사람 비만세포주(HMC-1)에서의 케모카인의 발현 양상

이희문 경희대학교 2000 국내박사

The mast cell is an essential effector cell in allergic inflammation through its capacity to respond to IgE dependent activation with release of both preformed and newly generated mediators. Mast cells also participate in the modulation of physiologic processes, but the role of mast cell in these processes is still unclear. Recently, the number of structurally defined chemoattractants for leukocytes has greatly increased, owing to largely to the identification of the chemokine superfamily. Chemokines are small proteins with molecular weights in the range of 8 to 12 kD and inducible in a number of pathophysiologic precesses. This study is aimed to examine the pattern of expression of chemokines in human mast cell line (HMC-1). HMC-1 were treated with PMA/ionophore and/or LPS and their induction of mRNA and protein were investigated by RT-PCR and ELISA. Messenger RNA of IL-8 , the representative CXC chemokine, was induced after PMA/ionophore treatment. All of the CC chemokines tested except eotaxin were induced after PMA/ionophore treatment and MIP-1α and MIP-1β were expressed their mRNA moderately in the resting state. CCR1, CXCR2, CXCR3 and CXCR4 were expressed in all test group regardless of activation. CCR3 was expressed only at 3 hours of activation. CCR2 and CXCR1 were not expressed in mast cell line. Production of chemokine protein was not detected in resting state and increased significantly after 3 hours of activation with PMA/ionophore. The effect of LPS treatment was negligible. MCP-1 protein was always produced without activation and accumulated in a time-dependent manner. These data suggest that the expression of mRNA and protein of chemokines and chemokine receptors are regulated transcriptionally and translationally. Human mast cell may respond to various stimuli by producing chemokines and their receptors to regulate their function and may act autonomously or through other inflammatory cell that they recruited.

Snythesis and biological evaluation of thiazolopyridine derivatives as c-KIT inhibitors

한정희 KU-KIST Graduate School of Converging Science and 2020 국내석사

c-KIT, which a type III receptor tyrosine kinase(RTK), has dysfunction signaling, result in tumorigenesis. More than 90% of gastrointestinal stromal tumor (GIST) involves c-KIT activating mutants which have been used as a diagnostic indicator of GIST. In this study, a selective and potent c-KIT inhibitor was rationally designed for GIST targeted therapy. Structure-activity relationship (SAR) study of 25 derivatives against c-KIT was conducted and N-(3-(2-aminothiazolo[5,4-b]pyridin-6-yl)-4-methylphenyl)-4-((4 methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)benzamide (7s) showed most potent inhibitory effect against c-KIT. 7s is comparable to imatinib in terms of enzymatic activity against c-KIT WT and anti-proliferative activity on GIST-T1 cells. 7s possesses great kinase selectivity against a panel of 371 kinases. It is worth noting that 7s exhibits a 10 fold higher anti-proliferative activity than imatinib on imatinib-resistant HMC (Human Mast Cell) 1.2. cells harboring both c-KIT-D816V and c-KIT-V560G. Molecular docking study reveals that H-bonding interaction between aminothiazole moiety of 7s and c-KIT-Cys673. Taken together, this study suggests that 7s could be a lead compound to override imatinib-resistant GIST. 위장관기질종양(GIST)의 90% 이상이 c-KIT 활성화 돌연변이종 (activating mutants)에 의해 발병되며 이는 GIST의 진단에 지표로 사용된다. 이 연구의 목적은 GIST를 타겟으로 하는 선택적이고 활성이 우수한 c-KIT 억제제를 합리적으로 설계하여 합성하였다. 대표화합물인 7s는 c-KIT WT에 대한 효소활성 및 GIST-T1 세포주에 대한 항 증식 활성이 이마티닙과 상응한다. 7s는 371개의 키나아제들 중에서 c-KIT에 대해 우수한 키나아제 선택성을 나타낸다. 주목할만한 점은 c-KIT-D816V 및 c-KIT-V560G 돌연변이를 갖는 이마티닙-내성을 갖는 HMC1.2 세포주에서 7s가 imatinib보다 10배 높은 항 증식 활성을 나타낸다. 분자 도킹(docking) 연구는 7의 아미노싸이아졸(aminothiazole) 부분과 c-KIT의 Cys673과의 수소결합 상호작용을 나타낸다. 종합적으로, 이 연구는 7s가 이마티닙 내성 GIST를 대체하는 주요 화합물일 수 있음을 제안한다.

