Miao Ying, Sun Qian, Wen Qian, Qiu Yue, Ge Yuan, Yu Miao-Miao, Fan Ting-Jun
Key Laboratory for Corneal Tissue Engineering, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, Shandong Province, China.
Int J Ophthalmol. 2014 Feb 18;7(1):14-21. doi: 10.3980/j.issn.2222-3959.2014.01.03. eCollection 2014.
To demonstrate the cytotoxic effect of betaxolol and its underlying mechanism on human corneal endothelial cells (HCE cells) in vitro and cat corneal endothelial cells (CCE cells) in vivo, providing experimental basis for safety anti-glaucoma drug usage in clinic of ophthalmology.
In vivo and in vitro experiments were conducted to explore whether and how betaxolol participates in corneal endothelial cell injury. The in vitro morphology, growth status, plasma membrane permeability, DNA fragmentation, and ultrastructure of HCE cells treated with 0.021875-0.28g/L betaxolol were examined by light microscope, 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay, acridine orange (AO)/ethidium bromide (EB) double-fluorescent staining, DNA agarose gel electrophoresis, and transmission electron microscope (TEM). The in vivo density, morphology, and ultrastructure of CCE cells, corneal thickness, and eye pressure of cat eyes treated with 0.28g/L betaxolol were investigated by specular microscopy, applanation tonometer, alizarin red staining, scanning electron microscope (SEM), and TEM.
Exposure to betaxolol at doses from 0.0875g/L to 2.8g/L induced morphological and ultrastructural changes of in vitro cultured HCE cells such as cytoplasmic vacuolation, cellular shrinkage, structural disorganization, chromatin condensation, and apoptotic body appearance. Simultaneously, betaxolol elevated plasma membrane permeability and induced DNA fragmentation of these cells in a dose-dependent manner in AO/EB staining. Furthermore, betaxolol at a dose of 2.8g/L also induced decrease of density of CCE cells in vivo, and non-hexagonal and shrunk apoptotic cells were also found in betaxolol-treated cat corneal endothelia.
Betaxolol has significant cytotoxicity on HCE cells in vitro by inducing apoptosis of these cells, and induced apoptosis of CCE cells in vivo as well. The findings help provide new insight into the apoptosis-inducing effect of anti-glaucoma drugs in eye clinic.
探讨倍他洛尔对体外培养的人角膜内皮细胞（HCE细胞）及体内猫角膜内皮细胞（CCE细胞）的细胞毒性作用及其潜在机制，为眼科临床安全使用抗青光眼药物提供实验依据。
通过体内和体外实验，探究倍他洛尔是否以及如何参与角膜内皮细胞损伤。采用光学显微镜、3-(4,5)-二甲基噻唑-2,5-二苯基四氮唑溴盐（MTT）法、吖啶橙（AO）/溴化乙锭（EB）双荧光染色、DNA琼脂糖凝胶电泳和透射电子显微镜（TEM），检测0.021875-0.28g/L倍他洛尔处理的HCE细胞的体外形态、生长状态、质膜通透性、DNA片段化及超微结构。采用镜面显微镜、压平眼压计、茜素红染色、扫描电子显微镜（SEM）和TEM，研究0.28g/L倍他洛尔处理的猫眼的CCE细胞体内密度、形态和超微结构、角膜厚度及眼压。
0.0875g/L至2.8g/L剂量的倍他洛尔可诱导体外培养的HCE细胞发生形态和超微结构变化，如细胞质空泡化、细胞收缩、结构紊乱、染色质凝聚及凋亡小体出现。同时，在AO/EB染色中，倍他洛尔可剂量依赖性地提高这些细胞的质膜通透性并诱导DNA片段化。此外，2.8g/L剂量的倍他洛尔还可导致体内CCE细胞密度降低，在倍他洛尔处理的猫角膜内皮中还发现了非六边形且收缩的凋亡细胞。
倍他洛尔在体外可通过诱导HCE细胞凋亡对其产生显著细胞毒性，在体内也可诱导CCE细胞凋亡。这些发现有助于为眼科临床抗青光眼药物的凋亡诱导作用提供新的见解。
