石斛菲醌衍生物抗肝纖維化活性之研究

Study of Dendrobium phenanthrenequinone derivatives in anti-hepatic fibrosis

陳姿惠(Zi-Hui Chen)

指導教授 ： 曾翠華

中山醫學大學/健康管理學院/應用化學系碩士班/碩士(2014年)

https://doi.org/10.6834/CSMU.2014.00093

摘要

石斛屬(Dendrobium SWARTZ)，隸屬於蘭科（Orchidaceae）植物，生長於高山溼潤之巖石或古木上。傳統中藥材石斛，多採用金釵石斛（Dendrobium Nobile Lindl.）、銅皮石斛 (Denbinobin moniliform)，或其多種同屬植物的莖為基原，早期文獻中也指出石斛具有抗氧化、抗發炎以及抑制腫瘤生成等藥理活性，然而有關於石斛菲醌類成分之活性研究卻仍是鮮少有較明確深入的探討結果。本研究透過合成方式取得銅皮石斛 (Denbinobin moniliform)中之菲醌成份Denbinobin及其衍生物 Moniliformediquinone (以下簡稱 MFD ) ，進行研究探討是否可以抑制肝纖維化之作用。
本研究初步使用肝臟星狀細胞株(HSC-T6)和正常小鼠肝細胞（BNL CL.2）進行體外實驗以 Denbinobin 和 MFD 處理細胞，透過細胞毒性分析(MTT assay)及細胞增生分析(BrdU assay)，偵測 Denbinobin 和 MFD 對細胞增生及毒性的影響，接著以DAPI染色、F-actin染色和AnnexinV-FITC/PI 雙染法分析MFD處理過後肝星狀細胞凋亡的現象和形態上的變化，並以西方墨點法分析細胞凋亡相關蛋白 Bcl-2 family，發炎相關蛋白包含轉化生長因子-β1（TGF-β1）、腫瘤壞死因子-α(TNF-α)和細胞間粘附分子-1（ICAM-1），及促纖維化相關蛋白包含平滑肌α-肌動蛋白（α-SMA），COL-1、CTGF、GFAP 和 TIMP-1 等蛋白表現以探討其分子機制。研究結果顯示，發現兩者皆能有效抑制肝星狀細胞(HSC-T6 cell)增生，但 MFD 卻對正常小鼠肝細胞（BNL CL.2）呈現較低毒性，細胞凋亡的現象隨著濃度上升凋亡的現象也就越顯著，也發現MFD會促進Bax表現，且負調控Bcl-2 family，在F-actin染色結果中亦可發現在形態上隨著MFD濃度上升，可降低偽足與觸角的生成或改變其形態，達到抑制活化的星狀細胞之遷移與沉積產生之纖維化現象。此外，利用西方墨點法探討纖維化之現象及分子機制，經MFD處理過的星狀細胞顯著的降低COL-1、CTGF、GFAP 和 TIMP-1 等促纖維化相關蛋白。在體內實驗中，利用四氯化碳(CCl4)誘導小鼠肝纖維化作用，期間以 0.1 mg/kg 及 0.5 mg/kg MFD 做治療。實驗結果顯示， MFD 顯著降低血液生化指標中天冬氨酸轉氨酶（AST），丙氨酸轉氨酶（ALT）和乳糖脫氫酶（LDH）以及肝組織中促發炎、纖維化相關蛋白如 TNF-α、TGF-β1，α-SMA 和 Col-1 等表現。透過病理分析，也發現到 MFD 可減輕四氯化碳誘導的肝毒性和肝纖維化。這些結果表示了 MFD 具有治療肝纖維的化潛力。

關鍵字

石斛屬 ； 肝纖維化 ； 轉型生長因子-β1 ； 腫瘤壞死因子-α

並列摘要

Plants of Dendrobium genus (Orchidaceae) are used in traditional herbal medicines in Asia, and studies have shown that they cotain a wide variety of medicinal properties including antioxidant, anti-inflammation and anti-cancer. In the present study, the anti-hepatic fibrosis potential of synthesized moniliformediquinone (MFD) and denbinobin, which have been isolated from Dendrobium moniliforme, are investigated. Since activated hepatic stellate cells are considered to be an essential part of hepatic fibrosis, the effects of MFD and denbinobin on activated hepatic stellate cells were evaluated first. The cytotoxicity of MFD and denbinobin in hepatic stellate cell line (HSC-T6) and normal mouse liver cells (BNL CL.2) was determined by MTT assay. It showed that both MFD and denbinobin inhibited cell viability of HSC-T6 cells in a dose dependent manner, but the MFD showed low toxicity in BNL CL.2cells. MFD also induced apoptosis and inhibited proliferation in HSC-T6 cells by Annexin V-FITC/PI double staining analysis and BrdU incorporation assay respectively. By western immunoblotting analysis, MFD and denbinobin up-regulated the expression of Bax and down-regulated the expression of Bcl-2. Furthermore, MFD and denbinobin inhibited the expression of inflammation-related proteins such as transforming growth factor -β1 (TGF-β1), tumor necrosis factor -α (TNF-α) and intercellular adhesion molecule - 1 (ICAM-1), and the expression of pro-fibrotic related proteins containing smooth muscle α- actin (α-SMA), COL-1, CTGF, GFAP and TIMP-1. In vivo, liver fibrosis was induced by administration of CCl4 twice weekly for 10 weeks in mice. The administration of MFD thrice weekly was started after 1 week of CCl4 administration. MFD significantly reduced the plasma aspartate transaminase (AST), alanine trasaminase (ALT) and lactose dehydrogenase (LDH) as well as hepatic expression of TGF-β1, α-SMA, and Col-1 in CCl4 –injected mice. By pathological analysis stained with H&E and Masson, MFD alleviated CCl4-induced hepatic toxicity and fibrosis. These results implicate that MFD possesses therapeutical potential for liver fibrosis.

並列關鍵字

Denbinobin ； Moniliformediquinone ； Dendrobium SWARTZ ； Hepatic fibrosis ； Transforming growth factor-β1 ； Tumor necrosis factor-α

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