[논문]미세먼지와 산화적 스트레스에 의한 인간 폐 상피 A549 세포에의 ROS 의존적 자가포식 유도

박범수; 김다혜; 황보현; 이혜숙; 홍수현; 정재훈; 최영현

doi:10.5352/jls.2022.32.4.310

미세먼지와 산화적 스트레스에 의한 인간 폐 상피 A549 세포에의 ROS 의존적 자가포식 유도
The Induction of ROS-dependent Autophagy by Particulate Matter 2.5 and Hydrogen Peroxide in Human Lung Epithelial A549 Cells 원문보기

생명과학회지 = Journal of life science, v.32 no.4, 2022년, pp.310 - 317

박범수 (동의대학교 항노화연구소) , 김다혜 (동의대학교 항노화연구소) , 황보현 (동의대학교 항노화연구소) , 이혜숙 (부산대학교 의과대학 융합의학교실) , 홍수현 (동의대학교 항노화연구소) , 정재훈 (부산대학교 분자생물학과) , 최영현 (동의대학교 항노화연구소)

초록
AI-Helper

최근 인체에 유해한 요인으로 대기오염의 주성분인 미세먼지에 대한 관심이 증가하고 있다. 특히 직경이 2.5 ㎛ 미만인 PM_2.5는 인간의 폐 상피세포에서 자가포식을 동반한 산화적 스트레스를 유발하는 것으로 알려져 있다. 그러나 PM_2.5가 산화적 스트레스 하에서 자가포식을 증가시키는지와 이 과정이 ROS 의존적인지에 대한 연구는 충분하지 않은 실정이다. 본 연구에서는 PM_2.5가 인간 폐 상피 A594 세포에서 ROS 생성을 통해 자가포식을 촉진하는지를 조사하였다. 우리의 결과에 의하면, PM_2.5와 H₂O₂를 함께 처리한 세포에서는 각각이 단독 처리된 세포에 비하여 세포 생존력이 유의적으로 감소하였으며, 이는 전체 및 미토콘드리아 ROS 생성의 증가와 관련이 있었다. 또한, PM_2.5와 H₂O₂의 동시 처리는 Cyto-ID 염색을 통해 확인된 바와 같이 자가포식 유도의 증가와 LC3, p62 및 beclin 1과 같은 자가포식 바이오 마커 단백질의 발현을 증가시켰다. 그러나 NAC의 전처리에 의하여 ROS의 생성을 인위적으로 차단하였을 경우, PM_2.5와 H₂O₂의 동시 처리에 의한 세포 생존율의 감소와 자가포식 유도는 현저하게 억제되었다. 따라서, PM_2.5에 의해 유도된 ROS 생성이 A549 세포에서 자가포식 유도에 중요한 역할을 할 것으로 추측되며, 이는 PM_2.5에 의해 유도될 수 있는 폐 기능 손상이 산화적 스트레스 하에서 더욱 증가할 수 있음을 의미한다.

Abstract ▼ AI-Helper

Recently, interest in the harmful factors of particulate matter (PM), a major component of air pollution, has been increasing. In particular, PM2.5 with a diameter of less than 2.5 ㎛ is well known to induce oxidative stress accompanied by autophagy in human lung epithelial cells. However, studies on whether PM2.5 increases autophagy under oxidative stress and whether this process is reactive oxygen species (ROS)-dependent are insufficient. Therefore, in this study, we investigated whether PM2.5 promotes autophagy through the generation of ROS in human alveolar epithelial A594 cells. According to our results, cells co-treated with PM2.5 and hydrogen peroxide (H2O2) showed a lower cell viability than cells treated with each alone, which was associated with increased total and mitochondrial ROS production. The co-treatment of PM2.5 and H2O2 also increased autophagy induction, which was confirmed through Cyto-ID staining, and the expression of autophagy biomarker proteins increased. However, when ROS generation was artificially blocked by N-acetyl-L-cysteine pretreatment, the reduction in cell viability and induction of autophagy by PM2.5 and H2O2 co-treatment were markedly attenuated. Therefore, the present results suggest that PM2.5-induced ROS generation may play a critical role in autophagy induction in A549 cells.

주제어

표/그림 (4)

그림 Fig. 1. Effects of PM_2.5 and H₂O₂ on cell viability of A549 cells. Cells were treated with the indicated concentrations of PM_2.5 and/or H₂O₂ for 48 hr. (A-C) Cell viability was measured by the MTT assay. Data were presented as mean ± SD. All experiments were performed in triplicate (*p<0.05, **p<0.01 and ***p<0.001 vs untreated cells; ^##p<0.01 and ^###p<0.001 vs 25 μg/ml and 50 μg/ml PM_2.5-treated cells, respectively). (D) Cells were observed under an inverted phase contrast microscope (x200) and representative images were presented.
그림 Fig. 2. Generation of ROS and induction of autophagy by PM_2.5 and H₂O₂ in A549 cells. Cells were treated with the indicated concentrations of PM_2.5 and/or H₂O₂ for 1 hr (A and B) or 48 hr (C). (A and B) Representative fluorescence images of cells stained with DCF-DA (A) and MitoSOX (B) visualized by a fluorescence microscope. (C) Representative fluorescence images of cells showing autophagic vesicles stained with Cyto-ID green visualized by a fluorescence microscope. DAPI was used to counterstain the nuclei (blue).
그림 Fig. 3. Effects of PM_2.5 and H₂O₂ on the expression of autophagy-regulatory proteins in A549 cells. Cells were treated with the indicated concentrations of PM_2.5 and/or H₂O₂ for 48 hr. (A) Total proteins were isolated from cells and Western blot analysis was performed using the indicated antibodies. Actin was used as a loading control. (B) Density quantification was performed and the relative expression ratio for actin was calculated. Data were presented as mean ± SD (***p<0.001 vs 25 μg/ml and 50 μg/ml PM_2.5-treated cells).
그림 Fig. 4. Effects of ROS on the inhibition of cell viability and induction of autophagy by PM_2.5 and H₂O₂ in A549 cells. Cells were cultured in media containing PM_2.5 and H₂O₂ for 48 hr, or pre-treated with 10 mM NAC for 1 hr, followed by exposure to PM_2.5 and H₂O₂ for an additional 48 hr. (A) Cell viability was measured by the MTT assay. (B) Cells were observed under an inverted phase contrast microscope (x200) and representative images were presented. (C) Total proteins were isolated from cells and Western blot analysis was performed using the indicated antibodies. Actin was used as a loading control. (D) Density quantification was performed and the relative expression ratio for actin was calculated. (A and D) Data were presented as mean ± SD (*p<0.05 and ***p<0.001 vs untreated cells; ^##p<0.01 and ^###p<0.001 vs PM_2.5 and H₂O₂-treated cells).

AI 본문요약
AI-Helper

데이터처리

실험 결과의 유의성을 판정하기 위하여 GraphPad Prism® version 5.0 (Graphpad Inc., San Diego, CA, USA)의 one-way ANOVA를 사용하였으며, Tukey's test로 사후 검정하여 p< 0.05 값을 유의한 값으로 처리하였다.

이론/모형

미토콘드리아 ROS의 생성 정도를 관찰하기 위해서는 형광 probe인 MitoSOX를 사용하였다. 이를 위하여 상기와 동일 조건에서 배양된 세포를 PBS로 수세 후 5 μM의 MitoSOX 용액 (Thermo Fisher Scientific, Waltham, MA, USA)을 처리하여 37℃에서 20분간 반응시켰다.

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저자의 다른 논문 :

표제어: PCR

동의어: Packet Collision Rate

용어 설명 출처 목록 (6)

용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

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