The Suppressive Effects of Salicornia europaea on Dextran Sulfate Sodium (DSS)-Induced Ulcerative Colitis in vivo and in vitro = 퉁퉁마디의 DSS 유도 궤양성 대장염 조절 효능

국문 초록 (Abstract)

궤양성 대장염은 심각한 염증과 조직 손상을 특징으로 하는 만성 염증성 장 질환으로 최근 한국 내 발병률이 지속적으로 증가하고 있다. 현재 주요 치료법으로 면역 조절제와 코르티코스테로이드가 사용되고 있으나, 이들은 장기간 투여 시 다양한 부작용이 동반된다는 한계가 있다. 염생식물(Halophyte)인 퉁퉁마디(Salicornia europaea, SE)는 전통 의학에서 염증치료를 위해 사용되어 온 약재로, 본 연구에서 안전한 천연물 제재로서의 치료 효능과 그 기저 분자 기전을 확인해보고자 한다.
추출물의 성분 분석을 위해 HPLC 분석을 수행하였으며, 그 결과 추출물 내의 주요 활성 성분으로 flavonoid계통의 성분인 isorhamnetin을 확인하였다. in vivo 실험에서는 C57BL/6N 수컷 마우스에 dextran sulfate sodium(DSS) 자연 음수를 통해 궤양성 대장염을 유도하였다. 이후 SE 추출물을 병행 투여하여 체중 감소, 질병활동 지수 (DAI)를 평가하였다. 염증지표 (myeloperoxidase(MPO), tumor necrosis factor(TNF)-α, interferon(IFN)-γ, interluekin(IL)-6, IL-8, prostaglandin(PG)E2를 ELISA로 분석하고, mitogen activated protain kinase(MAPK) 신호 전달 경로 활성화를 Western Blot으로 확인하였다. in vitro 실험에서는 Lipopolysaccharide(LPS)로 자극된 RAW 264.7 세포에 SE 추출물을 처리하여 세포 사멸율, nitric oxide(NO) 생성 억제, 주요 염증성 mRNA 및 단백질 발현 변화를 분석하였다.
연구 결과, SE 추출물은 DSS로 유도된 마우스에서 DAI 상승, 체중 감소 완화, 대장 길이 축소 등의 임상 증상을 용량 의존적으로 유의하게 개선하였다. 또한, 대장 조직 손상 및 MPO 활성을 현저히 감소시키고, 혈액 내 염증성 사이토카인 (TNF-α, IFN-γ, IL-6) 수준을 억제하였다. 기전적으로, SE 추출물은 in vivo 및 in vitro 모델 모두에서 염증 활성의 핵심인 c-Jun N-terminal Kinase (JNK) 및 Extracellular signl-Regulated Kinase(ERK)의 인산화를 효과적으로 차단하였다. 이러한 MAPK 억제는 NO 생성과 염증성 유전자 (IL-6, TNF-α)의 mRNA 발현 하향 조절로 이어졌다.
결론적으로, SE 추출물은 MAPK 신호 전달 경로 억제를 통해 강력한 항염증 및 항대장염 효능을 발휘하며, 염증성 장 질환 치료제 개발을 위한 안전하고 효과적인 소재로서의 잠재력을 지닌다.

다국어 초록 (Multilingual Abstract)

Ulcerative colitis (UC) is a chronic inflammatory bowel disease(IBD) characterized by severe inflammation and tissue damage, with a steadily increasing incidence in Korea. Current treatments involve immunomodulators and corticosteroids, which are associated with side effects during long-term administration. SE, a halophyte, has been used in medicine for treating inflammation. This study aimed to investigate the therapeutic efficacy and underlying molecular mechanisms of SE extract as a safe natural product for UC.
First, HPLC analysis was performed to standardize the extract, isorhamnetin was detected as a major bioactive constituent, which identified isorhamnetin as a major bioactive constituent. in the In vivo model, UC was induced in C57BL/6N male mice by oral administration of DSS in drinking water. Subsequently, SE extract was co-administered to evaluate changes in body weight and Disease Activity Index (DAI). Inflammatory biomarkers (MPO, TNF-α, IFN-γ, IL-6, and PGE2) were analyzed by ELISA, and the activation of the MAPK signaling pathway was probed by Western Blot. For In vitro experiments, RAW 264.7 cells stimulated with LPS were treated with SE extract to analyze cytotoxicity, inhibition of NO production, and changes in the expression levels of key pro-inflammatory mRNA and proteins.
SE extract significantly and dose-dependently ameliorated clinical symptoms, including elevated DAI, attenuated weight loss, and prevented colon shortening, in DSS-induced mice. Furthermore, it markedly reduced colonic tissue damage and MPO activity (neutrophil infiltration). SE treatment effectively suppressed the systemic levels of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-6). Mechanistically, SE extract was found to effectively block the phosphorylation of JNK and ERK, which are central to inflammatory activation, in both in vivo and in vitro models. This MAPK suppression led to the downregulation of NO production and pro-inflammatory gene expression (TNF-α, IL-6) at the mRNA level.
SE extract demonstrates potent anti-colitic and anti-inflammatory effects by inhibiting the MAPK signaling pathway. These findings suggest that SE extract is a safe and effective natural candidate for the development of therapeutic agents against IBD.

목차 (Table of Contents)

• I. INTRODUCTION 1
• II. MATERIALS AND METHODS 4
• 1. Reagents 4
• 2. Preparation of SE extract for experiments 4
• 3. Animal 5
• 4. DSS-induced Colitis Model 6
• 5. Disease activity index (DAI) score 7
• 6. Sample Collection and Processing 8
• 7. H&E staining 8
• 8. HPLC analysis 9
• 9. ELISA 10
• 10. Cell culture 11
• 11. Cell viability 12
• 12. Measurement of nitric oxide (NO) 13
• 13. RNA Extraction and RT-PCR 14
• 14. Western Blot 16
• 15. Statistical analysis 18
• III. RESULTS 19
• 1. Increasing trend of domestic UC patients in South Korea (2020-2024) 19
• 2. SE extract ameliorates clinical symptoms and DAI in DSS-induced Colitis 21
• 3. SE treatment promotes recovery of colon length in DSS-induced colitis 25
• 4. SE extract ameliorates histopathological colonic injury 28
• 5. SE extract inhibits MPO activity and pro-inflammatory mediators in colonic Tissue 30
• 6. SE extract reduces systemic pro-inflammatory cytokine levels in serum 34
• 7. SE extract suppresses MAPK signaling pathway activation 37
• 8. Phytochemical characterization of SE extract via HPLC analysis 40
• 9. Effect of SE extract on cell viability and NO production in RAW 264.7 macrophages 43
• 10. SE extract suppresses mRNA expression of pro-inflammatory cytokine in macrophages 46
• 11. SE extract down-regulates phosphorylation of MAPK signaling pathway in macrophages 49
• 12. SE extract inhibits secretion of pro-inflammatory cytokines in macrophages 51
• IV. DISCUSSION 55
• V. CONCLUSION 58
• REFERENCES 59