Department of Ophthalmology, The First Affiliated Hospital, Sun-Yat-sen University, Guangzhou, China.
Department of Otolaryngology, The First Affiliated Hospital, Sun-Yat-sen University, Guangzhou, China.
Invest Ophthalmol Vis Sci. 2021 Jul 1;62(9):7. doi: 10.1167/iovs.62.9.7.
To investigate the role of elastase in corneal epithelial barrier dysfunction caused by the exoproteins secreted by Pseudomonas aeruginosa.
Exoproteins obtained from Pseudomonas aeruginosa culture supernatant were analyzed by shotgun proteomics approach. In vitro multilayered rabbit corneal epithelial barrier model prepared by air-liquid interface technique (CECs-ALI) were treated with 2 µg/ml exoproteins and/or 8 mM elastase inhibitor. Then the epithelial barrier function was evaluated by transepithelial electrical resistance (TEER) assay and tight junction proteins immunofluorescence. Cell viability and the apoptosis rate were examined by CCK8 assay and flow cytometry. TNF-α, IL-6, IL-8, and IL-1β levels were measured by ELISA. Mice cornea treated with exoproteins and/or elastase inhibitor were evaluated in vivo and in vitro.
Elastase (24.2%) is one of the major components of exoproteins. After 2 µg/ml exoproteins were applied to CECs-ALI for two hours, TEER decreased from 323.2 ± 2.7 to 104 ± 6.8 Ω/cm2 (P < 0.001). The immunofluorescence results showed a distinct separation in tight junction and significant degradation of ZO-1 and occludin (P < 0.05). Elastase inhibitor (8 mM) alleviated the decrease in TEER value (234 ± 6.8 Ω cm2) induced by exoproteins. Inhibition of elastase decreased the apoptosis rate of CECs treated with exoproteins from 30.2 ± 3.8% to 7.26 ± 1.3% and the levels of inflammatory factors (P < 0.05). Mice corneal epithelium defect could be induced by exoproteins and protected by elastase inhibitor.
Elastase plays a critical role in corneal epithelial barrier dysfunction caused by Pseudomonas aeruginosa exoproteins via damaging tight junctions. The inhibition of elastase could protect the corneal epithelial barrier via reducing virulence and inflammation.
研究弹性蛋白酶在铜绿假单胞菌外蛋白引起的角膜上皮屏障功能障碍中的作用。
采用鸟枪法蛋白质组学方法分析铜绿假单胞菌培养上清液中的外蛋白。采用气液界面技术（CECs-ALI）制备体外多层兔角膜上皮屏障模型，用 2μg/ml 外蛋白和/或 8mM 弹性蛋白酶抑制剂处理，然后通过跨上皮电阻（TEER）测定和紧密连接蛋白免疫荧光法评估上皮屏障功能。通过 CCK8 测定和流式细胞术检测细胞活力和凋亡率。通过 ELISA 测定 TNF-α、IL-6、IL-8 和 IL-1β 水平。体内和体外评价外蛋白和/或弹性蛋白酶抑制剂处理的小鼠角膜。
弹性蛋白酶（24.2%）是外蛋白的主要成分之一。外蛋白（2μg/ml）作用于 CECs-ALI 2 小时后，TEER 从 323.2±2.7 降至 104±6.8Ω/cm2（P<0.001）。免疫荧光结果显示，紧密连接明显分离，ZO-1 和 occludin 明显降解（P<0.05）。弹性蛋白酶抑制剂（8mM）可减轻外蛋白引起的 TEER 值（234±6.8Ωcm2）下降。抑制弹性蛋白酶可降低外蛋白处理的 CECs 凋亡率（从 30.2±3.8%降至 7.26±1.3%）和炎症因子水平（P<0.05）。外蛋白可诱导小鼠角膜上皮缺损，弹性蛋白酶抑制剂可保护。
弹性蛋白酶通过破坏紧密连接在铜绿假单胞菌外蛋白引起的角膜上皮屏障功能障碍中起关键作用。抑制弹性蛋白酶可通过降低毒力和炎症来保护角膜上皮屏障。
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