在卵巢切除及睪丸切除的大鼠中對心臟肥大及凋亡療效的探討 第一部分Diosgenin對卵巢切除的大鼠心臟肥大及凋亡的影響 第二部分睪固酮與運動訓練對睪丸切除的大鼠心臟肥大及凋亡的影響

Therapeutic Effects of Cardiac Hypertrophy and Apoptosis in Ovariectomized and Orchiectomized rats PartⅠ.Effects of diosgenin on cardiac hypertrophy and apoptosis in ovariectomized rats PartⅡ.Effects of testosterone replacement and exercise training on cardiac hypertrophy and apoptosis in orchiectomized rats

蔡靜怡(Ching-Yi Tasi)

指導教授 ： 劉哲育

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2007年)

摘要

第一部份
女性罹患心臟疾病的時間比男性晚了10至15年左右，過去內生性的雌性素對粥狀動脈硬化及冠狀動脈心血管疾病應具有一定的保護功能，研究也證實停經後的婦女使用荷爾蒙替代療法確實能降低心血管疾病的發生，但卻會有極嚴重的副作用，例如：乳癌。Diosgenin 是結構類似雌性素的植物類固醇皂素，因此我們希望能取代雌性素用在卵巢切除鼠，探討Diosgenin 是否能保護心臟並了解其中的機制？將5-7周齡的Wistar大鼠分成假手術控制組及卵巢切除組，而卵巢切除組則施行卵巢切除一個月，再分別每天每公斤體重餵食0,10,50,100 mg的Diosgenin一個月，再探討心肌細胞的凋亡、存活及肥大等路徑，首先從H&E 染色的心臟切片中發現Diosgenin對卵巢切除鼠的心肌細胞有減緩傷害的情形，而且利用Western Blot也看到卵巢切除所活化的肥大指標蛋白ANP及BNP，又因添加Diosgenin而使蛋白表現下降，另外在誘導肥大的路徑p38、JNK、IL6-MEK5-ERK5等訊息路徑皆可看到相同的結果。在TUNEL assay中觀察到Diosgenin亦可去抑制卵巢切除所產生的心肌細胞凋亡的情形，還利用Western Blot證明心肌細胞凋亡是透過Death receptor及Mitochondria 這兩條路徑，而Diosgenin也會活化ERβ及PI3K/Akt這條路徑來保護卵巢切除的心肌細胞。由以上研究結果我們相信適量Diosgenin具有保護心肌細胞的作用，也許在不久的將來，可以應用在臨床上，並取代雌性素的荷爾蒙替代治療。
第二部份
男性在老化過程中，體內各種荷爾蒙分泌量會逐漸減少，包括雄性素。研究顯示在年老男性體內睪固酮的含量較低，常與冠狀心血管疾病及心肌梗塞的發生有著密切關聯。因此本研究想探討缺乏睪固酮的大鼠中能否藉由運動與補充睪固酮的方式來調控心肌細胞的肥大及凋亡？實驗方法：將7-8周齡的Wistar雄性大鼠，分成進行假手術的控制組（C）及睪丸切除一個月後又分成三組，包括有睪丸切除的控制組（CO）、每日以游泳為運動訓練三小時的睪丸切除組（EO），及運動後30分鐘皮下注射每公斤體重0.2 mg睪固酮(Testosterone) 的睪丸切除組（ETO）。經2週後，待老鼠兩天休息後犧牲，取得心臟並利用心臟切片染色及Western Blotting等實驗進行分析。首先，H&E 染色的心臟切片結果發現，運動訓練與睪固酮補充會降低睪丸切除所誘導的心肌細胞傷害的情形，也可降低被睪丸切除所誘導的心臟肥大指標蛋白BNP的降低，另外在誘導肥大的IL6-MEK5-ERK5訊息路徑也看到相同的結果。此外還發現睪丸切除是透過Death receptor及Mitochondria 這兩條路徑中活化下游Caspase 蛋白的表現來造成心肌細胞凋亡，運動訓練與睪固酮補充也會降低這路徑的蛋白，同時對於MAPK pathway中的p38α及p38β對心肌細胞凋亡與存活亦有相同的影響。因此本研究證實適量的運動訓練及睪固酮有防止心肌細胞的肥大與凋亡，進而達到保護心臟的作用。

關鍵字

薯蓣皂配基 ； 睪固酮 ； 卵巢切除 ； 睪丸切除 ； 心臟肥大 ； 心臟凋亡

並列摘要

Part1.
Background. Very limited information regarding the protective effects of diosgenin on cardiac hypertrophy and apoptosis after post-menopause or bilateral oophorectomy in women was available. The purpose of this study was to evaluate the effects of diosgenin on bilateral ovariectomy induced cardiac hypertrophic and apoptotic pathways. Methods. Forty-six female Wistar rats at 5-7 weeks of age randomly were divided into sham-operated group (Sham),bilateral ovariectomized group (Ovx), and Ovx groups with 10mg/kg, 50mg/kg,or 100mg/kg diosgenin spp.daily (Ovx10, 50, 100) for 1 month. The excised hearts were measured by histopathological analysis, western blotting and RT-PCR, and positive TUNEL assays. Results. Diosgenin spp. Decreased Ovx-induced cardiac abnormalities including abnormal myocardial architecture, enlarged interstitial spaces, and more cardiac TUNEL-positive apoptotic cells. Diosgenin spp. decreased Ovx-induced Fas ligand, Fas death receptors, FADD. Diosgenin spp. decreased Ovx-induced Bad, Bax, activated caspase 9 and activated caspase 3, but increased ERβ, IGF1 receptor, pAkt. Ovx100 exerted less anti-apoptotic than Ovx10 and Ovx50. Conclusions. Diosgenin spp. suppressed ovariectomy-induced cardiac hypertrophy and death receptor-dependent and mitochondria-dependent apoptotic pathway with dose-associated manners. The findings may provide one of possible therapeutic approach for potentially treating or preventing cardiac abnormalities in post-menopause or bilateral oophorectomized women.
Part 2
Background. Aging in men is associated with a progressive decline in the production of several hormones, including androgen. Coronary disease and myocardial infarction often appear in aging men at low levels of testosterone. Objective. To determine the effects of testosterone replacement with exercise training on cardiac hypertrophy and apoptosis. Methods. In this study, twenty-eight male Wistar rats at 7-8 weeks of age randomly were divided into sham-operated control group (C), bilateral orchiectomized group (CO), and CO group with exercise group (EO) and exercise group treated with testosterone (ETO). Rats in exercise group were exercised in 3h/day, 5day/week swimming model and some of them were injected testosterone injections(0.2mg/kg/day) for 2 weeks. The execised hearts were measured by histopathological analysis, western blotting. Results. Exercise training and testosterone spp. decreased CO-induced cardiac abnormalities including abnormal myocardial architecture, enlarged interstitial spaces. Exercise training and testosterone spp. decreased CO-induced BNP, IL6, MEK5 and ERK5, but no significant differences in EO group. Exercise training and testosterone spp. decreased CO-induced TNF , activated caspase 8, tBid, Fas, FADD, Bad, Bak, caspase 9, caspase 3 and p38α. Another exercise training and testosterone spp. increased pBad and p38β. Conclusions. Exercise training and testosterone spp. suppressed orchiectomy-induced cardiac Death receptor-dependent and mitochondria- dependent apoptotic pathway. The findings may provide possible therapeutic approach for potentially treating or preventing cardiac abnormalities in testosterone deficiency men.

並列關鍵字

Diosgenin ； testosterone ； ovariectomy ； orchiectomy ； cardiac hypertrophy ； cardiac apoptosis

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