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

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(1)國立臺灣師範大學生命科學專業學院生命科學系博士論文 Department of Life Science, School of Life Science. National Taiwan Normal University Doctoral Dissertation. 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 博士研究生：吳宮頡 Ph.D Student：Wu, Kung-Chieh 指導教授：鄭劍廷博士 Adviser：Chien, Chiang-Ting Ph.D. 中華民國 108 年 12 月 December 2019.

(2) Contents 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 I.

(3) 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 II.

(4) 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. III.

(5) 4-8 Future Works ……………………………………………………….32. Reference …………………….……………………………….33. IV.

(6) Figure Content Figure 1. Experimental protocol, the ITG extract active component from HPLC analysis, and effect of ITG, Daidzin, and Daidzein on scavenging H2O2 …………………...……….……………..………47. Figure 2. The original graph and statistic data of the effect of ITG or Daidzin treatment on CYP-induced bladder hyperactivity in five groups of rats ….……………………………………………..……50. Figure 3. The effect of ITG and Daidzin on voiding reflex including micturition. reflex. and. EUS-EMG. in. five. groups. of. rats ….……………………………………………………………..53. Figure 4. The expression of purinergic and muscarinic receptor, P2X2 and P2X3 and M2 and M3 proteins, in control (Con) group, CYP-induced (CYP) groups, CYP+ITG group, CYP+Daidzin group, and ITG+CYP pretreatment group ……………………….……….57. V.

(7) Figure 5. The effects of ITG on hematoxylin and eosin and masson’s of the rat urinary bladder in control (Con) group, CYP-induced (CYP) groups, CYP+ITG group, CYP+Daidzin group, and ITG+CYP pretreatment group ………….…………….……………………….58. Figure 6. The effects of ITG on immunohistochemical characterization of the rat urinary bladder in control (Con) group, CYP-induced (CYP) groups, CYP+ITG group, CYP+Daidzin group, and ITG+CYP pretreatment group ………...………………………………………60. Figure 7. Effect of ITG extract or Daidzin on CYP-induced bladder ROS production in vivo of the rat urinary bladder. The original data (A) and statistic data (B) of ROS from the urinary bladder in the control, CYP-treated,. CYP+ITG,. and. CYP+Daidzin. groups. are. indicated ……………………………………...……………...……63. VI.

(8) Figure 8. The expression of 3NT and NOX4 proteins. The expression of 3NT (A), NOX4 (B), in Control, CYP, CYP+ITG, CYP+Daidzin groups: All the statistic bars were obtained from n = 3 in each group ……………………………………..…………….…………65. Figure 9. A: The typical expression of cytokine antibody array including MMP-8, and TIMP-1 proteins, in the control group, CYP-induced (CYP) groups, CYP+ITG group, CYP+Daidzin group …….……..67. Figure 10. Summary diagram for depicting ITG extract or Daidzin on CYP-induced bladder dysfunction of the rat urinary bladder ….…68. Supplemental Figure. The statistic data of the effect of ITG, Daidzin or Daidzein treatment on CYP-induced bladder hyperactivity in six groups of rats …………………….………………………………..69. VII.

(9) 致謝時光荏苒，在這一路求學歷程中受多位恩師用心指導和鼓勵之下，從國立屏東教育大學體育學系畢業後，順利攻讀國立台灣師範大學生命科學系生理組，這是一個完全新的領域，還記得剛入學時對於新領域一竅不通的我，經歷不斷百翻苦讀和師長的用心指導下宮頡受益良多，直到至今我勇於嘗試新事物的批判和研究分析，並看見自己在博士班歷經磨練後的成長，也體悟到學習是永無止境的，這些試煉豐碩了我的學習視野。能順利完成本論文，皆承蒙恩師指導鄭劍廷教授，記得我當初剛踏入實驗室時，對於生命科學神經生理學撲朔離迷的我，從 Paper 閱讀和 ppt 呈現及報告表達方式，老師總是不厭其煩地用心教導與鼓勵，且在論文寫作上給予指正與批改，非常感謝您。更感謝口試委員余宏政教授、廖俊厚教授、林瑞興教授、吳忠信教授的悉心審核、批閱與精闢開釋，提供許多寶貴的建議，讓本論文更加的完整。也非常感謝實驗室學長姊們給予的幫忙和細心教導，當我文章或實驗遇到問題，伊婷、威毅總是會細心講解整個脈絡，來完成每次實驗及文章修正。. VIII.

(10) 其在分生實驗中非常感謝伊婷、超哥鼎力的幫忙，尤其伊婷當我文章需補做分生 data 時，總是在百忙中撥空大力幫忙讓我的文章順利完成通過，非常感恩，再來感謝怡慧學姐，在實驗室各項的經費事項幫忙處理，讓實驗室學弟妹們能夠專心於學業和實驗上。最後就是實驗室的各位夥伴們幃尹、晉容、威琳、怡安、佳錞、冠葦、孟哲、庭睿、麗雯、毛毛，在實驗室期間一起分擔的工作及平時生活中給予支持與勉勵，非常感謝你們願意幫助我。最感謝家人在我這一路求學歷程中給予精神上的支柱，謹以此論文獻給我最敬愛的爸爸、媽媽和姐姐的支持與關心，讓我安心攻讀博士，且順利獲取博士學位，使我倍感無比的溫馨。願將這份喜悅，分享給所有親朋好友們和關心我的人。. IX.

