(The) effect of Sophora flavescens Aiton on TNF-α/IL-4-stimulated BEAS-2B cells treated with particulate matter (PM), and a PM-exacerbated asthma animal model

국문 초록 (Abstract)

연구배경 및 목적: 미세먼지는 기도와 폐에 침투하여 염증을 유발하고, 전신적인 질병 악화에 영향을 미쳐 뇌졸중 및 심혈관 질환을 유발함으로써 사망에 이르게 하는 원인으로 대두되고 있다. 특히, 기도와 폐는 외부 물질에 대한 일차적인 방어선으로 작용하기 때문에 미세먼지로 인한 호흡기 질환 악화는 그 중요성이 더욱 부각되고 있다. 이에 미세먼지가 천식을 악화시키는 기전 및 기관지 세포에서 염증 매개 핵심지표에 대한 연구가 매우 중요하다. Sophora flavescens Aiton (SFA, 苦蔘, 고삼)는 전통적으로 열을 내리고 습을 건조시키는 효능이 있으며, 최근 연구에 따르면 항천식, 항염증, 항종양 효능이 보고되었다. 이 논문의 목적은 미세먼지에 의해 특이적으로 상승 또는 저하되는 지표들에 대한 분석을 토대로 SFA의 효과에 대한 기전을 규명하는데 있다.
방법: 기관지 상피세포인 BEAS-2B 세포에 tumor necrosis factor (TNF)-α/interleukin (IL)-4 (TI) 및 미세먼지를 동시에 자극한 후, SFA가 염증성 사이토카인 수준과 DNA 손상 정도에 미치는 영향은 ELISA와 형광 염색을 통해 분석하였다. 또한, MAPK 및 AKT/mTOR 신호전달경로와 LC3 Ⅱ 단백질 수준은 western blot으로 측정하였다. 미세먼지 악화 천식 동물 모델은 mouse에 ovalbumin (OVA)과 미세먼지를 노출시켜 확립하였으며, SFA는 10일 간 경구투여 하였다. 위 모델에서 혈청 IgE 수준과 폐 조직 내 사이토카인 수준은 ELISA를 통해 측정하였으며, 폐 조직 내 평활근 두께, PAS 및 LC3 II 양성 영역 수준은 H&E, PAS 염색 및 면역조직화학적 염색을 통해 분석하였다.
결과: 미세먼지는 TI로 자극한 BEAS-2B 세포에서 염증성 사이토카인인 IL-6, GM-CSF 및 MCP-1의 수준을 증가시켰으며, SFA는 이를 유의하게 억제시켰다. 또한, SFA는 DNA 손상을 감소시켰으며, JNK 및 AKT의 인산화 수준과 LC3 II 단백질 수준을 유의하게 감소시켰다. 미세먼지 악화 천식 동물 모델에서 SFA는 혈청 내 IgE와 OVA 특이적 IgE 수준을 유의하게 감소시켰으며, 폐 조직 내 IL-4, TNF-α 및 IFN-γ 수준을 유의하게 억제시켰다. 또한, 폐 조직 내 평활근 두께와 LC3 II 양성 영역을 유의하게 감소시켰다.
결론: 이러한 결과는 SFA가 MAPK와 자가포식 경로를 조절하고 조직병리학적 특징을 완화함으로써 미세먼지에 의해 유발된 천식 악화를 효과적으로 개선할 수 있음을 의미한다. 이는 SFA가 미세먼지 악화 천식 치료에 있어 안전하면서도 효과적인 약물로 활용될 수 있음을 시사한다.

다국어 초록 (Multilingual Abstract)

