臺灣博碩士論文加值系統

糖尿病是當今常見的一種代謝性疾病，而且，隨著人類壽命之延長，生活品質的提昇及物質充裕，糖尿病的病例也日益增多。因此，研發糖尿病的治療藥物乃刻不容緩之課題。於是，針對葛根根部純化物「葛根素」(puerarin)進行降血糖機轉的探討，希望有助於降血糖新藥之研發。
Puerarin乃純化自葛根Pueraria lobata Willd. (豆科)的一種成份。在活體實驗 (in vivo) 方面，靜脈注射puerarin到用streptozotocin誘發，類似人類第一型糖尿病或胰島素依賴型 (insulin-dependent DM，IDDM)，的大白鼠可產生劑量相關性的降血糖作用。同時，對於外來的葡萄糖刺激試驗 (glucose challenge test)，puerarin也可增加大白鼠對葡萄糖的利用率。另外，puerarin亦能促進IDDM大白鼠骨骼肌對葡萄糖的吸回作用(glucose uptake)及增強肝臟合成肝醣(glycogen synthesis)的能力。
另一方面，以C2C12小鼠肌母細胞(mouse myoblast cell line)進行體外實驗來瞭解puerarin的作用方式。結果，puerarin能以劑量相關的方式增強C2C12小鼠肌母細胞對葡萄糖的吸回；這項作用可被a1-adrenoceptor(a1-AR)阻斷劑的prazosin所解消。同時，puerarin隨著濃度的增加亦可逐漸取代a1-AR的[3H] parzosin在C2C12肌母細胞的結合。而且，以phospholipase C (PLC) 阻斷劑的U73122，或protein kinase C (PKC) 阻斷劑的chelerythrine將a1-AR活化的相關訊息阻斷後，亦可解消puerarin原先可增強C2C12肌母細胞對葡萄糖吸回的作用。另外，a1-AR的阻斷劑會依劑量相關的型式阻斷puerarin原先在IDDM大白鼠所產生的降血糖效果。因此，在沒有胰島素存在的情況下，puerarin的降血糖作用與a1-AR的活化有關。可是，這項作用的有效濃度仍不會引致血壓變化。
使用enzyme-linked immunosorbent assay (ELISA)技術，檢測血中的腦內啡(b-endorphin)時，在IDDM大白鼠發現，puerarin會以濃度相關性的方式增加血中的β-endorphin含量；這項作用可被a1-AR阻斷劑的prazosin所拮抗。同時，嗎啡μ型受體阻斷劑的naloxone亦會依劑量相關的型式阻斷puerarin在IDDM大白鼠的降血糖效果。另外，在嗎啡μ型受體剔除( knockout )的糖尿病小鼠，靜脈注射給予能產生降血糖濃度的puerarin，卻看不到原先的降血糖作用了。在離體的腎上腺髓質(adrenal medulla)，puerarin也會隨著劑量的增加來增強β-endorphin的釋放；此項作用亦被a1-AR阻斷劑的prazosin抑制。而且，靜脈注射給予puerarin至兩側腎上腺摘除(bilateral adrenalectomy)的糖尿病大白鼠，發現其降血糖作用也因而消失了。由此可知，puerarin會活化腎上腺的a1-AR，進而促使β-endorphin釋放來產生降血糖效果。
為了瞭解puerarin降低血糖的作用機轉，就以尾靜脈注射的方式，注射有效量的puerarin到IDDM大白鼠，每日三次，連續三天。除了明顯地降低了血糖之外，亦可增強大白鼠骨骼肌之葡萄糖轉移蛋白 (GLUT4) mRNA及蛋白質的表現。同時，puerarin也會降低IDDM大白鼠肝臟之解糖酵素 (PEPCK) mRNA及蛋白質的表現；以上兩種作用皆可被naloxone或naloxonazine所解消。
綜合所得的結果可知，在IDDM大白鼠，puerarin的降血糖效果乃是藉由活化位於腎上腺髓質的a1-AR，啟動PLC-PKC這條訊息傳遞路徑，促使腎上腺髓質釋放β-endorphin。釋出的β-endorphin會作用於周邊組織的嗎啡μ型受體，來增強糖尿病大白鼠骨骼肌和肝臟對葡萄糖的吸回作用，使過多的葡萄糖能有效地自細胞外吸入到細胞內儲存，因而降低了糖尿病大白鼠的高血糖現象。

