散血草降低四氯化碳誘導小鼠肝臟纖維化; Ajuga bracteosa Reduces Liver Fibrosis Induced by Carbon Tetrachloride in Mice

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Title:	散血草降低四氯化碳誘導小鼠肝臟纖維化;Ajuga bracteosa Reduces Liver Fibrosis Induced by Carbon Tetrachloride in Mice
Authors:	劉鈺亭;Yu-Ting Liu
Contributors:	中國醫藥大學：基礎醫學研究所
Keywords:	巨噬細胞;肝臟纖維化;四氯化碳;脂多醣;散血草;macrophage;liver fibrosis;carbon tetrachloride;lipopolysaccharide;Ajuga bracteosa
Date:	2009-07-11
Issue Date:	2009-08-11 14:35:50 (UTC+8)
Abstract:	散血草為民間用藥應用於護肝。本研究主要探討散血草50%酒精萃取物對四氯化碳誘導小鼠肝臟纖維化的抑制效果。肝臟纖維化之誘導，小鼠每週以管灌餵食兩次四氯化碳 (10％ 0.1ml/10g BW) 連續八週。實驗分為正常對照組及四氯化碳三組。四氯化碳三組每日一次經口投予散血草酒精萃出物(300 mg/kg、1000 mg/kg)或去離子水。四氯化碳誘導肝纖維化組顯著增加血液中丙氨酸轉移酶活性 (Alanine aminotransferase, ALT)，肝臟脂質過氧化程度及羥基脯胺酸含量，和四氯化碳組比較，高劑量散血草酒精萃物能降低血液中丙氨酸轉移酶活性，肝臟脂質過氧化程度及羥基脯胺酸含量。散血草可顯著降低四氯化碳誘導之肝臟之IL-10、INF-γ的增高。組織染色之分析發現散血草酒精萃取物能顯著緩解四氯化碳誘導肝臟纖維化、壞死，及減少CD14及CD68的表現。RT-PCR的分析也顯示散血草酒精萃取物能明顯降低肝臟 lipopolysaccharide-binding protein(LBP),CD14,CD68,α-smooth muscle actin (α-SMA), collagen (α1)(I), tu mor necrosis factor-α (TNF-α) mRNA的表現。另外，在大鼠肝臟庫氏細胞以LPS誘導NO模式中，發現散血草酒精萃取物可顯著降低LPS所誘導NO的產生量。在RAW264.7 巨噬細胞株研究發現，散血草不同分層萃取物皆有抑制LPS 所誘導之NO 的產生，其中以CHCl3 萃取物(ABCE)最為顯著。LPS 可誘導iNOS、p-IκBα、NF-κB p65、p50 及MAPK (p-ERK、 p-JNK、pp38)的表現，而ABCE 能顯著抑制LPS 所誘導之NF-κB 及 MAPK 的磷酸化，在LPS 誘發24 小時急性發炎後，其細胞上清液中也發現其TNF-α、IL-6 的表現量顯著增加，ABCE 可顯著降低此發炎反應。 ABCE能抑制NF-κB及MAPK的活化，進而影響其下游相關發炎因子如NO、TNF-α等。因此推測口服散血草酒精萃取物能明顯緩解四氯化碳誘導的肝臟纖維化，可能是經由抗發炎作用。 Ajuga bracteosa (Labiatae) has been used in traditional folk medicine for its hepatoprotective agent. The purpose of this study was to investigate the effect of 50% ethanol A. bracteosa extract (ABEE) on liver fibrosis induced by carbon tetrachloride (CCl4) in mice. Hepatic fibrosis was produced by CCl4 (10%; 0.1 ml/10 g body weight, p.o.) twice a week for 8 weeks in mice. Mice in the three CCl4 group were treated daily with distilled water and ABEE (300 or 1000 mg/kg) via gastrogavage throughout the experimental period. CCl4 caused liver fibrosis, featuring increase in plasma transaminase, hepatic malondialdehyde (MDA) and hydroxyproline (HP) contents. Compared with CCl4 group, ABEE (1000 mg/kg) treatment significantly decreased the activities of transaminase, hepatic MDA and HP contents. CCl4 treated mice significantly increased the hepatic contents of interferon-γ、interleukin-10, while ABEE suppressed these mediators of inflammation in liver damage. Histological evaluations showed that ABEE could attenuate the liver fibrosis, necrosis, and expressions of CD14 and CD68 that were induced by CCl4. In addition, RT-PCR analysis also showed that ABEE treatment decreased hepatic mRNA expressions of lipopolysaccharide-binding protein, α-smooth muscle actin, collagen (α1)(I), CD14, CD68 and tumor necrosis factor-α. In addition, ABEE was found to inhibit LPS-induced NO production in rat Kupffer cell. Various fractions of A. bracteosa, especially chloform fraction, were found to inhibit LPS-induced NO production in RAW 264.7 macrophage.A. bracteosa chloroform fraction (ABCE) inhibited the protein expression of iNOS. ABCE reduced phosphorylation of IκBα, NF-κB p65 and p50 protein expressions. In addition, ABCE inhibited phosphorylation mitogen activated protein kinase (MAPKs) protein expressions, including ERK1/2, JNK, and p38. ABCE suppressed the activation of NF-κB and MAPKs, then regulated downstream inflammatory factors such as NO and TNF-α. It is reasonable to suggest that oral administration of ABEE significantly reduced CCl4-induced hepatic fibrosis in mice probably through the anti-inflammatory action.
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