(11) 中文摘要一條根（ 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 X.

(12) 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。. 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. XI.

(13) ABSTRACT 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. XII.

(14) 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.. Key words: I-Tiao-Gung, daidzin, overactive bladder, cyclophosphamide, muscarinic receptor, purinergic receptor, oxidative stress XIII.

(15) Abbreviation Full Form. Abbrev.. 3-Nitrotyrosine. 3-NT. Acetylcholine. ACH. Adenosine triphosphate. ATP. Chemiluminescent. CL. Contractile amplitude ( MVP-BP= AM). AM. Control. CON. Cyclophosphamide. CYP. Double-distilled water. ddH2O. Extracellular matrix. ECM. Extraurethral sphincter-electromyogram. EUS-EMG. Hematoxylin and eosin. H&E. XIV.

(16) High-performance liquid chromatography. HPLC. Horseradish peroxidase. HRP. Hydrogen peroxide. H2O2. Immunohistochemistry. IHC. Intercontraction interval. ICI. Intravesical pressure. IVP. I-Tiao-Gung. ITG. Lower urinary tract symptoms. LUTS. Malondialdehyde. MDA. Matrix metalloproteinases. MMPs. Maximal voiding pressure. MVP. Micturition time. MT. NADPH oxidase 4. NOX4. XV.

(17) Overactive bladder. OAB. Potassium choloride. KCl. Reactive oxygen species. ROS. Standard error of the mean. SEM. Threshold pressure for triggering micturition. PTH. Tissue inhibitor of metalloprotease. TIMPs. XVI.

(18) Chapter 1. Introduction and Literature review. 1.

(19) 1-1 Traditional Chinese Medicince of I-Tiao-Gung I-Tiao-Gung (Glycine tomentella Hayata, ITG), a traditional Chinese herbal medicine, has been widely used in the treatment of rheumatic diseases and soreness in Taiwan.1 ITG extract contains soy isoflavones, phytoestrogens, flavonoids, and phenolic substances that can diminish proinflammatory cytokines, scavenge free radicals, and inhibit lipid peroxidation.1 Furthermore, soy isoflavones replacement has been reported to benefit detrusor overactivity in ovariectomized rats.2 Therefore, ITG rich in soy isoflavones may have a potential to prevent and improve CYP-induced bladder hyperactivity.. 1-2 CYP-induced cystitis and bladder dysfunction Cyclophosphamide (CYP) is a chemotherapeutic medicine that has been clinically used in the treatment of cancer and B-cell malignancies, such as lymphoma and myeloma; it is also used in non-malignant diseases such as rheumatoid arthritis.3 This chemotherapeutic agent has highly toxic metabolites that cause acute or chronic damage to the bladder, inducing hemorrhagic cystitis.4 Lower urinary tract symptoms (LUTS), including urine storage and voiding dysfunction, pain sensation, urgency and post-micturition dribble, are a highly prevalent condition that 2.

(20) influences the quality of life.5,6 Among these symptoms, urine storage dysfunction is believed to be a major problem.7,8 Overactive bladder (OAB), a widespread condition and easily ignored, is a symptom complex syndrome characterized by urine storage dysfunction. The syndrome includes symptoms such as urinary urgency, additional daytime frequency, and nocturia all with the absence of urinary tract infection, tumor or urolithiasis.7 According to epidemiological reports, the lower urinary tract symptoms in men is about 62%, and women around 67%, incidence in adults over the age of 60 will respectively rise to 81 and 79%. This dysfunction can cause acute urinary incontinence which leads to harassment, depression, or even lower their self-esteem and influence their quality of life.9 The factors lead to OAB are urethral outlet obstruction, bacterial infection and urothelial damage. These factors are as well considered as the causes of urinary tract injury and chronic inflammation.10 The stimulations of type 2 and 3 muscarinic receptors (M2 and M3) on bladder smooth muscle cells produce detrusor contraction. Anti-muscarinic agents are used in the treatment of bladder hyperactivity by the reduction of acetylcholine-induced contraction of the detrusor muscle.11,12 Accumulating evidence implicates that a non-cholinergic activation via purinergic receptors may be a new therapeutic target to 3.

(21) neurogenic bladders, outflow obstruction, idiopathic detrusor instability, and interstitial cystitis, as well as in the aging bladder.13 Adenosine triphosphate (ATP), a purinergic neurotransmitter, released from efferent nerves excites bladder smooth muscle, whereas ATP released from urothelial cells can activate afferent nerves and urothelial cells. 14 Bladder afferent nerve activity is markedly excited during bladder inflammation and contributes to bladder hyperactivity.12,15 Purinergic P2X receptors, particularly P2X2 and P2X3 receptors, are primarily responsible for the sensitivity of lower urinary tract.11,16 The enhanced release of ATP from bladder urothelium was associated with an increase of P2X3 receptor expression during chronic bladder inflammation.14 Bladder inflammation cause the production of reactive oxygen species (ROS), the main character in oxidative stress, and may eventually lead. to. bladder. dysfunction,17,18. and. bladder. fibrosis.. Matrix. metalloproteinases (MMPs) are a family of enzymes involved in different processes such as modulation of inflammation, tissue remodeling and collagen processing.19 The abnormal of MMPs and tissue inhibitor of metalloprotease (TIMPs) and/or dysregulation in MMPs/TIMPs are responsible for the process of fibrosis or proteolytic activity19-23 and possibly contributes to the bladder diseases. MMPs establish a complex network in which different enzymes may play opposite roles. In addition, specific family members may play different roles in different time points 4.

