개자(芥子)가 인간 기관지 상피세포에서 미세먼지 유도 염증 및 DNA 손상에 미치는 영향

다국어 초록 (Multilingual Abstract)

ABSTRACT
Effects of ethanol extract of Brassica juncea Czern. et Cosson on particulate matter-induced inflammation and DNA damage in BEAS-2B human bronchial epithelial cells

Hong Seok Choi
Department of Korean Medicine,
Master of Korean Medicine
Graduate School
Kyung Hee University
Seoul, Korea
(Directed by Professor Hyuk-Sang Jung K.M.D., Ph.D.)

Background and Purposes: Particulate Matter(PM) is composed of various components such as polycyclic aromatic hydrocarbons, metal components, ionic components, and carbon components. As particulate matter is inhaled into the human body, it can pass through the bronchial tubes and bronchioles and reach the alveoli. PM acts on airway epithelial cells and macrophages to induce reactive oxidative stress and inflammatory responses. As a result, PM is known to increase the prevalence of asthma, pneumonia, influenza, and bronchitis. Brassica Juncea(BJ), the dried seed of Brassica juncea(L.) Czern. et coss, has has traditionally been used in Eastern medicine for its alleviating phlegm and cough. Given the promising results of BJ extract in treating asthma and BJ’s known antibechic and apophlegmatic properties, this study sought to investigate the potential effectiveness of BJ ethanol extract(BJC) on particulate matter-induced inflammation and DNA damage in BEAS-2B human bronchial epithelial cells.

Materials and methods: In the in vitro study, BJC was divided into three groups, with concentrations of 100, 200, and 400 μg/mL, and added to TNF-α/IL-4+PM stimulated BEAS-2B cells to assess levels of ROS, IL-6, MCP-1, GM-CSF, MAPKs and LC3 II/LC3 I. ROS was measured by fluorescence microscopy after DCF-DA treatment. IL-6, MCP-1, GM-CSF was investigated by ELISA. MAPKs and LC3 II/LC3 I were examined using Western blot.

Results: In the in vitro model, It was confirmed that the ROS production decreased in dependent to the concentration of BJC. Among the inflammatory cytokines in BEAS-2B cells, GM-CSF level showed a significant decrease in dependent to BJC concentration (BJC 100μg/mL : p<0.001, BJC 200μg/mL : p<0.001, BJC 400μg/mL : p<0.001). Notably, there was a significant reduction in IL-6 level (BJC 200μg/mL : p<0.01, BJC 400μg/mL : p<0.001). Furthermore, phosphorylated MAPKs, including ERK (BJC 100μg/mL : p<0.001, BJC 200μg/mL : p<0.05) and p38 (BJC 400μg/mL : p<0.5) were also decreased. In addition, there was a significant reduction in the level of LC3 II/LC3 I (BJC 200μg/mL : p<0.05, BJC 400μg/mL : p<0.05), in dependent to
BJC concentration.

Conclusions: This study demonstrates that Brassica juncea Czern. et cosson (BJ) significantly alleviated the reactive oxidative stress and inflammatory responses on Particulate Matter induced inflammation in BEAS-2B human bronchial epithelial cell.

Keywords: Brassica juncea, Particulate Matter, Asthma, Epithelial cell, anti-inflammatory, ROS, BEAS-2B cell, MAPKs

목차 (Table of Contents)

• I. 서론 1
• II. 재료 및 방법 3
• 1. 시약 및 재료 3
• 2. 개자 에탄올 추출물(ethanol extract of Brassica juncea Czern. et Cosson, BJC) 제조 3
• 3. 세포 배양 3
• 4. 세포 독성 평가 4
• 5. ELISA 분석 4
• 6. 세포 내 ROS 수준 측정 4
• 7. Western blot 분석 4
• 8. 통계처리 5
• III. 실험 결과 6
• 1. BJC가 기관지 상피세포의 세포 생존율에 미치는 영향 6
• 2. BJC가 기관지 상피세포의 ROS 생성에 미치는 영향 8
• 3. BJC가 기관지 상피세포의 염증성 사이토카인 분비에 미치는 영향 10
• 4. BJC가 기관지 상피세포에서 MAPK 신호 경로에 미치는 영향 12
• 5. BJC가 기관지 상피세포에서 LC3 II/LC3 I 발현에 미치는 영향 14
• IV. 고 찰 16
• V. 결 론 19
• 참 고 문 헌 20
• ABSTRACT 24