Further mechanistic studies demonstrated that both Radix Rubiae and realgar were capable of inducing cellular apoptosis on HaCaT cells in a dose- and time-dependent manner as shown by morphological inspection, DNA fragmentation, TUNEL assay, cell cycle analysis, annexin V---PI staining and Western blot analysis. HPLC fingerprintings were constructed for quality control of the Radix Rubiae extract using mollugin as the chemical marker. Further phytochemical study found that ethyl acetate fraction of this herb possessed potent growth inhibition on HaCaT cells, with IC50 of 0.9 microg/ml. However, the chemical compounds obtained from commercial sources including mollugin, alizarin, purpurin, and quinizarin failed to induce growth inhibition. Meanwhile, arsenic trioxide, arsenic pentoxide and arsenic iodide, three arsenic salts presented in realgar, had significant anti-proliferative effect on HaCaT cells, with IC50 values of 2.4, 16 and 6.8 microM, respectively; and cellular apoptosis was found to be the underlying mechanism for the observed growth inhibitory activity. Furthermore, Radix Rubiae, realgar and arsenic compounds were also revealed to possess growth inhibition when evaluated in a PHA-activated PBMC model, and all of the substances except arsenic pentoxide significantly attenuated the release of inflammatory cytokines such as IFN-y, TNF-alpha and IL-2 in PBMC, indicating an anti-inflammatory effect. The in vivo mouse tail model experiments demonstrated that arsenic trioxide, arsenic pentoxide and arsenic iodide were able to markedly induce mouse tail keratinocyte differentiation, while such differentiation-modulating effect observed in the fraction of Radix Rubiae was only marginal. / In summary, Radix Rubiae and realgar extracts and three arsenic compounds have been identified and characterized as potential anti-psoriatic agents. The discoveries from the present PhD project not only help put the traditional use of these medicinal substances for psoriasis treatment on a scientific footing, but also open up new opportunities for their development into novel anti-psoriatic therapies. / Psoriasis, a chronic inflammatory skin disorder affecting approximately 2-3% of the population worldwide, is characterized histologically by hyperproliferation and aberrant differentiation of epidermal keratinocytes. Many conventional therapies are offered for psoriasis treatment but there exist problems such as unsatisfactory efficacy, side effects and drug resistance. Many patients therefore turn to alternative and complementary medicines for help. Traditionally, Chinese herbal medicine has been extensively used to treat psoriasis and produced promising clinical results. The present PhD study was conducted to investigate psoriasis-treating Chinese herbal medicines with an aim to identify effective anti-psoriatic agents. Sixty Chinese medicinal materials were selected for the screening project based on their ethnomedical use in psoriasis. The ethanolic extracts of these medicinal substances were evaluated for their anti-proliferative action on cultured HaCaT human keratinocytes using microplate SRB and MTT assays. Among them, the root of Rubia cordifolia L. (Radix Rubiae) and realgar were found to have significant anti-proliferative effects, with IC50 values of 1.4 and 6.6 microg/ml, respectively as measured by MTT assay, while they exerted mild significant cytotoxicity on the human fibroblast Hs-68 cell line. / Tse, Wai Pui. / Advisers: C. T. Che; Z. X. Lin. / Source: Dissertation Abstracts International, Volume: 70-09, Section: B, page: . / Thesis submitted in: October 2008. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 298-340). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

