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影響全球數百萬的患者的慢性傷口，以其持續性過度發炎，纖維細胞增殖放緩，及血管生成受損為表徵。藥用草藥地黃已證明在大鼠糖尿足模型上有顯著傷口癒合作用。然而，關於地黃的炮製及其活性成分對此等傷口癒合的活動主要是未知的。我們首先在地黃的炮製中，以抗一氧化氮（NO）和纖維細胞增殖實驗，確定了乾地黃表現出有效的傷口癒合活動。採用多方位生物活性導引分離（BGF），我們進一步研究乾地黃在抗炎，纖維細胞增殖，血管生成的活性成分，分別以抗NO產生，纖維細胞增殖，和TG(fli1:EGFP)y1/+(AB)斑馬魚芽血管生成模型為生物測定。此等具傷口癒合效果的活性成分將會作進一步研究。此外，我們會以電子細胞基質阻抗判斷(ECIS)的技術，對名為NF3(含RR的中草藥配方)在人類血管內皮細胞(HECV)上作體外血管生成及其信息的研究。通過抗NO測定導引分離，我們證明活性萃取物C3比其粗提物擁有100倍更有效的抗炎效果。C3中含地黃苦甙元。地黃苦甙元可顯著地抑制NO的產生。C3中地黃苦甙元的存在可能是其具抗炎的原因。C3進一步以抑制一氧化氮合酶(iNOS)，環氧合酶-2(COX-2)和細胞白介素第六因子(IL-6)的基因，蛋白質，及/或中介物的表達，證明其舒緩炎症的作用。因此，C3可能對慢性傷口癒合中的炎症有治療作用。此外，我們發現先從水提的RR粗提物，再以乙酸乙酯提取的活性萃取物C2-B4，證明此萃取物在纖維細胞增殖中具最有效及劑量依存作用。斑馬魚芽誘導模型導引分離顯示，C1-1萃取物在RR中具有最有效的血管生成作用。為斑馬魚芽誘導度身設計的30個血管生成相關基因顯示，C1-1廣泛地引發血管生成中的基因差異表達，特別在生長因子和血管穩定方面。就促進血管生成，C1-1進一步以體外人類微血管內皮細胞的血管生成檢測進行研究，結果發現C1-1具細胞移動及類血管生成能力。此外，降毛荚醛(norviburtinal)，在地黃提取物中首次被發現和具有血管生成作用。如此，C1-1和降毛荚醛為RR中血管生成作用的活性成分。此外，應用ECIS技術，含RR配方的NF3能通過磷酯肌醇激酶(PI3K)及WiskottAldrich氏症候群神經元蛋白 (N-WASP) 路徑在人類血管內皮細胞（HECV）上誘導內皮細胞粘附，遷移及類血管生成。西方墨點法分析表明，NF3 在HECV上激活Akt和有絲分裂活化蛋白質激酶(MAPKs)的表達。這些誘導在各方面促進血管生成。因此，這顯示NF3能在分子及功能上激活血管生成作用的複雜性。此外，我們進一步支持ECIS在血管內皮細胞篩選傷口癒合劑中的高靈敏度。總括而言，通過靶向抗炎，纖維細胞增殖增長和改善血管生成，各地黃生物活性導引化合物和萃取物，及其含RR的配方，可以在治療慢性傷口癒合上發揮功效。Chronic wounds, which influence millions of patients worldwide, are manifested with its sustained hyperinflammation, slackened fibroblast proliferation, and impaired angiogenesis. Agents retrieving these activities could facilitate the healing. Medicinal herb, Rehmanniae Radix (RR) demonstrated profound wound healing effect in rat diabetic foot model. However, the subtypes and the active components behind RR for such wound healing activities were largely unknown.Here we firstly identified that dried RR, among its subtypes, exhibited potent wound healing activities through nitric oxide (NO) anti-inflammatory and fibroblast proliferation assays. Using multi-directional bioassay-guided fractionation (BGF), we further studied the active component(s) of dried RR in anti-inflammation, fibroblast proliferation, and angiogenesis, respectively, by anti-NO production, fibroblast proliferation, and TG(fli1:EGFP)[superscript y]¹/+(AB) zebrafish sprout angiogenesis model. Active component(s) of such wound healing effects were further characterized. Furthermore, with a RR-containing herbal formula, NF3, the in vitro angiogenic activities and its underlying signaling of NF3-treated human vascular endothelial cells (HECV) were studied using electric cell-substrate impedance sensing (ECIS) technology.Via anti-NO assay-guided fractionation of dried RR, we demonstrated that the sub-fraction C3, possessed 100-fold more potent anti-inflammatory effect than that of the crude extract. Characterization of C3 showed that the anti-inflammatory activity could be partly due to the presence of rehmapicrogenin, which could significantly inhibit NO production. C3 was further demonstrated in blocking inflammation by inhibiting gene, protein, and/or mediator expression of inducible NO synthase, COX-2 and IL-6. Hence, C3 could be useful in treating inflammation in chronic wound healing. Additionally, we revealed that an active sub-fraction, C2-B4, from the ethyl acetate