Production and regulation of inflammatory cytokines in human mast cell line (HMC-1)

김미선 부산대학교 2002 국내박사

Cytokine production signaling and differentiation in human mast cell line (HMC-1) : 사람 비만세포주 (HMC-1)에서 세포분화와 싸이토카인 생성의 신호전달 경로

김지영 成均館大學校 大學院 2003 국내박사

본 연구실은 기니픽의 폐조직으로부터 분리된 비만세포 활성화시 ROS와 매개체 유리에 대한 상호관계는 이미 보고하였다. 그러나 사람 비만세포주에서 ROS와 싸이토카인의 생성에 관한 상호 연관성은 보고되지 않았다. 그러므로 본 연구는 PMA-자극 사람 비만세포주 (HMC-1)에서 생성되는 ROS에 의한 싸이토카인 (IL-8과 TNF-a) 생성에 있어서의 신호전달기전을 연구하는데 목적이 있었다. HMC-1 세포를 25ng/ml PMA로 자극 시 ROS 생성과 FceRI 발현은 FACS scan으로, 싸이토카인 생성은 ELISA 방법으로 측정하였다. MAP kinase (ERK, p38, JNK)의 인산화는 western blotting 방법, 싸이토카인의 mRNA 발현정도와 전사인자 (NF-κB, AP-1)의 DNA 결합정도는 각각 RT-PCR과 EMSA 방법으로 측정하였다. PMA-자극 HMC-1 세포는 30초 이내에 세포 내 ROS를 생성하였으며, 생성된 ROS는 항-산화제인 catalase와 DMTU에 의해서 모두 억제되었다. PMA-자극 HMC-1 세포는 30분부터 싸이토카인 생성을 시작하여 3∼5시간 사이에 최고치에 도달하였고, 이는 항-산화제 및MAP kinase에 특이적인 억제제들에 의해서 억제되었다. Catalase, DMTU 및 SB203580은 MAP kinase 중 p38의 인산화를 강하게 억제하였다. DMTU는 싸이토카인의 mRNA 발현정도와 전사인자 (NF-κB, AP-1)의 DNA 결합 정도를 모두 억제하였으나 catalase는 영향을 미치지 않았다. 이상의 결과로부터 PMA-자극 HMC-1세포에서 생성되는 ROS는 p38과 NF-κB, AP-1 신호전달 기전을 통하여 염증에 관여하는 싸이토카인의 생성을 유도하며 catalase와 DMTU 두 항-산화제의 신호전달기전은 서로 다르거나 효능의 차이로 추정할 수 있다. HMC-1세포는 백혈병을 갖는 환자의 말초 혈액으로부터 만들어진 미성숙 세포주로 알려져 있다. HMC-1세포에 murine 섬유 아세포주 (L 세포)와 사람 피부 섬유 아세포, 사람 각질세포의 배양 상층액, NGF를 가하여 배양하면 FceRI의 발현, 세포 내 tryptase, 히스타민 증가 등 비만세포의 특징을 증가 시킨다는 보고가 있다. 그러므로 NGF에 의한 HMC-1의 분화에 관련된 성장인자로서의 단백질들과 싸이토카인들의 발현을 규명하는데 목적이 있었다. HMC-1 세포분화 연구에서, 세포 배양 시 10ng/ml NGF를 첨가하여 10일 동안 배양하였다. 세포 내 과립형성과 히스타민 양의 증가는 각각 전자 현미경과 히스타민 분석기를 통하여 관찰하였다. 단백질과 싸이토카인의 발현은 각각 2-D 전기영동법, cDNA microarray로 관찰하였다. NGF를 처리한 HMC-1 세포에서 발현이 증가된 9개의 단백질, pyruvate kinase, annexin I, heat shock protein, enolase, myosin, squamous cel carcinoma antigen I, gamma actin, 을 얻었다. 이들 단백질 중 비만세포의 분화와 관련된 단백질인 pyruvate kinase와 annexin I은 다시 western blotting에 의해서 확인하였다. cDNA microarray에 의해서 발현이 증가된 싸이토카인 유전자는7개로 cytokine subfamily A member 2 (SCYA2), MCP-1, TNF-a, SCYA1, insulin receptor, metalloprotein III, TGF-β2, TGF-TGF-β이며 이중 2배 이상의 증가를 보인 MCP-1과 TNF-a의 발현은 RT-PCR을 통하여 확인하였다. 이상의 결과로부터 NGF에 의해 발현된 단백질과 싸이토카인들이 서로 상호작용을 하여 비만세포 분화에 영향을 줄 것으로 추정이된다. In previous studies, I reported the relationship of reactive oxygen species (ROS) and mediator release in guinea pig lung mast cell activation. However, the relationship of ROS and cytokines production in human mast cells had not yet been reported. This study aimed to examine the signal pathway in the production of cytokines (IL-8 and TNF-a) by ROS generated from PMA-stimulated HMC-1 cells (human mast cell line-1). HMC-1 cells were stimulated with 25ng/ml phorbol myristate acetate (PMA), and the ROS generation and production of cytokines (IL-8 and TNF-a) were measured by FACS and ELISA method, respectively. Phosphorylation of MAP kinase family (ERK, p38, and JNK) was detected by western blotting. The