銀屑病是一種免疫相關的慢性炎症性皮膚病，其發病率約占世界人口的1-3%，而現今仍然缺乏有效安全的根治方法。國內外使用中草藥治療銀屑病取得較好的療效，但目前缺少對其進行系統研究和開發。我們研究小組之前對61種常用治療銀屑病中藥進行篩選, 發現中藥茜草根和南蛇藤的乙醇提取物具有強大的抑制表皮細胞增生的作用，本博士研究課題的目的是確定新的安全有效的用于治療銀屑病的中藥化學成分, 並闡明其作用機制。 / 本研究篩選了28種存在于這兩種中藥中的化學單體成分，采用體外培養永生化的人類皮膚良性角質形成細胞株HaCaT, 應用MTT法, 繪制細胞生長曲線，獲得抑制50%細胞生長所需藥物濃度(IC50)。實驗結果發現1-羟基-3-甲基蒽醌（HMA）， 1，4-二氨基-2，3-（2-苯氧基乙氧基）蒽醌 （DBA）和南蛇藤表現了強大的抗表細胞生長作用，其48小時培養後的IC50分別爲17.9，15.8，1.1 μM. 值得一提的是這些化合物對正常人表皮角質細胞HEK和人類成纖維細胞Hs68只有相對輕微細胞毒性。 / 隨後進行的機理研究，通過熒光染色，DNA凝膠電泳，細胞周期檢測，流式細胞計檢測及Western blot 分析結果表明， HMA和南蛇藤素是通過誘導細胞凋亡作用抑制HaCaT細胞生長。其中南蛇藤素通過線粒體凋亡和死亡受體介導的兩種通路誘導細胞凋亡， 其誘導細胞凋亡作用與其抑制核因子-κB在HaCaT細胞中的表達和活化有關。 / 另一方面，DBA 抑制人體表皮角質細胞生長的作用機理在于其對角質細胞終末分化的誘導作用。DBA與HaCaT和HEK細胞共同培養96小時後，能顯著促進細胞角質化外膜形成，同時上調角蛋白K1/10，人體套膜蛋白，轉谷氨酰胺酶-1表達和下調角蛋白K5/14表達。而利用小鼠尾部鱗片表皮模型對HMA的外用制劑進行測試，結果顯示HMA誘導角質細胞終末分化能力較弱。 / 總而言之，本研究課題從兩種中藥中成功發現三個具有較強的抗銀屑病活性的化學單體成分，這些來自中藥的天然産物具有很好的開發成新的銀屑病治療外用制劑的應用前景。 / Psoriasis is an immunologically-mediated chronic inflammatory disease of the skin and joints affecting approximately 1-3% of the world’s population. Traditionally, Chinese medicine has been extensively used both inside and outside China for treating psoriasis with promising clinical results. Based on the promising findings in our previous screening project on 61 psoriasis-treating Chinese medicines which showed the root of Rubia cordifolia L. (Rubiae Radix) to have potent anti-psoriatic action, the present study aimed to identify active anti-psoriatic chemical constituents derived from Rubiae Radix and another Chinese herb namely Celastrus orbiculatus Thunb. and to elucidate the underlying mechanisms of action. / Microplate MTT assay was performed to evaluate the anti-proliferative actions of 28 selected Rubiae Radix-derived anthraquinones and other chemical ingredients on cultured HaCaT keratinocytes. Among them, 1-hydroxy-3-methyl-anthraquinone (HMA) and 1,4-diamino-2,3-bis(2-phenoxyethoxy)anthraquinone (DBA), as well as celastrol, a Celastrus orbiculatus-derived triterpene, were found to possess significant anti-proliferative action on HaCaT cells, with IC₅₀ value of 17.9, 15.8 and 1.1 μM, respectively. All DBA, HMA and celastrol showed only mild to moderate toxic effects on normal human keratinocyte HEK cells and human fibroblast Hs68 cells. / Mechanistically, celastrol and HMA was found to induce apoptosis in a dose-dependent manner in HaCaT cells as characterized by DNA fragmentation, phosphatidyl-serine externalization and activation of caspase 3. Further studies by flow cytometric and western blot analyses demonstrated that the celastrol-induced apoptosis on HaCaT cells was associated with the inhibition of NF-κB pathway and through caspase-related apoptotic pathway as characterized by activation of caspase proteins, regulation of Bcl-2 family proteins and depolarization of mitochondrial potential. / On the other hand, DBA showed an ability to induce terminal differentiation in cultured human keratinocytes and this capability is believed to be responsible for its growth inhibitory effects. DBA significantly accentuated the cornified envelope formation in HEK and HaCaT keratinocytes together with the augmentation of K1/K10, involucrin and transglutaminase 1 protein levels and decrease of expression of K5/K14 protein in DBA-treated cells. However, the subsequent in vivo study using a mouse tail model showed that HMA did not have significant effects on modulating keratinocyte terminal differentiation. / Taken together, our present PhD project successfully identified DBA, HMA and celastrol to have potent anti-psoriatic action on in vitro models, and the experimental findings render these naturally-occurring chemicals to be promising candidates for further development into anti-psoriatic pharmaceutical agents. