肝纖維化之分子機制探討與治療藥物評估

Abstract

肝星狀細胞 (hepatic stellate cell, HSC)活化後引發纖維增生，以因應外在傷害，是一種創傷癒合的正常反應；若纖維過度累積則會導致肝纖維化的發生。已知正腎上腺素 (norepinephrine, NE)為一可促進HSC活化的分子，其為多巴胺β羥化酶 (dopamine beta-hydroxylase, DBH)酵素反應後的產物。本篇研究目的在硫代乙醯胺 (thioacetamide, TAA)誘發的肝纖維化模式中，觀察NE如何誘發纖維增生。Balb/c小鼠經腹腔注射TAA後，其肝臟組織發生纖維化現象，DBH與纖維化相關的基因表現量明顯上升，由免疫組織染色發現DBH蛋白質在肝實質細胞中也明顯增加。在體外，以肝細胞株HepG2與肝星狀細胞株LX-2作為細胞模式，研究由肝細胞分泌出的NE是否能夠誘發HSC活化。實驗結果發現HepG2細胞會表現DBH與分泌NE，且HepG2條件培養基 (conditioned medium of HepG2 cells, HCM)能夠誘發LX-2細胞內的collagen增生；而TAA能夠促進DBH表現量上升，也使得細胞培養液中的NE分泌增加；以TAA處理過的HepG2條件培養基 (T-HCM)培養LX-2細胞，與HCM處理過的細胞相比，發現collagen增生情形更為明顯，藉由以DBH的siRNA降低HepG2細胞中DBH或是在細胞培養液中添加NE拮抗劑能夠抑制collagen增生現象。此外，也發現TAA在HepG2細胞中引發的DBH表現與引發細胞內的氧化壓力有關。由以上體外實驗結果推論，當肝細胞受到TAA刺激後，細胞中的氧化壓力增加而誘發DBH表現，並製造出NE而活化HSC，使得HSC細胞中的collagen累積，進而導致肝纖維化發生，未來阻斷肝細胞中DBH/NE的訊息傳遞可做為肝纖維化的治療策略之一。 本研究建立了一個TGF-β1誘發HSC活化的LX-2細胞模式，以找出具有潛力的抗肝纖維化天然物。首先以兩個已被報導具有抗肝纖維化作用的天然物-白藜蘆醇 (Resveratrol)與赤芝粗萃物 (Ganoderma lucidum extract, GLE)來評估此細胞模式的可行性，結果發現，不論是Resveratrol或是GLE皆可抑制由TGF-β1誘發的collagen表現量；接著由Ganoderma colossum中萃取出一種三萜類-五味子內酯A (Schisanlactone A, SA)，發現其能夠抑制LX-2細胞模式中的collagen增生，顯示其具有抗肝纖維化作用，同時SA也能抑制小鼠巨噬細胞RAW264.7中的發炎反應；進一步發現，在TAA誘發的肝纖維化小鼠活體模式中，SA也能夠降低肝臟中collagen增生與發炎反應。以上結果顯示，TGF-β1誘發LX-2活化細胞模式能夠用來初步評估藥物是否具有抗肝纖維化的能力，且三萜類SA是個有潛力的抗肝纖維化天然物，未來可繼續開發研究並用於肝纖維化的治療。

Collagen production by activated hepatic stellate cell (HSC) to encapsulate injury is a wound-healing response in injured liver. HSC activation could be mediated by norepinephrine (NE), a reaction product of dopamine beta-hydroxylase (DBH). In this study, the paracrine role of NE in thioacetamide (TAA)-induced hepatic fibrosis was investigated. In TAA-treated Balb/c mice, the fibrotic liver tissue showed significant increases in the expression of DBH and collagen. Further, immunohistochemical staining indicated DBH increases in parenchymal cells of the fibrotic liver. The hypothesis that the NE secreted by hepatocytes plays a paracrine role to promote HSC activation was thus proposed and verified using hepatoblast cell line HepG2 and HSC cell line LX-2. HepG2 cells express DBH and secrete NE, and that the conditioned medium of HepG2 cells (HCM) could promote collagenesis in LX-2 cells. TAA treatment increased the cellular DBH expression as well as the NE in the culture medium. The conditioned medium of TAA-treated HepG2 cells (T-HCM) was used to culture LX-2 cells, and was found to increase collagen expression in LX-2 cells. The collagenesis induction activity was diminished by pre-treating the HepG2 cells with siRNA of DBH or adding NE antagonists in the conditioned medium. Further, TAA-induced oxidative stress in HepG2 cells was found to account for the induction of DBH expression. This study suggests that chemical intoxication induces the parenchymal release of NE to activate fibrogenesis in HSC. The interruption of parenchymal DBH/NE induction may be a therapeutic strategy to inhibit liver fibrosis. In order to assess the effects of anti-fibrotic agents, TGF-β1-induced activation of HSC cell line LX-2 was employed and evaluated using two previously reported anti-fibrotic agents, resveratrol and Ganoderma lucidum extract (GLE). Both compounds inhibited the expression of Col(I) and other fibrogenic genes induced by TGF-β1. Schisanlactone A (SA), a triterpenoid purified from Ganoderma colossum, was found to possess anti-fibrotic ability by inhibiting collagenesis in LX-2 cells; moreover, the inflammation induced by lipopolysaccharide in mouse macrophage RAW264.7 cells was also inhibited by SA. Further, SA decreased fibrogenesis and inflammation in the mouse of TAA-induced liver fibrosis. These results suggest that TGF-β1-induced LX-2 cell activation is able to assess the fibrotic activity of natural compounds, and the triterpenoid SA is a potential candidate for the therapy of liver fibrosis.