珊瑚屬於腔腸動物門、珊瑚蟲綱 (Anthozoa)，它們是廣泛分佈於熱帶和亞熱帶的海洋動物。在藥物開發研究上，珊瑚已被認為是可能尋找新藥先導化合物的重要來源之ㄧ。為了評估分離自Briareum excavatum 珊瑚品種的briarane 型雙萜化合物之藥用活性，樹突狀細胞 (dendritic cell)，佛波醇誘導性皮膚炎 (TPA-induced dermatitis) 及第1 型糖尿病(type 1 diabetes) 的動物模型等三個實驗系統做為本研究的技術平台，比較性地來評估分析briarane 型雙萜化合物的抗發炎及免疫調節之活性。本研究結果顯示，briarane 型雙萜化合物excavatolide K 會有效影響人類樹突狀細胞的功能，尤其能顯著抑制白介素-12 (IL-12) 的表現。這種抑制作用與 rictor-mTOR/Akt 等磷酸激酶參與的訊息網絡受到干擾，進而抑制Erk1/2 磷酸激酶及NF-kB 轉錄因子的活化而有所關聯。本研究亦顯示，briarane 型雙萜化合物的8, 17-環氧基對於上述的生物抑制活性可扮演著重要的角色，而第12個碳上羥基長鏈酯的取代又會使得此抑制活性逐漸下降。此外， excavatolide B也可以有效抑制小鼠骨髓衍生樹突狀細胞白介素-12的表現，因而抑制此樹突狀細胞誘發的抗原特異性 Th1的反應。在小鼠非肥胖型糖尿病/重度聯合免疫缺陷(non-obese diabetic/severe combinedimmunodeficiency, NOD/SCID)動物模式下，excavatolide B可有效預防自體反應性T細胞誘發糖尿病(autoreactive T cell-mediated diabetes) 的發生。在佛波醇誘導性皮膚炎動物模式下，excavatolide B可抑制血管通透和水腫的產生，亦可抑制發炎指標酵素，這包括第二型環氧化酶 (COX-2)，誘導型一氧化氮合酶 (iNOS) 及基質金屬蛋白酶(MMP-9)的表現。進一步研究顯示，Akt/NF-kB 訊息傳導之干擾是與此抑制活性有所關聯。總結研究顯示，成果自海洋珊瑚分離出briaran型雙萜化合物excavatolide B及其結構類似物可以有效調節樹突狀細胞的功能，並抑制佛波醇誘導性皮炎和預防第一型糖尿病的產生。這些研究可能有助於未來珊瑚天然藥物之開發。

Corals are marine animals from the class Anthozoa and are widely distributed in
tropical and subtropical seawaters. They are considered as an important source of lead
compounds for drug discovery. For evaluating the medicinal activities of briarane-type
diterpenoids (BrDs) from marine coral Briareum excavatum, the regulation of a group of briarane-type diterpenoids (BrDs) on dendritic cell (DC) function, TPA-induced dermatitis and type 1 diabetes was investigated. The results show that the BrD excavatolide K (BrD2) remarkably suppressed the activation of human DCs, especially the expression of IL-12 p40. This inhibitory effect was mediated apparently by interference with the rictor-mTOR/Akt-mediated signaling network, resulting in persistent-phase activation of NF-kB and Erk1/2 signalings. In addition, the 8,17-epoxide of BrDs was observed to play a crucial role in inhibition of IL-12 p40 expression. Replacement of the C-12 hydroxyl group with longer esters in BrDs gradually decreased this inhibitory activity in human DCs. BrD excavatolide B (BrD1) effectively suppressed the capacity of mouse bone marrow-derived DCs to induce an antigen-specific Th1, response via the inhibition of IL-12 expression. Moreover, excavatolide B prevented the onset of autoreactive T cell-mediated diabetes in NOD/SCID mice. Furthermore, excavatolide B remarkably suppressed TPA-induced vascular permeability and edema in test skin tissues. At the biochemical level, excavatolide B inhibited TPA-induced expression of cyclooxygenase-2, inducible nitric oxide synthase and matrix metalloproteinase-9, the key indicators of cutaneous inflammation. This inhibition is apparently mediated by interference with the Akt/NF-kB-mediated signaling network. Together, these studies demonstrate that BrDs from specific marine corals can effectively regulate defined molecular and cellular functions of dendritic cells, suppress TPA-induced dermatitis, and prevent type 1 diabetes in murine models suggesting that BrDs may warrant further investigation as natural immunomodulatory agents or therapeutics.

