박해심 김서희 2023-02 32714 https://aurora.ajou.ac.kr/handle/2018.oak/24368 학위논문(박사)--아주대학교 일반대학원 :의생명과학과,2023. 2 배경 약물 재창출은 기존의 새로운 약물 개발 접근 방식에 비해 시간과 비용을 절약하며 기존 약물의 새로운 용도를 발견할 수 있는 방법으로, 최근 많은 주목을 받고 있다. 특정한 질병과 관련된 약물은 다른 질병과의 상호 의존 및 유사한 메커니즘으로 인해 다른 질병에도 사용될 수 있다. Tirofiban 은 심혈관 및 항 혈전 효과에 널리 사용되는 GPIIb/IIIa 수용체 억제제이며, 혈소판과 백혈구 간의 접촉 의존적 상호작용의 주요 인자로도 알려져 있다. 지금까지 천식에서 혈소판과 백혈구 사이의 직접적인 접촉 의존적인 상호작용에 대한 연구는 거의 없다. 또한, flavonoid 는 야채, 과일 음료수에 존재하는 폴리페놀 대사사물로 aryl hydrocarbon receptor (AhR)에 대한 억제효과를 통해 항산화 및 면역조절기능이 있다. 천연 flavonoid 인 apigenin 은 AhR 과 다양한 화합물의 결합을 억제하여 디젤 호기 입자 및 미세먼지와 같은 인공물질로 인해 유발되는 알레르기 염증을 약화시키는 것으로 알려져 있다. 목적 첫째 tirofiban 투여가 천식에서 혈소판과 호산구 응집의 억제를 통해 알레르기 염증과 기도과민성 감소를 유도하는지을 평가하였다. 두번째로 천식을 포함하는 알레르기 질환에서 환경호르몬이 미치는 영향을 평가하고, 알레르기 천식 모델에서 잠재적 치료제로서 apigenin 의 효과를 평가했다. 마지막으로, 사람 비만세포와 두드러기 환자에서 환경호르몬이 미치는 영향 확인했다. 재료 및 방법 Tirofiban 의 효과를 조사하기 위해 BALB/c 마우스를 0 일과 14 일에 OVA으로 복강 내 주사로 감작시킨 후 28일에서30일까지 3일동안 흡입기를 통해 OVA 를 투여하였다. 마우스에서 기도 과민성과 기도염증 및 혈소판과 백혈구의 응집도에 대해 평가했으며, 혈소판과 호산구의 활성화 수준을 평가했다. 마우스 폐 조직과 혈액에 대한 혈소판와 호산구의 발현 수준은 형광 염색과 유세포 분석을 통해 평가하였다. Apigenin 의 투여효과를 알아보기 위해서 MnBP 에 의해 악화되는 호산구 천식 마우스 모델을 구축하였다. MnBP 는 실험기간 동안 음용수 투여되었고, 길항제인 apigenin 은 ovalbumin 흡입기 투여 전 14 일 동안 경구투여 되었다. 마우스의 기도과민성, 기관지 폐포 세척액의 세포 수, 사이토카인을 측정하고 MnBP 와 apigenin 의 효과를 호산구, 상피세포, T 세포주에서 평가했다. 또한 10 명의 건강한 대조군과 두드러기 증상이 있는 20 명의 성인 환자에서 환경호르몬의 노출과 두드러기 사이의 연관성을 조사했다. 소변 내의 환경호르몬의 수치를 고성능 액체 크로마토그래피 질량분석기를 이용하여 측정하였고, 환경호르몬을 처리 후 사람 비만세포에서 유래물질의 양을 측정하였다. 결과 Tirofiban 투여는 기관지폐포 세척액에서 기도과민성과 호산구 염증 지표들을 감소시켰을 뿐만 아니라 인터루킨-4, -5, -13 의 발현을 감소시켰다. 또한 조직학적 분석에서도 기도염증을 감소시켰다. Tirofiban 을 통한 GPIIb/IIIa 수용체의 차단은 양성 대조군 마우스보다 혈액 및 폐 조직 모두에서 혈소판과 호산구의 응집도를 유의하게 감소시켰으며, 호산구와 혈소판의 활성이 모두 감소되었다. 환경호르몬 수용체로 알려진 aryl hydrocarbon receptor 의 발현은 MnBP 투여 시 현저하게 증가하였고, 기도과민성, 호산구를 포함한 기도 염증 세포, 2 형 사이토카인의 발현을 모두 증가시켰다. 그러나 apigenin 처리는 AhR 발현과 모든 천식 특징들을 감소시켰다. MnBP 는 호산구, 상피세포, T 세포의 활성화를 유도하는 반면, apigenin 처리는 MnBP 의 활성화로 인한 모든 효과를 감소시켰다. 특히 환경호르몬의 대사산물 중 DEHP 의 발현 정도는 건강한 대조군과 비교하여 두드러기 환자의 소변에서 유의하게 증가하였다. 사람 비만 세포에서 beta-hexosaminidase 의 분비 또한 음성대조군과 비교하여 환경호르몬 처리시 더 크게 증가하였으며, MECCP 를 가장 낮은 농도로 비만세포에 처리했을 때에 가장 높은 분비량을 보였다. 결론 특정 알레르기 질환에 맞는 동물모델을 통해서 tirofiban 과 apigenin 의 약물 재창출 가능성을 확인하였다. Tirofiban 으로 GPIIb/IIIa 수용체 차단 시 혈소판과 호산구의 응집 및 활성화 억제를 통해 기도과민성과 기도염증을 약화시키고, flavonoid 는 AhR 발현을 억제하여 환경호르몬으로 인한 악화된 천식에서 잠재적인 치료제가 될 수 있음을 확인하였다. 또한 환경호르몬은 호산구 염증 및 만성 두드러기의 위험인자 중 하나로 작용할 수 있음을 확인하였다. The effect of tirofiban on the mouse model of asthma 1 I. INTRODUCTION 2 II. MATERIALS AND METHODS 4 A. Mouse 4 B. Ethics statement· 4 C. Eosinophilic asthma mouse model and tirofiban treatment 4 D. Measurement of airway resistance and sample collection 4 E. Harvest of the BALF and measurement of differential cell counts 5 F. Measurement of cytokine levels 5 G. Identification of platelet eosinophil aggregation (PEA) in whole blood 5 H. Detection MAC-1 of by western blotting 6 I. Immunofluorescence staining 6 J. Antibodies and reagents 7 K. Statistical analysis 7 III. RESULTS 8 A. The effects of tirofiban on airway resistance and inflammation 8 B. Staining in the lung tissues 9 C. The percentage of PEA in whole blood of mice 10 D. Tirofiban inhibits both platelet and eosinophil activation in the BALF 11 E. The levels of EPX and PSGL-1 in the lung tissues 11 F. Tirofiban decreases adhesion molecules on eosinophil surface 12 IV. DISCUSSION 14 The effect of phthalates as an aggravating factor on allergic diseases 16 I. INTRODUCTION 17 II. MATERIALS AND METHODS 19 A. Mouse 19 B. Eosinophilic asthma mouse model, MnBP and apigenin treatment 19 C. Evaluation of airway resistance, cytokines in the BALF, and the lung histology 20 