extract of the aqueous extract of RR, demonstrated the most potent and dose-dependent fibroblast proliferative effect.Zebrafish sprout-inducing model-guided fractionation suggested C1-1 sub-fraction possessed the most potent angiogenesis effect in RR. A 30 tailor-made angiogenesis-associated gene panel designed for zebrafish sprout angiogenesis revealed that C1-1 triggered differential gene expression across wide angiogenic events, particularly concerned with those of growth factors and vessel stabilization. The pro-angiogenic activity was further supported by in vitro human microvascular endothelial cell-based angiogenesis assays, with C1-1 being pro-motogenic and tubule inducing. Also, norviburtinal was, for the first time, found in the extract of RR and possessed novel angiogenesis effect. Thus, C1-1 and norviburtinal were the active components responsible for the pro-angiogenesis effect of RR.Moreover, RR-containing formula (NF3), which induced endothelial cell attachment, migration, and tubule formation in human vascular endothelial cell (HECV), could be mediated through PI3K and N-WASP pathways. Activated Akt and MAPK kinases expression in western blot analysis were also demonstrated in NF3-treated HECV. These inductions would promote angiogenesis at various levels. Hence, the complexity of angiogenesis effect activated by the NF3 treatment molecularly and functionally was shown, and we further supported the high sensitivity of ECIS in the screening of wound healing agents with endothelial cells.In conclusion, through targeting anti-inflammation, elevated fibroblast proliferation and improved angiogenesis, our respective bioassay-guided active fractions and compounds in Rehmanniae Radix, and the RR-containing formula, could play beneficial uses in treating chronic wound healing.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.Detailed summary in vernacular field only.Liu, Cheuk Lun.Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.Includes bibliographical references (leaves 221-249).Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.Abstract also in Chinese.Table of Contents --- p.iAbstract (in English) --- p.viAbstract (in Chinese) --- p.ixStatement of Originality --- p.xiiiAcknowledgements --- p.xivPublications --- p.xivList of Tables --- p.xviiList of Figures --- p.xviiiList of Abbreviations --- p.xxiChapter Chapter 1: --- Literature Review and Study ObjectivesChapter 1.1 --- Overview on wound healingChapter 1.1.1 --- Normal wound healing --- p.1Chapter 1.1.2 --- Chronic wound healingChapter 1.1.2.1 --- Venous ulcers --- p.8Chapter 1.1.2.2 --- Diabetic ulcers --- p.11Chapter 1.1.2.3 --- Mechanism of chronic wound healing --- p.13Chapter 1.1.2.4 --- Current treatments for chronic wound healing --- p.19Chapter 1.2 --- Rehmanniae Radix (RR) overviewChapter 1.2.1 --- RR and its subtypes --- p.29Chapter 1.2.2 --- Chemistry of RR --- p.34Chapter 1.2.3 --- Pharmacology of RR --- p.38Chapter 1.2.3.1 --- RR and chronic wound healingChapter 1.3 --- Study objectives --- p.42Chapter Chapter 2: --- comparison of wound healing effect of the subtypes of rrChapter 2.1 --- Introduction --- p.43Chapter 2.2 --- MethodsChapter 