(22) of the disease. For example, MMP-8, also known as collagenase-2, can digest native collagen, but its function in vivo seems to be more related to the control of the inflammatory response.24 By the ability to cleave different cytokines and chemokines, MMP-8 promotes the initial onset and the later clearance of the neutrophilic inflammatory response and mice lacking MMP-8 show a delayed wound healing25 and a decreased lung fibrosis.26 Earlier evidence has indicated that Cyclophosphamide (CYP) not only causes abnormal ECM deposition, fibrosis but leads to smooth muscle hyperreflexia in the bladder.27 CYP was intraperitoneally injected to induce bladder hyperactivity in the rat model in other research.28,29 Therefore, we used CYP to induce bladder inflammation and fibrosis in a rat model of our study to explore the response of MMP-8 expression.. 1-3 Reseach Aims Our recent report has first demonstrated that ITG extract through its active component Daidzin effectively improved CYP-induced bladder contractile dysfunction by the action of restoring Phase 2 activity and inhibiting the expressions of purinergic P2X2, P2X3, and muscarinic M3 receptors.30 However, to further explore the other protective mechanisms, we considered the antioxidant soy isoflavones-rich ITG may diminish ROS-evoked bladder oxidative stress, inflammation, and fibrosis leading 5.

(23) to ameliorate CYP-induced hemorrhagic cystitis.. 6.

(24) Chapter 2. Materials and Methods. 7.

(25) 2-1 I-Tiao-Gung I-Tiao-Gung (ITG) belongs to Glycine tomentella Hayata grown from the Xiguo mountain of Kinmen area in Taiwan and is provided by Kinmen County Agriculture Research Institute. We used the biennial dried main root of ITG in the present study.. 2-2 Preparation of I-Tiao-Gung extract The method for extracting ITG was described previously.1 In brief, the dry root of ITG was grounded, added ethanol, heated at 70°C for 2 hours and filtrated. After doing the previous step twice, the combined liquid extracts were lyophilized and obtained ITG extract. The ITG extract was then stored at 4°C until use.1. 2-3 HPLC analysis The ITG extract was analyzed by a high-performance liquid chromatography (HPLC, Hitachi system) consisting of a LaChrom Pump L-2130, a Programmable Autosampler L-2200, an Interface D-7000 and a LaChrom Diode Array Detector L-2455 (Merck, Vienna, Austria). The reagents of genistin, genistein, daidzin, daidzein, glycitin, glycitein were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Acetonitrile 8.

(26) (HPLC grade) and DMSO (analytical reagent grade) were obtained from Merck (Darmstadt, Germany).. 2-4 Animals Thirty female Wistar rats (200-250 g body weight) were purchased from BioLASCO (Taiwan Co. Ltd., Taipei) were housed with a consistent light cycle (light from 07:00 to 18:00). Food and water were provided ad libitum. All the surgical and experimental procedures were approved by the Institutional Animal Care and Use Committee of the National Taiwan Normal University and were in accordance with the guidelines of the National Science Council of Republic of China (NSC 1997). The rats were anesthetized with intraperitoneal urethane (1.2 g/kg body weight). Urethane was chosen for lacking ganglionic blocking properties.13 The maintenance of deep anesthesia was determined by the persistence of miotic pupils as judged from frequent inspection. 13 Body temperature was maintained at 37°C with a heat lamp. After experiments, the anesthetized animals were sacrificed by intravenous KCl. Thirty rats were divided into control rats (Control, n=6), CYP rats (CYP, n=6), CYP treated rats with ITG (CYP+ITG, n=6), CYP treated rats with Diadzin (CYP+Diadzin, n=6), and one week of ITG preconditioning with CYP rats (ITG+CYP, n=6). The detailed protocols were indicated in Figure 1A. 9.

(27) 2-5 ITG and Daidzin given dose Previous studies showed the safety dose of ITG extract was below 2 g/kg/day in rats for 28 days.31 Our preliminary data has evaluated the dosage at intragastrical 0.117 g/kg, 0.585 g/kg, 1.17 g/kg and 3.34 g/kg on CYP-induced voiding dysfunction. We found ITG extract at 1.17 g/kg displayed the most efficient action on the improvement CYP-induced overactive bladder. The intragastric dosage of 3.34 g/kg increased non-voiding contractions in the bladders implicating a possible toxic effect. Therefore, we applied the dosage of ITG extract intragastrically at 1.17 g/kg/day to the animal. Our ITG extract contained 1.07% Daidzin and 0.77% Daidzein. The ITG extract mixed with 1 mL of distilled water was applied to animals intragastrically. ITG 1.17 g contains 12. 5 mg of Daidzin. Daidzin mixed with 1 mL of distilled water (12.5 mg/kg/day) was applied to animals intragastrically. Control rats were given distilled water.. 2-6 Antioxidant activity of ITG, Daidzin and Daidzein A major reactive oxygen species generated from activated leukocytes via the myeloperoxidase system is hydrogen peroxide (H 2O2), which can initiate inflammation.32 We evaluated the antioxidant H2O2. 10.

