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| 題 名 | 利用基因微陣列圖譜與動物之離體及活體模式評估中藥ZC008抗肝臟纖維化之功效(全程總報告)=Microarray Profiling Delineates Molecular Portrait of the Anti-fibrosis Effects of a Chinese Herbal Medicine, ZC008, by Using in Vivo and in Vitro Models (Final Report) |
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| 作 者 | 徐士蘭; 黃奇英; | 書刊名 | 中醫藥年報 |
| 卷 期 | 29:8 2011.09[民100.09] |
| 頁 次 | 頁51-92 |
| 專 輯 | 中醫藥基因體相關研究 |
| 分類號 | 414.32 |
| 關鍵詞 | 肝炎; 肝纖維化; 肝纖維細胞; 肝Kupffer細胞; 中草藥; ZC008; Hepatitis; Cirrhosis; Hepatic stellate cells; Kupffer cell; TNF-α; IL-6; IL-10; Herbal medicine; Egr1; Cyr61; Id1; α-smooth muscle actin; |
| 語 文 | 中文(Chinese) |
| 中文摘要 | 研究目的：雖然醫療技術一直在進步，但台灣地區因罹患肝疾病而致死之人數仍然高居不下。在肝疾病中除肝癌外，最常發現的即為病毒性肝炎及肝硬化。而肝纖維化是各種慢性肝病進展為肝硬化的必經途徑， 其實質是肝內細胞外基質(Extracellular matrix)的合成與降解失去平衡，導致細胞外基質尤其是膠原蛋白的過度沈積和分佈異常。隨著肝纖維化程度加重，異常增生的纖維束包纏形成假小葉，使正常肝臟的小葉結構和功能被破壞，即形成肝硬化。肝纖維化是可逆轉的，因此，抗肝纖維化治療是十分重要的。體內、外研究證實肝星狀細胞(Hepatic stellate cell)及其活化形式即轉變成纖維細胞是細胞外基質的主要來源，活化的肝星狀細胞，釋放TGF-β1 等多種細胞因子，從而促進細胞外基質的異常合成，抑制其降解。故肝星狀細胞的活化是肝纖維化形成過程的中心環節。此外，肝受損發炎時，活化的Kupffer 細胞分泌TGF-β、TNF-α及PDGF 等因子，促進了肝星狀細胞的轉化、增生及合成細胞外基質，形成肝纖維化。因此，若能抑制Kupffer細胞的活化，也可有效降低肝纖維化的形成。據此，人們提出了針對形成肝星狀細胞活化的多個環節進行治療的策略，及針對抑制Kupffer 細胞活化的治療的策 略。目前國內外有關肝纖維化治療的研究多處於實驗階段，尚乏有效療法。而治療肝炎的藥物，除病毒性肝炎可使用抗病毒藥物外，其他如毒性物質或酒精中毒等非病毒性之肝炎，也尚無有效治療之藥劑。中草藥的使用已有幾千年的臨床經驗的累積為尋找有特殊療效之醫療資源庫。但因缺乏科學化之研究及驗證無法取 信於國際醫學界。中醫藥的生物科技化研究是使中醫藥躍上國際生物醫學界的舞台，必走之途徑，也是目前世界潮流之趨勢。近年政府對中醫藥及天然藥物之研發工作日趨重視，也明訂中藥科學化與新藥研究開發列為國家重點發展項目。更選定中草藥為具有發展潛力之生物科技優先支持項目。本研究計畫是針對過去本研究室發現之具有治療肝硬化功效之中草藥代號ZC008，基因晶片分析、細胞實驗及疾病動物實驗等方法，探討ZC008 抗肝纖維化作用之分子機轉。期能藉由建立之離體細胞培養模式，及並動物模式及基因晶片分析，給予中草藥科學化之驗證。 研究方法：本研究將以初代培養之為研究模式，以細胞數目測定、TUNEL 凋亡分析、Caspase 活性分析、西方點墨蛋白質分析、及免疫螢光染色分析等技術，探討肝星狀細胞處理ZC008 後其細胞的活化轉型之抑制及細胞凋亡等分子作用。又以初代培養之大鼠肝Kupffer 細胞利用LPS 激活此免疫細胞為研究模式，以細胞數目測定、細胞素ELISA 分析、西方點墨蛋白質分析、RT-PCR 基因mRNA 表現分析及免疫螢光染色分析等技術，探討被LPS 激活的肝Kupffer 細胞中與發炎相關之細胞素及mediators 之活性及表現，是否會被ZC008 抑制，藉此離體培養方式快速檢測ZC008 是否具有抗發炎之功效，並且找尋ZC008 抗發炎之分子作用機制。同時使用大鼠肝纖維化疾病動物模式，驗證ZC008 治療肝炎及肝硬化之效果。並使用基因微陣列分析與肝發炎或肝纖維化相關之基因，並以Q-PCR 確認其準確 性。又以基因選殖技術，將特定的基因序列(Egr1、Cyr61、Id1)植入載體。或以siRNA 序列Knockdown 基因的表現。探討這些基因的生物功能，及在肝疾病中扮演的角色。 結果與討論：中草藥ZC008 在低濃度時具抑制肝星狀細胞生長之能力，而高濃度ZC008處理可促進肝星狀細胞凋亡之作用。此ZC008 誘發之肝星狀細胞凋亡，是經由活化內生性的粒腺體致死路徑，因為活化了Caspase-3 及-9。同時ZC008 處理肝星狀細胞可抑制alpha smooth muscle actin 及collagen I 分子的表現。但是同樣的濃度對肝細胞的生長及存活卻無影響。而ZC008 水層萃取物，是可以抑制LPS 刺激的肝Kupffer 細胞之發炎相關cytokines(TNF-α, IL6)及mediator (nitric oxide)之產生。LPS 誘發之肝細胞活化是經由活化ERK/NFkB 路徑。而ZC008 可抑制ERK/NFkB 分子的活性，造成抑制cytokines(TNF-α, IL6)及mediator (nitric oxide) 之產生，進而達到抑制肝發炎的功效。肝臟中之星狀細胞被刺激活化轉型而成為肌纖維化細胞，此細胞大量在肝臟中增殖，破壞了正常肝組織的結構，使肝臟功能不足，導致患者死亡。若肝臟中之Kupffer 細胞被活化，除造成肝發炎外，其所釋放的發炎物質，也會刺激肝星狀細胞的轉型而成為肌纖維化之細胞，造成肝纖維化。因此若能有效的抑制肝Kupffer 細胞的活化，及抑制肝星狀細胞的活化轉型及增生，是可以有效的防止肝纖維化。目前國內外有關肝纖維化的治療，尚乏有效藥物。而有關肝炎治療的藥物可用的也不多，僅病毒性肝癌有抗病毒之藥物可用，但療效只有五、六成。其他如酒精性肝炎、藥物中毒性肝炎、免疫疾病造成的肝炎、及代謝異常性肝炎，仍缺乏有效療法。本研究探討已知有抗肝炎及抗肝纖維化作用之中藥ZC008，是可藉由抑制肝星狀細胞之凋亡及抑制肝Kupffer細胞之發炎反應，達到治療肝炎及肝纖維化之效應。以大鼠肝纖維化動物模式配合離體初代培養的細胞模式，以現代化之科學技術，結合基因微陣列分析，對ZC008 治療肝纖維化之功效給予科學化之驗證，此研究結果可使ZC008 之醫藥價值受到國際醫界之認同。本研究計畫實驗模式之建立，亦可當作是中藥科學化平台架構之範本。目前進ㄧ步構築基因載體，將基因晶片分析所得與疾病及治療相關的三個分子Egr1、Cyr61、及Id1，的功能做進ㄧ步探討。 |