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhou, Linli. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 213-244). / 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) --- p.i / Abstract (Chinese) --- p.iii / Publications --- p.v / Acknowledgements --- p.vii / Table of Contents --- p.viii / List of Figures --- p.xvii / List of Tables --- p.xxi / List of Abbreviations --- p.xxii / Chapter Chapter One --- General Introduction / Chapter 1.1 --- Psoriasis --- p.2 / Chapter 1.1.1 --- Structure of skin --- p.2 / Chapter 1.1.2 --- Epidemiology of psoriasis --- p.3 / Chapter 1.1.3 --- Pathogenesis --- p.5 / Chapter 1.1.4 --- Classification --- p.8 / Chapter 1.1.4.1 --- Nonpustular (plaque type) psoriasis --- p.9 / Chapter 1.1.4.2 --- Guttate psoriasis --- p.9 / Chapter 1.1.4.3 --- Pustular psoriasis --- p.9 / Chapter 1.1.4.4 --- Erythrodermic psoriasis --- p.10 / Chapter 1.1.4.5 --- Nail psoriasis --- p.11 / Chapter 1.1.4.6 --- Psoriatic arthritis --- p.11 / Chapter 1.1.5 --- Comorbidities --- p.13 / Chapter 1.2 --- Treatment of Psoriasis --- p.16 / Chapter 1.2.1 --- Conventional treatment for psoriasis --- p.16 / Chapter 1.2.1.1 --- Topical therapy --- p.16 / Chapter 1.2.1.2 --- Phototherapy --- p.19 / Chapter 1.2.1.3 --- Systemic therapy --- p.21 / Chapter 1.2.2 --- Targeted immunotherapy --- p.24 / Chapter 1.2.3 --- Combination, rotational and sequential therapy --- p.25 / Chapter 1.2.4 --- Complementary treatment --- p.26 / Chapter 1.3 --- Traditional Chinese Medicine for Psoriasis --- p.30 / Chapter 1.3.1 --- Prescriptions for psoriasis based on pattern differentiation --- p.30 / Chapter 1.3.2 --- Clinical and experimental study of TCM for psoriasis --- p.34 / Chapter 1.3.3 --- Possible action mechanisms of Chinese herbs for psoriasis --- p.34 / Chapter 1.3.4 --- Previous studies on TCM for psoriasis conducted by our research group --- p.35 / Chapter 1.4 --- Aims and Objectives of the Present Study --- p.38 / Chapter Chapter Two --- Phytochemical and Apoptotic Studies of Rubiae Radix-derived Anthraquinones and Other Related Compounds / Chapter 2.1 --- Introduction --- p.41 / Chapter 2.2 --- Selection and Screening of Rubiae Radix-derived anthraquinones and Other Related Compounds for Anti-proliferative Action on Cultured HaCaT Human Keratinocytes --- p.43 / Chapter 2.2.1 --- Introduction --- p.43 / Chapter 2.2.2 --- Materials and methods --- p.45 / Chapter 2.2.2.1 --- Procurement of Rubiae Radix-derived anthraquiones and other related compounds --- p.45 / Chapter 2.2.2.2 --- Purification of anthraquinones from Rubiae Radix --- p.50 / Chapter 2.2.2.3 --- General cell culture --- p.54 / Chapter 2.2.2.4 --- SRB assay --- p.55 / Chapter 2.2.2.5 --- MTT assay --- p.56 / Chapter 2.2.2.6 --- Assessment of synergistic or antagonistic effects between two active anthraquiones --- p.56 / Chapter 2.2.2.7 --- Statistical analysis --- p.57 / Chapter 2.2.3 --- Results --- p.57 / Chapter 2.2.3.1 --- Anti-proliferative effects of 35 Rubiae Radix fractions on HaCaT cells by SRB assay --- p.57 / Chapter 2.2.3.2 --- Anti-proliferative effects of the 27 anthraquinones and related compounds on HaCaT cells by