (A-C) Relative mRNA expression of (A) α-SMA, (B) collagen I and (C) fibronectin. HIFs were pretreated with PFD for 1 h prior to TGF-β1 stimulation for 48 h. (D) Western blotting and (E) quantitative analysis of α-SMA, collagen I and fibronectin protein expression. HIFs were pretreated with PFD for 1 h prior to TGF-β1 stimulation for 48 h. Data are presented as the mean ± standard deviation of triplicate experiments. *P<0.05 vs. control group; #P<0.05 vs. TGF-β1 group. PFD, pirfenidone; HIFs, human intestinal fibroblasts; TGF, transforming growth factor; α-SMA, α-smooth muscle actin; COL1A1, Collagen I α 1.
Figure 3. PFD inhibits HIFs differentiation and the collagen deposition induced by TGF-β1. (A-C) Relative mRNA expression of (A) α-SMA, (B) collagen I and (C) fibronectin. HIFs were pretreated with PFD for 1 h prior to TGF-β1 stimulation for 48 h. (D) Western blotting and (E) quantitative analysis of α-SMA, collagen I and fibronectin protein expression. HIFs were pretreated with PFD for 1 h prior to TGF-β1 stimulation for 48 h. Data are presented as the mean ± standard deviation of triplicate experiments. *P<0.05 vs. control group; #P<0.05 vs. TGF-β1 group. PFD, pirfenidone; HIFs, human intestinal fibroblasts; TGF, transforming growth factor; α-SMA, α-smooth muscle actin; COL1A1, Collagen I α 1.CC BY-NC-ND · PubMed Central
Figure 4. PFD exerts antifibrotic effects by inhibiting Smad and PI3K/AKT signaling activation in HIFs incubated with TGF-β1. (A) Western blotting analysis of Smad2/3, p-Smad2/3, PI3K, AKT and p-AKT protein expression. HIFs were pretreated with PFD for 1 h prior to TGF-β1 stimulation for 48 h. (B-D) Quantitative analysis of (B) p-Smad2/3 to Smad2/3 ratio, (C) PI3K and (D) p-AKT to t-AKT ratio protein expression. Data are presented as the mean ± standard deviation of triplicate experiments. *P<0.05 vs. control group; #P<0.05 vs. TGF-β1 group. PFD, pirfenidone; HIFs, human intestinal fibroblasts; TGF, transforming growth factor; Smad, mothers against decapentaplegic homolog; p-, phosphorylated; AKT, protein kinase B; PI3K, phosphoinositide 3-kinase; t-, total.
Figure 4. PFD exerts antifibrotic effects by inhibiting Smad and PI3K/AKT signaling activation in HIFs incubated with TGF-β1. (A) Western blotting analysis of Smad2/3, p-Smad2/3, PI3K, AKT and p-AKT protein expression. HIFs were pretreated with PFD for 1 h prior to TGF-β1 stimulation for 48 h. (B-D) Quantitative analysis of (B) p-Smad2/3 to Smad2/3 ratio, (C) PI3K and (D) p-AKT to t-AKT ratio protein expression. Data are presented as the mean ± standard deviation of triplicate experiments. *P<0.05 vs. control group; #P<0.05 vs. TGF-β1 group. PFD, pirfenidone; HIFs, human intestinal fibroblasts; TGF, transforming growth factor; Smad, mothers against decapentaplegic homolog; p-, phosphorylated; AKT, protein kinase B; PI3K, phosphoinositide 3-kinase; t-, total.CC BY-NC-ND · PubMed Central

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Citations

Cites

Research works printed but not linked: 31

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Metadata

Authors
Sun, Y., Zhang, Y., & Chi, P.
Affiliations
Union Hospital (CN)
Corresponding author
Chi, Pan
ORCID
Venue
Molecular Medicine Reports
Publisher
Spandidos Publications
Published
2018-08-22
Volume
18
Issue
4
Pages
3907-3913
ISSN
  • 1791-2997 (ISSN-L)
  • 1791-3004
DOI
10.3892/mmr.2018.9423
PMC
PMC6131636
OpenAlex
W2888293125
Cited by
50
References
1
Citations by year
2026: 3 · 2025: 7 · 2024: 3 · 2023: 7 · 2022: 5 · 2021: 10 · 2020: 10 · 2019: 5
FWCI
2.22
Type
journal article
Language
en
Open access
Licence
cc-by-nc-nd
Locations
  • Molecular Medicine Reports (publishedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
Funders
  • Fujian Provincial Department of Science and Technology (2015Y0058)
  • Fujian Medical University
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › TGF-β signaling in diseases
  • Health Sciences › Medicine › Gastroenterology › Inflammatory Bowel Disease
  • Health Sciences › Medicine › Pulmonary and Respiratory Medicine › Interstitial Lung Diseases and Idiopathic Pulmonary Fibrosis
Keywords
pirfenidone, intestinal fibrosis, intestinal fibroblasts, TGF-β1, Smad signaling, PI3K/Akt signaling, cell proliferation, apoptosis, myofibroblast differentiation, alpha-smooth muscle actin, type I collagen, fibronectin, antifibrotic therapy
MeSH terms
Cell Differentiation, Collagen, Fibroblasts, Humans, Intestines, Pyridones, Signal Transduction, Apoptosis, Phosphatidylinositol 3-Kinases, Cell Proliferation, Proto-Oncogene Proteins c-akt, Smad Proteins, Transforming Growth Factor beta1

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