D. Western blotting to detect aryl hydrocarbon receptor and eosinophilic granular proteins 20 E. cDNA preparation and real time polymerase chain reaction 21 F. Mononuclear cell isolation and ex vivo cytokines measurement 21 G. In vitro cells preparation and culture 21 H. A549 cell by siRNA transfection 22 I. Measurement on β-hexosaminidase release in LAD2 cells 22 J. Measurement of phthalates in urine of patients with chronic urticaria and healthy controls 23 K. Statistical analysis 24 III. RESULTS 25 A. The expression of aryl hydrocarbon receptor in the MnBP-treated allergic asthma 25 B. The effects of MnBP and apigenin on allergic asthma mouse 26 C. The effects of MnBP and apigenin on eosinophilic activation on allergic mouse model 28 D. Ex vivo effects of MnBP and apigenin on type II cytokine productions on the splenic MNCs 29 E. In vitro effects of MnBP and apigenin on type II cytokine productions using Jurkat cells 30 F. The effects of MnBP and apigenin on eosinophilic activation on Eol-1 cells 30 G. The inhibitory effects of aryl hydrocarbon receptor using siRNA and its antagonist in vitro epithelial cells 31 H. Stimulatory effects of beta-hexosaminidase release on phthalates stimulated LAD 2 cells 32 I. The comparison of concentrations of phthalate metabolites in urine 33 IV. DISCUSSION 35 V. CONCLUSION 39 REFERENCES 40 국문요약 49 eng The Graduate School, Ajou University 아주대학교 논문은 저작권에 의해 보호받습니다. The utilization of drug repositioning in allergic disease Thesis 아주대학교 대학원 Seo Hee Kim 일반대학원 의생명과학과 2023-02 Doctor https://dcoll.ajou.ac.kr/dcollection/common/orgView/000000032714 Asthma Eosinophil Flavonoid Phthalate Tirofiban Background Drug repositioning has attracted a lot of attention due to its potential for discovering new indications for existing drugs and due to its efficiency in saving time and cost over the traditional de novo drug development approaches. Drugs related to a specific disease may work on other diseases due to the interdependence and similar mechanisms between these different diseases. Tirofiban is a GPIIb/IIIa receptor inhibitor widely used for cardiovascular and antithrombotic effects. The GP IIb/IIIa receptor is also known as a major factor for contact-dependent interactions between platelets and leukocytes. There have been few studies on direct contact-dependent interactions between platelets and leukocytes in asthma. Flavonoids are polyphenolic metabolites present in vegetables, fruits, and beverages, and have antioxidant and immune-modulating potencies through an inhibitory effect on aryl hydrocarbon receptor (AhR) which acts as a systemic environmental sensor. The natural flavonoid, apigenin, is known to attenuate artificial material induced allergic inflammation such as diesel exhaled particles and particulate matter through inhibition of binding between AhR and these kinds of diverse compounds. Objective Firstly, I investigated whether tirofiban treatment can attenuate allergic inflammation and airway hyperresponsiveness through inhibition of platelet–eosinophil aggregation (PEA) in asthma. Secondly, I evaluated the contribution of endocrine disruptors to the exacerbation of inflammatory diseases including asthma and evaluated the effect of apigenin as potential therapeutics on allergic asthma model. Thirdly, I confirmed the effect of phthalate on human mast cells and urticaria patients. Materials & Methods To investigate the effect of tirofiban, BALB/c mice were sensitized by intraperitoneal injection of ovalbumin (OVA) on days 0 and 14, followed by 3 nebulized OVA challenges on days 28–30. On each challenge day, 5 mg/kg tirofiban was administered intraperitoneally 30 min before