SRB assay --- p.59 / Chapter 2.2.3.3 --- Confirmation of the anti-proliferative action of 8 active pure compounds using MTT assay --- p.61 / Chapter 2.2.3.4 --- Cytotoxic effects of 1-hydroxy-3-methyl-anthraquinone and1,4-diamino-2,3-bis(2-phenoxyethoxy)anthraquinone on the growth of HEK and Hs68 cells --- p.64 / Chapter 2.2.3.5 --- Drug interactions between different active anthraquinones --- p.67 / Chapter 2.2.4 --- Discussion --- p.69 / Chapter 2.3 --- Investigations of the Apoptotic Effects of DBA and HMA on HaCaT cells --- p.71 / Chapter 2.3.1 --- Introduction --- p.71 / Chapter 2.3.2 --- Materials and methods --- p.76 / Chapter 2.3.2.1 --- Chemicals --- p.76 / Chapter 2.3.2.2 --- General cell culture methods --- p.76 / Chapter 2.3.2.3 --- Cell cycle analysis with PI staining --- p.76 / Chapter 2.3.2.4 --- Hoechst fluorescence staining for morphological evaluation --- p.77 / Chapter 2.3.2.5 --- DNA fragmentation assay --- p.77 / Chapter 2.3.2.6 --- Detection of apoptosis by flow cytometry --- p.78 / Chapter 2.3.2.7 --- Prepare cytosol fraction of HaCaT cells --- p.79 / Chapter 2.3.2.8 --- Western blot analysis --- p.79 / Chapter 2.3.2.9 --- Statistical analysis --- p.80 / Chapter 2.3.3 --- Results --- p.76 / Chapter 2.3.3.1 --- Action of DBA and HMA on cell cycle progression --- p.80 / Chapter 2.3.3.2 --- Alteration of cellular morphology --- p.84 / Chapter 2.3.3.3 --- Detection of DNA fragmentation --- p.86 / Chapter 2.3.3.4 --- Quantitative analysis of apoptotic cells by annexin V-PI staining --- p.88 / Chapter 2.3.3.5 --- Activation of procaspase-3 and release of cytochrome c protein --- p.91 / Chapter 2.3.4 --- Discussion --- p.94 / Chapter 2.4 --- General Discussion --- p.97 / Chapter Chapter Three --- Effects of Rubiae Radix and Its-derived Anthraquinones on Keratinocyte Terminal Differentiation / Chapter 3.1 --- Introduction --- p.100 / Chapter 3.2 --- Materials and Methods --- p.105 / Chapter 3.2.1 --- Chemicals --- p.105 / Chapter 3.2.2 --- General cell culture --- p.105 / Chapter 3.2.3 --- Cornified envelope (CE) formation assay --- p.106 / Chapter 3.2.4 --- Western blot analysis --- p.107 / Chapter 3.2.4 --- Statistical analysis --- p.107 / Chapter 3.3 --- Results --- p.108 / Chapter 3.3.1 --- EA fraction of Rubiae Radix, DBA and HMA stimulates CE formation --- p.108 / Chapter 3.3.2 --- EA fraction of Rubiae Radix, DBA and HMA regulated TG1 expression and involucrin production in cultured human keratinocytes --- p.112 / Chapter 3.3.3 --- Regulation of cytokeratins by EA fraction of Rubiae Radix, DBA and HMA --- p.118 / Chapter 3.4 --- Discussion --- p.128 / Chapter Chapter Four --- Anti-psoriatic Action of Celastrol from Celastrus orbiculatus / Chapter 4.1 --- Introduction --- p.136 / Chapter 4.2 --- Anti-proliferative Action of Celastrol on Cultured Human Keratinocytes and Other Cell Types --- p.138 / Chapter 4.2.1 --- Introduction --- p.138 / Chapter 4.2.2 --- Materials and methods / Chapter 4.2.2.1 --- Chemicals --- p.138 / Chapter 4.2.2.2 --- General cell culture --- p.139 / Chapter 4.2.2.3 --- MTT assay --- p.139 / Chapter 4.2.2.4 --- Statistical analysis --- p.139 / Chapter 4.2.3 --- Results --- p.142 / Chapter 4.2.3.1 --- Anti-proliferative effect of celastrol on cultured cells --- p.142 / Chapter 4.2.4 --- Discussion --- p.145 / Chapter 4.3 --- Induction of Apoptosis by Celastrol on Human Keratinocytes --- p.146 / Chapter 4.3.1 --- Introduction --- p.146 / Chapter 4.3.2 --- Materials and methods --- p.146 / Chapter 4.3.2.1 --- Chemicals --- p.146 / Chapter 4.3.2.2 --- General cell culture --- p.147 / Chapter 4.3.2.3 --- Cell cycle analysis with PI staining --- p.147 / Chapter 4.3.2.4 --- Detection of apoptosis by flow cytometry --- p.147 / Chapter 4.3.2.5 --- Measurement of the mitochondrial membrane potential (ΔΨm) --- p.148 / Chapter 4.3.2.6 --- Western blot analysis --- p.148 / Chapter 4.3.2.7 --- Statistical analysis --- p.148 / Chapter 4.3.3 --- Results --- p.149 / Chapter 4.3.3.1 --- Induction of sub-G1 phase by celastrol on HaCaT cells --- p.149 / Chapter 4.3.3.2 --- Quantitative analysis of apoptotic cells by Annexin V-PI staining --- p.151 / Chapter 4.3.3.3 --- Alteration of ΔΨm --- p.153 / Chapter 4.3.3.4 --- Activation of caspase family protein --- p.155 / Chapter 4.3.3.5 --- Celastrol regulates the Bcl-2 family members --- p.159 / Chapter 4.3.4 --- Discussion --- p.161 / Chapter 4.4 --- Inhibition of NF-κB Transcription Factor Activation by Celastrol --- p.164 / Chapter 4.4.1 --- Introduction --- p.164 / Chapter 4.4.2 --- Materials and methods --- p.165 / Chapter 4.4.2.1 --- Chemicals --- p.165 / Chapter 4.4.2.2 --- General cell cultrue --- p.165 / Chapter 4.4.2.3 --- Western blot analysis --- p.165 / Chapter 4.4.2.4 --- Detect nuclear p65 by ELISA assay --- p.166 / Chapter 4.4.2.5 --- Statistical analysis --- p.166 / Chapter 4.4.3 --- Results --- p.167 / Chapter 4.4.3.1 --- Celastrol inhibited the NF-κB activation --- p.167 / Chapter 4.4.4 --- Discussion --- p.170 / Chapter 4.5 --- Induction of Terminal Differentiation by Celastrol --- p.173 / Chapter 4.5.1 --- Introduction --- p.173 / Chapter 4.5.2 --- Materials and methods --- p.174 / Chapter 4.5.2.1 --- Chemicals --- p.174 / Chapter 4.5.2.2 --- General cell culture --- p.174 / Chapter 4.5.2.3 --- CE formation assay --- p.174 / Chapter 4.5.2.4 --- Western blot analysis --- p.174 / Chapter 4.5.2.5 --- Statistical analysis --- p.174 / Chapter 4.5.3 --- Results --- p.175 / Chapter 4.5.3.1 --- Regulation of CE formation by celastrol --- p.175 / Chapter 4.5.3.2 --- Modulation of terminal differentiation markers by celastrol --- p.178 / Chapter 4.5.4 --- Discussion --- p.181 / Chapter 4.6 --- General Discussion --- p.183 / Chapter Chapter Five --- In vivo Anti-psoriatic Effects of Topical Preparation of 1-hydroxy-3-methyl-anthraquinone / Chapter 5.1 --- Introduction --- p.187 / Chapter 5.2 --- Material and Methods --- p.191 / Chapter 5.2.1 --- Chemicals --- p.191 / Chapter 5.2.2 --- Formulation of topical preparation containing HMA --- p.191 / Chapter 5.2.3 --- Mouse tail model --- p.192 / Chapter 5.2.4 --- Histopathological evaluation --- p.193 / Chapter 5.2.5 --- Statistical analysis --- p.194 / Chapter 5.3 --- Results --- p.195 / Chapter 5.3.1 --- Body weight profile --- p.195 / Chapter 5.3.2 --- Histological resutls --- p.197 / Chapter 5.4 --- Discussion --- p.201 / Chapter Chapter Six --- General Conclusions and Future Perspectives / Chapter 6.1 --- General Conclusions --- p.205 / Chapter 6.2 --- Future Perspectives --- p.210 / References / References by alphabetical order --- p.213

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

