나복자(蘿?子)는 십자화과 식물에 속하는 무 (Raphanus sativus Linne)의 씨앗이다. 나복자는 전통적으로 消化不良, 痰飮, 咳嗽, 氣喘을 치료하기 위하여 사용되어 왔다. 나복자는 alkaloids, anthraquinon glycosides, terpenoids glycosides, steroids, tannins, carbohydrates, fats, oils, flavonoids 등을 함유하고 있고, 최근 나복자 추출물의 항균, 항염, 항산화, 항고혈압 및 항당뇨 작용이 보고되었다. 그 중 항염 작용과 관련하여 천식 마우스 모델에서의 항염, 항천식 효능, 염증성 장질환 동물모델 및 인간 비만세포에서 나복자의 함유 성분인 allyl isothiocyanate의 항염 효능에 대한 연구가 있었다. 그러나 인간의 각질형성세포와 가장 유사한 HaCaT cell에서의 항염 기전에 대해서는 아직 밝혀진 바가 없다. 따라서 본 연구에서는 인간피부각질형성세포 (HaCaT keratinocytes) 모델을 TNF-α와 IFN-γ로 자극하여 아토피 피부염과 유사한 환경을 조성한 후 나복자의 항염 효능에 대한 연구를 진행하였다. 나복자 에탄올 추출물 (RSE)은 HaCaT keratinocytes에서 TNF-α와 IFN-γ자극에 의하여 증가된 IκBα, ERK의 인산화를 억제하였으며 또 다른 전사인자인 JAK2와 STAT1, STAT6의 인산화를 억제하였다. 뿐만 아니라 periostin과 TSLP의 발현을 감소시켰다. 모든 결과를 종합하면, 나복자 에탄올 추출물 (RSE)은 HaCaT cell에서 pro-inflammatory cytokines 및 transcription factors의 발현을 감소시켜, 만성 염증성 질환에서 나복자의 사용 가능성을 확인하였다.
Raphani semen is a seed of Raphanus sativus Linne which belongs to the family Cruciferae. Traditionally, Raphani semen has been used to treat indigestion, abdominal distension, and constipation, and contains alkaloids, anthraquinon glycosides, terpenoids glycosides, steroids, tannins, carbohydrates, fats, oils, and flavonoids. There have been studies about Raphani semen’s antibacterial, anti-inflammatory, and anti-hypertensive effects. About anti-inflammatory effects, there have been studies on the asthma mouse model, experimental ulcerative colitis rat models, and human mast cells. However, inflammatory effects in HaCaT cells, which are most similar to human keratinocytes, have not been revealed. Therefore, in this study, we stimulated HaCaT cells with TNF-α and IFN-γ to create an environment similar to atopic dermatitis skin, then checked the anti-inflammatory efficacy of Raphani semen ethanol extracts (RSE). As a result, RSE inhibited phosphorylation of IκBα and ERK induced by TNF-α and IFN-γ stimulation in HaCaT keratinocytes, and suppressed phosphorylation of JAK2, STAT1, and STAT6. In addition, the expression of TSLP and periostin protein was reduced. Taken together, RSE showed the possibility of use in chronic inflammatory diseases through inhibition of pro-inflamatory cytokines and transcription factors in HaCaT cells.
Ⅰ. Introduction 1Ⅱ. Materials and Methods 31. Reagents 32. Preparation of RSE 43. Cell culture and sample treatment 44. MTT assay 55. Western blot analysis 56. Statistical analysis 6Ⅲ. Results 71. Effects of RSE on cell viability in HaCaT keratinocytes 72. Effects of RSE on p-IκBα expression in TNF-α and IFN-γ stimulated HaCaT keratinocytes 93. Effects of RSE on p-ERK expression in TNF-α and IFN-γ stimulated HaCaT keratinocytes 114. Effects of RSE on p-JAK2 expression in TNF-α and IFN-γ stimulated HaCaT keratinocytes 135. Effects of RSE on p-STAT1 expression in TNF-α and IFN-γ stimulated HaCaT keratinocytes 156. Effects of RSE on p-STAT6 expression in TNF-α and IFN-γ stimulated HaCaT keratinocytes 177. Effects of RSE on periostin expression in TNF-α and IFN-γ stimulated HaCaT keratinocytes 198. Effects of RSE on TSLP expression in TNF-α and IFN-γ stimulated HaCaT keratinocytes 21Ⅳ. Discussion 23Ⅴ. Conclusions 29Ⅵ. References 30ABSTRACT 39