探討α-solanine 在大鼠心臟及血管細胞誘發程式凋亡之機轉

Study the Mechanism of α-Solanine-Induced Apoptosis in Rat Cardiomyoblasts And Vascular Smooth Muscle Cells

黃健原(Chien-Yuan Huang)

指導教授 ： 葉竹來

高雄醫學大學/醫學院/藥理學科研究所/碩士(2010年)

https://doi.org/10.6832/KMU.2010.00046

摘要

α-Solanine是一種含有固醇骨架的醣基生物鹼 (glycoalkaloid)，普遍地在馬鈴薯 (Solanum tuberosum L.) 的塊莖以及茄科 (Solanaceae) 中的茄屬 (nightshade) 植物，任何部分都可以發現到α-Solanine的存在。馬鈴薯是日常生活中最常見的食材之一，亦是α-solanine使人類最常見的中毒來源之一，當人類誤食過多未成熟馬鈴薯塊莖的時候，即會產生一連串的中毒現象，如：胃腸障礙、心跳加速、溶血現象以及神經毒性等等，嚴重者甚至有可能造成死亡。到目前為止，在文獻中，α-solanine在心臟及血管細胞中，所引起的毒性影響及機轉仍然尚不明確。
在本研究中將使用兩種細胞，分別是H9c2心肌細胞以及大鼠胸主動脈血管平滑肌細胞，來確認α-solanine所引起的細胞毒性可能機轉。在細胞存活 (MTT assay) 以及細胞損傷 (LDH assay) 的試驗結果顯示細胞的損傷跟死亡皆呈現劑量相關性。在顯微鏡下觀察給藥24小時後的細胞型態，隨著α-solanine的劑量增加造成細胞型態萎縮，在H2DCF-DA染色觀察中，α-solanine會造成細胞產生大量自由基。在進一步的實驗，如：Hoechst 33342染色觀察以及染劑染色後進行流式細胞儀 (flow cytometry) 相關試驗，α-solanine所引起的細胞毒性從實驗結果中顯示有程式細胞凋亡 (programmed cell death, PCD) 的傾向。而在西方點墨法的分析結果顯示α-solanine會劑量相關地降低Bcl-2/Bax的相對分率；亦會誘發caspase-9、caspase-3以及MAPK (mitogen activation protein kinase) 等蛋白質的表現增加。
在離體動物實驗方面，分別用離體大鼠自律性跳動的右心房、電刺激引起收縮反應的左心房以及胸主動脈的血管環，加入不同濃度的α-solanine，來觀察α-solanine對心房以及胸主動脈的影響。從結果發現，α-solanine會增強對心房產生收縮力且同時降低心跳速率；對血管也會產生收縮力增強作用。
在此次的研究中，我們得到的結論是α-solanine會透過自由基的產生和調節Bcl-2家族、caspase家族以及MAPK家族的蛋白質表現來促使心肌細胞 (H9c2) 以及大鼠胸主動脈血管平滑肌細胞走向程式細胞凋亡的路徑。在離體組織實驗，α-solanine會產生強心作用且同時造成心跳速率抑制作用以及對血管產生收縮作用。

關鍵字

α-solanine ； 馬鈴薯 ； 心肌 ； 血管 ； 程式凋亡

並列摘要

α-Solanine, a naturally occurring steroidal glycoalkaloid, is found mainly in potato sprouts (Solanum tuberosum Linn.) and in the whole plant of the nightshade (Solanum nigrum Linn.) of the family Solanaceae. However, the most common source of α-solanine-induced poisoning, which at high levels may induce toxic effects in humans, has been the tuber of potato. Up to now, there is a few data about the effects and toxic molecular mechanism of α-solanine in rat cardiomyoblasts and vascular smooth muscle cells (VSMCs). This study was aimed to examine the possible mechanism of α-solanine-induced cytotoxicity in H9c2 cells and rat aortic VSMCs.
Here, we report that cell death of these cells was induced concentration-dependently with α-solanine by using the MTT and LDH assays. Furthermore, we observed α-solanine induced the destructive morphology and apoptosis of these cells as evidenced by microscopy, Hoechst 33342 stain and flow cytometry assay, respectively. In addition, incubation of these cells with α-solanine significantly increased the intracellular reactive oxygen species (ROS) level according to fluorescent microscopic observation using H2DCF-DA as a fluorescent substrate. Similarly, observation of Western blot showed that α-solanine decreased Bcl-2/Bax ratio concentration-dependently. Finally, we found that α-solanine-induced downstream signaling, resulting in activation of caspase-9 and caspase-3 and increased MAPK cascades expression.
In the other part, the effect of α-solanine on physical function in the atria and in the artery was studied in isolated left atrium, right atrium and isolated ring segement of the rat aorta. We found positive inotropic and negative chrontropic response in the isolated atria treated with α-solanine. In the isolated aorta, α-solanine could produce vasoconstriction concentration-dependently and Ca2+- dependently.
In conclusion, all these data suggest that α-solanine induces apoptosis in these cells, and that these effects are mediated, at least in part, by ROS, Bcl-2 related family, caspases and MAPK pathways. In addition, α-solanine can induce positive inotropic and negative chrontropic response in the isolated atria and vasoconstriction in the isolated ring segement of the rat aorta.

並列關鍵字

α-solanine ； H9c2 cells ； vascular smooth muscle cells ； apoptosis

參考文獻

Allan LA & Clarke PR (2009) Apoptosis and autophagy: Regulation of caspase-9 by phosphorylation. FEBS J 276, 6063-6073.

Barceloux DG (2009) Potatoes, tomatoes, and solanine toxicity (Solanum tuberosum L., Solanum lycopersicum L.). Dis Mon 55, 391-402.

Dalvi RR (1985) Comparative assessment of the effect of solanine administered orally and intraperitoneally on hepatic dysfunction in male rats. Nippon Juigaku Zasshi 47, 657-659.

Dao L & Friedman M (1994) Chlorophyll, Chlorogenic Acid, Glycoalkaloid, and Protease Inhibitor Content of Fresh and Green Potatoes J. Agric. Food Chem. 42, 633~639.

Dhein S, Schott M, Gottwald E, Muller A & Klaus W (1995) The contribution of neutrophils to reperfusion arrhythmias and a possible role for antiadhesive pharmacological substances. Cardiovasc Res 30, 881-888.