O tratamento de pacientes apresentando doenças inflamatórias imunomediadas com drogas anti-TNF-alfa aumenta o risco da reativação da tuberculose. Isso sugere que tais drogas possam afetar a imunidade celular destes. No entanto, há dados conflitantes sobre se esse tratamento suprime as respostas para o teste tuberculínico (TT) e os ensaios de liberação de interferon-gama (IGRAs) e poucos dados em pacientes com psoríase. O presente estudo avaliou pacientes com psoríase moderada a grave enfocando os efeitos do tratamento com infliximabe em suas respostas imunológicas celulares. Foram avaliadas as respostas imunes celulares de doze pacientes antes e durante o tratamento com infliximabe. As células mononucleares do sangue periférico (PBMC) foram estimuladas com a fito-hemaglutinina (PHA), o superantígeno enterotoxina B (SEB), um lisado de citomegalovírus (CMV), e antígenos de Mycobacterium tuberculosis, e a ativação de linfócitos foi avaliada por ELISPOT para enumerar células secretoras de IFN-y, por ELISA para detecção da secreção de IFN-y, e através da incorporação de[3H] timidina para medir a proliferação. O tratamento com infliximabe não levou à redução de INF-y e da resposta linfoproliferativa nos pacientes. Pelo contrário, aumentou a liberação desta citocina em culturas de PBMC estimulados com PHA e SEB por 12 h. Este efeito foi mais notado no pico do efeito clínico do agente anti-TNF (7 semanas de tratamento) e menos proeminente no seu nadir (logo antes da infusão da próxima dose). Reatividade imunitária ao CMV também não foi significativamente afetada, notando-se leve aumento pelo agente anti-TNF. É de se notar que secreção de IFN-y e resposta proliferativa a Mtb dos dois pacientes TT positivos foram, também, visivelmente aumentadas na semana 7, declinando quando infliximabe atingiu o seu nadir. Os efeitos deletérios do bloqueio do TNF em pacientes com psoríase grave, submetidos ao tratamento com infliximabe parecem ser atenuados, pelo menos parcialmente, por um efeito de aumento na imunidade mediada por células desses pacientes, possivelmente pela diminuição da ação imunossupressora decorrente do excesso de TNF / Treatment of patients with immune-mediated inflammatory diseases with anti-TNF agents increases the risk of tuberculosis reactivation, suggesting that it may affect their cellular immune response. However, there are conflicting data on whether anti-TNF treatment suppresses the responses to tuberculin skin test (TST) and interferon-y release assays and no information regarding psoriasis patients on anti-TNF treatment. The present study evaluated patients with moderate to severe psoriasis focusing on the effects of treatment with infliximab on their cellular immune responses. Cellular immune responses of twelve patients were evaluated before and during infliximab treatment. Peripheral blood mononuclear cells (PBMC) were stimulated with phytohemaglutinin (PHA), the superantigen enterotoxin B (SEB), a cytomegalovirus lysate (CMV), and Mycobacterium tuberculosis antigens, and the lymphocyte activation was evaluated by ELISPOT for enumeration of IFN-y-secreting cells, ELISA for detection of secreted IFN-y, and by [3H]thymidine incorporation for proliferation measurement. Treatment with infliximab does not lead to reduction in the INF-y and lymphoproliferative responses of patients. It rather increased the overnight release of this cytokine in PBMC cultures stimulated with PHA and SEB. This effect was most noted at the peak of the clinical effect of the anti-TNF agent (week 7 of treatment) and less prominent at its nadir (just before infusion of the next dose). Immune reactivity to CMV was also either unaffected or slightly increased by the TNF blocking agent. Of note, the IFN-y and proliferative responses to Mtb from the two TST-responder patients were also remarkably increased at week 7, declining when infliximab reached its nadir. The deleterious consequences of TNF blockade in patients with severe psoriasis undergoing infliximab treatment may be in part attenuated by an enhancing effect on the cell mediated immunity of the patients, possibly due to the abbreviation of the immunosuppressive effect of TNF overexpression