expression of cytokine mRNAs was measured by RT-PCR, and DNA binding activity of transcription factors (NF-κB and AP-1) were detected by EMSA. PMA-stimulated HMC-1 cells immediately generated ROS. The generated ROS was inhibited by anti-oxidants (catalase and DMTU). The production of cytokines in PMA-stimulated HMC-1 cells reached the maximun at 3-5 hr, and was inhibited by anti-oxidants and each specific inhibitor. Catalase, DMTU, and SB203580 strongly inhibited the p38 phosphorylation of MAP kinase pathways. DMTU inhibited the expressed cytokines mRNA level and the increased DNA binding activity of transcription factors, NF-κB and AP-1, in PMA-stimulated HMC-1 cells, but catalase did not affect. These data suggest that ROS generated from PMA-stimulated HMC-1 cells leads to produce inflammatory cytokines via p38 kinase-NF-κB/AP-1 and that signaling pathway of both anti-oxidants differs from each other. HMC-1 cell line has been established from the peripheral blood of patients with mast cell leukemia and has been known as immature mast cell line. In the previous studies for human mast cell differentiation, it has been reported that supernatant of murine fibroblast cell line (L-cells), normal human skin fibroblasts, human keratinocyte, and nerve growth factor (NGF) are able to up-regulate mast cell characteristics such as FcεRI expression and intracellular trpytase, as well as histamine levels, but it is controversial. Therefore, this study aimed to confirm the HMC-1 cells differentiation, FceRI expression and granule generation, by NGF tratmnet in our system, and to screen the proteins and cytokines related to the mast cell differentiation by NGF. NGF (10ng/ml) was added to the basic culture medium for 10days. After NGF treatment, I observed increasement of intracellular histamine level by total histamine content and granules generation by electron microscopy but FceRI expression was not observed. Various proteins and cytokines increased by NGF treatment were detected by two-dimensional gel electrophoresis and cDNA microarray, respectively. I found nine proteins which was significantly increased in NGF treated cells including pyruvate kinase (subtype), annexin I, heat shock protein, enolase, myosin, squamous cell carcinoma antigen I, and gamma actin. It was confirmed by western blotting because two of these proteins, pyruvate kinase and annexin I, were known that related to the mast cell differentiation. Enhanced expression level of cytokine genes included cytokine subfamily A member 2 (SCYA2); MCP-1, TNF-a, SCYA1, insulin receptor, metalloprotein III, TGF-b2, and TGF-b. MCP-1 and TNF-a expression level were increased more than 2-fold, it was further confirmed by RT-PCR. These data suggest that various proteins and cytokines increased by NGF may complexly affect the mast cell differentiation.

