臺灣博碩士論文加值系統

活性氧分子(ROS)是細胞有氧代謝的副產物與免疫反應中細胞相互傳遞訊號的分子。不平衡的氧化壓力可能會造成諸多疾病，包含神經性疾病與自體免疫疾病等。菸醯胺腺嘌呤二磷酸氧化酶(NADPH oxidase)為免疫系統中產生ROS的重要，在近年的研究中發現，NADPH oxidase影響自體免疫疾病的發展與疾病程度。以免疫介導的關節炎為例，在實驗室先前的研究中發現，當老鼠體內白血球缺乏了NADPH oxidase的情況下，以血清誘發關節炎後，會有大量白血球驅化激素與介白素-1產生，造成發炎反應明顯比起wild type老鼠來的嚴重。由此推論，氧化壓力可能扮演著調控發炎反應的角色。目前臨床上所治療關節炎的藥物，尚未有以調節ROS為治療目標的藥物。
本實驗以篩選具有調控白血球活性氧分子為目標，找尋具有潛在能力的治療藥物。我們以美國食品藥物管理局核可的藥物，建立高通量篩選平台，找出具有調節ROS的藥物。首先我們利用致關節炎血清於wild type老鼠與NADPH oxidase缺失鼠中誘發關節炎，結果如預期發現白血球NADPH oxidase缺失鼠有較嚴重的發炎反應，並且證明在這些缺失鼠中關節的氧化壓力也較低。在過去的研究中已知維生素K3 (menadione)能刺激白血球產生ROS，因此我們給予10mg/kg與25mg/kg的menadione以腹腔注射給予治療，發現相較於為治療的組別，給予維生素K3治療的血清誘發關節炎NADPH oxidase缺失鼠，關節的發炎反應有明顯的減緩，證明ROS能減緩關節的發炎反應。接著我們利用H2DCF-DA為檢測ROS產生的指示劑，並偵測嗜中性白血球所釋放的螢光值。發現在給予phorbol 12-myristate 13-acetate (PMA)與menadione皆能刺激白血球產生ROS，並且產生ROS的來源分別為依賴與非依賴NADPH oxidase路徑。我們以類嗜中性白血球細胞為模型建立MetaXpress高通量篩選平台，篩選促進氧化壓力之分子與抑制氧化壓力之分子。在此平台我們以menadione為促ROS正對照組，而PMA為給予ROS生成的刺激物進行篩選。在LOPAC1280 drug library 240隻藥物中以single-shot、3個濃度的方式進行初篩，並利用Z-factor (Z`)為quality control，normalized percentage(Np)與strictly standardized mean difference (SSMD)為計算方式篩選出”hit”。於促氧化合物的結果中，我們以Np≥3和/或SSMD≥3為基準，在240隻藥物中挑選出總共18 藥物。而在抗氧化合物中，隨著劑量而抑制ROS生成化合物為篩選標準，從中找尋到12支藥物。未來計畫近一步進行二次篩選，並將二篩後的藥物在關節炎老鼠中進行試驗。由於篩選之促氧或抑氧藥物為臨床用藥，因此我們可以利用已知的作用機制治療不同疾病，或是進行藥物包裹(如奈米包裹藥物)進行投藥，達成更為精準的治療。此研究結果能在未來臨床治療上，為氧化壓力失調的疾病提供多一個藥物治療的可能性。

Reactive oxygen species (ROS) are byproducts of cellular metabolism which may play important roles for signal transduction in immune cells. Although ROS were thought to cause toxic effects associated with various pathologies, previous studies indicated that lack of ROS production might raise the severity of the autoimmune disease. Our previous studies showed that in Ncf1-/- mice, which lack of leukocyte NADPH oxidase (NOX2), ROS act as negative feedback to regulate IL-1β-mediated inflammation and enhance the severity of inflammation in a model of arthritis joint than in wild-type mice. Thus, we hypothesize that modulates the leukocyte ROS production may regulate the pathogenicity of immune-mediated arthritis. However, there were no clinical drugs which regulate ROS as target for treatment in immune-mediated arthritis.
In this study, we aim to set up a high-content screening system with FDA approve drugs to screen redox modulators. We developed an image-based high-throughput screening platform on differentiated neutrophil-like HL-60 (dHL-60) to identify anti- or pro-oxidant molecules. We also performed the experiment to characterize the level of ROS in the WT and Ncf1-/- serum-induced arthritis.
We found that serum-induced joint inflammation was more severe but the ROS level was lower in Ncf1-/- mice compared with WT mice. The severity of inflammation attenuated in the treatment of giving 10 mg/kg or 25 mg/kg menadione which may regulate the redox level in arthritic joints in Ncf1-/- mice. ROS level was stained with H2DCF-DA and measured with flow cytometry, we found that phorbol 12-myristate 13-acetate (PMA) and menadione could stimulate neutrophils to produce ROS by NOX2-dependent and NOX2-independent pathway, respectively. We set up the high-content screening system with MetaXpress acquisition system to screen anti- or pro-oxidants. We screened 240 compounds from LOPAC1280 library in a single-shot, 3-point interpolate titration (final concentrations: 5,10 and 20 M). Z-factors were considered as the quality control readouts while Strictly standardized median difference (SSMD) ≥ 3 and normalization ≥0.3 were used as the criterion for hit selection in the assay. We found 18 of the drugs enhanced the ROS production while 12 of the compounds established anti-oxidant.
As the compounds we discovered were FDA approved drugs which were already used in clinical treatment, we could further classify their mechanism and delivered this compounds with different packing components such as nanoparticles to enhance the specificities of treatment. These compounds will further undergo a secondary screening and animal disease model. The potential drugs we discovered in this project may establish a new strategy for treating redox-related inflammatory diseases.