| 英文摘要 | Aim: Hepatic fibrosis, a precursor of cirrhosis, is a consequence of severe liver damage that occurs in many patients with chronic liver disease, and involves the abnormal accumulation of extracellular matrix (ECM), particularly collagens. It has been shown that the activation of stellate cells in injured livers leads to their proliferation and transformation into myofibroblast-like cells. The activated hepatic stellate cell is now well established as the key cellular element involved in the development of hepatic fibrosis. In addition, accumulating evidence indicates that inflammation plays a central role in the current paradigm of liver fibrosis. Kupffer cells, which represent the largest population of resident macrophages in the liver, are uniquely positioned as the predominant primary inflammatory effector cells to initiate the inflammatory cascade leading to tissue remodeling and fibrosis. For this reason, the presence of increased population of kupffer cells together with the bulk release of inflammatory mediators by these macrophages are considered to be critical events during the early stages of liver inflammation and fibrosis. Herbal medicines that have been used in China for thousands of years are now being manufactured in China as drugs with standardized quality and quantity of ingredients. In previous study, we found that ZC008, a Chinese herbal medicine which exhibits the beneficial effects for treatment of patients with liver cirrhosis, reduces inflammatory effect and induces apoptotic cell death in activated hepatic stellate cells. The goal of this study is to evaluate the hepatoprotective effect of ZC008 on in vitro cultured cells and in vivo disease animal model. Methods: Hepatic stellate cells from rat liver were treated with various concentrations of ZC008 extract for different duration. Cell death were assessed by direct cell number counting and TUNEL assay. Caspase activity was measured using specific fluorogenic substrates. The expression and cellular distribution of alpha smooth muscle actin and collagen I were examined by Western blotting and immunostaining. Dimethylnitrosamine (DMN)-induced liver fibrosis model was performed. Liver samples were immediately removed after sacrifice and prepared for histopathological staining. Blood samples, collected from the animals at autopsy, were used to measure serum concentrations or activity of biomarkers. Gene expression profilings were examined by Affymetrix GeneChip system (RG-U34A chip) and analysis by GeneSpring solfware 7.3. Moreover, LPS-stimulated Kupffer cells were treated with various concentrations of ZC008 water soluble extract for different