(28) activity of ITG extract, Daidzin and Daidzein on the enhanced chemiluminescent signals from the H2O2-luminol mixture as described previously.32. 2-7 CYP-induced cystitis and bladder hyperactivity CYP-induced hemorrhagic cystitis and bladder hyperactivity were performed with intraperitoneal CYP at a dose of 200 mg/kg, while a control group received intraperitoneal saline.. 2-8 Transcystometric model We utilized a transcystometric technique to evaluate voiding response to CYP.13 These parameters of bladder responsiveness were measured: intercontraction interval (ICI, the time interval between two micturition cycles identified with active contractions [> 10 mmHg]), baseline bladder pressure (BP), threshold pressure for triggering micturition (PTH), maximal voiding pressure (MVP) and contractile amplitude (MVP-BP =AM) (Figure 2A).. 2-9 Recording of extraurethral sphincter-electromyogram (EUS-EMG) activity For measurement of EUS-EMG activity, epoxy-coated stainless steel 11.

(29) wire (50 mm; M. T. Giken Co., Tokyo, Japan) EMG electrodes were placed percutaneously in the extraurethral sphincter (EUS) by using a 30-gauge needle with a hooked EMG electrode positioned at the tip. The needle was inserted into EUS approximately 5–10 mm lateral to the urethra and then withdrawn, leaving the EMG wires embedded in the muscle.13. 2-10 In vitro and in vivo chemiluminescence recording for ROS activity The major reactive oxygen species generated from activated leukocytes via the myeloperoxidase system is hydrogen peroxide (H2O2), which can initiate inflammation.33 We evaluated the antioxidant H2O2 activity of ITG extract and Daidzin on the enhanced chemiluminescent (CL) signals from the H2O2-luminol mixture as described previously.33. 2-11 Western blot The expression levels of respective antibody against M2 (#bs-0441R, Biosynthesis Biotechnology, Littleton, CO, USA), M3 (#ab87199, Abcam's RabMb® technology, Cambridge, UK), P2X2 (#ab10266, Abcam's RabMb ® technology, Cambridge, UK), and P2X3 (#RA14139, Neuromics, Edina, MN, USA), and 3-Nitrotyrosine (3-NT, #ab61392,. 12.

(30) Abcam's RabMb® technology, Cambridge, UK) and NADPH oxidase 4 (NOX4, #MBS176126, Mybiosource, San Diego, USA) in the whole bladder homogenates were analyzed by Western blot.34 Horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (#406401, Biolegend , San Diego, CA, USA) was used as secondary antibody. The band density was determined semi-quantitatively by densitometry using an image analyzing system (Alpha Innotech, San Leandro, CA, USA). Lipid peroxidation indicated by malondialdehyde (MDA) was determined with the MDA Assay Kit (#ab118970, Abcam's RabMb® technology, Cambridge, UK).. 2-12 Multiple cytokine antibody arrays In response to toxicity, several inflammatory mediators such as cytokines. and. chemokines. could. be. released. by. activated. macrophages/Kupffer cells in the damaged bladder. Therefore, multiple cytokine expression levels were simultaneously detected and identified with the aid of RayBio®rat cytokine protein array (RayBiotech, Inc., Norcross, GA, USA) according to the manufacturer's instructions.. 2-13 Histology Five-µm sections of formalin-fixed bladders were stained with. 13.

(31) hematoxylin and eosin (H&E) for evaluating leukocytes infiltration, hemorrhage and edema, toluidine blue staining for mast cell labeling 32, and Masson’s trichrome for fibrosis. The leukocytes, hemorrhage and edema in the bladder tissue were analyzed and scored as described. 35 Bladder sections were immunohistochemically stained with the specific antibody as described in Western blot. For speific immunostaining, the tissue sections were incubated overnight at 4°C with specific antibody. A biotinylated secondary antibody (Dako, Botany, NSW, Australia) was applied followed by streptavidin conjugated to HRP. The chromogen used was Dako Liquid diaminobenzene. Twenty fields were randomly selected for each section, and the value of each positive stain was counted.. 2-14 Statistical Analysis All data were expressed as means ± standard error of the mean (SEM). Data were subjected to one way of analysis of variance, followed by Duncan’s multiple-range test for assessment of the difference among groups. p<0.05 was considered to indicate statistical significance.. 14.

(32) Chapter 3. Results. 15.

(33) 3-1 ITG-HPLC analysis Our results showed 1.07% of Daidzin and 0.77% of Daidzein (Figures 1B-1D). Thus, Daidzin was selected as a major component and was applied in the animal experiment for evaluating CYP-induced bladder hyperactivity. ITG dose-dependently and significantly reduced H2O2 amount (Figure 1E). Daidzin inhibited H2O2 amounts. Daidzin is more efficient than Daidzein in scavenging H2O2 amount (Figure 1F).. 3-2 The effect of ITG on voiding parameters Daily ITG or Daidzin treatment in control rat did not display abnormal voiding parameters (unpresented data). Our results showed that the level of PTH in CYP group was significantly decreased as compared to the control group. The decreased PTH by CYP was significantly recovered by ITG treatment (Figure 2B). A significant decrease of ICI and a significant increase of urinary frequency were found in CYP group vs. control group (Figures 2C and 2D). The. 16.