치자의 HMC-1 비만세포에 대한 항염증효과

문지홍 경희대학교 대학원 2023 국내박사

Abstract Anti-inflammatory effect of Gardenia Jasminoides extract (GJ) in HMC-1 human mast cell Moon, Jihong Dept. of Korean Medicine Graduate School Kyung Hee University Seoul, Korea Supervised by Prof. Ko, Seong-Gyu, K.M.D., Ph.D. Objective: In this study, the anti-inflammatory effects of Gardenia Jasminoides were investigated in HMC-1 human mast cell. To identify which mechanism is involved in the anti-inflammatory mechanism, we investigated the inhibitory effect of GJ on PMA and Ionomycin-induced inflammation. Methods: The effect of GJ was analyzed by RT-PCR, ELISA and Western blotting in PMA and Ionomycin-activated HMC-1, human mast cells. Results: We found that GJ significantly inhibited the phosphorylation of NF-κB, ERK, JNK, and P38. In addition, GJ decreased nuclear translocation of NF-κB and the mRNA expression of IL-6, IL-8 and IL-17A. GJ also suppreseed the expression of pro-inflammatory cytokines such as IL-6 and TNF-α. Conclusion: These results suggest that GJ has an anti-inflammatory effect through the phosphorylation of the NF-κB and MAPK proteins, thereby decreasing nuclear translocation of NF-κB and production of pro-inflammatory cytokines in HMC-1 mast cell. Key words: Gardnia Jasminoides; anti-inflammatory; HMC-1; mast cell;

Anti-inflammatory effect of Tonggyu-tang via interukin and NF-κB pathway in HMC-1 and HaCaT

김효인 경희대학교 대학원 2017 국내석사

배경: 알레르기성 비염, 천식 및 아토피성 피부염을 비롯한 알레르기성 염증질환이 전 세계적으로 증가하는 추세이다. 현재 알레르기 반응을 치료에 주로 사용되는 약물인 항 히스타민제와 코르티코이드 스테로이드제는 지속시간이 짧다는 점뿐만 아니라 여러 부작용을 나타내고 있어 사용을 제한하고 있어 이를 보완 및 대체할 수 있는 의약품을 찾기 위해 노력하고 있다. 목적: 알레르기성 비염환자를 치료하기 위해 한의학에서 처방하는 통규탕의 항염증 기전을 확인하고자 한다. 방법: 본 연구에서는 HMC-1 (Human mast cell-1)세포와 피부각질세포인 HaCaT 세포를 RT-PCR 및 ELISA를 이용하여 염증관련 interukin(IL)의 mRNA 발현량과 cytokine 방출량을 측정하였다. 또한, 염증관련 다양한 cell signaling pathway 에서의 단백질 생성량 확인을 통하여 통규탕이 항염효과를 확인하였다. 결과: 본 연구는 통규탕이 HMC-1과 HaCaT 세포에서 IL-4, IL-6, IL-8, IL-13, TNF-α 와 같은 염증성 cytokine 의 발현 및 생성을 효과적으로 감소시킴을 확인하였다. 또한, MAPK pathway, PI3K-AKT pathway, NF-κB pathway 에서 염증반응에 주요하게 작용하는 경로를 효과적으로 억제함을 확인하였다. 결론: 통규탕은 HMC-1과 HaCaT 세포에서 염증관련 경로에 관여하여 염증성 cytokine의 발현을 억제함으로써, 알레르기성 염증 치료에 효과적임을 나타내었다. Allergic inflammatory diseases including allergic rhinitis (AR), asthma and atopic dermatitis (AD) are increasing worldwide. Current medications commonly used to treat allergies reactions are anti-histamines and corticosteroids but they have their own limitations such as short duration and severe side effects. Thus, complementary and alternative medicine is gaining an interest in the field of allergic diseases. In traditional Korean Medicine, several herbal medicines have been used for allergic rhinitis treatment. Especially, among such medicines, 通竅湯 (Tonggyu-tang, TGT) composed of 14 herbs has shown anti allergic effects in patients with nasal disorders. HMC-1 (Human mast cell-1) and HaCaT cells are essential reaction components for allergic reactions which are located in almost all tissues, influencing inflammation by releasing histamine. Excessive activation of these inflammatory cells leads to histamine degranulation, which results in hypersensitive allergic reactions. In my study, I identified the mechanisms for allergic reactions using TGT in these two representative immune cells, HMC-1 cells and HaCaT cells Activated HMC-1 and HaCaT cells were treated with various concentration of TGT, and I investigated pro-inflammatory cytokines and inflammation-related cell signaling pathways.