2.2.1 --- Preparation and authentication of subtypes of RR --- p.46Chapter 2.2.2 --- RAW 264.7 murine macrophage culture and sample treatment protocol, and cell viability test --- p.47Chapter 2.2.3 --- Nitric oxide inhibitory assay --- p.47Chapter 2.2.4 --- Hs27 human fibroblast culture and sample treatment protocol --- p.48Chapter 2.2.5 --- Fibroblast proliferation assay --- p.48Chapter 2.2.6 --- Statistical analysis --- p.49Chapter 2.3 --- Results --- p.63Chapter 2.3.1 --- Nitric oxide anti-inflammatory effect of the subtypes of RR --- p.49Chapter 2.3.2 --- Fibroblast proliferative effect of the subtypes of RR --- p.52Chapter 2.4 --- Discussion --- p.54Chapter Chapter 3: --- Fibroblast proliferative effect of RRChapter 3.1 --- Introduction --- p.57Chapter 3.2 --- MethodsChapter 3.2.1 --- Preparation of aqueous extracts of RR --- p.61Chapter 3.2.2 --- Hs27 human fibroblast culture and sample treatment protocol --- p.61Chapter 3.2.3 --- Hs27 human fibroblast proliferation assay --- p.61Chapter 3.2.4 --- Bioassay-guided fractionation of RR --- p.61Chapter 3.2.5 --- LC-MS analysis of bioassay-guided fraction, C2-B4 --- p.65Chapter 3.2.6 --- Statistical analysis --- p.66Chapter 3.3 --- ResultsChapter 3.3.1 --- Fibroblast proliferative effect of RR aqueous crude extract and its bioassay-guided fractions --- p.67Chapter 3.3.2 --- Chemical structure of the isolated compounds --- p.70Chapter 3.3.3 --- LC-MS analysis of bioassay-guided fraction, C2-B4 --- p.72Chapter 3.4 --- Discussion --- p.73Chapter Chapter 4: --- Anti-inflammatory effect and its underlying mechanism of RRChapter 4.1 --- Introduction --- p.77Chapter 4.2 --- MethodsChapter 4.2.1 --- Preparation of aqueous extracts of RR --- p.82Chapter 4.2.2 --- RAW 264.7 murine macrophage culture and sample treatment protocol, and cell viability test --- p.82Chapter 4.2.3 --- Assay for nitric oxide inhibitory effect using RAW264.7 cells --- p.83Chapter 4.2.4 --- Bioassay-guided fractionation of RR --- p.83Chapter 4.2.5 --- Ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/QTOF-MS) analysis of sub-fraction, C3 --- p.85Chapter 4.2.6 --- Prostaglandin E2 (PGE2) and interleukin-6 (IL-6) assay --- p.87Chapter 4.2.7 --- Real-time PCR --- p.87Chapter 4.2.8 --- Western blot analysis --- p.89Chapter 4.2.9 --- Statistical analysis --- p.91Chapter 4.3 --- ResultsChapter 4.3.1 --- Nitric oxide inhibitory effects of L-NMMA, RR aqueous crude extract and its bioassay guided fractions --- p.92Chapter 4.3.2 --- LC-MS analysis of bioassay-guided fraction, C3 --- p.96Chapter 4.3.3 --- Suppression of inflammation-related mRNA expression level in macrophages by C3 --- p.97Chapter 4.3.4 --- Suppression of protein expression of inducible nitric oxide synthase and COX-2 in macrophages by C3 --- p.99Chapter 4.3.5 --- Inhibition of release of inflammatory cytokine in macrophages by C3 --- p.102Chapter 4.4 --- Discussion --- p.103Chapter Chapter 5: --- Angiogenesis effect and its underlying mechanism of RRChapter 5.1 --- Introduction --- p.111Chapter 5.2 --- MethodsChapter 5.2.1 --- Preparation of aqueous extracts of RR --- p.113Chapter 5.2.2 --- Zebrafish culture --- p.113Chapter 5.2.3 --- Collection