title: Benzalkonium chloride induces subconjunctival fibrosis through the COX-2-modulated activation of a TGF-β1/Smad3 signaling pathway.
abstract: OBJECTIVE
  The purpose is to investigate the mechanism of subconjunctival fibrosis caused by benzalkonium chloride (BAC), which is the most common preservative in ophthalmic preparations.
  
  
  METHODS
  The left eyes of male Sprague-Dawley rats were topically treated with 0.01% BAC or PBS twice daily for 1 month. Primary conjunctival fibroblasts (CFs) were exposed for 24 hours to 0.00005% BAC, 0.000075% BAC, 0.000075% BAC + LY2157299 (a selective transforming growth factor β receptor type I inhibitor); 0.000075% BAC + NS-398 (a selective cyclooxygenase-2 inhibitor) and PBS, respectively. The pathological changes of the bulbar conjunctival tissue of rats were examined using hematoxylin-eosin (HE), Van Gieson's (vG), periodic acid-Schiff (PAS) stains, or immunohistochemisty (IHC). The expression of the extracellular matrix (ECM), the transforming growth factor β (TGF-β) signaling pathway-related molecules, and cyclooxygenase-2 (COX-2) in bulbar conjunctival tissues and CFs were detected using Western blot (WB) and quantitative real-time RT-PCR (qRT-PCR).
  
  
  RESULTS
  Rats treated with 0.01% BAC exhibited a slight increase of the fibroblast density and a more compact collagen deposition in the bulbar subepithelial connective tissues in comparison with rats treated with PBS. Western blot and qRT-PCR analyses showed that the expression of ECM, TGF-β signaling pathway-related molecules, and COX-2 were markedly increased in the bulbar conjunctival tissues of rats exposed to 0.01% BAC and in CFs exposed to 0.00005% and 0.000075% BAC. In conjunctival fibroblasts, BAC-induced ECM expression was clearly decreased by LY2157299, while the BAC-induced activation of the TGF-β1/Smad3 signaling pathway was greatly attenuated by NS-398.
  
  
  CONCLUSIONS
  Subconjunctival fibrosis BAC-induced is a consequence of excessive ECM production of CFs through the COX-2-modulated activation of a TGF-β1/Smad3 signaling pathway.
authors.affiliation: Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China.
authors.forename: Caihong
authors.initials: C
authors.lastname: Huang
authors.affiliation: Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China.
authors.forename: He
authors.initials: H
authors.lastname: Wang
authors.affiliation: Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China.
authors.forename: Juxin
authors.initials: J
authors.lastname: Pan
authors.affiliation: Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Zhou
authors.affiliation: Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China Affiliated Xiamen Eye Center of Xiamen University, Xiamen, Fujian, China.
authors.forename: Wensheng
authors.initials: W
authors.lastname: Chen
authors.affiliation: Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China Affiliated Xiamen Eye Center of Xiamen University, Xiamen, Fujian, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.affiliation: Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China.
authors.forename: Yongxiong
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Eye Institute of Xiamen University, Fujian Provincial Key Laboratory of Ophthalmology and Visual Science, Xiamen, Fujian, China Affiliated Xiamen Eye Center of Xiamen University, Xiamen, Fujian, China.
authors.forename: Zuguo
authors.initials: Z
authors.lastname: Liu
mesh_terms: D000818:Animals; D001548:Benzalkonium Compounds / Q000009:adverse effects*; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D003228:Conjunctiva / Q000166:cytology; D003229:Conjunctival Diseases / Q000139:chemically induced / Q000378:metabolism*; D051546:Cyclooxygenase 2 / Q000378:metabolism*; D052246:Cyclooxygenase 2 Inhibitors / Q000494:pharmacology; D004195:Disease Models, Animal; D005109:Extracellular Matrix / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects; D005355:Fibrosis / Q000139:chemically induced / Q000378:metabolism*; D008297:Male; D011310:Preservatives, Pharmaceutical / Q000009:adverse effects*; D011720:Pyrazoles / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D051900:Smad3 Protein / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D001548:Benzalkonium Compounds; D015415:Biomarkers; D052246:Cyclooxygenase 2 Inhibitors; D011310:Preservatives, Pharmaceutical; D011720:Pyrazoles; D011804:Quinolines; D051900:Smad3 Protein; D053773:Transforming Growth Factor beta1; C557799:LY-2157299; D051546:Cyclooxygenase 2
keywords: benzalkonium chloride; cyclooxygenase-2; extracellular matrix; subconjunctival fibrosis; transforming growth factor-β