牛樟芝菌絲體甲醇萃取物抗血癌及抗發炎活性及其分子機制之研究

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题名:	牛樟芝菌絲體甲醇萃取物抗血癌及抗發炎活性及其分子機制之研究 The anti-leukemia and anti-inflammatory effects and their molecular mechanisms of methanol extract of Antrodia cinnamomea mycelia
作者:	文紀鑾;Wen, Chi-Luan
贡献者:	中國藥學暨中藥資源學系博士班
关键词:	牛樟芝;CCAAT/enhancer-binding protein (C/EBPβ);分化;Extracellular signal-regulated kinase(ERK);血癌;抗發炎;誘導性一氧化氮合成?一氧化氮;環氧酵素二型;丙二醛;腫瘤壞死因子-α;過氧化氫?超氧化物歧化?;Antrodia cinnamomea;CCAAT/enhancer-binding protein (C/EBPβ);CD14;Differentiation;Extracellular signal-regulated kinase(ERK);Leukemia;Anti-inflammation;Inducible Nitric oxide synthase (iNOS);nitric Oxide (NO);cyclooxygenase2 (COX2);Malondialdehyde;Tumor necrosis factor alpha(TNF-a);Catalase;Superoxide dismutase
日期:	2012-07-25
上传时间:	2012-08-31 16:10:26 (UTC+8)
出版者:	中國醫藥大學
摘要:	牛樟芝(Antrodia cinnamomea) 是台灣特有的傳統藥用菌類，具有許多的生理活性，尤其是具有抗腫瘤及抗發炎的特性。本論文的研究分為兩個部分探討液態發酵牛樟芝菌絲之甲醇萃取物之藥理活性，第一部分為抗血癌，第二部分為抗發炎的活性，並進一步探討其作用之分子機制。在第一部分的研究中，我們是第一個發現牛樟芝對人類急性骨髓瘤HL60細胞具有促進分化的能力，處理牛樟芝菌絲甲醇萃取物(MEMAC)可以抑制HL60細胞之增生，使細胞週期停至於G1期，進而誘導血癌細胞分化為單核球。實驗的結果顯示MEMAC是藉由extracellular signal-regulated kinase (ERK)的磷酸化，促進其下游路徑的活化，上調轉錄因子CCAAT/enhancer binding protein β (C/EBPβ)的表現及活性。這結果証明MEMAC誘導血癌細胞分化為單核球是經由活化ERK的路徑，促使C/EBPβ活化在核內與CD14基因啟動子附近的調控區結合，進而增加CD14的表現。由於CD14的表現是單核球分化的指標分子之一，因此本實驗之結果顯示，MEMAC是具有促進人類血癌細分化潛力的誘導劑。在第二部分的研究中，我們利用離體細胞培養及活體動物模式評估MEMAC抗發炎的活性。在細胞抗發炎的試驗中，將小鼠巨噬細胞(RAW264.7 cells)，先預處理MEMAC，再分別以lipopolysaccharide (LPS)、palmitoyl-3-cysteine-serine-lysine-4 (Pam3CSK4)、poly -inosine–polycytidylic acid (PolyIC)處理，誘導發炎細胞活化，分析MEMAC對發炎相關的細胞素及介質釋放之影響。結果顯示MEMAC可抑制分別經由LPS、Pam3CSK4、PolyIC誘導之小鼠巨噬細胞的發炎反應，降低發炎細胞素如腫瘤致死因子(TNF-α) 和白介素 (IL-6)，以及一氧化氮(NO)及前列腺素E2(PGE2)的釋放，並且抑制一氧化氮合成酶(iNOS)和環氧合酵素-2 (COX2) 的基因表現。在活體動物試驗是利用λ-角叉菜膠誘導小鼠足蹠腫脹作為一種被誘發的動物發炎模式。以腹腔注射MEMAC及indomethacin觀察急性足蹠腫脹之現象。試驗結果顯示，MEMAC可明顯抑制λ-角叉菜膠誘導小鼠足蹠腫脹，MEMAC (400 mg/kg)也可以減少λ-角叉菜膠誘導白血球轉移之能力(50.92 ± 5.71%)。同時，MEMAC處理也可增加小鼠肝臟中catalase (CAT)、superoxide dismutase (SOD)、及 glutathione peroxidase (GPx)的活性，和降低血清中λ-角叉菜膠所造成之一氧化氮(NO)及腫瘤致死因子(TNF-α)之含量。 Antrodia cinnamomea (named as Niu-chang-chih), a well-known Taiwanese folk medicinal mushroom, has a broad-spectrum of pharmacological effects, especially with anti-tumor and anti-inflammatory properties. In this study, we attempted to assess the anti-leukemia and anti-inflammatory activities and mode of action of the methanol extract from liquid cultured mycelia of A. cinnamomea (MEMAC). In part 1, we examined the potential role and the underlying mechanisms of MEMAC in the cell growth and differentiation of human acute myeloid leukemia HL60 cells. We found firstly that MEMAC inhibited proliferation and induced G1 cell cycle arrest in HL60 cells. Moreover, MEMAC could induce differentiation of HL60 cells into the monocytic lineage. In addition, MEMAC activated the extracellular signal-regulated kinase (ERK) pathway and increased CCAAT/enhancer-binding protein β (C/EBPβ) expression. These findings demonstrate that MEMAC-induced HL60 cell monocytic differentiation might be via the activating ERK signaling pathway, and upregulating the downstream transcription factor C/EBPβ, consequently enhancing the differentiation marker CD14 gene expression. These observations suggest that MEMAC might be a potential differentiation-inducing agent for treatment of leukemia. In part 2, we evaluated the anti-inflammatory effect of MEMAC in both in vitro and in vivo systems. The in vitro anti-inflammatory activity of MEMAC was judged by the measurement of the levels of pro-inflammatory cytokines and mediators in lipopolysaccharide (LPS)-, palmitoyl-3-cysteine-serine-lysine-4 (Pam3CSK4)-, polyinosine–polycytidylic acid (PolyIC)-stimulated RAW264.7 cells, respectively. The results showed that MEMAC inhibited the production of LPS, Pam3CSK4, PolyIC-induced pro-inflammatory cytokines(TNF-α and IL-6), and mediators (NO and PGE2). Moreover, MEMAC decreased the levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2) protein expression in LPS-stimulated RAW264.7 macrophages. The in vivo anti-inflammatory activity of MEMAC was carried out using carrageenan-induced hind paw edema in mice. The data indicated that MEMAC exhibited significant (p < 0.05) anti-inflammatory activity by reducing the edema volume in carrageenan-induced paw edema in mice. MEMAC (400 mg/kg) also reduced the carrageenan-induced leukocyte migration (50.92 ± 5.71%). Furthermore, MEMAC also increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in the liver tissue and decreased the levels of serum NO and TNF-α after carrageenan administration. Taken together, our findings reveal that MEMAC has the anti-AML and anti-inflammatory properties.
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