Ying-Jen Chen1,2, Shih-Ming Huang1,3, Ming-Cheng Tai1,2, Jiann-Torng Chen1,2, An-Rong Lee4, Ren-Yeong Huang5, Chang-Min Liang6,7. 1. Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China. 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. 3. Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China. 4. School of Pharmacy, National Defense Medical Center, Taipei, Taiwan, Republic of China. 5. Department of Periodontology, School of Dentistry, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, Republic of China. 6. Graduate Institute of Medical Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China. doc30875@yahoo.com.tw. 7. 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. doc30875@yahoo.com.tw.
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.
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.
Entities:
Keywords:
2,4-Diamino-5-(1-hydroxynaphthalen-2-yl)-5H-chromeno[2,3-b]pyridine-3-carbonitrile; Corneal fibrosis; Cyclooxygenase-2; Human primary corneal fibroblasts