2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38761Diabetic bladder dysfunction (DBD) is a most common complication of diabetes mellitus (DM), and is seen in 50-80% of diabetic patients. The mechanism involved is still under debate and treatment options are limited. Recently many studies implicated that oxidative stress contributes to the pathophysiology of many diseases, and suggested that the oxidative damage of detrusor muscle cells may be a contributory factor in DBD. However, there are no experimental study reports about the curative effects of electro-acupuncture (Ea) at Zusanli (ST-36) acupoints and green tea polyphenols (GTP) therapies in DBD. The purpose of this study is to investigate whether Ea treatment and GTP administration could increase the antioxidant capabilities or protective effects in streptozotocin (STZ) induced diabetic rats with DBD. Female Sprague-Dawley rats are dosed with STZ (65 mg/kg) or vehicle and divided into three groups containing 10 animals each: Control (with needling at Zusanli without stimulation), STZ-induced DM and STZ-DM-Ea (Ea is applied to bilateral Zusanli for 30 min every day following the induction of DM) group. Five weeks after treatment, functional changes are assessed by cystometry and the bladder is then removed for oxidative status (superoxide dismutase activity, the levels of dinitrophenyl and nitrotyrosine), isometric contractile and molecular studies. The levels of expression of the actin regulatory proteins are determined at the mRNA and protein levels by reverse transcriptase-polymerase chain reaction and Western blotting, respectively. The experimental objective of 2nd year is to investigate the antioxidant effects of GTP on DBD. The rats are separated into 5 groups. They are control, DM, DM with GTP oral administration (400 mg/kg/day) only, DM with Ea stimulation only, and DM with GTP and Ea treatments simultaneously. Five weeks later, the bladders are dissected. Oxidative status, isometric contractile, histological studies and actin regulatory protein expression are measured. The first aim at 3th year we are interesting is using immunostaining methods and confocal microscope to objectively monitor pathological changes and cellular distributions of actin regulatory proteins (calponin, caldesmon and tropomyosin) in cells dissociated from diabetic hypertrophy bladders (with/without Ea or/and GTP treatment) and primary cultured overnight. Another purpose is to study the antioxidant capabilities of GTP in normal/high glucose treated detrusor cells following 40μg/ml GTP treatment ± 1mM H2O2 (oxidant agent). Cell viability, apoptosis and oxidative status are evaluated. The levels of expression of the actin regulatory proteins were determined at the mRNA and protein levels by reverse transcriptase-polymerase chain reaction andWestern blotting, respectively. Ea at Zusanli acupoints and GTP administration should be considered as alternative methods for improving hyperglycermic oxidative stress induced by STZ in rats.We hope such a therapeutic technique has potential in clinical practice.有50~80% 糖尿病(diabetic mellitus, DM) 患，深受糖尿病膀胱功能異常(diabetic bladder dysfunction, DBD) 症狀之困擾，但其詳細致病機轉仍不清楚。陸續有研究認為高 血糖環境會引發過氧化壓力(oxidative stress)，在DBD 的致病過程扮演重要角色。因此 本實驗目的主要探討在動物的足三里穴位(Zusanli acupoint) 進行電針刺激 (electroacupuncture, Ea) 及投餵綠茶多酚(green tea polyphenols, GTP)，觀察抗氧化處理 對streptozotocin (STZ)-DBD 的保護或改善效應。 本研究先建立STZ-誘發DM 動物模式，第一年擬監測電針刺激的抗氧化能力：將實 驗動物分成控制組(未注射STZ，穴位進針但不通電刺激)、DM 組及DM-Ea 處理三組， 誘發DM 3 天後，每天在DM-Ea 動物兩側足三里穴進行電針刺激(100 Hz, 0.5mA, 1.5- 2ms) 30 分鐘。在誘發DM 後第5 週，每組各取10 隻動物，探討膀胱組織中的氧化狀 態(包括超氧歧化酶活性、dinitro phenyl 及Nitrotyrosine 的濃度變化)、膀胱逼尿肌肌束 的收縮能力以及肌肉中肌動蛋白調控蛋白(actin regulatory protein) 的表現量的變化。 第二年擬觀察GTP 對DM 動物膀胱氧化損傷的改善效應，與電針刺激足三里穴的效 果進行比較；或DM 動物同時接受GTP 與電針刺激處理，觀察DBD 的改善程度。將動 物分成五組：控制組；DM 組；DM-GTP 組；DM-Ea 組及DM-GTP-Ea 組。動物每天依組 別以管餵方式餵食GTP (400mg/kg) 或電針處理30 分鐘，實驗處理5 週後，觀察DM 膀 胱的型態及神經支配變化、膀胱組織中的氧化狀態、逼尿肌肌束收縮能力以及肌動蛋白 調控蛋白的表現量的變化或改善。 第三年的實驗分二個實驗主題：首先探討在膀胱逼尿肌細胞(detrusor smooth muscle cell; DSM) 中之收縮性肌絲相關蛋白的表現及分佈：動物的分組及處理與第二年 相同，方法是動物在依組別處理後，分別將各組動物之膀胱逼尿肌肌(detrusor smooth muscle , DSM) 細胞游離出來進行初代培養(primary culture)，利用免疫多重染色方法及 共軛焦顯微鏡(confocal microscope)，實際觀察並比較細胞內肌動蛋白調控蛋白(如 Calponin、Caldesmon 及Tropomyosin) 與肌動蛋白在處理前後的表現及在空間上的互動 關係。第二個研究主題擬直接在細胞層面觀察高糖份(50 mM glucose) 和H2O2 對正常 DSM 細胞所造成的氧化傷害程度，並探討GTP 抗氧化處理對DSM 細胞肌動蛋白調控蛋 白表現及細胞凋亡程度的影響。 藉由本實驗設計期望能釐清DM 的氧化壓力造成DBD 的機制；並更進一步了解電針 刺激足三里穴及GTP 抗氧化能力改善膀胱功能異常的潛能和分子機轉，期能對為DBD 所苦的病患提供更有效的替代療法。糖尿病膀胱功能異常足三里穴電針刺激肌動蛋白調控蛋白氧化壓力逼尿肌綠茶多酚初代培養共軛焦顯微鏡Project