Diabetes mellitus (DM ) is one of the metabolic disorders. It has been established that the patients of DM increased in the development of countries. However, drugs for the handling of DM seem not enough. Thus, R & D of new agent is required in recent.
Puerarin is one of the active principles in the root of Pueraria lobata Willd.( Leguminosae ). In the present study, the antihyperglycemic mechanism of puerarin was investigated. A dose-dependent lowering of plasma glucose was observed in the both fasting streptozotocin (STZ)-induced diabetic rat and Wistar rats, after intravenous injection of puerarin at 30 min. Puerarin at an effective dose (15 mg/kg, i.v.) significantly attenuated the increase of plasma glucose induced by intravenous glucose challenge test in Wistar rats. Stimulatory effects of puerarin on the glucose uptake and glycogen synthesis were also obtained in soleus muscles and hepatocyte isolated from STZ-diabetic rats indicating an increase of glucose utilization by puerarin which was not dependent on insulin.
Puerarin enhanced the uptake of radioactive glucose into mouse myoblast cell line C2C12 cells in a concentration-dependent manner, which was abolished by α1-adrenoceptor(α1-AR) blocker prazosin pretreatment. Effect of puerarin on a1-adrenoceptors was further supported by the displacement of [3H] prazosin binding in C2C12 cells. The plasma glucose lowering effect of puerarin in STZ-diabetic rat was also abolished by pretreatment prazosin. Pharmacological inhibition of phospholipase C (PLC) by U73312 resulted in a concentration-dependent decrease in puerarin-stimulated uptake of radioactive glucose into C2C12 cells, although, the inactive congener, U73343, failed to block puerarin-stimulated glucose uptake. Moreover, chelerythrine diminished the action of puerarin at concentration sufficient to inhibit protein kinase C (PKC).The obtained data suggest that an activation of a1-AR may play an important role in the plasma glucose lowering action of puerarin in the absence of insulin. Actually, puerarin failed to modify blood pressure at the used doses.
Injection of puerarin at the effective dose increased the plasma b-endorphin concentration in STZ-diabetic rats and antagonist of a1-AR abolished this action. The plasma glucose lowering effect of puerarin was also abolished by pretreatment with naloxone at doses sufficient to block opioid m-receptors. Plasma glucose lowering action of puerarin at the dose effective in diabetic rats was disappeared in opioid m-receptors knockout mice, while the plasma glucose lowering response to puerarin was still observed in wild-type mice.
Also, puerarin enhanced the b-endorphin release from the isolated adrenal medulla in a concentration-dependent manner and this effect was abolished by the antagonists of a1-AR . Bilateral adrenalectomy resulted in the loss of plasma glucose lowering effect of puerarin .
The mRNA and protein levels of glucose transporter subtype 4 form (GLUT4) in soleus muscle was raised by puerarin after repeated treatment for 3 day in STZ-diabetic rats. Otherwise, similar repeated treatment with puerarin reversed the elevated mRNA level of phosphoenolpyruvate carboxykinase (PEPCK) in liver of STZ-diabetic rats to the normal level. Pharmacological inhibition of opioid m-receptors deleted these effect of puerarin.
The obtained results suggest that b-endorphin released from the adrenal gland seems responsible for the lowering of plasma glucose in STZ-diabetic rats by puerarin through an activation of a1-AR. Activation of opioid m-receptors by b-endorphin can increase the utilization of glucose and/or decrease hepatic gluconeogenesis to lower plasma glucose in diabetic rats lacking insulin.

中文摘要(P.1)英文摘要(P.5)縮寫表(P.9)第一章 緒論第一節前言(P.12)第二節葛根素（Puerarin）的特性(P.17)第二章 實驗材料與方法一、實驗動物及細胞(20)二、實驗材料(一)實驗試劑(P.21)(二)實驗器材(P.27)(三)實驗方法(P.29)第三章 實驗結果(P.56)第四章 討論(P.76)第五章 結論(P.86) 參考文獻(P.89)附圖表(P.99)

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