[논문]인간 망막 색소상피 세포에서 산화적 스트레스에 대한 천연 및 합성 화합물들의 세포 보호 효과 비교

김다혜; 김정환; 박세광; 정지원; 김미영; 남수완; 이혜숙; 최영현

doi:10.5352/jls.2021.31.2.126

인간 망막 색소상피 세포에서 산화적 스트레스에 대한 천연 및 합성 화합물들의 세포 보호 효과 비교
Comparison of the Cytoprotective Effects of Several Natural and Synthetic Compounds against Oxidative Stress in Human Retinal Pigment Epithelial Cells 원문보기

생명과학회지 = Journal of life science, v.31 no.2, 2021년, pp.126 - 136

김다혜 (동의대학교 스마트 바이오헬스학과) , 김정환 ((주)비지엔케어 연구개발전담부서) , 박세광 ((주)비지엔케어 연구개발전담부서) , 정지원 (BGN밝은눈안과 병원) , 김미영 ((주)비지엔케어 연구개발전담부서) , 남수완 (동의대학교 의생명공학과) , 이혜숙 (동의대학교 항노화연구소) , 최영현 (동의대학교 항노화연구소)

초록
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산화적 스트레스에 의한 망막 색소상피 세포의 손상 및 퇴화는 시력 소실을 포함한 다양한 망막질환의 원인으로 알려져 있다. 본 연구에서는 천연물 유래 14종 단일 성분과 합성 화합물 2종을 대상으로 망막질환 개선 가능성을 평가하기 위해, 사람 유래 망막 색소상피 세포를 대상으로 산화적 스트레스에 의한 세포 손상에 대한 보호 효능을 평가하였다. 그 결과, 16종의 후보물질 중 H2O2에 의해 유발된 세포독성을 개선시키는 효능을 보인 5종(auranofin, FK-509, hemistepsin A, honokiol 및 spermidine)을 선별하였다. 이를 기반으로 H2O2에 의한 미토콘드리아 기능 손상 및 활성 저하 억제능을 평가하였으며, auranofin을 제외한 4종에서 유의한 세포 보호 효능이 관찰되었다. 더불어 H2O2에 의해 유도된 DNA 손상에 미치는 영향을 조사한 결과, FK-506, honokiol과 spermidine이 DNA 손상을 개선시켰음을 확인하였다. 그러나 5종의 후보물질이 활성 산소종의 생성에는 뚜렷한 억제 효능을 나타내지 않았으며, 이는 5종의 후보 물질이 ROS-비의존적 기전을 통해 세포 보호효과를 나타낼 것으로 사료된다. 이상의 결과를 바탕으로, 조사 대상 후보물질 중에서 spermidine이 산화적 스트레스로부터 망막 색소상피 세포의 보호 효능이 가장 우수한 천연물 유래 단일 물질임을 규명하였으며, 동시에 합성물질로는 FK-506이 망막 세포 보호 효능이 우수한 것으로 나타났다. 비록 작용 기전에 대한 추가 연구가 필요하겠지만, 본 연구 결과는 spermidine과 FK-506이 산화적 스트레스에 의한 망막질환의 위험을 억제할 가능성이 있음을 시사한다.

Abstract ▼ AI-Helper

Oxidative stress causes injury to and degeneration of retinal pigment epithelial (RPE) cells. It is involved in several retinal disorders and leads to vision loss. In the present study, we investigated the effect of 14 kinds of natural compounds and two kinds of synthetic compounds on oxidative stress-induced cellular damage in human PRE cell lines (ARPE-19). From among them, we selected five kinds of compounds, including auranofin, FK-509, hemistepsin A, honokiol, and spermidine, which have inhibitory effects against hydrogen peroxide (H2O2)-mediated cytotoxicity. In addition, we found that four kinds of compounds (excluding auranofin) have protective effects on H2O2-induced mitochondrial dysfunction. Furthermore, the expression of phosphorylation of histone H2AX, a sensitive marker of DNA damage, was markedly up-regulated by H2O2, whereas it was notably down-regulated by FK-506, honokiol, and spermidine treatment. Meanwhile, five kinds of candidate compounds had no effect on H2O2-induced intracellular reactive oxygen species (ROS) levels, suggesting that the five candidate compounds have protective effects on oxidative stress-induced cellular damage through the ROS-independent pathway. Taken together, according to the results of H2O2-mediated cellular damage―such as cytotoxicity, apoptosis, mitochondrial dysfunction, and DNA damage―spermidine and FK-506 are the natural and synthetic compounds with the most protective effects against oxidative stress in RPE. Although further studies on the identification of the mechanism responsible are required, the results of the present study suggest the possibility of using spermidine and FK-506 to suppress the risk of retinal disorders.