O tratamento de pacientes apresentando doenças inflamatórias imunomediadas com drogas anti-TNF-alfa aumenta o risco da reativação da tuberculose. Isso sugere que tais drogas possam afetar a imunidade celular destes. No entanto, há dados conflitantes sobre se esse tratamento suprime as respostas para o teste tuberculínico (TT) e os ensaios de liberação de interferon-gama (IGRAs) e poucos dados em pacientes com psoríase. O presente estudo avaliou pacientes com psoríase moderada a grave enfocando os efeitos do tratamento com infliximabe em suas respostas imunológicas celulares. Foram avaliadas as respostas imunes celulares de doze pacientes antes e durante o tratamento com infliximabe. As células mononucleares do sangue periférico (PBMC) foram estimuladas com a fito-hemaglutinina (PHA), o superantígeno enterotoxina B (SEB), um lisado de citomegalovírus (CMV), e antígenos de Mycobacterium tuberculosis, e a ativação de linfócitos foi avaliada por ELISPOT para enumerar células secretoras de IFN-y, por ELISA para detecção da secreção de IFN-y, e através da incorporação de[3H] timidina para medir a proliferação. O tratamento com infliximabe não levou à redução de INF-y e da resposta linfoproliferativa nos pacientes. Pelo contrário, aumentou a liberação desta citocina em culturas de PBMC estimulados com PHA e SEB por 12 h. Este efeito foi mais notado no pico do efeito clínico do agente anti-TNF (7 semanas de tratamento) e menos proeminente no seu nadir (logo antes da infusão da próxima dose). Reatividade imunitária ao CMV também não foi significativamente afetada, notando-se leve aumento pelo agente anti-TNF. É de se notar que secreção de IFN-y e resposta proliferativa a Mtb dos dois pacientes TT positivos foram, também, visivelmente aumentadas na semana 7, declinando quando infliximabe atingiu o seu nadir. Os efeitos deletérios do bloqueio do TNF em pacientes com psoríase grave, submetidos ao tratamento com infliximabe parecem ser atenuados, pelo menos parcialmente, por um efeito de aumento na imunidade mediada por células desses pacientes, possivelmente pela diminuição da ação imunossupressora decorrente do excesso de TNF / Treatment of patients with immune-mediated inflammatory diseases with anti-TNF agents increases the risk of tuberculosis reactivation, suggesting that it may affect their cellular immune response. However, there are conflicting data on whether anti-TNF treatment suppresses the responses to tuberculin skin test (TST) and interferon-y release assays and no information regarding psoriasis patients on anti-TNF treatment. The present study evaluated patients with moderate to severe psoriasis focusing on the effects of treatment with infliximab on their cellular immune responses. Cellular immune responses of twelve patients were evaluated before and during infliximab treatment. Peripheral blood mononuclear cells (PBMC) were stimulated with phytohemaglutinin (PHA), the superantigen enterotoxin B (SEB), a cytomegalovirus lysate (CMV), and Mycobacterium tuberculosis antigens, and the lymphocyte activation was evaluated by ELISPOT for enumeration of IFN-y-secreting cells, ELISA for detection of secreted IFN-y, and by [3H]thymidine incorporation for proliferation measurement. Treatment with infliximab does not lead to reduction in the INF-y and lymphoproliferative responses of