(34) improvement of bladder hyperactivity was noted in the CYP+ITG, CYP+Daidzin,. ITG+CYP. groups. (Figures. 2C. and. 2D). and. CYP+Daidzein group (Supplemental figure). The BP in CYP, CYP+ITG and CYP+Daidzin group was significantly reduced vs. control group (Figure 2E). MT (time spent from Phase 1 to Phase 4 contraction as described in Figure 3F) in CYP group was significantly increased vs. control group (Figure 2F). The CYP-increased MT was significantly depressed by ITG or Daidzin treatment (Figure 2F). MVP in CYP, CYP+ITG and ITG+CYP was significantly decreased vs. control group (Figure 2G). AM in CYP group was significantly reduced vs. control group (Figure 2H). The significant improvement of AM was found in the CYP+Daidzin group vs. CYP group (Figure 2H).. 3-3 The effect of ITG and bladder hyperactivity The voiding frequency in CYP group (Figure 3B) was significantly increased vs. control group (Figure 3A). CYP-enhanced bladder. 17.

(35) hyperactivity was significantly depressed in the CYP+ITG (Figure 3C), CYP+Daidzin (Figure 3D) and ITG+CYP (Figure 3E) group.. 3-4 The effect of ITG on voiding cycle and EMG activity An increased voiding frequency associated with a depressed EUS-EMG response were found in CYP rats (Figure 3G) vs. control rats (Figure 3F). These responses were partly recovered in CYP+ITG (Figure 3H), CYP+Daidzin (Figure 3I) and ITG+CYP (Figure 3J) rats. A normal voiding cycle includes four voiding contractile phases (Figure 3F). CYP efficiently depressed the response of Phase 2 duration, peak EUS-EMG activity and silent/active component ratio (Figures 3K-3M). These depressed responses were partly recovered by ITG or Daidzin treatment.. 3-5 The effect of ITG on purinergic receptors The expression of P2X2 (Figure 4A) and P2X3 (Figure 4B) was significantly upregulated in CYP bladders. The upregulation of P2X2 and P2X3 by CYP was significantly depressed by ITG or Daidzin treatment.. 18.

(36) 3-6 The effect of ITG on muscarinic receptors The M2 expression was similar among these five groups (Figure 4C). CYP significantly enhanced M3 expression, whereas the CYP-enhanced M3 expression was significantly depressed by ITG or Daidzin treatment (Figure 4D).. 3-7 The effect of ITG and bladder inflammatory and fibrosis and MDA The increased hemorrhage (Figure 5A), edema (Figure 5B) and leukocytes infiltration (Figure 5C) were found in the CYP bladders. CYP+ITG,. CYP+Daidzin,. and. ITG+CYP. bladders. significantly. decreased hemorrhage (Figure 5E), edema (Figure 5F) and leukocytes infiltration (Figure 5G). Masson’s trichrome staining showed increased fibrosis in CYP bladder and decreased fibrosis in CYP+ITG, CYP+Daidzin, and ITG+CYP bladders (Figure 5D). CYP significantly increased bladder MDA level, whereas the increased MDA level wase significantly reduced by ITG or Daidzin treatment (Figure 5H).. 19.

(37) 3-8 The effect of ITG and purinergic and muscarinic receptors and mast cell and hematuria M2 expression was similar among these five groups (Figures 6A and 6G). CYP enhanced M3 expression, whereas CYP-enhanced M3 expression was decreased by ITG or Daidzin treatment (Figures 6B and 6H). CYP significantly enhanced the expression of P2X2 (Figures 6C and 6I) and P2X3 (Figures 6D and 6J), whereas the enhanced expression of P2X2 and P2X3 was significantly inhibited by ITG or Daidzin treatment (Figures 6I and 6J). Toluidine blue staining showed increased mast cells in CYP group vs. control group (Figure 6E). The mast cells number wase significantly reduced in CYP+ITG, CYP+Daidzin, and ITG+CYP groups vs. CYP group (Figure 6K). The appearance of hematuria was found in the CYP group and the response was inhibited in CYP+ITG, CYP+Daidzin, and ITG+CYP groups (Figure 5J).. 20.

(38) 3-9 In vivo chemiluminescence recording for bladder ROS parameters The ROS amounts in CYP group was significantly increased compared with the control group. CYP-induced bladder ROS was decreased significantly in the CYP+ITG and CYP+Daidzin groups (Figures 7 A and B).. 3-10 The effect of ITG on 3-NT and NOX4 3-NT expression was significantly enhanced in CYP group, whereas 3-NT expression was lower in the ITG extract or Daidzin treated group (Figure 8A). Additionally, the NOX4 expression was significantly elevated in CYP group as compared to control group and was enormously depressed by ITG extract or Daidzin treatment in CYP+ITG and Daidzin+CYP groups (Figure 8B).. 21.

(39) 3-11 Cytokine array MMP-8 expression was significantly enhanced in the CYP group as compared to control group (Figure 9A), whereas the enhanced MMP-8 expression was significantly reduced in ITG extract or Daidzin treated group. Additionally, TIMP-1 expression was significantly higher in CYP group and was significantly depressed in the ITG+CYP group or Daidzin+CYP group (Figure 9B).. 22.

(40) Chapter 4. Discussion and Conclusion. 23.