Protective effects of deacetylasperulosidic acid on Atopic dermatitis by regulating Th1/Th2 Immune Balance and restoring skin barrier function in HaCaT, HMC-1, and EOL-1 Cells

오진수 경희대학교 대학원 2021 국내석사

이전 연구를 통하여, 발효 노니가 아토피성 피부염을 유발한 동물 모델에서 효능이 있음이 입증되었다. 본 연구에서는 발효 노니로부터 분리된 단일성분의 항아토피 효능 및 기전연구를 통해 효능성분을 규명하고자 하였다. 발효 노니의 HPLC-PDA 분석 결과, deacetylasperulosidic acid (DAA)와 asperulosidic acid (AA)가 지표성분으로 밝혀졌으므로 아토피성 피부염에 중요한 역할을 하는 각질세포, 비만세포, 호산구 세포를 이용하여 기능(지표)물질을 규명하고자 하였다. DAA와 AA의 효능을 비교하기 위해 TNF-α 와 IFN-γ 처리한 인간 각질세포주 HaCaT 세포에서 생산되는 TSLP 와 IL-33의 분비량, PMACI를 처리한 인간 비만세포주 HMC-1 세포에서 생산되는 histamine과 IL-4의 분비량, HDM 처리된 인간 호산구세포주 EOL-1 세포에서 생산되는 MCP-1 과 IL-5의 분비량을 비교하였다. DAA와 AA는 세 세포주 모두에서 아토피성 피부염과 관련된 사이토카인 및 케모카인을 유사하게 억제하였다. DAA와 AA의 최고농도에서 임상 치료제로 사용되는 dexamethasone (DEX)와 유사한 효능을 보였다. 따라서, 함량이 가장 많고 발효에 의해서도 함량이 상대적으로 높아지는 DAA를 기능(지표)성분으로 선정하였다. DAA를 이용하여 항아토피 효능을 규명하기 위해 세 세포에서 아토피성 피부염과 관련된 사이토 카인 및 케모카인의 발현 및 분비 수준을 비교하였다. DAA는 HaCaT 세포에서 IL-1β, IL-6, IL-8, TNF-α, MCP-1 유전자 발현 및 분비를 억제하였다. 또한, Th2 세포의 직간접적인 활성과 관련된 IL-25, TARC, MDC, RANTES의 유전자 발현 및 분비를 억제하였다. DAA는 HMC-1 세포에서 아토피성 피부염과 관련된 IL-1β, IL-6, IL-8, TNF-α, TSLP, 및 MCP-1의 유전자 발현 및 분비를 억제하였다. EOL-1 세포에서도 IL-6, IL-8, TNF-α 및 MCP-1의 유전자 발현 및 분비를 억제하여 항아토피 효능을 나타냄을 규명할 수 있었다. DAA의 작용기전을 규명하기 위해 HaCaT, HMC-1, 및 EOL-1 세포에서 MAPK pathway에 관여되는 ERK, JNK, P38의 인산화 비율을 비교하였으며 NF-kB의 발현 정도를 비교하기 위해 IkBα의 분해 및 핵내로의 NF-kB의 전위를 비교하였다. DAA는 세 세포 모두에서 MAPK pathway 및 NF-kB의 발현 억제를 통해 항 아토피 효능을 나타냄을 규명할 수 있었다. 임상적으로 가장 많이 사용되는 스테로이드 치료제의 단점인 피부장벽 손상에 대한 효능을 보기 위해, DAA가 HaCaT 세포에서 피부장벽기능을 담당하는 filaggrin (FLG) 및 involucrin (IVL)의 단백질 발현에 대해 어떤 영향을 주는 지를 확인하였다. DEX는 FLG 및 IVL의 발현 수준을 회복시키지 못한 반면, DAA는 농도의존적으로 증가시켰으며 최고농도에서 정상치로 회복시켰다. 결론적으로 DAA는 Th1/Th2 면역 불균형의 회복 및 피부장벽 기능의 개선을 통해 부작용이 적은 아토피 성 피부염의 신약개발 후보 물질이 될 수 있음을 시사하였다. The medicinal plant Noni (Morinda citrifolia) is widely dispersed throughout Southeast Asia, the Caribbean, and Australia. We previously reported that fermented Noni could alleviate atopic dermatitis (AD) by recovering Th1/Th2 immune balance and enhancing skin barrier function induced by 2,4-dinitrochlorobenzene. Noni has a high deacetylasperulosidic acid (DAA) content, whose concentration further increased in fermented Noni as an iridoid constituent. This study aimed to determine the anti-AD effects and mechanisms of DAA on HaCaT, HMC-1, and EOL-1 cells. DAA inhibited the gene expression and secretion of AD-related cytokines and chemokines including interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-25, IL-33, thymic stromal lymphopoietin, tumor necrosis factor-alpha, monocyte chemoattractant protein-1, thymus and activation-regulated chemokine, macrophage-derived chemokine, and regulated upon activation, normal T cell expressed and secreted, in all cells, and inhibited histamine release in HMC-1 cells. DAA controlled mitogen-activated protein kinase phosphorylation levels and the translocation of nuclear factor-kappa light chain enhancer of activated B cells into the nucleus by inhibiting IκBα decomposition in all the cells. Furthermore, DAA increased the expression of proteins involved in skin barrier functions such as filaggrin and involucrin in HaCaT cells. These results confirmed that DAA could relieve AD by controlling immune balance and recovering skin barrier function.