duration. Cell viability were assessed by direct cell number counting. Inflammatory cytokines (TNF- α, IL-6, and IL10) or mediator (nitric oxide, NO) production was measured using ELISA assay and Gress reagent analysis. The expression of inducible NO synthase (iNOS) and phosphorylated ERK were examined by Western blotting. DNA- binding activity of NFkB was determined by electrophoretic mobility shift assay. Results & Discussion: We found that ZC008 extract is the most potency drug among the tested drugs to treat of hepatic stellate cells. Treatment with ZC008 decreased the expression of alpha smooth muscle actin and collagen I in primary cultured stellate cells. DNA fragmentation and caspase activation were detected in ZC008-treated stellate cells, suggesting that ZC008 extract triggered a apoptotic cell death through a intrinsic mitochondrial pathway in this primary cultured hepatic stellate cells. The current results indicate that curing the liver fibrosis by ZC008 may be due to direct elimination of the activated fibrotic cells by the agent. However, ZC008 water soluble extract is the most potency fraction among the tested fractions to treat of LPS-stimulated Kupffer cells. Treatment with ZC008 decreased the LPS-induced TNA-α, IL6 and NO production in primary cultured Kupffer cells, while increased the LPS-enhanced IL10 expression. Moreover, ZC008 inhibited the LPS-induced ERK phosphorylated activation and NFkB-DNA binding activity in Kupffer cells, suggesting that water soluble extract of ZC008 exhibited an anti-inflammtory effect through an ERK/NFkB-dependent signaling pathway in this primary cultured Kupffer cells. Based on our previous and the current results, we suggest that curing the liver fibrosis by ZC008 may be due to direct elimination of the activated fibrotic cells and inhibition of Kupffer cell activation by the agent. This study intends to build a hypothesis-driven research on liver fibrosis at the system level by implementation of diverse research approaches with the goal to elucidate novel insights into the effect of ZC008 on liver fibrosis. We used dimethylnitrosamine (DMN) to induce rat necroinflammatory and hepatic fibrosis in a 6-week time course. Using the Affymetrix RG-U34A chip, 256 differentially expressed genes, including 44 necroinflammatory-related and 62 fibrosis-related, were demonstrated from the liver injury tissues and ZC008-treated cultured hepatic stellate cells and hepatic Kupffer cells. Among them, Egr1, Cyr61 and Id1 were cloned and functionally analyzed in hepatic stellate cells and Kupffer cells. Taken together, ZC008 was validated to be an excellent anti-liver damage candidate drug by histopathological, biochemical and microarray analysis. Our proposed research model may provide a new exploratory modality for traditional Chinese medicine research. |
本系統中英文摘要資訊取自各篇刊載內容。