주제어

표/그림 (7)

그림 Fig. 1. Structure of 16 kinds of candidate compounds.
그림 Fig. 2. Effect of 16 kinds of candidate compounds on the cell viability in ARPE-19 cells. ARPE-19 cells were treated with the indicated concentration of candidate compounds for 24 hr. And then cell viability was measured by an MTT assay. Data are expressed as the mean ± SD (n=6). The statistical analyses were conducted using analysis of variance (ANOVA-Tukey's post-hoc test) between groups. *p<0.05, **p<0.01 and ***p<0.001 when compared to control.
그림 Fig. 3. Effect of 16 kinds of candidate compounds on the cell viability in H₂O₂-stimulated ARPE-19 cells. ARPE-19 cells were pre-treated with the indicated concentration of candidate compounds for 3 hr, and then treated with 300 μM H₂O₂ for 12 hr. Subsequently cell viability was measured by an MTT assay. Data are expressed as the mean ± SD (n=6). The statistical analyses were conducted using analysis of variance (ANOVA-Tukey's post-hoc test) between groups. ***p<0.001 when compared to control. #p<0.05, ##p<0.01 and ###p<0.001 when compared to H₂O₂-treated cells.
그림 Fig. 4. Effect of 5 kinds of candidate compounds on the intracellular ROS production in H₂O₂-stimualted ARPE-19 cells. ARPE-19 cells were pre-treated with candidate compounds for 3 hr, and then treated with 300 μM H₂O₂ for 30 min. The cells were incubated at 37℃ in the dark for 20 min with a culture medium containing 10 μM DCF-DA to monitor ROS production. (A) The degree of ROS production was measured with a flow cytometer. (B) Quantitative data of DCF-DA intensity of 5 kinds of candidate compounds-treated cells with or without 300 μM H₂O₂. Data are expressed as the mean ± SD (n=3). The statistical analyses were conducted using analysis of variance (ANOVA-Tukey's post-hoc test) between groups. ***p<0.001 when compared to control.
그림 Fig. 5. Effect of 5 kinds of candidate compounds on H₂O₂-induced apoptosis in ARPE-19 cells. The cells were treated with candidate compounds for 3 hr, and then incubated with or without 300 μM H₂O₂ for 24 hr. (A) Flow cytometry analysis was performed by annexin V and PI staining. Representative histograms are presented. (B) The percentages of apoptotic cells were determined by expressing the numbers of annexin V+ cells as percentages of all cells. Data are expressed as the mean ± SD (n=3). The statistical analyses were conducted using analysis of variance (ANOVA-Tukey's post-hoc test) between groups. *p<0.05, **p<0.01 and ***p<0.001 when compared to control. ###p<0.001 when compared to H₂O₂-treated cells.
표 Table 1. Summary of the effect of candidates on H₂O₂-induced cellular damages in ARPE-19 cells
그림 Fig. 6. Effect of 5 kinds of candidate compounds on mitochondrial and DNA damages in H₂O₂-stimulated ARPE-19 cells. The cells were pretreated with candidate compounds for 3 hr, and then incubated with or without 300 μM H₂O₂ for 24 hr. (A) The cells were stained with JC-1 dye, and were then analyzed by a flow cytometer to evaluate the changes in MMP. Representative photographs are presented. (B) The percentages of monomeric cells were determined by expressing the numbers of JC-1 green as percentages of all cells. (C) Cells were stained with 50 nM MitoTracker® Red (scale bar; 75 μm). (D) Cell were stained with γH2AX antibody and DAPI was used to counterstain the nuclei (blue), then the cells were visualized using a fluorescence microscope. Representative fluorescence microscopy images, including a merged image, are shown (γH2AX; red, DAPI; blue, scale bar; 25 μm).

AI 본문요약
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문제 정의

따라서, 본 연구에서는 천연물에서 유래한 14종의 유효 단일 성분(coptisine chloride, cordycepin, diallyl trisulfide (DATS), fucoidan, genistein, glutathione, hemistepsin A, honokiol, morin, nargenicin A1, quercetin, schisandrin A, spermidine 및 sulforaphane)과 합성 화합물 2종(auranofin 및 FK-506)의 망막질환 개선 가능성을 평가하기 위해, 사람 유래 망막 색소상피 세포(ARPE-19)를 대상으로 산화적 스트레스 조건 하에서 유발된 세포 손상에 대한 보호 효능을 평가하고자 하였다.

데이터처리

GraphPad Prism® version 5.0 (Graphpad Inc., San Diego, CA, USA)의 one-way ANOVA를 사용하여 통계분석을 실시하였으며, Tukey's test로 사후 검정하여 p<0.05 값을 유의한 값으로 보고 통계 처리하였다.

성능/효과

2의 결과에서 알 수 있듯이, auranofin, coptisine, fucoidan, morin, nargenicin A1 및 quercetin은 처리 농도 1 μM까지는 세포독성을 나타내지 않았으나 그 이상의 농도에서 유의적인 세포 생존율 감소를 보였다

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