Chinese Abstract………………………………………………… ix
Abstract…………………………………………………………….. x
Chapter 1 Introduction……………………………………………… 1
Chapter 2 Materials and Methods…………………………………... 11
2.1 Reagents……………………………………………...……….... 11
2.2 Mice……………………………………………………………. 11
2.3 Preparation of 12-acyloxyl analogues of excavatolide B…………….... 11
2.4 Culture of DCs from human peripheral blood mononuclear cells……… 13
2.5 Culture of DCs from mouse bone marrow cells……………………… 14
2.6 Flow cytometry………………………………………………….. 14
2.7 Measurement of cytokines……………………………………….... 14
2.8 Primer design, RT-PCR and Real time PCR…………………….…... 15
2.9 BMDC-mediated antigen-specific T-cell proliferation/differentiation.…. 16
2.10 NOD/SCID diabetes model…………..……………….…………. 16
2.11 Histopathological analysis………………………… …….……... 17
2.12 Measurement of vascular permeability………………………….… 18
2.13 Western blot analysis………………………….………….....….. 18
2.14 Statistical analysis…………………………………………….… 19
Chapter 3 Results………………………………………….…………. 20


3.1 Briarane-type diterpenoids from marine coral effectively regulate
dendritic cell functions and prevent type 1 diabetes in murine model............. 20
3.1.1 Excavatolide K (BrD2) efficiently suppresses DC activation……….. 20
3.1.2 Excavatolide K (BrD2) inhibits activation of rictor-mTOR/Akt
signaling pathway…………………………………………… 26
3.1.3 Structure-activity relationship of BrDs that underlies inhibition of
LPS-induced IL-12 p40 expression………………………………. 30
3.1.4 The 12-acyloxyl substituents reduces the bioactivity of briarane-type
diterpenoids…………………………………… 31
3.1.5 Excavatolide B (BrD1) suppresses the capacity of DCs to induce
Ag-specific Th1 responses via inhibition of IL-12 expression............. 33
3.1.6 Excavatolide B (BrD1) prevents onset of autoreactive T cell-mediated
diabetes……………………………………………… 37
3.2 Topical application of marine briarane-type diterpenoids effectively
Inhibits 12-O-tetradecanoylphorbol-13-acetate-induced inflammation
and dermatitis in murine skin…………………………… 39
3.2.1 Excavatolide B (BrD1) effectively inhibits TPA-induced vascular
permeability in mouse skin……………………………………… 39
3.2.2 BrD1 inhibits TPA-induced edema……………………………… 41
3.2.3 BrD1 suppresses TPA-induced COX-2 and iNOS expression in mouse
skin……………………………………………………… 43
3.2.4 BrD1 inhibits TPA-induced MMP-9 expression in mouse skin……… 45
3.2.5 BrD1 inhibits TPA-stimulated NF-kB and Akt activation in mouse skin 47
3.2.6 BrD1 inhibits LPS-induced IL-6 and TNF-a expression in mouse bone
marrow-derived dendritic cells…………………………… 49
3.2.7 Structure-activity relationship of BrDs that underlies inhibition of
LPS-induced IL-6 expression…………………………………… 51
3.2.8 The 12-acyloxyl substituents reduces the inhibitory bioactivity of
briarane-type diterpenoids…………………………… 54
Chapter 4 Discussion…………………… ………………………… 56
References………………………………… 63

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