the challenge. Mice were assessed for airway hyperresponsiveness (AHR), airway inflammation, and the degree of PEA. Finally, the activation levels of platelets and eosinophils were evaluated. The expression levels of platelets and eosinophils aggregation on the lung tissues and mice blood were evaluated by fluorescence staining and flow cytometry. To assess the therapeutic effect of apigenin, I established on eosinophilic asthma model exacerbated by mono-n-butyl phthalate (MnBP). MnBP was administered through drinking water administration throughout the study period, and its antagonist, apigenin, was orally treated for 14 days before OVA challenges. Mice were assessed for airway AHR, differential cell count and type 2 cytokines in the bronchoalveolar lavage fluid (BALF) were measured in vivo, and the effects of MnBP and apigenin were evaluated on eosinophils, epithelium, and T cell-line in vitro. I also investigated the association between phthalate exposure and urticaria in 10 healthy controls and 20 adult patients with active urticarial symptoms. The urinary levels of mono-n-butyl phthalate ( MnBP), mono-2-ethyl-5-carboxypentyl phthalate (MECPP), mono-(2-ethyl-5-oxohexyl) phthalate, and mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) were measured by using high performance liquid chromatography tandem mass spectrometry, and human mast cell releasability was determined after phthalate treatment. Results Tirofiban treatment decreased AHR and eosinophilic inflammation in the BALF. This treatment also reduced the levels of interleukin (IL)-4, IL-5, and IL-13 in the BALF and airway inflammatory cell infiltration in histological evaluation. Moreover, the blocking of the GP IIb/IIIa receptor was significantly reduced PEA in both blood and the lung tissues in tirofiban-treated mice than in those of the positive control mice, and both eosinophilic and platelet activations were attenuated in tirofiban-treated mice. The expression of the aryl hydrocarbon receptor known as a receptor of phthalate was markedly increased when MnBP was administered, and MnBP treatment increased AHR, airway inflammatory cells including eosinophil, and type 2 cytokines following OVA challenge compared to those of vehicle-treated mice. However, apigenin treatment reduced all asthma features such as AHR, airway inflammation, type 2 cytokines, and the expression of aryl hydrocarbon receptor in MnBP augmented eosinophilic asthma. Additionally, in an in vitro study, MnBP induced activation of eosinophil, epithelial cell, and T cell, in contrast, apigenin treatment reversed all effects of MnBP. The levels of phthalate metabolites, especially di-ethylhexyl phthalate (DEHP), are significantly increased in the urine of patients with urticaria compared to the healthy controls. The release of β-hexosaminidase in human mast cells is more significantly increased by MnBP, mono-benzyl phthalate, mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), and mono-(2-ethyl-5-oxohexyl) phthalate (MECPP) compared to the negative controls; interestingly, the highest secretion of β-hexosaminidase is observed after the lowest stimulation of MECPP. Conclusions I confirmed the possibility of drug repositioning in substances such as tirofiban and apigenin through a disease-specific asthma model. The blocking of GP IIb/IIIa receptor with tirofiban can attenuate AHR and airway inflammation through the inhibition of PEA. The inhibition of AhR using flavonoid can be potential therapeutic targets in endocrine disrupting chemical-aggravated asthma. Phthalates may be a risk factor for aggravating eosinophilic inflammation and symptoms of chronic spontaneous urticaria.