臺灣博碩士論文加值系統

四氫薑黃素（tetrahydrocurcumin；THC）是薑黃素的主要代謝產物，與薑黃素相比具有更好的生物活性和生物利用率。已有研究顯示THC具有抗肝纖維化的作用，但其分子機制尚未被探索。本研究利用體外（in vitro）和體內（in vivo）試驗研究THC調控肝星狀細胞的自噬以達到逆轉肝纖維化。在體外試驗中，THC顯著抑制TGF-β1誘導活化態HSC-T6細胞中α-SMA蛋白質表現量和脂滴（lipid droplet；LD）的降解；進一步的實驗證實，THC能藉由抑制TGF-β1誘導活化態HSC-T6細胞中的自噬後期，使自噬基質CX43和p62降解減少。此外活化態HSC-T6細胞共同處理THC和自噬後期抑制劑chloroquine（CQ）後，其α-SMA蛋白質表現量減少、LD降解及抑制活化態HSC-T6細胞中的自噬產生更為顯著；上述結果顯示，THC可能透過抑制自噬後期使TGF-β1所誘導活化的HSC-T6細胞回復為不活化態。在體內試驗中，SD大鼠先以四氯化碳（carbon tetrachloride；CCl4）誘導肝纖維化共12週，於第9週開始持續4週每天管餵THC（10 mg/kg和50 mg/kg）及Silymarin（200 mg/kg）。實驗結果顯示THC 10 mg/kg能顯著改善由CCl4誘導的肝臟變性（hepatic degeneration）及橋狀膠原纖維（bridge collagen fibers）形成，並抑制肝臟中α-SMA和LC3 II蛋白的表達；以免疫螢光染色的結果顯示，餵食THC 10 mg/kg可顯著降低大鼠肝臟中活化態肝星狀細胞的自噬。綜合上述結果，THC可能透過抑制自噬逆轉肝纖維化。我們的研究顯示THC具有逆轉肝纖維化的潛力。

Tetrahydrocurcumin (THC) is a major metabolite of curcumin and shows better biological activities and bioavailability than curcumin. Previous research has demonstrated that THC has anti-fibrotic effects, but the molecular mechanism is unknown. This study is design to investigate the reversal effect of THC in hepatic fibrosis by regulating autophagy in hepatic stellate cells in vitro and in vivo. In in vitro, THC significantly attenuated expression of α-SMA and the degradation of lipid droplet in TGF-β1 activated HSC-T6 cells. Further experiments showed that THC prevented CX43 and p62 degradation by suppressing autophagy in TGF-β1 activated HSC-T6 cells. CQ combined with THC showed more significantly effects in the above experiments. We investigated the number of intracellular lysosomes by Lysotracker suggested that THC may have the effect on inhibit lysosomal acidification. These results shown that THC reversed TGF-β1 activated HSC-T6 cells by inhibiting autophagy. In in vivo, hepatic fibrosis was induced by intraperitoneal injection of CCl4 twice per week for 12 weeks. THC (10 mg/kg and 50 mg/kg) and Silymarin (200 mg/kg) administered via gavage daily for 4 weeks since 8th week. We found THC 10 mg/kg significantly improved CCl4-induced hepatic degeneration, the formation of bridging collagen fibers and suppressed the α-SMA and LC3 II protein expression in the liver. The results of immunofluorescence staining also showed that feeding THC 10 mg/kg significantly reduced autophagy in activated hepatic stellate cells. All the results suggest that THC may be able to reverse liver fibrosis by inhibit autophagy with different molecular mechanism observed in vitro and in vivo. Therefore, THC shows the therapeutic potential on treating hepatic fibrosis.

目錄 I
附表目錄 III
附圖目錄 III
表目錄 IV
圖目錄 IV
縮寫全名對照表 V
縮寫全名對照表（續） VI
摘要 VII
英文摘要 IX
第一章、文獻整理 1
一、肝纖維化的病理機制 1
二、肝纖維化病程的關鍵–HSCs的活化 5
三、肝纖維化的中樞調節劑–TGF-β 10
四、抑制肝纖維化策略 13
五、細胞自噬（autophagy） 17
六、自噬在肝臟中的功能與角色 22
七、薑黃中活性成分Curcumin的主要代謝物–THC 24
第二章、研究目的 29
第三章、實驗架構 30
第四章、實驗材料與方法 31
一、儀器與廠牌 31
二、藥品與廠牌 32
三、實驗方法 35
第五章 結果與討論 52
一、細胞實驗 52
1.1　不同濃度THC對HSC-T6細胞株之毒性影響 52
1.2　THC對活化態HSC-T6細胞中纖維化蛋白質表現量之影響 52
1.3　THC對活化態HSC-T6中自噬蛋白質表現量及AVOs的影響 53
1.4　CQ合併THC處理對活化態HSC-T6細胞中自噬作用與脂滴分解的影
響 54
1.5　CQ合併THC處理調節活化態HSC-T6細胞自噬recycling期 57
二、動物實驗 58
2.1　THC post-treatment對CCl4誘導肝纖維化大鼠體重之影響 58
2.2　THC post-treatment對CCl4誘導肝纖維化大鼠臟器外觀及重量之影響 59
2.3　THC post-treatment對CCl4誘導肝纖維化大鼠血清生化指標之影響 60
2.4　THC post-treatment對CCl4誘導肝纖維化大鼠肝臟病理變化之影響 61
2.5　THC post-treatment對CCl4誘導大鼠肝纖維化之影響 61
2.6　THC post-treatment對CCl4誘導纖維化大鼠肝臟中HSCs活化和自噬蛋
白質表現之影響 62
2.7　THC post-treatment對CCl4誘導纖維化大鼠肝臟aHSCs自噬之影響 63
第六章、結論 66
第七章、結果圖表 68
第八章、參考文獻 84

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