應用硫代乙醯胺建立肝纖維化齧齒類動物模式與探討水飛薊預防肝纖維化之分子機制

Abstract

肝臟纖維化與肝硬化為慢性肝病，主要由於慢性肝損傷造成，像是酗酒或慢性肝炎等。肝臟損傷會導致傷疤組織在健康的肝臟中累積，若造成肝纖維化或肝硬化的病因持續進行，肝臟最終將會走向肝硬化而使肝臟功能喪失。以往醫學研究認為肝硬化是不可逆的，然而在肝硬化形成初期，即肝纖維化初始時，其病情是仍處於可逆轉的情況。因此，本研究之目的是建立肝纖維化動物模式並找尋可能用來治療或預防肝纖維化的機制。本研究利用硫代乙醯胺(thioacetamide, TAA)以腹腔注射來誘發實驗動物肝臟纖維化。同時評估雄性ICR小鼠、BALB/c小鼠以及Wistar大鼠中哪種實驗動物較適合用於肝纖維化動物模式。初步結果顯示給予每公斤體重100毫克的TAA，可以誘發明顯的肝臟纖維化，並造成肝重體重比 (1.3-1.5倍)以及使血液中AST (1.6-2.4倍) 與ALT (3-4倍) 的上升。因此，應用TAA可成功的在三種實驗動物誘發肝臟損傷並造成肝纖維化。此外，本研究也比較了雄性與雌性ICR小鼠在誘發肝纖維化因性別所造成的差異，初步結果顯示雄性小鼠作為誘發肝纖維化動物模式為較佳的選擇。為了找尋治療或預防肝纖維化的機制，我們應用此動物模式評估薑黃素 (curcumin)、白藜蘆醇 (resveratrol) 以及水飛薊 (silymarin) 的護肝功效，就先前文獻已知此三種植化素已被證實具有抗發炎、抗氧化以及抗癌等特性。本試驗初步結果顯示，薑黃素、白藜蘆醇以及水飛薊均有預防肝損傷以及肝纖維化的功效，而水飛薊在本試驗中發現效果較為顯著，並已是現今臨床用藥且具有治療潛力；因此選擇此藥物進行後續肝纖維化預防之分子機制探討，本試驗利用半定量反轉錄聚合酵素連鎖反應分析NF-κB、AP-1以及KLF6等轉錄因子的mRNA量，結果發現TAA的處理可顯著增加此三種轉錄因子的表現，而在TAA與水飛薊共處理組中發現水飛薊處理可以減少AP-1以及KLF6的表現。肝細胞中的KLF6可調控其下游基因TGF-β1表現分泌，並刺激肝臟星狀細胞的活化並使細胞外基質堆積在肝臟中。這些結果顯示水飛薊保護肝臟減緩纖維化的機制可能是經由減少KLF6轉錄因子的表現，來抑制肝臟星狀細胞的活化以及減少肝臟纖維化的程度。總結以上結果，本試驗已成功建立肝纖維化齧齒類動物模式，並可應用於評估具預防肝纖維化功效之健康食品。而薑黃素、白藜蘆醇以及水飛薊應用於此一動物模式中均有預防肝損傷以及肝纖維化的功效。此外，水飛薊預防肝纖維化的機制可能經由抑制KLF6轉錄因子的表現，進而減少TGF-β1的表現量來抑制星狀細胞的活化並且減少肝臟組織中纖維的堆積。但其調控機制需要更進一步的探討。

Liver fibrosis and cirrhosis are chronic and progressive liver diseases that most often caused by chronic liver injuries, including alcoholism, hepatitis infection and other illnesses. Damage to the liver causes the development of scar tissue in the healthy liver tissue. If the underlying cause of liver fibrosis or cirrhosis continues, liver fibrosis and cirrhosis will progress and liver function will diminish. Cirrhosis is irreversible, but it is demonstrated liver fibrosis in animals and humans can be restored when collagen synthesis is inhibited. For this reason, the aim of this study is to establish an animal model of liver fibrosis and then find out the therapy pathway to cure liver fibrosis. The liver damage and fibrosis of animals were induced by intraperitoneal administration of thioacetamide (TAA), associated with increased oxidative stress and activation of hepatic stellate cells. Male ICR mice, BALB/c mice and Wistar rats were used to test which species may be more suitable to develop liver fibrosis. Preliminary results showed that intraperitoneal administration of 100 mg/kg bw TAA dosage would cause obvious liver fibrosis according to Masson’s Trichrome staining and increase liver weight (1.3 - 1.5 times), serum AST (1.6 - 2.4 times) and ALT (3 - 4 times) levels compared to those without TAA. This animal model successfully induced liver damage and fibrosis. Male and female ICR mice were also compared in this study for testing the gender differences on the development of iver fibrosis. In order to compare the therapy ability on liver fibrosis, curcumin, resveratrol and silymarin were used to evaluate the beneficial effects in this animal model. These phytochemicals were all reporterd with anti-inflammatory, anti-oxidant and anti-cancer properties. Our results showed that curcumin, resveratrol and silymarin all had beneficial effects on liver injury and fibrosis. Besides, silymarin had the most beneficial effects compared to curcumin and resveratrol, and it normalize liver weight, serum AST and ALT levels to those of animals without TAA treatments. In the late stage of liver fibrosis in this animal model, the mRNA levels of NF-κB, activator protein-1 (AP-1) and Kruppel-like factor 6 (KLF6) were detected by semi-quantitative RT-PCR. It showed that TAA treatments increased the expression of these 3 transcription factors. Taking of silymarin decreased the mRNA expression of AP-1 and KLF6, but not NF-κB, where KLF-6 would up-regulate the expression of TGF-β1 in hepatocyte. TGF-β1 then stimulated the activation of hepatic stellae cell and then caused extra cellular matrix accumulation in the liver. These results showed that silymarin may prevent liver fibrosis and reduce KLF6 pathway in mRNA levels, and then inhibit the activation of hepatic stellate cell and minimize hepatic fibrosis. In conclusion, the animal models of liver fibrosis were successfully established in ICR mice, BALB/c mice and Wistar rats. These models were helpful for further studies to test whether the health food have a hepatic protection or not. Curcumin, resveratrol and silymarin all had the beneficial effects in mouse model of liver fibrosis. Silymarin can prevent liver fibrosis and reduce KLF6 pathway. So far, this is the first finding in silymarin associated with KLF6 pathway, but the detailed pathway needs a further study to clarify.