Introduction: Particulate matter (PM) is increasingly recognized as a cause of death due to its ability to enter the airways and lungs, trigger inflammation, and lead to systemic diseases such as stroke and cardiovascular disease. Particularly, the airways and lungs are the first line of defense against external agents, making the aggravation of respiratory diseases due to PM even more important. Therefore, it is very essential to study the mechanisms by which PM exacerbates asthma and the key markers of inflammation in bronchial cells. Sophora flavescens Aiton (SFA, 苦蔘, gosam) is traditionally used for its heat-clearing and damp-drying properties, and recent studies have reported anti-asthmatic, anti-inflammatory, and antitumor activities. The aim of this article is to identify the mechanisms behind the effects of SFA based on the analysis of indicators that are specifically elevated or degraded by PM.
Methods: BEAS-2B cells, bronchial epithelial cells, were co-stimulated with tumor necrosis factor (TNF)-α/interleukin (IL)-4 (TI) and PM. The effects of SFA on inflammatory cytokine levels were analyzed by ELISA and DNA damage was identified by fluorescence staining. In addition, MAPK and AKT/mTOR signaling pathways and LC3 Ⅱ protein levels were measured by western blot. An animal model of PM-exacerbated asthma was established by exposing mice to ovalbumin (OVA) and PM, and SFA was administered orally for 10 days. In the above models, serum IgE levels and cytokine levels in lung tissue were measured by ELISA. Smooth muscle thickness, PAS and LC3 II positive area levels in lung tissue were analyzed by H&E, PAS staining and immunohistochemistry staining.
Results: PM increased the levels of proinflammatory cytokines IL-6, GM-CSF, and MCP-1 in TI-stimulated BEAS-2B cells, which were significantly inhibited by SFA. Furthermore, SFA reduced DNA damage and significantly decreased phosphorylation levels of JNK, AKT and protein levels of LC3 II. In an animal model of PM-exacerbated asthma, SFA significantly reduced the levels of IgE and OVA-specific IgE in serum, and markedly inhibited the levels of IL-4, TNF-α, and IFN-γ in lung tissue. In addition, it significantly reduced smooth muscle thickness and LC3 II-positive areas in lung tissue.
Conclusion: These results indicate that SFA can effectively ameliorate asthma exacerbation induced by PM by modulating MAPK and autophagy pathways and alleviating histopathologic features. This suggests that SFA could be utilized as a safe and effective drug for the treatment of asthma exacerbated by PM.

목차 (Table of Contents)

• Contents · ⅰ
• List of Figures · ⅲ
• List of Table · ⅳ
• Abbreviations · ⅴ
• 1. INTRODUCTION · 1
• 2. MATERIALS AND METHODS · 4
• 2.1. Reagents · 4
• 2.2. Sample preparation · 5
• 2.3. Cell culture · 5
• 2.4. Cell viability · 5
• 2.5. ELISA analysis · 6
• 2.6. DNA damage analysis · 6
• 2.7. Western blot analysis · 6
• 2.8. Animals management · 9
• 2.9. Application of OVA, PM, and drugs in the allergic asthma models · 9
• 2.10. Measurement of IgE, OVA-specific IgE, aspartate aminotransferase (AST), and alanine transferase (ALT) levels in Serum · 11
• 2.11. Analysis of inflammatory cytokine levels in the lung tissue · 11
• 2.12. Histological staining · 12
• 2.13. Immunohistochemical (IHC) staining · 12
• 2.14. High-preparation liquid chromatography (HPLC) analysis of SFA 13
• 2.15. Statistical analyses 13
• 3. RESULTS · 14
• 3.1. Effect of SFA on viability of BEAS-2B cells · 14
• 3.2. Effect of SFA on pro-inflammatory cytokines level in BEAS-2B cells stimulated with TI+PM · 16
• 3.3. Effect of SFA on DNA damage in BEAS-2B cells stimulated with TI+PM · 18
• 3.4. Effect of SFA on MAPK signaling pathway in BEAS-2B cells stimulated with TI+PM · 20
• 3.5. Effect of SFA on AKT/mTOR signaling pathway in BEAS-2B cells stimulated with TI+PM · 22
• 3.6. Effect of SFA on LC3 Ⅱ/LC3 Ⅰ level in BEAS-2B cells stimulated with TI+PM · 24
• 3.7. Effect of SFA on organ weight and hepatotoxicity in PM-exacerbated asthma model · 26
• 3.8. Effect of SFA on serum IgE and OVA-specific IgE levels in a PM-exacerbated asthma model · 28
• 3.9. Effect of SFA on inflammatory cytokine levels in lung tissue in a PM-exacerbated asthma model · 30
• 3.10. Effect of SFA histopathologic and immunohistochemical changes in the lung in a PM-exacerbated asthma model · 32
• 3.11. HPLC analysis of SFA · 34
• 4. DISCUSSION · 36
• 5. CONCLUSION · 43
• REFERENCES · 44
• ABSTRACT · 49
• ABSTRACT IN KOREAN · 51