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벤잘코니움의 장기 노출이 섬유주세포의 노화에 미치는 영향

Effect of Chronic Benzalkonium Chloride Exposure on Senescence in Trabecular Meshwork Cells

이수진|이규원|김재찬|박지혜
대한안과학회 (The Korean Ophthalmological Society)|2019.01|대한안과학회지 (Journal of The Korean Ophthalmological Society)|제60권 제1호|pp. 55-61

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SenescenceTrabecular meshworkBenzalkonium chloride
주제분류안과학

초록

목적: 섬유주를 저농도의 벤잘코니움(BAK)에 장기간 노출하였을 때, 섬유주세포에 어떤 변화가 유발되는지 알아보고, 그와 관련된 경로를 분석하였다. 대상과 방법: 섬유주세포에 0.0005%, 0.00075%, 0.001%, 0.0025% BAK를 10분간 처리한 후, 새로운 배양액에 24시간 배양하는 과정을 3회 반복하였다. MTT = 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assay를 통해 세포생존율을 측정하여 세포사멸이 일어나지 않는 농도를 결정하였다. BAK를 처리한 후 섬유주세포를 노화특이적인 Senescence-associated (SA)-β-gal 염색을 통해 푸르게 염색된 세포 수를 음성 대조군과 비교하였고, Western blot를 통해 세포주기 조절 관련된 표지자들의 발현 정도를 확인하였다. 결과: 섬유주세포의 생존에 영향을 미치지 않는 BAK 농도는 0.0005%와 0.00075%로 확인하였고, 이 농도들을 실험에 사용하였다. 섬유주세포에 SA-β-gal 염색을 하였을 때 세포노화는 음성 대조군에 비해 0.0005% BAK 처리군에서 28% (± 2.08), 0.00075% BAK 처리군에서 37% (± 2.08)로 유의하게 증가하였다(p<0.05). BAK에 의한 세포주기 조절인자의 분자적 분석을 위해 시행한 Western blot에서 cyclin E/CDK2의 발현이 억제되었고, 그 상위기전 조절인자인 p53와 p21의 발현이 증가하였다. 결론: 저농도 BAK의 만성적인 처리는 세포주기 억제를 통한 세포의 조기노화를 유발할 수 있을 것이다. 노화된 섬유주는 방수유출기능이 저하되어 녹내장을 악화시킬 수 있기 때문에, 녹내장 환자에게 BAK가 포함된 안약을 장기적으로 사용할 시 이를 고려해야 할 것이다.

Abstract

Purpose: To determine the possible effects of chronic exposure of low dose benzalkonium chloride (BAK) on trabecular meshwork cells, and to characterize the pathways involved in the effects. Methods: Trabecular meshwork cells were treated with 0.0005%, 0.00075%, 0.001%, and 0.0025% BAK for 10 minutes; then, the cells were transferred to a new medium for 24 hours. This process was repeated three times. Cell survival was assessed using the MTT assay to determine the non-apoptotic BAK concentration. Senescence-associated (SA)-β-gal staining was performed to compare quantitatively the cellular senescence of BAK-treated cells with the control group. Cells treated with BAK were analyzed by western blot to determine whether the expressions of cell cycle regulators were affected. Results: Two concentrations (0.0005% and 0.00075%) showed persistent cell viability and were chosen for further experiments. After SA-β-gal staining, cells treated with 0.0005% and 0.00075% BAK showed 28% (± 2.08), 37% (± 2.08) increases in cellular senescence expression, respectively, when compared with control cells (p < 0.05). To identify the molecular pathways involved in cell cycle arrest via BAK, western blot analysis was performed on trabecular meshwork cells, resulting in decreased expressions of cyclin E/CDK2, and increased expressions of the upper stream control molecules, p53 and p21. Conclusions: Chronic exposure to low dose BAK accelerated cell senescence through cell cycle arrest. Because senescent cells of the trabecular meshwork can inhibit its outflow pathway function and ultimately worsen the glaucomatous process, long-term usage of topical glaucoma medications containing BAK should be conducted with caution.

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