(41) 4-1 The treatment of ITG and Daidzin on CYP-induced bladder hyperactivity CYP significantly increased bladder hyperactivity. The increased voiding frequency was found 2 days after intraperitoneal injection with CYP28,36 and a significant reduction in voiding frequency was observed by ITG or Daidzin treatment. Dietary Daidzin can efficiently improve detrusor overactivity of ovariectomized rats.2 In our prepared ITG extract, Daidzein is also contained in ITG extract at approximately 70% level of Daidzin. Our in vitro data showed that the capability of anti-oxidant activity in Daidzin was more efficient than in Daidzein (Figure 1F). Our in vivo data showed that the recovery of the shorten ICI level was significantly higher in Daidzin than in Daidzein (70% value of Daidzin in Supplemental figure) implicating Daidzin is more efficient than Daidzein in improvement of CYP-induced bladder hyperactivity. We found that Daidzin alone is more effective than ITG extract (mixture of Daidzin and Daidzein) in improving CYP-induced bladderhyperactivity possibly due. 24.

(42) to. some. components. affecting. purinergic. and. cholinergic. neurotransmission, proinflammatory or pro-oxidant signaling existing in the ITG extract. Therefore, based on these data, we suggest that Daidzin may play a more important role than Daidzein in ITG extract to ameloriate CYP-induced bladder dysfunction. In addition, ITG+CYP also demonstrated similar improvement with CYP+ITG. We suggest that the use of ITG extract can ameliorate CYP-induced bladder dysfunction through the preconditioning or conditioning route in future.. 4-2 The effect of ITG and Daidzin on CYP-induced voiding dysfunction and EUS-EMG activity The efficient voiding function depends on the co-ordinated activity of smooth muscles (urinary bladder and urethra) and striated muscles (EUS) in the lower urinary tract and can be evaluated by the techniques of transcystometrogram and EUS-EMG recording.13,37 A micturition cycle can be regulated by purinergic receptors-mediated Phase 1 and muscarinic receptors-mediated Phase 2 contraction.13 The increased IVP. 25.

(43) in Phase 1 is associated with increased tonic pelvic efferent nervous activity and tonic EUS-EMG activity. Phase 2 characterized by the bursting of pelvic efferent nervous activity and EUS-EMG activity is responsible for the bladder emptying. Our data delineated CYP-induced voiding dysfunction is ascribed to the bursting EUS activity being converted to tonic EUS activity and the depressed silent/active component ratio possibly by the CYP toxicity to the pelvic and pudenal nerves and muscles. Increase in active period of EUS-EMG is likely to increase MT in CYP group compared to control group. ITG and its active component, Daidzin, can efficiently restore Phase 2 contraction, EUS-EMG activity and silent/active component ratio to ameliorate CYP-induced bladder dysfunction.. 4-3 The effect of CYP-induced bladder hyperactivity in purinergic receptors and protein expression ATP plays a role in sensory neurotrnamission of purinergic receptor, which in turn causes voiding reflexes.12 The exaggerated release of ATP. 26.

(44) in the urothelial epithelium of neurogenic bladder leads to the activation of P2X2 and P2X3 receptors resulting in further neuronal activation and bladder hyperactivity.37,38 Increased P2X3 expression is also found in several types of neurogenic hyperactive bladder.11,39,40 The expressions of P2X2 and P2X3 receptors were significantly increased in our CYP bladders. The expressions of P2X3 and P2X2/3 receptors were increased significantly in the neurogenic bladders.41,42 Additionally, upregulation of P2X2 receptor promoted detrusor hyperactivity.43 ITG extract or Daidzin efficiently and completely inhibited the upregulation of P2X2 and P2X3. We suggest the application of ITG and Daidzin can reduce the expression of P2X2 and P2X3 subsequently improving bladder hyperactivity.. 4-4 The effect of CYP-induced bladder hyperactivity in muscarinic receptors and protein expression The expectable treatment is to suppress the exaggerated contractility of detrusor and to inhibit bladder hyperactivity.11,44 Mirabegron, a β3-adrenoceptor agonist, has been tested in the clinical trials for the. 27.

(45) treatment of overactive bladder for its action to relax detrusor. 45 M2 seems to play no role in CYP induced bladder hyperactivity because M2 expression is similar among these five groups. Overexpression of M3 receptors in detrusor contributes to bladder hyperactivity.34 Our data showed CYP enhanced M3 receptor expression and the enhanced M3 expression was significantly and partially depressed by ITG or Daidzin.. 4-5 Effect of CYP-induced bladder inflammation triggered 3-NT or NOX4 expression and ROS accumulation Exaggerated ROS production may induce oxidative injury, inflammation and voiding dysfunction in the bladders. 33,46 NOX4 is an pro-oxidant enzyme for triggering ROS production and 3-NT, a product of tyrosine nitration mediated by ROS, is identified as an indicator or marker of cell damage, oxidative stress and inflammation.47-49 Previous studies have shown that CYP induced bladder inflammation triggered 3-NT and ROS accumulation in the urinary bladder, and further led to bladder hyperactivity.47 Our study also showed higher 3-NT expression in. 28.

(46) the CYP group and a lower 3-NT expression in the ITG and Daidzin treated bladders. In addition, activation or high expression of NOX4 exaggerated ROS production.48 CYP-treated bladder displayed the higher expression of NOX4 possibly and sesquentially stimulating higher ROS formation in the CYP-treated urinary bladder.49 Our data indicated CYP elevated NOX4 and 3-NT expression that was significantly attenuated through ITG extract or Daidzin treatment in the damaged bladders implicating the therapeutic potential of ITG in reducing oxidative injury.. 4-6 Effect of CYP-induced bladder inflammation triggered MMP-8 or TIMP-1 expression Moreover, studies have shown ROS can activate MMPs, function as basement membrane and extracellular matrix degradation,50 and can downregulate the endogenous inhibitors of MMPs (TIMPs).51 One previous report stated that some MMPs are anti-fibrotic, while other MMPs can have pro-fibrotic functions. Some studies have reported that CYP is related to MMPs and TIMPs expression.52,53 Zhang et al. (2013). 29.