Content
Abstract I
中文摘要 III
致 謝 V
Abbreviations VI
Chapter 1 Introduction 1
1.1 Innate immune response and phagocytes 2
1.2 Respiratory burst and leukocyte NADPH oxidase 3
1.2.1 Respiratory burst 3
1.2.2 Leukocyte NADPH oxidase system 4
1.3 Reactive oxygen species and redox homeostasis 5
1.3.1 Reactive oxygen species 5
1.3.2 ROS homeostasis 6
1.4 Regulation of ROS in immune-mediated disease 7
1.5 Drug repurposing and high-content screening 9
1.5.1 Drug repurposing 9
1.5.2 High-content screening system 10
1.6 The aim of this study 11
Chapter 2 Materials and Methods 12
2.1 Reagents 13
2.2 Mice 13
2.3 Serum-induced arthritis 14
2.4 Preparation of protein extracts from wrists and ankles 14
2.5 Measured the ROS level of joints 14
2.5.1 Evaluation of the lipid peroxidation by Malondialdehyde assay 14
2.5.2 Assessment the level of protein oxidation 15
2.6 Cell culture 15
2.7 Granulocytes isolation 16
2.8 ROS measurement by FACS analysis 16
2.9 Preparation for the primary screening of LOPAC 1280 drug library 17
2.10 High-throughput screening for ROS-modulators in the LOPAC 1280 drug library 17
2.11 Data analysis for ROS assays on the high-content platform for LOPAC1280 screening 18
2.12 Statistical analysis 19
Chapter 3 Results 20
3.1 Arthritogenic serum transfer induced more severe joint inflammation and lower levels of tissue oxidation in the NOX2-deficient mice 21
3.2 PMA and Menadione induced ROS production through NADPH oxidase-dependent and independent pathways in dHL-60 cells 21
3.3 The development and optimization of the ROS assays on the image-based high-throughput high-content acquisition system 22
3.4 Establishment of methods measuring ROS production by leukocytes 23
3.5 Verification of the performance of a high-content screening assay with known redox-active compounds 23
3.6 Identification of compounds with redox modulating activity on leukocytes from 240 drugs of LOPAC1280 24
3.6.1 Identification of 18 Pro-oxidants 24
3.6.2 Identification of 12 Anti-oxidants 25
3.6.3 Identification of priming substances 25
3.7 Menadione treatment decreased the severity of serum-induced arthritis in Ncf1-/- mice 26
Chapter 4 Discussion 27
4.1 The mechanism of regulating ROS production in the compounds of primary screening 29
4.2 Nanoparticles package the ROS modulators for specific drug delivery 32
Chapter 5 Tables and Figures 34
Table 1. Multiple cell scoring analysis and top-hat segmentation method of cell definition 35
Table 2. Pro-oxidants screened from 240 drugs of LOPAC1280 36
Table 3. The anti-oxidants screened from 240 drugs of LOPAC1280 library 37
Figure 1. 38
Figure 1. Flowchart of assays to assess the ROS modulator activity and the design of the platform for high-throughput screening. 39
Figure 2. 40
Figure 2. The severity of arthritis on Ncf1-/- mice was higher while the ROS production in the inflamed joint was lower when compared with those in WT mice 41
Figure 3. 42
Figure 3. Differentiated HL-60 cells produced more ROS after PMA stimulation when compared with undifferentiated HL-60 cells. 43
Figure 4. 44
Figure 4. Neutrophils treated with PMA and menadione produce ROS through NOX2-dependent and independent pathways respectively. 45
Figure 5. 46
Figure 5. Menadione induced the ROS formation in neutrophil-like differentiated HL-60 (dHL-60) cells measured with the high-content screening assay. 47
Figure 6. 48
Figure 6. ROS production measured with high-content screening assays for fMLP-primed neutrophil-like differentiated HL-60 (dHL-60) cells stimulated with PMA showed apparent priming effects of fMLP. 49
Figure 7. 50
Figure 7. The multiple scoring analysis and the pre-test of a high-content screening assay for redox-active compounds. 51
Figure 8. 52
Figure 8. The “hit” compounds which induce the dHL-60 cells to enhance ROS production in the primary screening. 53
Figure 9. 54
Figure 9. The “hit” compounds which reduce the dHL-60 cell ROS production after 50 nM PMA treatment in the primary screening. 55
Figure 10. 56
Figure 10. Menadione treatment attenuated the swelling level of serum-induced arthritic joints in Ncf1-/- mice. 57
Chapter 6 References 58

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