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The anti-fibrotic and anti-inflammatory effects of 2,4-diamino-5-(1-hydroxynaphthalen-2-yl)-5H-chromeno[2,3-b] pyriine-3-carbonitrile in corneal fibroblasts

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Abstract

Background

Although several studies had addressed the anti-inflammatory effects of derivatives of 4H-chromene and chromeno[2,3-b]pyridine in the different types of cells, whether these derivatives would exert beneficial anti-fibrotic effects during corneal fibrotic scar formation was unclear.

Methods

We examined the cyclooxygenase-2 (COX-2) expression of 2,4-diamino-5-(1-hydroxynaphthalen-2-yl)-5H-chromeno[2,3-b]pyridine-3-carbonitrile (N1) in the human corneal fibroblasts (HCFs) under the treatment TGF-β1. Signaling pathways underlying the mechanism of the N1 effect on the HCFs were determined.

Results

Application of N1 significantly decreased COX-2 expression after 2 h and 4 h in the HCFs stimulated with TGF-β1. Notably, reduced production of extracellular matrix proteins under N1 treatment was found, including fibronectin, collagen I, and matrix metallopeptidase 9. Immunoblot analysis showed that treatment with N1 significantly attenuated phosphorylation of both STAT3 and Smad 2 in the TGF-β1-stimulated HCFs. Upregulated mRNA of Smad2 and downregulated mRNA of Smad3 were observed using the quantitative real-time polymerase chain reaction. In addition, N1 induced significant increases in HO-1 and Nrf2 expression, but inhibited phosphorylation of NF-κB in the HCFs treated with TGF-β1.

Conclusions

Our findings show for the first time that N1 exerts anti-fibrotic and anti-inflammatory effects through suppression of COX-2, Smad2, STAT3, iNOS and NF-κB expressions as well as upregulation of Nrf2 and HO-1 expressions, which suggests they are potential therapeutic targets in the treatment of corneal fibrosis.

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Abbreviations

COX-2:

Cyclooxygenase-2

DMEM:

Dulbecco’s modified Eagle’s medium

EGF:

Epidermal growth factor

FGF:

Fibroblast growth factor

FBS:

Fetal bovine serum

HCF:

Human corneal fibroblast

iNOS:

Inducible nitric oxide synthase

IGF-I:

Insulin-like growth factor I

KGF:

Keratinocyte growth factor

MMP-9:

Matrix metallopeptidase 9

MAPK:

p38 Mitogen-activated protein kinase

N1:

2,4-Diamino-5-(1-hydroxynaphthalen-2-yl)-5H-chromeno[2,3-b]pyridine-3-carbonitrile

PDGF:

Platelet-derived growth factor

PCR:

Polymerase chain reaction

TGF:

Transforming growth factor

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Funding

This study was supported in part by grants from the Tri-Service General Hospital (TSGH-C 108-220) and the Ministry of Science and Technology (MOST 107-2314-B-016-035), Taiwan, ROC.

Author information

Authors and Affiliations

  1. Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China

    Ying-Jen Chen, Shih-Ming Huang, Ming-Cheng Tai, Jiann-Torng Chen & Chang-Min Liang

  2. Department of Ophthalmology, School of Medicine, Tri-Service General Hospital, National Defense Medical Center, Number 325, Section 2 Chang-gong Rd, Nei-Hu District, 114, Taipei, Taiwan, Republic of China

    Ying-Jen Chen, Ming-Cheng Tai, Jiann-Torng Chen & Chang-Min Liang

  3. Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China

    Shih-Ming Huang

  4. School of Pharmacy, National Defense Medical Center, Taipei, Taiwan, Republic of China

    An-Rong Lee

  5. Department of Periodontology, School of Dentistry, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China

    Ren-Yeong Huang

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  1. Ying-Jen Chen
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Contributions

Conceived and designed the analysis: YJC, SMH, MCT, JTC, CML. Collected the data: YJC, SMH, MCT, JTC, ARL, RYH, CML. Performed the analysis: YJC, SMH, MCT, JTC, CML. Wrote the paper: YJC. Contributed analysis tools: YJC, SMH, MCT, JTC, ARL, RYH, CML.

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Correspondence to Chang-Min Liang.

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43440_2019_26_MOESM1_ESM.pdf

Supplementary Figure S1. In vitro characterization of HCFs (A) Cellular morphology of different participants by microscopy. (B) Immunoblotting analysis showed expression of vimentin in the HCFs of different participants. (C) COX-2 activity was measured with or without N1 (10 nM) and TGF β1 (10 ng/mL) stimulation. (PDF 564 kb)

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Chen, YJ., Huang, SM., Tai, MC. et al. The anti-fibrotic and anti-inflammatory effects of 2,4-diamino-5-(1-hydroxynaphthalen-2-yl)-5H-chromeno[2,3-b] pyriine-3-carbonitrile in corneal fibroblasts. Pharmacol. Rep 72, 115–125 (2020). https://doi.org/10.1007/s43440-019-00026-9

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  • DOI: https://doi.org/10.1007/s43440-019-00026-9

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