富含三萜類之山苦瓜葉萃取物對於四氯化碳誘導小鼠肝臟纖維化之保護效應

Abstract

肝臟纖維化是由於慢性肝損傷造成大量的基質蛋白堆積在肝臟組織，為肝臟疾病最後一個可逆的階段，肝纖維化可能導致肝硬化或肝癌。山苦瓜具有多種藥理活性，其活性成分有酚類、黃酮類和三萜類等化合物。但山苦瓜三萜類化合物對於緩解肝纖維化的功效未明，因此本研究探討富含三萜類化合物之山苦瓜葉萃取物對於四氯化碳 (CCl4) 誘導肝纖維化的保護能力。 取得台灣山苦瓜 (花蓮一號) 並製備山苦瓜葉乙醇萃物 (bitter melon leaf extract, BMLE) 和富含三萜類之區分物 (triterpenoid-enriched extract, TEE)，以 LC/MS 鑑定 TEE 含有 Kuguacin R、Charantadiol A 以及3β, 7β, 25-trihydroxycucurbita-5, 23-dien-19-al (TCD) 葫蘆烷型三萜類化合物。給予 ICR 小鼠40% CCl4 (1 mL/kg) 和分別餵食 BMLE (100 mg/kg) 或 TEE (100 mg/kg) 八周，由肝臟組織切片結果發現 BMLE 與 TEE 均可減少肝臟細胞壞死、肝細胞氣球樣變性、發炎細胞聚集和膠原纖維蛋白沉積等現象。為進一步探討 TEE 的保護效應，給予 ICR 小鼠40% CCl4 (1 mL/kg) 和分別餵食 TEE (25、50、100、150 mg/kg) 與 silymarin (200 mg/kg) 作為對照組，為期九周，並於注射 CCl4 前一周預先給予 TEE 和 silymarin。 實驗結果顯示餵食 TEE (25、50、100、150 mg/kg) 均顯著降低 CCl4 誘導血清 AST、ALT 和肝脂堆積。肝臟組織染色結果觀察餵食 TEE (100、150 mg/kg) 組的肝臟門脈周邊橋連壞死 (periportal +/- bridging necrosis)、門脈發炎和纖維化的評分值顯著低於 CCl4 組，TEE (150 mg/kg) 組的肝小葉內變性和局部壞死的評分值顯著低於 CCl4 組，且隨著 TEE 劑量增加而抑制 α-SMA 的表現。整體 TEE (100、150 mg/kg) 組的組織學活性指數總評分顯著低於 CCl4組。從西方墨點法的結果觀察餵食 TEE (150 mg/kg) 顯著抑制肝臟纖維化蛋白質的 TGF-β1 和 α-SMA 表現。定量聚合酶連鎖反應的結果觀察餵食 TEE (25、50、100、150 mg/kg) 顯著抑制肝臟發炎的 TLR4 基因表現，TEE (25、50、150 mg/kg) 顯著抑制肝臟纖維化的 TIMP-1 基因表現。此外隨著 TEE 劑量增加而有逐漸減少發炎型 Ly6Chi 單核球聚集的趨勢，具抗發炎潛力。 綜合言之，TEE 具有緩解 CCl4 誘導小鼠肝纖維化之功效、減少肝臟發炎壞死，且隨著 TEE 劑量增加而有提升保護效應的趨勢，顯示富含三萜類化合物之山苦瓜葉萃取物具有抑制肝纖維化的潛力，是值得繼續研究開發的護肝素材。Liver fibrosis results from chronic damage to the liver with excessive extracellular matrix deposition and fibrous scar formation. Liver fibrosis has been shown to be reversible after the removal of causative agents and often progresses to cirrhosis, liver failure, and hepatocellular carcinoma. Wild bitter melon (WBM, Momordica charantia Linn. var. abbreviata Ser.) possesses various biological functions. WBM is rich in bioactive chemical constituents like cucurbitane type triterpenoids, triterpene glycosides, phenolic acids, flavonoids, essential oils, and saponins. However, the protective effect of WBM and its components on liver fibrosis is still unknown. In this study, we investigated the protective effects of WBM leaf extract (BMLE) and its triterpenoid-enriched extract (TEE) on carbon tetrachloride (CCl4)-induced liver fibrosis in mice. In experiment 1, BMLE (100 mg/kg) and TEE (100 mg/kg) were administered to 6-week-old male ICR mice by oral gavage daily during I.P. injection of 40% CCl4 (1 mL/kg) twice per week for eight weeks. The results showed that the administration of BMLE or TEE alleviated hepatocyte damage, necrosis, ballooned degeneration, inflammatory foci and collagen deposition. In experiment 2, TEE (25, 50, 100, 150 mg/kg) and silymarin (200 mg/kg) were given to 6-week-old male ICR mice by oral gavage daily for ten weeks starting one week before the start of I.P. injection of 40% CCl4 (1 mL/kg) twice per week. The results revealed that TEE (25, 50, 100, 150 mg/kg) significantly reduced serum aspartate transaminase (AST), alanine transaminase (ALT) and hepatic triglyceride deposite. According to tissue staining, TEE (100, 150 mg/kg) significantly reduced periportal+/- bridging necrosis, portal inflammation and fibrosis. TEE (150 mg/kg) also significantly reduced intralobular degeneration and focal necrosis. Overall, TEE (100, 150 mg/kg) groups had lower histological activity index than other groups. Western blot analysis indicated that TEE (150 mg/kg) significantly decreased the expression of transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA). Besides, qPCR analysis indicated that TEE (25, 50, 100, 150 mg/kg) significantly decreased the toll like receptor 4 (TLR4) mRNA. TEE (25, 50, 150 mg/kg) also significantly decreased the tissue inhibitor of metalloproteinases-1 (TIMP-1) mRNA. According to flow cytometry, the number of intrahepatic inflammatory Ly6Chi monocyte slightly decreased with the concentration of TEE increased. Our findings suggested that BMLE and TEE exhibit hepatoprotective effects on CCl4-induced liver fibrosis in mice, and might be promising anti-fibrotic options for preventing chronic liver diseases.