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研究生： 吳宮頡
Wu, Kung-Chieh
論文名稱： 一條根萃取物活性成分改善環磷醯胺誘導膀胱功能障礙與病理機制之大鼠模式
I-Tiao-Gung Extract through its Active Component Improves Cyclophosphamide-Induced Bladder Dysfunction and Mechanisms in Rat Model
指導教授： 鄭劍廷
Chien, Chiang-Ting
學位類別： 博士
Doctor
系所名稱： 生命科學系
Department of Life Science
論文出版年： 2019
畢業學年度： 108
語文別： 英文
論文頁數： 69
中文關鍵詞： 一條根 、黃豆戒 、膀胱過動 、環磷醯胺 、蕈鹼受體 、嘌呤受體 、氧化壓力
英文關鍵詞： I-Tiao-Gung, daidzin, overactive bladder, cyclophosphamide, muscarinic receptor, purinergic receptor, oxidative stress
DOI URL： http://doi.org/10.6345/NTNU202000039
論文種類： 學術論文
相關次數： 點閱：134  下載：3 
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    • 一條根（I-Tiao-Gung）在金門當地已經普遍被廣泛使用於治療風濕性疾病以及痠痛之傳統中草藥。而金門一條根屬於豆科植物的闊葉大豆（Glycine tomentella Hayata），且豐含天然植物雌激素黃酮類及酚類物質，有研究指出一條根具有抗發炎及抗氧化能力之功效。
    膀胱過動症（OAB）是一種很常見且又很容易被忽略的疾病。膀胱過動症的起因是由多重因素所造成的，如尿道出口阻塞、細菌感染及尿路上皮受損等症狀，在病生理學上，當膀胱過動症確診後，會引起尿道損傷及慢性發炎的症狀。環磷醯胺（Cyclophosphamide, CYP）是一種化療藥物，具有高毒活性代謝物，在尿液中對膀胱產生急性或慢性之損傷，包含出血性膀胱炎。我們使用環磷醯胺腹腔注射誘導膀胱過動症的大鼠模式，當膀胱發炎時機械敏感性的傳入神經變得較敏感，將導致膀胱過動（Bladder hyperactivity）。膀胱發炎會引起活性氧（ROS）的產生，而活性氧是氧化壓力形成的原因之一，且其最終可能會導致膀胱功能障礙。
    因此，我們探究內服給予中藥一條根萃取物中黃豆戒（Daidzin）活性成分對環磷醯胺誘導膀胱炎、氧化壓力、纖維化和發炎及膀胱過動症之治療潛力。本研究使用Wistar 大鼠，其CYP給予方式為腹腔注射，一條根及黃豆戒為口服管餵方式。我們透過西方墨點法檢測，蕈鹼受體M2和M3和P2X2和P2X3嘌呤能受體以及3-硝基酪氨酸（3-NT）和NADPH氧化酶4（NOX4）的表現，以及動物膀胱內壓與尿道外括約肌電圖相關之檢測。此外，我們透過超靈敏化學發光分析儀，從而確定了膀胱活性氧（ROS）的量，以及透過細胞因子陣列來確認多種細胞因子譜的表現包含在內的MMP-8和TIMP-1。我們結果顯示，一條根萃取中黃豆戒活性成可有效改善環磷醯胺誘導膀胱炎和恢復第二階段的活性作用（EUS-EMG），並抑制P2X2，P2X3，M3受體，3-NT，NOX4的表達。結論，一條根萃取成分和其主要活性成分黃豆戒可降低環磷醯胺誘導氧化壓力且可抑制環磷醯胺造成之MMP-8、TIMP-1、發炎和纖維化。

