- 學位論文
葒草對肝星狀細胞功能及生長抑制之研究
The investigation of Polygonum orientale L. on cell function and growth inhibition in hepatic stellate cell.
摘要
慢性肝病及肝硬化久居國人十大死因，而肝纖維化則是造成肝硬化及慢性肝病的必經過程。肝臟中的肝星狀細胞(hepatic stellate cell，HSC)活化及增生是造成肝纖維化的重要因素。近年來的研究證明中藥在對抗肝纖維化具有一定療效的。葒草(Polygonum orientale L)，源自於蓼科，具有擴張大鼠冠狀動脈並且對組織胺所引起的支氣管痙攣具有拮抗作用，是民間治療肝疾病之中草藥。本研究以體外細胞培養的模式，探討葒草在抗纖維化方面的效用與可能機制。為了測試葒草是否對肝星狀細胞具有生長抑制作用，首先進行細胞型態之觀察，結果顯示在藥物處理組中，隨著葒草培養時間的增加，肝星狀細胞有生長抑制以及細胞懸浮之現象。接著在細胞毒殺測試（MTT assay）發現葒草能抑制肝星狀細胞的增生，此生長抑制與劑量及處理時間具有相關性。流式細胞儀分析細胞週期的結果則進一步顯示葒草使肝星狀細胞停滯於G1期，而S期有被明顯的抑制的情形。另一方面，TUNEL assay 中，也可以明顯觀察到DNA片段化的現象，且與細胞凋亡相關的 caspase-3 及caspase-9 活性皆有明顯增加的現象。以上結果證實葒草具有抗肝星狀細胞增生及誘導細胞凋亡之效果。另外，在低濃度(0.3 mg/ml)葒草處理之下，肝星狀細胞遷移能力即明顯被抑制； Q-PCR的檢測則發現藥物濃度高達 2 mg/ml 時，肝星狀細胞分泌第一型膠原蛋白、第三型膠原蛋白及結締組織生長因子的能力會被明顯抑制；但對金屬基質蛋白酶MMP-2的產生卻沒有明顯抑制的效果。這些結果顯示，葒草藉由抑制肝星狀細胞增生與誘導肝星狀細胞凋亡，並降低肝星狀細胞遷移與分泌細胞激素及膠原蛋白的功能，故能減緩肝星狀細胞所造成纖維化。
並列摘要
The chronic liver disease and liver cirrhosis for a long time occupy the top ten leading causes of death. Liver fibrosis and cirrhosis represent the final common pathway of virtually all chronic liver diseases. Activation of hepatic stellate cell (HSC) is a critical event in the pathogenesis of liver fibrosis. Recent studies suggest traditional Chinese medicine is successful in the treatment of liver fibrosis. Polygonum orientale L (POL), derived from Polygonaceae, is known to expand the coronary artery and antagonize histamine-induced bronchospasm in rat, and is applied to treat liver diseases. This study focused on its anti-fibrosis effect and the possible mechanisms in vitro. Herein, rat HSC was used as the model of liver fibrosis in vitro. The data from phase-contrast microscopic examination of POL-treated cells showed that POL induced growth inhibition and detachment from the culture plate after 48hr of incubation. The POL cytotoxic effect on HSC was determined by MTT assay. The results showed that POL promoted HSC death in a dose- and time-dependent manner. Cell cycle analysis by fluorescence activated cell-sorting (FACS) demonstrated POL induced G1 arrest on HSC. Data from in situ terminal transferase-mediated dUTP-fluorescein nick end-labeling (TUNEL) assay showed DNA fragmentation. It suggested that POL provoked apoptotic cell death in HSC. The activities of caspase-3 and caspase-9 were apparently increased. These results suggest that POL inhibits HSC proliferation and induces HSC apoptosis. Moreover, even low-dose (0.3 mg/ml) of POL could prevent HSC from migration. Besides, followed by high-dose (2 mg/ml) of POL treatment, secretion of collagen type I, collagen type III and connective tissue growth factor (CTGF) were suppressed. But matrix metalloproteinase-2 activity did not change significantly in zymographic assay. All of above suggest that POL possibly has a good effect on anti-fibrosis through suppression of HSC proliferation, induction of HSC apoptosis and inhibition of HSC functions, including cell migration and secretion of CTGF and collagens.