Trypsin으로 자극한 HMC-1으로부터 염증성 매개물질의 분비에 대한 Curcumin의 억제효과

백옥선 又石大學校 大學院 2003 국내석사

Curcumin은 울금 (Curcuma longa L., Gingiberaceae)의 황색색소 성분으로서 특유의 향미를 지니고 있어 음식의 재료나 화장품에도 사용된다. 또한 항염증, 항암활성, 항산화작용 등을 발휘함으써 전통적으로 아시아 지역에서 치료약으로서 사용되어왔다. 단백질 활성 수용체-2 (PAR-2)의 agonist인 trypsin은 염증상태에 있어서 중요한 역할을 하며, Human leukemic mast cell (HMC-1)이 PAR-2를 발현하도록 유도한다. 본 연구는 trypsin으로 자극한 HMC-1으로부터의 tryptase와 tumor necrosis factor-α (TNF-α)의 생성과 분비에 대한 curcumin의 효과에 관한 실험이다. Trypsin으로 자극한 HMC-1에서 curcumin (1-100μM)은 tryptase와 TNF-α의 mRNA와 단백질의 합성을 용량의존적 방법으로 억제하였다. 더욱이 curcumin (1-100μM)은 trypsin으로 자극한 HMC-1에서 extracellular signal regulated kinase (ERK)의 인산화를 현저히 억제하였다. 반면에 curcumin (1-100μM)은 c-Jun N-terminal kinase (JNK)의 인산화와 p38 mitogen-activated protein kinase (p38 MAPK) 경로의 활성에는 영향을 미치지 않았다. 또한 trypsin의 활성에 대한 curcumin의 직접적인 영향은 나타나지 않았다. 이러한 결과를 통해, curcumin이 ERK 경로를 통하여 tryptase와 TNF-α의 합성을 억제하는 것을 알 수 있다. 앞으로 curcumin의 염증성장질환에 대한 치료제로의 개발을 기대할 수 있겠다. Curcumin, a major yellow pigment and active component of turmeric powder extracted from Curcuma longa L. (Gingiberaceae), has been shown to possess anti-inflammatory and anti-cancer activities. Proteinase-activated receptor-2 (PAR-2) agonist trypsin plays a role in inflammation and human leukemic mast cells (HMC-1) express PAR-2. In the present study, the effect of curcumin on tryptase and tumor necrosis factor-α (TNF-α ) secretion from trypsin-stimulated HMC-1 was examined. Curcumin(1-100 mM) inhibited the mRNA and protein production of tryptase and TNF-α in a dose-dependent manner in trypsin-stimulated HMC-1. We previously showed that trypsin induced phosphorylation of ERK without phosphorylation of JNK and p38. Moreover, curcumin did not affect the trypsin activity even 100 mM. These results suggested that curcumin may inhibit the tryptase and TNF-a production by not inhibiton of trypsin activity but inhibition of ERK phosphorylation.