    I-Tiao-Gung (ITG) is a traditional Chinese herbal medicine which has been widely used in the treatment of rheumatic diseases and soreness in Kinmen. The ITG belongs to Glycine tomentella Hayata, a family of soybean rich in natural phytoestrogens, such as flavonoids and phenolic substances. Studies have pointed out that ITG has anti-inflammatory and antioxidant effects.
    Overactive bladder (OAB), a very common disease but easily being ignored, is formed by multiple factors, including urethral obstruction, bacterial infection and urinary tract epithelial damage. In pathophysiology, once OAB is confirmed, it could further lead to urethral injury and chronic inflammation. Cyclophosphamide (CYP), a chemotherapeutic agent, but has highly toxic metabolites that cause acute or chronic damage to the bladder, inducing hemorrhagic cystitis. We used intraperitoneal injection of CYP to induce the bladder hyperactivity and inflammation in rats based on its harm for bladder as previously described. Mechanical sensitization of the afferent nerve becomes higher when bladder inflammation occurs, and then leads to bladder hyperactivity. Bladder inflammation causes the production of reactive oxygen species (ROS), the main character in oxidative stress, and might eventually lead to bladder dysfunction and fibrosis.
    Therefore, we explored the therapeutic potential of intragastric administration traditional Chinese medicine ITG extract and its active component Daidzin on CYP-induced cystitis, oxidative stress, fibrosis, inflammation and bladder hyperactivity in rats. In this study, CYP was intraperitoneal injected to Wistar rats and ITG or Daidzin was administrated by oral gavage. We determined the transcystometrogram associated with external urethral sphincter electromyogram, and the expression of M2, M3 muscarinic, P2X2, P2X3 purinergic receptors, 3-nitrotyrosine (3-NT) and NADPH oxidase 4 (NOX4) by Western blot in rats. In addition, we determined the bladder reactive oxygen species (ROS) amounts by an ultrasensitive chemiluminescence analyzer, the expression of multiple cytokine profiles including MMP-8 and TIMP-1 via cytokine array. In conclusion, these data suggest that ITG extract through its active component Daidzin effectively improved CYP-induced cystitis by the action of restoring Phase 2 activity（EUS-EMG）and inhibiting the expressions of P2X2, P2X3, M3 receptors, 3-NT, NOX4 and, oral intake ITG or Daidzin improved CYP-induced oxidative stress, inflammation and fibrosis through inhibiting the MMP-8, TIMP-1 and oxidative stress.

    中文摘要 X Abstract XII Abbreviation XIV Chapter 1. Introduction and Literature review 1 1-1 Traditional Chinese Medicince of I-Tiao-Gung 2 1-2 CYP-induced cystitis and bladder dysfunction 2 1-3 Research Aims 5 Chapter 2. Materials and Methods 7 2-1 I-Tiao-Gung 8 2-2 Preparation of I-Tiao-Gung extract 8 2-3 HPLC analysis 8 2-4 Animals 9 2-5 ITG and Daidzin given dose 10 2-6 Antioxidant activity of ITG, Daidzin and Daidzein 10 2-7 CYP-induced cystitis and bladder hyperactivity 11 2-8 Transcystometric model 11 2-9 Recording of extraurethral sphincter-electromyogram (EUS-EMG) activity 11 2-10 In vitro and in vivo chemiluminescence recording for ROS activity 12 2-11 Western blot 12 2-12 Multiple cytokine antibody arrays 13 2-13 Histology 13 2-14 Statistical Analysis 14 Chapter 3. Results 15 3-1 ITG-HPLC analysis 16 3-2 The effect of ITG on voiding parameters 16 3-3 The effect of ITG and bladder hyperactivity 17 3-4 The effect of ITG on voiding cycle and EMG activity 18 3-5 The effect of ITG on purinergic receptors 18 3-6 The effect of ITG on muscarinic receptors 19 3-7 The effect of ITG and bladder inflammatory and fibrosis and MDA 19 3-8 The effect of ITG and purinergic and muscarinic receptors and mast cell and hematuria 20 3-9 In vivo chemiluminescence recording for bladder ROS parameters 21 3-10 The effect of ITG on 3-NT and NOX4 21 3-11 Cytokine array 22 Chapter 4. Discussion and Conclusions 23 4-1 The treatment of ITG and Daidzin on CYP-induced bladder hyperactivity 24 4-2 The effect of ITG and Daidzin on CYP-induced voiding dysfunction and EUS-EMG activity 25 4-3 The effect of CYP-induced bladder hyperactivity in purinergic receptors and protein expression 26 4-4 The effect of CYP-induced bladder hyperactivity in muscarinic receptors and protein expression 27 4-5 Effect of CYP-induced bladder inflammation triggered 3-NT or NOX4 expression and ROS accumulation 28 4-6 Effect of CYP-induced bladder inflammation triggered MMP-8 or TIMP-1 expression 29 4-7 Conclusions 31 4-8 Future Works 32 Reference 33 Figure Contents 46

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