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섬유주세포에서 산화스트레스에 대한 Rho kinase 저해제의 세포 보호 효과에 관한 연구

Cytoprotective Effect of Rho Kinase Inhibitors against Oxidative Stress in Trabecular Meshwork Cells

백민주|손명서|김재우
대한안과학회 (The Korean Ophthalmological Society)|2020.04|대한안과학회지 (Journal of The Korean Ophthalmological Society)|제61권 제4호|pp. 394-399

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ApoptosisOxidative stressRho-associated kinase inhibitorTrabecular meshwork
주제분류안과학

초록

목적: 섬유주 유출 촉진 약제인 Rho kinase (ROCK) 저해제가 산화스트레스에 대한 섬유주세포 보호 효과를 비교해 보고자 하였다. 대상과 방법: 사람의 섬유주세포를 일차배양한 후 200 μM 과산화수소에 30분간 노출시킨 후 ROCK 저해제인 Y-27632, Ripasudil, fasudil에 각각 10, 20 μM씩 24시간 노출시켰다. Trypan blue를 이용하여 세포의 생존을 조사하였고, MTT assay를 이용하여 대사량을 측정하였다. H2DCFDA assay를 이용하여 반응성산소종의 생성량을 측정하였고, Annexin-propidium iodide 이중염색을 이용한 유세포분석으로 세포고사의 정도를 측정하였다. 결과: 섬유주 세포에서 과산화수소에 의한 산화스트레스에 대해 Y-27632는 반응성 산소종의 생성을 감소시켰다. Ripasudil과 fasudil 은 세포대사를 증가시켰으며 세포고사를 억제하였는데 그 중 fasudil이 더 유의한 세포보호 효과를 나타냈다. 결론: 섬유주 유출 촉진 약제인 ROCK 저해제는 산화스트레스에 의한 세포고사를 감소시켰으며 fasudil이 더 유의한 세포보호 효과를 나타냈다. 따라서 ROCK 저해제는 안압하강 효과뿐만 아니라 섬유주세포에서 산화스트레스에 대한 세포보호 효과도 나타낼 가능성이 있다.

Abstract

Purpose: We evaluated the protective effect of trabecular outflow drugs, Rho kinase (ROCK) inhibitors against oxidative stress in trabecular meshwork cells. Methods: Primary-cultured human trabecular meshwork cells (HTMCs) were exposed to ROCK inhibitors at 10 and 20 μM Y-27632, ripasudil or fasudil for 24 hours, after pretreatment with 200 μM hydrogen peroxide for 30 min. The cell viabilities and metabolic activities were assessed using the Trypan Blue dye exclusion test and MTT assay, respectively. Reactive oxygen species (ROS) production was measured using the H2DCFDA assay, and the degree of apoptosis was measured with flow cytometry using annexin-propidium iodide double staining. Results: In HTMCs, Y-27632 suppressed ROS production. Ripasudil and fasudil increased the metabolic activities and decreased the degrees of apoptosis. Fasudil showed the most cytoprotective effects among the three ROCK inhibitors tested. Conclusions: Against oxidative stress, ROCK inhibitors decreased apoptosis accompanied by decreased ROS production, especially fasudil. ROCK inhibitors may therefore have cytoprotective properties in addition to increasing trabecular outflow.

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