(47) demonstrated a upregulation of MMP-8 and/TIMP-1 expression in the left ventricles of rats with cardiac volume overload.53 It is suggested that specific family members of MMPs may play different roles in different time points of the disease and in in vitro or in vivo condition. MMP-8 can trigger proteolytic activity, but its function in vivo seems to be more related to the control of the inflammatory response.24 MMP-8 promotes the initial onset and the later clearance of the neutrophilic inflammatory response and mice lacking MMP-8 show a delayed wound healing25 and a decreased lung fibrosis26 MMP-8 is present in the initial stages of the acute inflammatory response and can therefore influence neutrophil recruitment.54 The dysregulated enhancement of MMP-8 and TIMP-1 expression was found in myocardium with cardiac volume overload53 and in our CYP bladders. Our present data was consistent with previous report that the elevation of MMP-8 and TIMP-1 contributed to inflammatory responses in the CYP group.55 However, the expression of MMP-8 and TIMP-1 was rarely observed in the ITG extract and Daidzin groups that indicated ITG extract and Daidzin could efficiently inhibit. 30.

(48) MMP-8 and TIMP-1 expression, reduce inflammation and subsequently improve bladder hyperactivity. The higher expression of TIMP-1 resulted in the inhibition of MMP activity and the accumulation of matrix proteins in extracellular space.56,57 These studies concluded CYP may lead to bladder fibrosis, the production of MMPs and TIMP in bladder tissue and the deposition of collagen in ECM. We confirmed CYP induced bladder fibrosis via Masson’s trichrome stain. However, significant recoveries were found in the bladders treated with ITG extract and Daidzin. Additionally, studies have shown that the thickening of bladder wall may result from excessive production of pro-fibrotic ECM and may provoke muscle growth, edema, and infiltration of immune cells and inflammatory substances.58-60. 4-7 Conclusions ITG can improve CYP-induced cystitis and bladder hyperactivity through the action of Daidzin to confer ant-oxidation, anti-inflammation, restore Phase 2 activity by maintaining the balance between detrusor and. 31.

(49) urethral and downregulate P2X2, P2X3, M3, MMP-8, TIMP-1, 3-NT and NOX4 expression in CYP bladders (Figure 10).. 4-8 Future Works ITG can inhibit inflammation through 5-lipoxygenase (5-LOX) and Cyclooxygenase-2 (COX-2) pathway61 and. reduce inflammatory. cytokines IL-1β, and IL-6 release.1 Our data presented the increased mast cells, leukocytes infiltration, hemorrhage and edema in the CYP bladders, but these inflammatory responses were all depressed by ITG or Daidzin treatment. We speculated that the inflammation can be reduced via the inhibition of LOX-COX-2-IL-1β-IL-6 pathway. However, the detailed mechanism for the reduction of leukocytes and mast cells are required determined further.. 32.

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(66) Figure 1. Experimental protocol, the ITG extract active component from HPLC analysis, and effect of ITG, Daidzin, and Daidzein on scavenging H2O2. (A) Experimental protocol and group design. CYP was administered at the time labed with a blue arrow for 48 hours. Conditioning ITG or Daidzin was administered two times (time 0 and 24 hours) during 48 hours of CYP treatment. One week of ITG was administered 7 times during one week preconditioning. (B) HPLC of six isoflavone standard, (C) HPLC of the ITG sample, (D) The percentage of the six isoflavone in our prepared ITG sample. (E) The antioxidant effect for scavenging H2O2 amount from ITG extract and Daidzin. (F) The antioxidant effect for scavenging H2O2 amount between Daidzin and Daidzein. *p < 0.05 when compared to 0 mg/mL or Daidzin.. 49.

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(69) Figure 2. The original graph (A) and statistic data of the effect of ITG or Daidzin treatment on CYP-induced bladder hyperactivity in five groups of rats (n=6 in each goup). (B) PTH: threshold pressure, (C) ICI: intercontraction interval, (D) Urinary frequency, (E) BP: baseline bladder pressure, (F) MT: micturition time (the time from Phase 1 to Phase 4), (G) MVP: maximal voiding pressure, and (H) AM: contractile amplitude (AM = MVP-BP). *p < 0.05 when compared to Control group. #P < 0.05 when compared to CYP group.. 52.

