PI3K/Akt路徑調控穿心蓮萃出物及穿心蓮內酯誘發 大鼠初代肝細胞pi屬麩胱甘肽硫轉移酶表現機制之探討

Role of PI3K/Akt pathway in up-regulation of Andrographis paniculata extracts- and Andrographolide-induced expression of pi class of glutathione S-transferase in rat primary hepatocytes

歐俞秀(Yu-Shiou Ou)

指導教授 ： 陳暉雯

中山醫學大學/健康管理學院/營養學研究所/碩士(2008年)

https://doi.org/10.6834/CSMU.2008.00102

摘要

現代人注重健康及養生，因此，近年來將傳統中草藥應用於日常保健養生或疾病療養上的人口越來越多。穿心蓮，拉丁學名 Andrographis paniculata (Burm. F.) Nees，是一種來源廣且毒性副作用低的中草藥，普遍使用於東南亞及中國大陸等國家。穿心蓮具有清熱解毒、涼血消腫、抗血小板凝集、降血糖和血壓、抗發炎、抗癌等生理功效，穿心蓮內酯是穿心蓮當中重要的活性成分之一。過去研究指出，穿心蓮內酯可藉由活化PI3K/Akt 訊息傳遞路徑而抑制內皮細胞的細胞凋亡反應；實驗室先前研究發現，穿心蓮乙醇、乙酸乙酯萃出物及穿心蓮內酯皆可顯著增加大鼠初代肝細胞生物轉換酵素系統pi屬麩胱甘肽硫轉移酶表現，預處理PI3K抑制劑wortmannin則會抑制穿心蓮誘發pi屬麩胱甘肽硫轉移酶表現，推論PI3K/Akt訊息傳遞路徑可能參與穿心蓮調控pi屬麩胱甘肽硫轉移酶表現。因此，本實驗利用大鼠初代肝細胞培養模式，以穿心蓮乙醇、乙酸乙酯萃出物、穿心蓮內酯及PI3K抑制劑wortmannin和LY294002，深入探討PI3K/Akt路徑是否參與穿心蓮誘發pi屬麩胱甘肽硫轉移酶表現的過程。大鼠初代肝細胞預處理PI3K抑制劑25 μM LY294002或1 μM wortmannin 1小時，再加入100 μg/ml 穿心蓮乙醇、乙酸乙酯萃出物或40 μM穿心蓮內酯繼續培養48小時，觀察GSTP蛋白質表現情形。結果顯示，LY294002及wortmannin皆可顯著降低穿心蓮誘發GSTP表現 (p < 0.05)。進一步探討穿心蓮萃出物及穿心蓮內酯是否影響細胞磷酸化Akt蛋白質表現，細胞處理100 μg/ml穿心蓮萃出物或40 μM穿心蓮內酯1小時，觀察磷酸化Akt (p-Akt)蛋白質表現情形。結果顯示，穿心蓮萃出物及穿心蓮內酯皆可顯著增加細胞磷酸化Akt蛋白質表現 (p < 0.05)，此外，以LY294002或wortmannin預處理1小時，可顯著降低穿心蓮誘發磷酸化Akt蛋白質表現 (p < 0.05)。綜合以上實驗結果得知，PI3K/Akt訊息傳遞路徑參與穿心蓮萃出物及穿心蓮內酯誘發細胞GSTP表現。

關鍵字

大鼠初代肝細胞 ； 穿心蓮 ； 穿心蓮內酯 ； pi屬麩胱甘肽硫轉移酶

並列摘要

Andrographis paniculata (Ap) has been widely used as a traditional medicine in Asia for the prevention and treatment of common cold, inflammation, cancer, hyperglycemia and hypertension. Our previous study showed that the ethanol (ApEE) or ethyl acetate (ApEAE) extracts of Ap and andrographolide, the major active diterpene lactone of Ap, induced pi class of glutathione S-transferase (GSTP) expression in rat primary hepatocytes. Also, the induction of GSTP protein expression by Ap extracts and andrographolide was significantly inhibited by wortmannin, an inhibitor of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway (p < 0.05). In the present study, we investigated the role of the PI3K/Akt pathway in GSTP induction by ApEE, ApEAE or andrographolide in rat primary hepatocytes. In the GSTP expression experiment, cells were pretreated with or without 25 µM LY294002 or 1 µM wortmannin for 1 h before the addition of 100 µg/ml of ApEE, ApEAE or 40 µM andrographolide for 48 h. In the Akt phosphorylation experiment, cells were pretreated with or without 25 µM LY294002 or 1 µM wortmannin for 1 h before the addition of 100 µg/ml ApEE, ApEAE or 40 µM andrographolide for 1 h. The total protein was collected and determined for GSTP and phospho-Akt (p-Akt) protein expression by Western blot analyses. The results showed that ApEE, ApEAE and andrographolide significantly induced GSTP and p-Akt protein expression (p < 0.05). In addition, the induction of GSTP and p-Akt protein expression by Ap extracts and andrographolide was significantly inhibited by LY294002 and wortmannin (p < 0.05). These results indicate that the PI3K/Akt pathway is involved in the regulation of pi class of glutathione S-transferase expression induced by Ap extracts and andrographolide in rat primary hepatocytes.

並列關鍵字

Andrographis paniculata (Ap) ； andrographolide ； pi class of glutathione S-transferase (GSTP) ； phosphatidylinositol 3-kinase (PI3K)/Akt ； LY294002 ； wortmannin ； rat primary hepatocytes

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