銀屑病是一種慢性炎症性皮膚病，其發病率約佔全球1-3%的人口。銀屑病的病理特徵包括角質細胞增殖和分化異常，同時伴隨炎症反應，白細胞聚集於真皮和表皮以及血管擴張。證據顯示角質細胞能參與及延續免疫反應，以達致維持或促進該病的作用。研究亦建議角質細胞減少凋亡是引致銀屑病的一個特定現象；因此，長期以來誘導角質細胞凋亡就被用作為治療銀屑病的一種有效策略。 / 根據銀屑病的嚴重程度，治療方法可分為三級：外用藥物主要用於比較輕微的病患，而光療適合中等程度的病患；對於嚴重病例則可使用系統性治療或生物製劑。基於大約75%的銀屑病患者屬於輕微至中度病患，外用藥物是目前應用最為廣泛的治療方法。在中國銀屑病治療的歷史中曾經使用過中草藥，研究亦表明，其治療機制可能通過抑制角質細胞增殖和誘導角質細胞凋亡。比較研究也指出，傳統中藥比西藥的副作用相對較少，及具有較長的舒緩期和較低的復發率。 / 我們先前的研究發現，茜草根提取物能夠抑制一個和銀屑病相關的HaCaT角質細胞增殖。本研究證實，茜草根的乙酸乙酯提取物（EA）能誘導HaCaT細胞凋亡，其抑制角質細胞增殖的作用比茜草根的乙醇提取物（EE）更為有效，並可和一個流行於歐洲國家的重要外用銀屑病治療藥地蒽酚相比。另外，透過不同的檢測，包括形態學觀察，細胞凋亡雙染（磷脂結合蛋白V-碘化丙啶）分析，細胞週期分析，去氧核醣核酸斷裂測試，原位末端轉移酶標記技術，免疫熒光染色以及西方墨點法，我們發現一種在茜草中的化合物，1,4-二羥基-2-萘甲酸（DHNA）能通過死亡受體介導，線粒體介導或不依賴胱天蛋白酶的途徑導致HaCaT細胞凋亡。同時，在其中一種銀屑病動物模型，小鼠鼠尾鱗片表皮上的初步研究顯示DHNA亦可誘導角質細胞分化。此外，在細胞水平（存活率，釋放白细胞介素-1α）和動物上（Draize動物皮膚刺激性試驗）的實驗結果表明DHNA比地蒽酚的刺激性較小。 / 總括而言，本研究透過人類皮膚細胞和動物實驗說明EA和DHNA的細胞凋亡機制，以及DHNA對皮膚的潛在刺激性。這些結果顯示EA和DHNA有潛能發展成為安全及能有效治療銀屑病的替代藥物。EA和DHNA可在一個連續療程中結合使用，其中EA藥效媲美地蒽酚，應能迅速清除銀屑病皮損；而DHNA比地蒽酚的刺激性小，則比較適合應用在這個連續療程中後來的維護保養階段 / Psoriasis is a chronic inflammatory skin disorder that affects approximately 1-3% of the population worldwide. It is characterized by epidermal hyperplasia or abnormal differentiation, infiltration of leucocytes into the dermis and epidermis, dilation of blood vessels in dermis and inflammation. Evidence indicates keratinocytes contributed to the disease, and keratinocytes also participate in maintaining the chronically perpetuating immune response that sustains psoriasis. Decrease in keratinocytes apoptosis is suggested to be a specific pathogenic phenomenon, and induction of keratinocytes apoptosis have long been considered as an effective anti-psoriatic strategy. / Treatment of psoriasis is based on disease severity. Topical agents are predominantly for mild conditions; phototherapy for moderate conditions and systemic treatment or biological agents for severe cases. Topical treatment remains the most widely used method as an estimated 75% of psoriatic patients have mild to moderate disease. Chinese herbs have been used for the treatment of psoriasis in China, and studies showed their mechanism on treating psoriasis may through inhibition of keratinocyte proliferation and induction of apoptosis. Comparison studies also show that traditional Chinese medicine has relatively fewer side effects than western therapeutic agents, with a longer remission time and lower recurrence rate. / The extract of the root of Rubia cordifolia L. (Rubiae Radix et Rhizoma) was previously found to inhibit keratinocyte proliferation using a psoriasis-relevant HaCaT cells model. In this study, the ethyl acetate extract of the root of Rubia cordifolia L. (EA) was confirmed to induce apoptosis on HaCaT cell, and the antiproliferative effect of EA is more potent than the ethanol extract of the herb (EE) and is comparable to dithranol, an important and popular topical treatment for psoriasis among Europe countries. Besides, we identified one of the components in Rubia cordifolia L., 1,4-dihydroxy-2-naphthoic acid (DHNA), could induce HaCaT keratinocyte apoptosis through the death receptor and mitochondria mediated pathway as well as in a caspase independent manner using various assays such as morphological examination, annexin V-PI staining, cell cycle analysis, DNA fragmentation, TUNEL assay, immunofluorescence staining and Western blot analysis. Moreover, DHNA was found to induce keratinocyte differentiation in a preliminary study using the in vivo mouse tail model of psoriasis. Furthermore, results from in vitro (cell viability, IL-1α release) and in vivo (Draize animal skin irritation test) experiments suggested DHNA have less irritation problems than dithranol. / In summary, this study describes the apoptotic mechanism of EA and DHNA, as well as the irritation potential of DHNA using different human skin cells and animal model. These results suggest EA and DHNA have the potential to develop as safe and effective therapeutic alternative for the treatment of psoriasis. EA and DHNA can be used together in a sequential therapy, in which EA is effective in rapid clearing of psoriatic lesions as its potency is comparable to dithranol; whereas DHNA is better suited for the later maintenance therapy for its milder irritation effect compared with dithranol. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Mok, Chong Fai. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 164-183). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract (English version) --- p.iv / Abstract (Chinese version) --- p.vi / List of Publication and Presentation --- p.viii / Acknowledgements --- p.ix / Table of Contents --- p.x / List of Tables --- p.xvi / List of Figures --- p.xvii / List of Abbreviations --- p.xx / Chapter Chapter 1: --- Introduction --- p.1 / Chapter 1.1. --- Psoriasis --- p.1 / Chapter 1.1.1. --- Histological features --- p.2 / Chapter 1.1.2. --- Role of keratinocytes in psoriasis --- p.4 / Chapter 1.1.3. --- Decrease in skin cell apoptosis --- p.8 / Chapter 1.2. --- Treatment of psoriasis --- p.9 / Chapter 1.2.1. --- Conventional treatment --- p.9 / Chapter 1.2.1.1. --- Mild disease --- p.10 / Chapter 1.2.1.1.1. --- Corticosteroids --- p.10 / Chapter 1.2.1.1.2. --- Vitamin D₃ analogs --- p.11 / Chapter 1.2.1.1.3. --- Tazarotene --- p.11 / Chapter 1.2.1.1.4. --- Anthralin --- p.12 / Chapter 1.2.1.1.5. --- Coal tar --- p.12 / Chapter 1.2.1.2. --- Moderate disease --- p.12 / Chapter 1.2.1.2.1. --- Phototherapy --- p.12 / Chapter 1.2.1.3. --- Severe disease --- p.13 / Chapter 1.2.1.3.1. --- Retinoids --- p.13 / Chapter 1.2.1.3.2. --- Methotrexate --- p.14 / Chapter 1.2.1.3.3. --- Cyclosporine --- p.14 / Chapter 1.2.1.3.4. --- Fumaric acid --- p.15 / Chapter 1.2.1.3.5. --- Biological agents --- p.15 / Chapter 1.2.2. --- Alternative treatment --- p.16 / Chapter 1.2.2.1. --- Traditional Chinese Medicine (TCM) --- p.17 / Chapter 1.3. --- Aims and objectives of the present study --- p.19 / Chapter Chapter 2: --- Apoptotic Action of Ethyl Acetate Fraction of the Root of Rubia cordifolia L. (Rubiae Radix et Rhizoma) on HaCaT Human Keratinocytes --- p.21 / Chapter 2.1. --- Introduction --- p.21 / Chapter 2.1.1. --- Rubia cordifolia L. --- p.21 / Chapter 2.1.2. --- Apoptosis --- p.22 / Chapter 2.1.3. --- Study objectives --- p.28 / Chapter 2.2. --- Materials and Methods --- p.30 / Chapter 2.2.1. --- Sources of medicinal materials --- p.30 / Chapter 2.2.2. --- Preparation of extracts --- p.30 / Chapter 2.2.3. --- Reagents --- p.31 / Chapter 2.2.4. --- Cell culture --- p.31 / Chapter 2.2.5. --- Proliferation assay --- p.32 / Chapter 2.2.6. --- Fluorescent staining for morphological evaluation --- p.33 / Chapter 2.2.7. --- Annexin V/propidium iodide staining --- p.33 / Chapter 2.2.8. --- JC-1 staining --- p.34 / Chapter 2.2.9. --- Statistical analysis --- p.35 / Chapter 2.3. --- Results --- p.36 / Chapter 2.3.1. --- EA inhibits proliferation of human epidermal HaCaT keratinocytes --- p.36 / Chapter 2.3.2. --- Alteration of cellular morphology --- p.39 / Chapter 2.3.3. --- EA increases phosphatidylserine externalization in HaCaT cells --- p.41 / Chapter 2.3.4. --- EA decreases MMP --- p.45 / Chapter 2.4. --- Discussion --- p.47 / Chapter Chapter 3: --- Identification of Pure Compound for Possible Apoptotic Action on HaCaT Human Keratinocytes and Detailed Mechanistic Study --- p.51 / Chapter 3.1. --- Introduction --- p.51 / Chapter 3.1.1. --- Anthraquinone --- p.51 / Chapter 3.1.2. --- Study objectives --- p.52 / Chapter 3.2. --- Materials and Methods --- p.54 / Chapter 3.2.1. --- Reagents --- p.54 / Chapter 3.2.2. --- Cell culture --- p.54 / Chapter 3.2.3. --- Proliferation assay --- p.55 / Chapter 3.2.4. --- Fluorescent staining for morphological evaluation --- p.56 / Chapter 3.2.5. --- Annexin