Effect of Polygala tenuifolia on TMA-induced atopic dermatitis aggravated by immobilization stress in mice

서봉준 경희대학교 대학원 2014 국내박사

Atopic dermatitis (AD) and stress create a vicious cycle: stress exacerbates atopic symptoms, and atopic skin disease elicits stress and anxiety. Increasing evidence suggests that stress-induced exacerbation of AD symptom is closely associated with the degranulation of skin mast cells via corticotropin-releasing factor (CRF) and substance P (SP) signaling triggered by stress. The aim of this study is to evaluate efficacy of the boiled water extract of Polygala tenuifolia Willd. (PTW) on immobilization (IMO) stress-exacerbated AD in the mouse and to identify the action mechanism at the molecular level. An in vitro model of stress-associated AD was developed by applying the stress hormone CRF (200 nM) to the human mast cell line (HMC)-1 following pre-treatment of SP (10 µM) for 48 h to induce inflammatory priming. The cells were treated with PTW (250, or 500 g/ml) 30 min before exposure to CRF. Mast cell degranulation was assessed by microscopic examination and tryptase assay 24 h after treatment. Specifically, the upstream signaling of protein kinase A (PKA) and protein kinase C (PKC) and the downstream phosphorylation of mitogen-activated protein kinase (MAPK) was examined. For the in vivo experiment, AD-like skin lesions were generated by repeated application of tetramethylammonium (TMA) to both ears of Balb/c mice. Sensitization was induced by application of 5% TMA to the dorsal skin, followed by application of 2% TMA to both ears on 5th days, and then, 1% TMA was applied once a day from 6th to 14th day (totally 9 days). The animals underwent 2 h-IMO stress after every application of 1% TMA from 9th to 14th day. Mice undergoing IMO stress received oral PTW (50, or 250 mg/kg) daily 30 min prior to the application of 1% TMA from 9th to 14th day, and the medicinal efficacy of PTW was assessed using inflammation of ear skin tissue, scratching behavior, water content of the ear tissue, lymph node weight, and serum histamine and immunoglobulin E (lgE) levels. The microscopic examination and tryptase activity assay in the in vitro experiment revealed that mast cell degranulation was significantly greater in the cells exposed to CRF to the HMC-1 cells after pre-treatment with SP for 48 h than in those exposed to either compound alone and both. Treatment with 500 g/ml PTW effectively inhibited mast cell degranulation and release of inflammatory cytokines in the HMC-1 cells. In the in vivo experiment, treatment with 250 mg/kg PTW showed a significant decrease in scratching behavior, morphological changes in ear thickness, water content, lymph node weight, and serum histamine and lgE levels. Furthermore, treatment with 250 mg/kg PTW significantly decreased the expression of tumor necrosis factor-alpha (TNF-α) and interleukin-4 (IL-4) cytokines in the mouse of IMO stress-induced AD exacerbation. Application of CRF to the HMC-1 cells after pre-treatment with SP for 48 h significantly activated upstream signaling of PKA and PKC; however, treatment with 500 g/ml PTW showed only result of decreasing of PKA activation. Moreover, the analysis of the downstream phosphorylation of MAPK revealed that p38 phosphorylation was significantly decreased by PTW. Taken together, the IMO stress-exacerbated AD was verified to be valid in an animal model and PTW inhibited mast cell degranulation in HMC-1 cell line and IMO stress-exacerbated atopic mouse. Anti-atopic effect of PTW was exerted via the modulation of p38 MAPK cell signaling rather than affecting intrinsic signaling of atopic inflammation itself. Thus, PTW may be a useful for the treatment of IMO stress-exacerbated AD. Moreover, these results provide a molecular basis for developing new therapeutics for treating various inflammatory diseases, especially those aggravated by stress.