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(72) Figure 3. The effect of ITG and Daidzin on voiding reflex including micturition reflex and EUS-EMG in five groups of rats. The simultaneous recordings of IVP and EUS-EMG are demonstrated in one Control (A), CYP (B), CYP+ITG (C), CYP+Daidzin (D) and ITG+CYP (E) rat. The integrated graph in a fast speed shows simultaneous recordings of IVP and EUS-EMG in control (F), CYP (G), CYP+ITG (H), CYP+Daidzin (I) and ITG+CYP (J) rat. Four phases of IVP are identifiable in a micturition reflex: Phase 1, an initial rise in IVP; Phase 2, a plateau of HFOs; Phase 3, a rebound pressure and Phase 4, a rapid fall in IVP. Note that a depressed phase 2 contraction and a loss of EUS-EMG activity in the CYP rat (G). ITG or Daidzin partly restores these depressed parameters in response to CYP treatment. (K) When compared to Control group, a significantly decreased Phase 2 contractile duration was found in the CYP rats. ITG, Daidzin or ITG preconditioning significantly recovered the Phase 2 contractile duration. (L) As compared to Control group, a significantly inhibited peak EUS-EMG activity was noted in CYP group. The depressed peak EUS-EMG activity was significantly and partly recovered in the CYP+ITG, CYP+Daidzin and ITG+CYP groups. (M) During Phase 2 contraction, silent component (S) was indicated with red arrow, whereas active component (A) was indicated with blue arrow. As compared to Control group, a significantly decreased silent/active component ratio was found in CYP group. The depressed silent/active 55.

(73) component ratio was significantly and partly recovered in the CYP+ITG, CYP+Daidzin and ITG+CYP groups. A statistical data was obtained from n=6 in each goup. MT (micturition time, the spent time from Phase 1 to Phase 4 contraction) *p < 0.05 when compared to Con group. #P < 0.05 when compared to CYP group.. 56.

(74) Figure 4. The expression of purinergic and muscarinic receptor, P2X2 and P2X3 and M2 and M3 proteins, in control (Con) group, CYP-induced (CYP) groups, CYP+ITG group, CYP+Daidzin group, and ITG+CYP pretreatment group. The expression of P2X2 (A), P2X3 (B), M2 (C) and M3 (D) in Con, CYP, CYP+ITG, CYP+Daidzin and ITG+CYP groups: All the statistic bars were obtained from n=3 in each group. *p < 0.05 when compared to Con group. #P < 0.05 when compared to CYP group.. 57.

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(76) Figure 5. The effects of ITG on hematoxylin and eosin and masson’s of the rat urinary bladder in control (Con) group, CYP-induced (CYP) groups,. CYP+ITG. group,. CYP+Daidzin. group,. and. ITG+CYP. pretreatment group. A-1-A-5：H&E staining of hemorrhage indicated with black arrows. B-1-B-5: H&E staining of edema indicated with black line. C-1-C-5：H&E staining of leukocyte infiltration by blue stains. D-1-D-5: Masson’s trichrome staining. The statistic data from the degree of hemorrhage (E), edema (F), leukocytes infiltration (G), mast cell numbers (H), MDA concentration were indicated and were obtained from n=6 in each group. *p < 0.05 when compared to Con group. #P < 0.05 when compared to CYP group.. 59.

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(79) Figure 6. The effects of ITG on immunohistochemical characterization of the rat urinary bladder in control (Con) group, CYP-induced (CYP) groups,. CYP+ITG. group,. CYP+Daidzin. group,. and. ITG+CYP. pretreatment group. A-1-A-5: M2 stain indicated by brown color. B-1-B-5: M3 stain indicated by brown color. C-1-C-5: P2X2 stain indicated by brown color. D-1-D-5：P2X3 stain indicated by brown color. E-1-E-5: Mast cells positive stained by toluidine blue (T blue) and indicated by black arrows. F-1-F-5: the appearance of hematuria in the CYP group, whereas less appearance in the ITG or Daidzin treated groups. The statistic data from the degree of M2 expression (G), M3 expression (H), P2X2 expression (I) and P2X3 expression (J) and mast cell numbers (K) were indicated and were obtained from n=6 in each group. *P < 0.05 when compared to Con group. #p < 0.05 when compared to CYP group.. 62.

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(81) Figure 7. Effect of ITG extract or Daidzin on CYP-induced bladder ROS production in vivo of the rat urinary bladder. The original data (A) and statistic data (B) of ROS from the urinary bladder in the control, CYP-treated, CYP+ITG, and CYP+Daidzin groups are indicated. *p < 0.05 when compared to the Control group. #p < 0.05 when compared to CYP group.. 64.

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(83) Figure 8. The expression of 3NT and NOX4 proteins. The expression of 3NT (A), NOX4 (B), in Control, CYP, CYP+ITG, CYP+Daidzin groups: All the statistic bars were obtained from n = 3 in each group. *p < 0.05 when compared to the Control group. #p < 0.05 when compared to CYP group.. 66.

(84) Figure 9. A: The typical expression of cytokine antibody array including MMP-8, and TIMP-1 proteins, in the control group, CYP-induced (CYP) groups, CYP+ITG group, CYP+Daidzin group. B: The statistical data of the expression of MMP-8 and TIMP-1 in Control, CYP, CYP+ITG, CYP+Daidzin groups: All the statistic bars were obtained from n = 3 in each group. *p < 0.05 when compared to Control group. #p < 0.05 when compared to CYP group.. 67.

(85) Figure 10. Summary diagram for depicting ITG extract or Daidzin on CYP-induced bladder dysfunction of the rat urinary bladder.. 68.

(86) Supplemental Figure. The statistic data of the effect of ITG, Daidzin or Daidzein treatment on CYP-induced bladder hyperactivity in six groups of rats. *p < 0.05 when compared to Control (Con) group. #p < 0.05 when compared to CYP group. ap < 0.05 CYP+Daidzin vs. CYP+Daidzein.. 69.

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