山藥和紅景天保護過氧化氫所引起的心臟細胞凋亡之機轉探討

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Title:	山藥和紅景天保護過氧化氫所引起的心臟細胞凋亡之機轉探討 Part I Diosgenin protects H2O2-induced myocardial cells apoptosis via activation of PI3K/Akt signaling through estrogen receptors Part II Rhodiola (salidroside) protects H2O2-induced H9c2 cardiomyoblast cells apoptosis involvement of ERK1/2
Authors:	黃上恩;Shang-En Huang
Contributors:	中醫學院中醫學系碩士班
Keywords:	停經;中醫藥;山藥;紅景天;氧化壓力;凋亡;Menopause;Traditional Chinese medicine (TCM);Diosgenin;Rhodiola;Oxidative stress;Apoptosis
Date:	2010
Issue Date:	2010-09-29 12:01:02 (UTC+8)
Abstract:	停經後婦女因體內雌激素的減少，而使其罹患心血管疾病的風險增加。近年來，這些使停經後婦女罹患心血管疾病的風險增加的機轉逐漸被研究得知，是因體內氧化壓力上升所造成的。雌激素在許多生物活體和細胞層次的實驗中，已皆被証實其有益於預防保護心臟疾病的發生。臨床上荷爾蒙替代療法亦已長時間使用於停經後婦女身上。但是卻導致罹患中風和乳癌等副作用的機會大大增加。因此我希望能自天然的果類和草藥中尋找出一種固醇類賀荷爾蒙的前驅物來取代原本用於停經後婦女的荷爾蒙替代療法，如此不但可以減少心臟疾病的發生並可減少副作用的產生。 Diosgenin，是一種固醇類的薯預皂苷元，其廣泛存在於薯預和山藥植物中。Diosgenin也是眾多荷爾蒙產物的組成材料，並且其結構非常相近於雌激素。已有實驗證實Diosgenin對於細胞毒殺性和抗氧化傷害均有許多有利於人體的作用。在本實驗中，我觀察並證實過氧化氫確實隨著時間的增加而引起心肌細胞的細胞毒殺性和凋亡，利用MTT assay和Hematoxylin-and-eosin staining來證明細胞毒殺性，並使用Western blotting和Immunofluorescence assay証實其凋亡是經由活化Fas-dependent、 mitochondria-dependent pathway和下游Caspase-3等凋亡蛋白表現。並且在JC-1 staining偵測下，過氧化氫亦能引起粒線體膜電位之不穩定。在後續的實驗結果中則證實diosgenin明顯有效地保護過氧化氫所引起心肌細胞的細胞毒殺性和凋亡之機轉。Diosgenin在不同濃度梯度下，透過抑制Fas-dependent、 mitochondria-dependent pathway和下游caspase-3等凋亡蛋白表現，同樣地我以MTT assay和Hematoxylin-and-eosin staining等方法證明diosgenin能保護過氧化氫所引起之細胞毒殺性，並利用Western blotting和 TUNEL assay證明其抗凋亡的途徑與功能。更進一步地我也用Western blotting和JC-1 staining發現diosgenin抗凋亡的機轉是透過活化IGF1途徑等存活蛋白的表現達到恢復粒線體膜電位的穩定。在深入探討其訊遞分子時我發現，diosgenin透過雌激素接受器活化磷酸化態PI3k/Akt和ERK1/2達到保護的效果。我更利用了雌激素接受器的拮抗劑等等藥物和siRNA的處理，以及MTT assay, Western blotting, TUNEL assay, 和Immunofluorescence assay等方法，逐一為我的發現做佐證。紅景天（Rhodiola）因對於環境有高適應性的特性，在傳統中醫用藥已廣泛被使用治療高原病。紅景天生長於亞洲及歐洲之高海拔區域，常用於增加器官之抗缺氧性和治療各種不同氧化壓力傷害的情況。Salidroside，是自植物性中藥紅景天中分離出的主要抗氧化成分。已有藥理學實驗證實salidroside 對於抗老化和抗氧化傷害等均有許多有利於人體的作用。在本實驗中，證實Rhodiola和salidroside皆有明顯有效地保護過氧化氫所引起心肌細胞的細胞毒殺性和凋亡之機轉。首先應用MTT assay和Hematoxylin-and-eosin staining來證明Rhodiola保護過氧化氫所引起之細胞毒殺性。再利用Western blotting證明Rhodiola在不同濃度梯度下，透過抑制Fas-dependent，HIF-1 alpha，mitochondria-dependent pathway和下游caspase-3等凋亡蛋白表現達到抗凋亡功能，並透過IGF1R活化磷酸化態ERK1/2達到保護的效果。接著同樣以MTT assay和Hematoxylin-and-eosin staining證實salidroside也確實地保護過氧化氫所引起之細胞毒殺性。再深入探討發現，salidroside亦須經由活化磷酸化態ERK1/2達到保護的效果，我成功地應用了ERK抑制劑並以MTT assay和Immunofluorescence assay驗證了我的發現。 Objectives: The risks of cardiovascular diseases in post-menopausal women were increased. Oxidative stress is one of important contributory factors to the etiology of many cardiovascular diseases. Diosgenin, a member of steroidal sapogenin, structurally similar to estrogen has been shown to have antioxidant effects. The study investigate whether diosgenin directly prevent oxidation-induced cardiomyocyte apoptosis. Methods: Apoptotic pathways and mitochondria membrane stabilizing potential were measured in H9c2 cardiomyoblast cells and neonatal cardiomyocytes cultured in serum-free medium for 12 h then pretreated diosgenin for 1 h, subsequently stimulated with H2O2. Results: In this study, H2O2 treated H9c2 cardiomyoblast cells induced cytotoxicity and apoptosis by activated Fas-dependent and mitochondria-dependent pathway. And it also induced instability of mitochondria membrane potential. Further experiment results demonstrated that diosgenin protected H2O2 induced H9c2 cell apoptosis through activated survival pathway (IGF1 signaling) and recovered mitochondria membrane stabilizing potential to suppress Fas-dependent, mitochondria-dependent pathway against apoptosis. And it also inhibited the H2O2-induced cytotoxicity and apoptosis through the estrogen receptors activated phosphorylation of PI3K/Akt and extracellular regulated protein kinases 1/2 (ERK1/2) in myocardial cells. Conclusion: Diosgenin prevented oxidation-induced cardiomyocytes cytotoxicity and apoptosis. Objectives: Oxidative stress is one of important contributory factors to the etiology of many cardiovascular diseases. Rhodiola widely used for its high capacity to increase the organism resistance to different oxidative stress conditions. Salidroside, a phenylpropanoid glycoside separated from a traditional Chinese medicinal plant Rhodiola rosea, and has a potent antioxidant property. Methods: Cell viability and apoptotic pathways and were measured in H9c2 cardiomyoblast cells cultured in serum-free medium for 12 h then stimulated with H2O2 for 4 h subsequently post-treated Rhodiola (salidroside) 24 h. Results: In this study, Rhodiola (salidroside) protected H2O2 induced cytotoxicity and apoptosis through IGF1R activated extracellular regulated protein kinases 1/2 (ERK1/2) to inhibited Fas-dependent, HIF-1 alpha, Bax and Bak related apoptotic proteins in H9c2 cells. Conclusion: Rhodiola (salidroside) protected oxidation-induced H9c2 cardiomyoblast cells cytotoxicity and apoptosis.
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