臺灣博碩士論文加值系統

Constitutive androstane receptor（CAR）是核受體的一員，也是一種轉錄因子，可受到內生性及外生性物質直接或間接的活化，進而影響下游基因像是代謝酵素UGT1A1等等的表現。CAR的活化也被證實與藥物代謝具有很大的相關性且活化CAR的訊息傳導可以改善某些疾病像是癌症、代謝性疾病、炎症及肝臟疾病的治療效果。研究指出投與CAR的致效劑可以促進膽紅素 (bilirubin)的代謝及排除，動物實驗證明活化CAR可增加胰島素的敏感性、降低肝醣生成而改善第二型糖尿病的高血糖狀況，同時有助於脂質代謝異常的疾病像是脂肪肝等等。有鑒於此，CAR是一個很好的藥物發展目標。然而，因為CAR活化訊息的複雜性及無體外簡便且快速篩選具有活化CAR能力之化合物的系統，因此臨床上並無有效的人類CAR的致效劑存在。因此，在此篇研究將建立一篩選平台可在體外快速篩選具有活化CAR之活性的藥物，並利用此平台篩選香豆素衍生物活化CAR的活性，同時利用已篩出最具活性之化合物(6,7-diprenoxycoumarin)進一步探討此類化合物之作用機轉。研究結果顯示，6,7-diprenoxycoumarin是經由抑制EGFR磷酸化、促進PP2Ac甲基化進而使CAR去磷酸化使得UGT1A1及MRP2表現量增加。

Constitutive androstane receptor (CAR), also known as nuclear receptor subfamily 1, group I, member 3 (NR1I3), is a transcriptional factor that can be directly/indirectly activated by endobiotics and xenobiotics. The CAR responses for regulating drug metabolism and activation CAR signaling pathway could improve the therapeutic efficacy toward various diseases such as cancer, diabetes, inflammatory, metabolic and liver diseases. The CAR also plays a key role in accelerating bile acid detoxification and bilirubin clearance through the induction of metabolizing enzymes and transporters such as UGT1A1 and MRP2. Recently, CAR was reported to increase insulin sensitivity and ameliorate lipid diseases. Although CAR is a potential drug target for various diseases, there are no effective CAR agonists for clinical use. Lacking clinical agents for regulating CAR activity might due to the complexity of CAR signaling and lack of robust biological assay methods. In present study, a new luciferase-based assay platform responsible for rapidly screening compounds that regulate CAR activity was established and the CAR activating activity of coumarin derivatives were tested. Furthermore, the molecular mechanism(s) which involved in coumarin derivatives mediated CAR activating was also investigated. The preliminary results showed that 6,7-diprenoxycoumarin, the most potent CAR activation compound, could down-regulate the phosphorylation of EGFR, block methylation of PP2Ac and increase CAR phosphorylation, resulting increase of UGT1A1 and MRP2 production.

Index
English abbreviations III
Table index V
Figure index VI
Chinese abstract VII
English abstract VIII
Chapter 1 Introduction 1
I. Constitutive androstane receptor (CAR) 1
II. The role of CAR in hyperbilirubinemia 2
III. The role of CAR in hyperglycemia 3
IV. CAR downstream signaling: UGT1A1 4
V. CAR downstream signaling: MRP2 5
VI. CAR agonists investigation 6
Chapter 2 Research motivations 8
Chapter 3 Research approach 9
I. Materials and Instruments 9
i. Chemicals 9
ii. Cultured cell lines 9
iii. Cell culture medium 9
iv. Primary antibodies 9
v. Secondary antibodies 10
vi. Reagents 10
vii. The analytic kits 11
viii. Instruments (Alphabetic order) 11
II. Methods 12
i. Cell culture 12
ii. Cell cytotoxicity assay of coumarin derivatives
13
iii. Reverse transcription polymerases chain reaction (RT-PCR) 13
iv. Quantitative real-time PCR analysis. 14
v. Cloning and transfection 14
vi. Luciferase assays 15
vii. Nuclear and cytoplasmic extraction 15
viii. Protein extraction 15
ix. Western blot 16
x. Immunofluorescence 17
xi. Oral glucose tolerance tests in vivo in experiments in fasted normal mice 17
Chapter 4 Results 18
I. Established CAR-activation screening system and validated CAR-activation screening system 18
II. Determined CAR-activation activity of coumarin derivatives by luciferase assay 19
III. CAR activation could be observed after 2 hrs treatment of 6,7-diprenoxycoumarin 19
IV. 6,7-diprenoxycoumarin inhibited EGFR phosphorylation 20
V. 6,7-diprenoxycoumarin increased the expression of methyl-PP2Ac 20
VI. 6,7-diprenoxycoumarin induced mRNA expression of CAR downstream target genes, UGT1A1 and MRP2 21
VII. 6,7-diprenoxycoumarin increased the protein expression of CAR downstream, UGT1A1 and MRP2 21
VIII. 6,7-diprenoxycoumarin decreased serum glucose level in vivo 21
Chapter 5 Discussions 23
References 26

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