patients. It rather increased the overnight release of this cytokine in PBMC cultures stimulated with PHA and SEB. This effect was most noted at the peak of the clinical effect of the anti-TNF agent (week 7 of treatment) and less prominent at its nadir (just before infusion of the next dose). Immune reactivity to CMV was also either unaffected or slightly increased by the TNF blocking agent. Of note, the IFN-y and proliferative responses to Mtb from the two TST-responder patients were also remarkably increased at week 7, declining when infliximab reached its nadir. The deleterious consequences of TNF blockade in patients with severe psoriasis undergoing infliximab treatment may be in part attenuated by an enhancing effect on the cell mediated immunity of the patients, possibly due to the abbreviation of the immunosuppressive effect of TNF overexpression

Background: About 2% of the German population are affected by psoriasis. A growing number of cost-intensive systemic treatments are available. Surveys have shown high proportions of patients with moderate to severe psoriasis are not adequately treated despite a high disease burden. Digital therapy recommendation systems (TRS) may help implement guideline-based treatment. However, little is known about the acceptance of such clinical decision support systems (CDSSs). Therefore, the aim of the study was to access the acceptance of a prototypical TRS demonstrator. - Methods: Three scenarios (potential test patients with psoriasis but different sociodemographic and clinical characteristics, previous treatments, desire to have children, and multiple comorbidities) were designed in the demonstrator. The TRS demonstrator and test patients were presented to a random sample of 76 dermatologists attending a national dermatology conference in a cross-sectional face-to-face survey with case vignettes. The dermatologist were asked to rate the demonstrator by system usability scale (SUS), whether they would use it for certain patients populations and barriers of usage. Reasons for potential usage of the TRS demonstrator were tested via a Poisson regression with robust standard errors. Results: Acceptance of the TRS was highest for patients eligible for systemic therapy (82%). 50% of participants accepted the system for patients with additional comorbidities and 43% for patients with special subtypes of psoriasis. Dermatologists in the outpatient sector or with many patients per week were less willing to use the TRS for patients with special psoriasis-subtypes. Dermatologists rated the demonstrator as acceptable with an mean SUS of 76.8. Participants whose SUS was 10 points above average were 27% more likely to use TRS for special psoriasissubtypes. The main barrier in using the TRS was time demand (47.4%). Participants who perceived time as an obstacle were 22.3% less willing to use TRS with systemic therapy patients. 27.6% of physicians stated that they did not understand exactly how the recommendation was generated by the TRS, with no effect on the preparedness to use the system. - Conclusion: The considerably high acceptance and the preparedness to use the psoriasis CDSS suggests that a TRS appears to be implementable in routine healthcare and may improve clinical care. Main barrier is the additional time demand posed on dermatologists in a busy clinical setting. Therefore, it will be a major challenge to identify a limited set of variables that still allows a valid recommendation with precise prediction of the patient-individual benefits and harms.