| 摘要: | 肝硬化乃是指肝臟的瀰漫性纖維化及伴發無數的肝實質再生結節，而肝纖維化則是肝細胞外間質沈積的結果，其和肝細胞的壞死、發炎後的修復及免疫細胞調控有關。肝纖維化的致病因，包括了病毒性、酒精性、藥物性、膽汁性等。本研究選擇膽道結紮 (Bile duct ligation, BDL) 的動物模型，來模擬次發膽道性肝纖維化。此模型會造成漸進性門脈纖維化，最後進展為肝硬化，但卻沒有因為肝毒性而造成肝細胞發炎、變性壞死，因此可以直接評估藥物在預防及對抗肝纖維化方面的療效。 本研究目的，在於證實中醫臨床上常用於治療肝病的小柴胡湯，對於膽道結紮的肝纖維化動物模型，是否有預防肝纖維化作用、是否有減少肝膠原纖維堆積作用，同時進一步探討中藥在肝纖維化機制中，對膠原酵素抑制劑 (TIMP-1)、肝生長因子 (TGF-β1, PDGF, EFG) 及對免疫介質的影響。將大白鼠膽道的兩端結紮並且中間截斷後，為期六週，評估小柴胡湯對肝功能生化指標的影響，並以肝組織膠原蛋白(HYP)的定量檢測為抗肝纖維化的標準。其後，以反轉錄─聚合脢連鎖反應去檢測小柴胡湯對第一、三型前膠原、膠原酵素抑制劑 (TIMP-1)、轉型生長因子(TGF-β1)、血小板衍生生長因子 (PDGF) 及表皮細胞生長因子 (EGF)等，是否有抑制作用，並以放射免疫法 (RIA) 評估血清中纖維化指標 (PⅢNP) 的變化，再以ELISA 方法評估對免疫介質 (IL-1, IL-6, TNF-α) 的調控。對肝纖維化的重要調控中心─貯脂細胞，則以組織免疫染色做定性觀察，並以西方墨點法評估貯脂細胞在蛋白質階層的表現。最後，檢測肝均勻漿的脂質過氧化產物 “丙二醛”，藉以評估小柴胡湯是否能對膽道結紮所造成的脂質過氧化有抑制作用，而達到抗肝纖維化的作用。 研究結果顯示：(1)小柴胡湯明顯減少膽道結紮所造成的膽汁鬱積 (2)小柴胡湯顯著減少肝組織膠原含量達50%，及血清中纖維化指標 PⅢNP含量達11% (3)小柴胡湯的預防及抗肝纖維化作用，可能是藉由其抑制第一、三型前膠原及組織膠原酵素制劑 (TIMP-1)等，在mRNA階層的表現而達成 (4)小柴胡湯顯著抑制肝纖維化作用，部分機轉是藉由其抑制膽道鬱積所造成的脂質過氧化而達成 (5)小柴胡湯的預防肝纖維化作用，部分是藉由抑制TGF-β1 mRNA的表現，而減少EGF, PDGF等肝生長因子的表現，進而減少膽管增生及膽管旁纖維化。此結果在TGF-β1 的免疫染色中，再次得到證實 (6)小柴胡湯對肝纖維化過程中的免疫調節，是藉由抑制IL-1β的上昇，誘導TNF-α的生成，進而抑制貯脂細胞的分裂增生及膠原沈積，以達到預防肝纖維化作用。; Liver cirrhosis defined as liver diffuse fibrosis and multiple regeneration nodules. Hepatic fibrosis is an over-accumulation of ECM (extra-cellular matrix), it is a result of imbalance between collagen synthesis and degradation. Matrix metalloproteinase (MMP) have degradative activity against collagen, but tissue inhibitors of metalloproteinase (TIMP) control the active forms of MMP by blocking the active site of MMP. In our study, we established bile duct ligated model (BDL) in rats to evaluate anti-fibrotic potential of Chinese medicine sho-saiko-to (TJ-9), which is used in the treatment of cirrhosis patients. The bile duct ligation model can in the end induce progressive portal fibrosis and cirrhosis. Thus, we assessed the drug’s potential in suppressing procollagen α1 (I) & (Ⅲ) mRNA expression, inhibiting of collagen accumulation (HYP & PIIINP), activated Ito cells, and regulating of TIMP-1. Furthermore, another objective of study was to determine whether the TJ-9 exerted its anti-oxidant effect and suppressing effect of protein level on Ito cell. Finally, we evaluated the TJ-9 potential in regulating the hepatic growth factor and cytokines during the process of fibrogenesis. After the administration of TJ-9, hyperbilirubinemia reduced about 4-fold when compared with BDL untreated group. TJ-9 also significantly reduced the collagen content and fibrogenic score, as well as to downregulate elevated procollagen α1 type (I), (Ⅲ) and TIMP-1 mRNA level. Finally, we concluded that: (1) TJ-9 at a dose of 0.5g/kg significantly reduced cholestasis in rats with bile duct ligation. (2) TJ-9 markedly reduced the collagen content by 50% and the serum fibrogenic marker PIIINP by 11%. (3) TJ-9 exerted its antifibrogenic effect by downregulating the expression of procollagenα1 type (I), (Ⅲ) and TIMP-1 mRNA in liver tissue. (4) TJ-9 prevented liver fibrosis partly through the mechanism of anti-oxidation and suppression of Ito cell. (5) TJ-9 exerted its antifibrotic effect by downregulating the mRNA expression of TGF-β1, EGF and PDGF growth factor, so as to reduce the bile duct proliferation and fibrosis. (6) TJ-9 inhibited the Ito cell’s mitosis by suppressing the IL-1β and enhancing the TNF-α level. |