V/propidium iodide staining --- p.56 / Chapter 3.2.6. --- JC-1 staining --- p.56 / Chapter 3.2.7. --- Cell cycle analysis --- p.56 / Chapter 3.2.8. --- Detection of DNA fragmentation --- p.57 / Chapter 3.2.9. --- Terminal Deoxynucleotidyltransferase-Mediated dUTP Nick End Labeling (TUNEL) assay --- p.57 / Chapter 3.2.10. --- Western blot analysis --- p.58 / Chapter 3.2.11. --- Immunofluorescence staining --- p.59 / Chapter 3.2.12. --- Statistical analysis --- p.60 / Chapter 3.3. --- Results --- p.61 / Chapter 3.3.1. --- DHNA inhibits proliferation of human epidermal HaCaT Keratinocytes --- p.61 / Chapter 3.3.2. --- Alteration of cellular morphology --- p.70 / Chapter 3.3.3. --- DHNA increases phosphatidylserine externalization in HaCaT cells --- p.72 / Chapter 3.3.4. --- DHNA decreases MMP --- p.76 / Chapter 3.3.5. --- DHNA causes G0/G1 cell cycle arrest in HaCaT cells --- p.78 / Chapter 3.3.6. --- DHNA increases DNA fragmentation --- p.81 / Chapter 3.3.7. --- DHNA increases TUNEL positive cells in HaCaT cells --- p.83 / Chapter 3.3.8. --- Western blot analysis --- p.85 / Chapter 3.3.9. --- DHNA induced Fas aggregation in HaCaT cells --- p.88 / Chapter 3.3.10. --- Caspase inhibition assay --- p.90 / Chapter 3.3.11. --- DHNA induced caspase independent apoptosis in HaCaT cells --- p.93 / Chapter 3.3.12. --- Effects of DHNA on MAPK in HaCaT cells --- p.96 / Chapter 3.3.13. --- MAPK inhibition assay --- p.100 / Chapter 3.4. --- Discussion --- p.104 / Chapter Chapter 4: --- Anti-Psoriatic Effects of Topical 1,4-Dihydroxy-2-naphthoic acid Formulation on in vivo Mouse Tail Experiments --- p.111 / Chapter 4.1. --- Introduction --- p.111 / Chapter 4.1.1. --- Keratinocytes differentiation process --- p.111 / Chapter 4.1.2. --- Animal model for psoriasis --- p.114 / Chapter 4.1.3. --- Study objectives --- p.119 / Chapter 4.2. --- Materials and Methods --- p.122 / Chapter 4.2.1. --- Reagents --- p.122 / Chapter 4.2.2. --- Formulation and preparation of topical drug --- p.122 / Chapter 4.2.3. --- Mice for in vivo experiments --- p.123 / Chapter 4.2.4. --- Treatment with topical preparations --- p.124 / Chapter 4.2.5. --- Statistical analysis --- p.125 / Chapter 4.3. --- Results --- p.126 / Chapter 4.3.1. --- Tail skin appearance after topical treatment --- p.126 / Chapter 4.3.2. --- Histological examination and findings --- p.128 / Chapter 4.4. --- Discussion --- p.132 / Chapter Chapter 5: --- Prediction of Skin Irritation Potential of 1,4-Dihydroxy-2-naphthoic acid by in vitro and in vivo Experiments --- p.135 / Chapter 5.1. --- Introduction --- p.135 / Chapter 5.1.1. --- Skin irritation --- p.135 / Chapter 5.1.2. --- Viability test and IL-1α release --- p.136 / Chapter 5.1.3. --- Animal irritation test --- p.139 / Chapter 5.1.4. --- Study objectives --- p.139 / Chapter 5.2. --- Materials and Methods --- p.141 / Chapter 5.2.1. --- Reagents --- p.141 / Chapter 5.2.2. --- Cell culture --- p.141 / Chapter 5.2.3. --- Viability test --- p.141 / Chapter 5.2.4. --- IL-1α release assay --- p.142 / Chapter 5.2.5. --- Animal irritation test --- p.142 / Chapter 5.2.6. --- Statistical analysis --- p.143 / Chapter 5.3. --- Results --- p.144 / Chapter 5.3.1. --- Viability test --- p.144 / Chapter 5.3.2. --- IL-1α release assay --- p.144 / Chapter 5.3.3. --- Animal irritation test --- p.147 / Chapter 5.4. --- Discussion --- p.152 / Chapter Chapter 6: --- General Discussion and Conclusions --- p.155 / References --- p.164
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328053 |
| Date | January 2012 |
| Contributors | Mok, Chong Fai., Chinese University of Hong Kong Graduate School. Division of Biomedical Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xxi, 183 leaves) : ill. (some col.) |
| Coverage | China, China |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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