藉由小鼠肝纖維化模式與細胞實驗探討烏龍茶多酚聚酯型兒茶素 A 抗肝纖維化之功效與相關分子機制

Abstract

肝纖維化為肝臟受到持續性慢性肝損傷時傷口修復並結疤的過程，這個過程若不斷進展會導致許多嚴重的併發症，包括肝臟與肝功能受損、肝硬化，甚至是肝癌。而肝臟星狀細胞 (hepatic stellate cells, HSCs) 為主要參與肝纖維化形成過程的細胞，當肝臟受到外來刺激而損傷時，肝臟星狀細胞會活化並轉型成纖維母細胞樣細胞的型態，活化的星狀細胞特徵為應力纖維 (stress fibres) 的累積，如 α-smooth muscle actin (α-SMA)，其也會產生許多胞外基質蛋白 (extracellular matrix proteins)，並且降低肝臟胞外基質被降解的速率。烏龍茶在亞洲與多數的東方國家被大量飲用，而烏龍茶中的主要多酚類物質之一為聚酯型兒茶素 (theasinensins)，其被認為是烏龍茶中一種重要的生理活性物質。聚酯型兒茶素 A 至 E (TSA, TSB, TSC, TSD, TSE) 五種中，TSA 是烏龍茶中主要的聚酯型兒茶素。根據先前的研究已經指出 TSA 具有抗氧化、誘發癌細胞凋亡、抑制基質金屬蛋白酶 (matrix metalloproteinases, MMPs) 的活性與抗發炎的能力。基於由茶中分離純化出特定的多酚類物質相當不容易，且產量不足以提供細胞與動物實驗使用，因此選擇以生物模擬方式合成 TSA，並且利用四氯化碳誘導小鼠肝纖維化之動物模式與大鼠肝臟星狀細胞株 HSC-T6探討 TSA 對於抗肝纖維化的功效與相關分子機制。結果顯示，在四氯化碳誘導小鼠肝纖維化模式下，TSA 可以減緩肝臟損傷情形與顯著降低肝臟中 α-SMA 與膠原纖維的累積，同時也可以抑制 TIMP-1 與 MMP-9 兩種肝纖維化與活化態星狀細胞重要指標之蛋白質的表現量；而細胞實驗結果顯示 TSA 可以透過抑制 TGF-β1 / Smad2/3 路徑降低 TIMP-1、MMP-2 與 MMP-9 的表現，進而抑制 HSC-T6 的活化與降低其爬行遷移的能力。由動物實驗與細胞實驗的結果顯示，TSA 對於肝纖維化的進展具有減緩與抑制的功效。

Liver fibrosis is a pathological scarring process in response to a variety of chronic stimuli. It leads to the development of severe complications including liver failure, liver cirrhosis and hepatocellular carcinoma (HCC). Hepatic stellate cells (HSCs) are the predominant cell type in the development of liver fibrosis. Following liver injury, HSCs are activated and transformed into a myofibroblast-like phenotype, characterized by the accumulation of stress fibres, such as α-smooth muscle actin (α-SMA), increased production of extracellular matrix (ECM) proteins, decreased matrix degradation. Oolong tea is consumed heavily in Asian and most Eastern countries. Oolong tea theasinensins are a group of tea polyphenols, and they are considered as bioactive compounds in Oolong tea. Among five types of theasinensins, theasinensin A (TSA) is the major theasinensins in oolong tea. Several studies have revealed that TSA have potential biological activities, such as antioxidative effects, apoptosis induction, inhibitory effect on MMPs activities and anti-inflammatory activities. Because chromatographic separation of tea polyphenols is too difficult to supply sufficient quantities of pure compounds for biological experiments, I decided to use biomimetic preparation of TSA. And we evaluated its anti-liver fibrosis effects in vivo and in vitro. In in vivo experiments, I used 6-week-old male ICR mice as animal model and gave them 40% CCl4/olive oil by I.P. injection to induce them liver fibrosis. The results revealed that TSA can ameliorate liver injury compared with CCl4+H2O group. Also, TSA can restrain the progression of liver fibrosis due to the down-regulation of α-SMA and collagen depositing in liver tissue. Meanwhile, TSA can inhibit the expression levels of TIMP-1 and MMP-9 which are both important markers of activated HSCs and liver fibrosis. The results of in vitro experiments showed that the expression levels of TIMP-1, MMP-2 and MMP-9 were decreased because TSA inhibited TGF-β1/Smad2/3 signaling pathway. Thus the activation and migrated ability of HSC-T6 cells were inhibited. In conclusion, TSA has the effects on retarding the progression of liver fibrosis.