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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(2-2)=Microarray Profiling Delineates Molecular Portrait of the Anti-fibrosis Effects of a Chinese Herbal Medicine, ZC008, by Using in Vivo and in Vitro Models (2-2) |
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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25-50 |
專 輯 | 中醫藥基因體相關研究 |
分類號 | 414.32 |
關鍵詞 | 肝炎; 肝Kupffer細胞; 中草藥; ZC008; Hepatitis; Kupffer cell; TNF-Α; IL6; Herbal medicine; Egr1; Cyr61; Id1; |
語 文 | 中文(Chinese) |
中文摘要 | 研究目的:Kupffer 細胞的活化,是肝損傷、病毒性肝炎、酒精性肝炎和自體免疫性肝炎發生的重要機制之一。當肝發炎時,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。若能抑制Kupffer 細胞的活化,即可有效降低肝纖維化的形成。據此,人們提出了針對抑制Kupffer 細胞活化的治療的策略。目前國內外有關肝炎治療的藥物,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,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。本研究計畫以培養之大鼠肝Kupffer 細胞以細菌內毒素LPS 刺激活化,建立離體的肝發炎細胞模式,探討ZC008 抗肝炎之效果及其分子作用機轉。期能藉由建立之離體細胞培養模式對ZC008 抗發炎之效果,給予科學化之實驗數據。 研究方法:本研究將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細胞騎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。同時使用基因微陣列分析之結果找到與肝發炎或肝纖維化相關之基因已Q-PCR 確認,並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 水層萃取物,可抑制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,即可有效的抑制肝纖維細胞的轉型及增生,可防止肝纖維化。目前國內外有關肝炎治療的藥物可用的不多,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,尚乏有效療法。本研究探討已知有抗肝炎及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。本研究計畫目前進ㄧ步構築基因載體,將分別在肝纖維細胞及肝kupffer 細胞中,探討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 上下游調控之相關性。 |
英文摘要 | Aim: 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. 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. It is hypothesis that ZC008 plays a role in Kupffer cell function and in the pathogenesis of liver inflammation and fibrosis. The goal of this study is to explore the anti-inflammatory effect of ZC008 in cultured Kupffer cells. Methods: 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 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-a, 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 chronic liver diseases. In addition, using the Affymetrix RG-U34A chip, 12 fibrosis-related genes were demonstrated from the liver injury tissues and cultured hepatic stellate cells and Kupffer cells. Among them, Egr1, Cyr61 and Id1 were cloned and functionally analyzed in hepatic stellate cells and Kupffer cells. |
本系統中英文摘要資訊取自各篇刊載內容。