of zebrafish embryos and herbal treatment protocol --- p.115Chapter 5.2.4 --- Microinjection of vascular endothelial growth factor (VEGF) to zebrafish embryos --- p.116Chapter 5.2.5 --- Screening of zebrafish embryos using fluorescence microscopy --- p.116Chapter 5.2.6 --- Bioassay-guided fractionation of RRChapter 5.2.7 --- Isolation and structure elucidation of compound C2A --- p.120Chapter 5.2.8 --- Gas chromatographymass spectrometry (GC-MS) analysis of bioassay-guided fraction of RR, C1-1 --- p.120Chapter 5.2.9 --- Detection of zebrafish mRNA expression level by real-time PCR (RT-PCR) --- p.122Chapter 5.2.10 --- Human microvascular endothelial cell (HMEC-1) culture and sample treatment protocol --- p.125Chapter 5.2.11 --- HMEC-1 proliferation assay --- p.125Chapter 5.2.12 --- HMEC-1 scratch assay --- p.126Chapter 5.2.13 --- HMEC-1 tubule formation assay --- p.126Chapter 5.2.14 --- Statistical analysis --- p.127Chapter 5.3 --- ResultsChapter 5.3.1 --- Angiogenesis effects of RR aqueous crude extract, its bioassay-guided fractions and isolated compound, in zebrafish model --- p.128Chapter 5.3.2 --- Chemical structure and angiogenesis effect of the isolated compound C2A, norviburtinal --- p.133Chapter 5.3.3 --- Components of C1-1 from GC-MS analysis --- p.135Chapter 5.3.4 --- Angiogenesis effect of C1-1 in angiogenesis-related mRNA expression level in zebrafish --- p.137Chapter 5.3.5 --- Effect of endothelial cell proliferation of C1-1 in HMEC-1 cell --- p.142Chapter 5.3.6 --- Cell migration effect of C1-1 in HMEC-1 cell --- p.143Chapter 5.3.7 --- Tubule formation of C1-1 in HMEC-1 cell --- p.145Chapter 5.4 --- Discussion --- p.147Chapter Chapter 6: --- Angiogenesis effect and its underlying mechanism of RR AND AR-containing two-herbs formula, NF3 using ecis modelChapter 6.1 --- Introduction --- p.165Chapter 6.2 --- MethodsChapter 6.2.1 --- Preparation and authentication of aqueous extracts of NF3 --- p.176Chapter 6.2.2 --- Human vascular endothelial cells (HECV) culture --- p.177Chapter 6.2.3 --- HECV cell proliferation assay --- p.178Chapter 6.2.4 --- Scratch assay --- p.178Chapter 6.2.5 --- Tubule formation assay --- p.179Chapter 6.2.6 --- Electric cell-substrate impedance sensing (ECIS)-based cell attachment and motility assay --- p.180Chapter 6.2.7 --- Western blot analysis --- p.181Chapter 6.2.8 --- Statistical analysis --- p.182Chapter 6.3 --- ResultsChapter 6.3.1 --- LC-MS analysis of NF3 --- p.184Chapter 6.3.2 --- Effects of NF3, AR and RR on cell viability and migration of HECV --- p.185Chapter 6.3.3 --- Tubule formation effect of NF3, AR and RR in HECV cell --- p.188Chapter 6.3.4 --- Effects of NF3, AR, and RR on HECV cell attachment using ECIS model --- p.190Chapter 6.3.5 --- Effects of NF3, AR and RR on HECV cell migration using ECIS model --- p.191Chapter 6.3.6 --- Effects of NF3 on HECV for MAPK and Akt protein activation --- p.195Chapter 6.4 --- Discussion --- p.197Chapter Chapter 7: --- General Discussion and ConclusionsChapter 7.1 --- General discussion and conclusions --- p.206Chapter 7.2 --- Limitation of the study --- p.215Chapter 7.3 --- Future work --- p.215Appendix --- p.218References --- p.22