Anti‐allergic effects of Chicoric acid of Taraxacum on allergic responses in human mast cell : 蒲公英에 함유된 chicoric acid의 항알레르기 효과

엄예진 상지대학교 일반대학원 2015 국내석사

Background and objectives: Currently, Many people are suffering from chronic allergic inflammatory diseases, such as atopic dermatitis, allergic rhinoconjunctivitis. Unfortunately, there is no definitive treatment of allergic disease. Chicoric acid is the component of Taraxacum. Although, anti‐inflammatory and anti‐oxidative properties of chicoric acid were investigated in several studies, anti‐allergic effects and mechanisms on late‐phase reaction of allergic response in human mast cells (HMC‐1 cells) have not been reported. The aim of this study is to investigate the basic mechanisms about anti‐allergic inflammation effects of chicoric acid in vitro. Methods: HMC‐1 cells were incubated with chicoric acid (6.25, 12.5, 25 and 50 µM) and 20 nM of phorbol 12‐myristate 13‐acetate plus 1 µM of A23187 (PMACI). Cytotoxicity was measured with a 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyl‐tetrazolium bromide (MTT) assay. Pro‐inflammatory cytokines productions were measured with enzyme‐linked immuno sorbent assay (ELISA). Expressions of mRNA and protein were measured with real‐time polymerase chain reaction (PCR) and Western blot analysis. Results and conclusions: Chicoric acid significantly suppressed the expression of tumor necrosis factor‐alpha (TNF‐α), interleukin‐6 (IL‐6) in PMACI‐activated HMC‐1 cells by inhibit mRNA expression. Furthermore, chicoric acid reduced the activation of nuclear factor‐kappa B (NF‐κB), mitogen‐activated protein (MAP) kinase pathway and caspase‐1. These results suggest that chicoric acid probably prove to be a useful agent for treating anti‐allergic inflammation diseases. Key words: Chicoric acid; Taraxacum; Anti‐allergic effects; Human mast cell (HMC‐1 cells) 최근 아토피성 피부염, 알레르기성 비염과 같은 만성 알레르기성 염증성 질환으로 고통 받는 인구가 증가하였으나 아직까지 알레르기 질환의 효과적인 치료방법은 없었다. 따라서 알레르기 질환 치료에 효과적인 성분을 찾는 것이 전 세계적인 관심사로 대두되고 있다. 포공영 뿌리의 주성분인 chicoric acid는 페놀 화합물의 일종으로써 항산화작용, HIV‐1 억제효과, 인슐린 분비 자극, 항염 작용 등이 보고되어 있으나 알레르기 염증 반응 조절기전에 대한 연구는 아직 보고된 바 없다. 본 연구에서는 염증 유발된 HMC‐1에서 chicoric acid가 알레르기 염증 반응을 효과적으로 억제하는가를 관찰하였다. 연구 결과 chicoric acid는 TNF‐α, IL‐6 와 같은 염증성 사이토카인의 발현을 감소시켰으며 pro‐caspase‐1의 분해 억제를 통해 caspase‐1의 활성을 효과적으로 억제하였다. 또한 이러한 염증성 사이토카인 발현 억제 효과는 IκBα의 분해 억제를 통한 NF‐κB의 핵 내 이동 및 활성화 억제와 JNK, ERK, p38의 인산화 억제를 통한 MAPK 신호전달경로의 억제 기전을 통한 것임을 확인하였다. 결론적으로, chicoric acid는 염증성 사이토카인의 발현을 차단하여 비만세포 유래 알레르기 염증 반응을 효과적으로 조절하는 것으로 확인되었다. 이러한 연구 결과를 바탕으로 포공영에 함유된 chicoric acid가 알레르기질환 치료에 효과적인 후보약물이 될 수 있을 것이라 사료된다.