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섬유주세포에서 황화수소가 산화스트레스와 세포노화에 미치는 영향

Effect of Hydrogen Sulfide on the Oxidative Stress and Senescence in Trabecular Meshwork Cells

김윤영|김재우|박현진
대한안과학회 (The Korean Ophthalmological Society)|2020.04|대한안과학회지 (Journal of The Korean Ophthalmological Society)|제61권 제4호|pp. 400-406

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키워드

Oxidative stressReactive oxygen speciesSenescenceHydrogen sulfideTrabecular meshwork
주제분류안과학

초록

목적: 섬유주세포에서 황화수소(hydrogen sulfide, H2S)가 산화스트레스와 세포노화에 미치는 영향을 알아보고자 하였다. 대상과 방법: 섬유주세포를 200 μM 과산화수소로 산화스트레스를 유발한 다음 황화수소(sodium hydrogen sulfide, NaHS) 0, 50, 100 μM에 24시간 노출시킨 후 MTT assay로 세포의 생존율을 측정하였고, 세포노화의 정도를 SA-β-galactosidase 염색을 시행하여 측정하였다. 반응성 산소종과 superoxide의 생성 정도를 dichlorofluorescein assay와 modified cytochrome c assay로 각각 측정하였다. Griess assay와 RT-PCR로 일산화질소의 생성과 endothelial nitric oxide synthase (eNOS) mRNA의 발현을 각각 측정하였다. 결과: 200 μM 과산화수소는 세포의 생존에 유의한 영향을 미치지 않았다. 50, 100 μM NaHS는 과산화질소에 노출된 대조군에 비하여 세포의 노화를 유의하게 감소시켰다. 과산화수소에 노출된 대조군에 비하여 50, 100 μM NaHS는 반응성 산소종의 생성을 감소시켰고100 μM NaHS는 superoxide의 생성을 저하시켰다. 100 μM NaHS는 일산화질소의 생성과 eNOS mRNA의 발현을 각각 유의하게 증가시켰다. 결론: 과산화수소로 유발된 산화스트레스에 대해 NaHS는 자유유리기의 생성을 감소시키면서 세포의 노화를 억제하였다. 따라서 황화질소는 산화스트레스를 감소시켜 섬유주세포의 노화를 감소시키는 작용을 나타낼 것으로 생각된다.

Abstract

Purpose: To investigate the effects of hydrogen sulfide (H2S) on the senescence and oxidative stress of cultured human trabecular meshwork cells (HTMCs). Methods: After inducing oxidative stress with 200 μM hydrogen peroxide (H2O2), primary cultured HTMCs were exposed to 0, 50, and 100 μM sodium hydrogen sulfide (NaHS) for 24 hours. Cell survival and senescence were measured using an MTT assay and SA-β-galactosidase staining, respectively. Generation of reactive oxygen species (ROS) and superoxide were measured using the dichlorofluorescein assay and modified cytochrome c assay. Production of nitric oxide (NO) and expression of eNOS mRNA were assessed using the Griess assay and reverse polymerase chain reaction. Results: Cell survival was not affected by 200 μM H2O2. Exposure to 50 and 100 μM NaHS significantly decreased cellular senescence, compared with the H2O2-exposed control. Exposure to 50 and 100 μM NaHS decreased the generation of ROS, and exposure to 100 μM NaHS decreased the generation of superoxide. Treatment with 100 μM NaHS increased the production of NO and expression of eNOS mRNA, respectively. Conclusions: NaHS decreased the cellular senescence associated with the decreased generation of free radicals. Thus, H2S could inhibit the senescence of HTMCs by reducing oxidative stress.

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