The Mechanism Of Benzalkonium Chloride Induced Subconjunctival Fibrosis

Purpose:Benzalkonium chloride (BAC) is the most common preservative in ophthalmic preparations. Large bodies of clinical and experimental studies have shown that long term use of topical drugs with BAC can induce a series of ocular surface disease, such as subconjunctival fibrosis. The purpose of this study is to investigate the mechanism underlying subconjunctival fibrosis caused by BAC.Methods:45Sprague-Dawley male rats were randomly assigned to control group, experimental group and cell culture group of15rats each. In control and experimental groups, the left eye of rats were treated topically with PBS or0.01%BAC twice daily for one month, respectively. In cell culture group, cells were isolated from left eye of rats and cultured. The primary conjunctval fibroblasts (CFs) were exposed for24hours to0.00005%BAC,0.000075%BAC,0.000075%BAC+LY2157299(a selective TGF-βR1inhibitor,200μM),0.000075%BAC+NS-398(a selective COX-2inhibitor,100μM) and PBS, respectively. The expression of extracellular matrix (alpha-1type I collagen and fibronectin-1), TGF-P signaling pathway-related molecules (TGF-J31, TGF-βR1, TGF-βR2, Smad2, Smad3and phosphorylated Smad3) and COX-2in bulbar conjunctival tissues and CFs were detected by Western blot (WB) and quantitative real-time RT-PCR (qRT-PCR). The pathological changes of bulbar conjunctival tissue of rats were examined by haematoxylin-eosin (H&E), Van Gieson’s, periodic acid-Schiff (PAS) and immunohistochemical staining.Results:Rats treated with0.01%BAC exhibited a slight increase of the fibroblast density and more compact collagen deposition in the bulbar subepithelial connective tissues compared with rats treated with PBS. While there were no difference in inflammatory cell infiltration and alteration of epithelium and goblet cells in conjunctival tissues between the two groups. WB and qRT-PCR analysis showed that the expression of extracellular matrix (ECM), TGF-β signaling pathway-related molecules and COX-2were markedly increased in the bulbar conjunctival tissues of rats exposed to0.01%BAC and in CFs exposed to0.00005%and0.000075%BAC. In CFs, BAC-induced ECM expression was obviously decreased by LY2157299, while the BAC-induced activation of TGF-β1/Smad3signaling pathway was greatly attenuated by NS-398.Conclusion:BAC-induced subconjunctival fibrosis is a consequence of excessive ECM expression of CFs through COX-2-mediated activation of TGF-β1/Smad3Signaling Pathway.