Benzalkonium chloride induces subconjunctival fibrosis through the COX-2-modulated activation of a TGF-β1/Smad3 signaling pathway.

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 þ LY2157299 (a selective transforming growth factor b receptor type I inhibitor); 0.000075% BAC þ 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 b (TGF-b) 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-b 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-b1/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-b1/Smad3 signaling pathway.