以TGF-β1處理之Hep 3B與HSC-T6模式探討「靈芝-豆科」發酵液之抑制肝纖維化效果

Abstract

肝纖維化是一種肝臟受損而進行修復的可逆性反應，損傷長期而持續發生時，細胞外間質（extracellular matrix）會過量累積而再進展至肝硬化。肝纖維化的發展與肝臟中氧化壓力的不平衡有關，因此近年來許多研究著眼於抗氧化方面，以期針對肝纖維化之病因找到預防或解決之道。前人研究發現，靈芝中的多醣體、三萜類等活性成分及豆科植物（如黑豆、黃耆）中富含之異黃酮素具有良好抗氧化效果。因此，本研究乃以人類肝癌細胞株（Hep 3B）與大鼠肝臟星狀細胞株（HSC-T6）為評估模式，探討不同發酵條件下利用豆科植物（黑豆、黃耆）為培養基質進行液態發酵培養靈芝所得之「靈芝-豆科」發酵產物其對氧化損傷之保護效果與抗肝纖維化之能力。 本研究中所使用之「靈芝-豆科」液態發酵產物分為發酵濾液與菌絲體兩大部分，經處理分別得到發酵濾液凍乾物（b）、發酵濾液去多醣物（be）、菌絲體熱水萃出物（mHW）、菌絲體乙醇萃出物（mE）及菌絲體甲醇萃出物（mM）等五種樣品，並與兩種原料（黑豆、黃耆）及赤芝（Ganoderma lucidum）子實體進行比較。模式一主要以TGF-β1誘導Hep 3B細胞走向細胞凋亡，模擬體內肝細胞處於高氧化壓力下可能促進肝纖維化的情形，分別利用MTT試驗得到之細胞存活力（cell viability）計算樣品之氧化損傷回復率（recovery ratio），進而以流式細胞儀（flow cytometer）探討前述活性樣品對細胞週期之影響與抑制細胞凋亡之能力。模式二則以TGF-β1誘導HSC-T6細胞活化表現平滑肌α-肌動蛋白（α-smooth muscle actin, α-SMA），以酵素連結免疫吸附（enzyme-linked immunosorbent assay, ELISA）分析α-SMA表現受抑制程度，評估樣品之抗肝纖維化效果。 結果顯示，在Hep 3B細胞模式中，添加豆科之發酵濾液凍乾物效果均優於未添加豆科者，而菌絲體部分則有熱水萃出物＞乙醇萃出物＞甲醇萃出物之趨勢。整體而言，此模式中以添加5％黑豆、2％黃耆、發酵溫度24 ℃、攪拌速度50 rpm、通氣量 0.375 vvm、發酵11天之發酵濾液凍乾物（GL-2b）與添加5％黑豆、2％黃耆、發酵溫度30 ℃、攪拌速度50 rpm、通氣量 0.75 vvm、發酵11天之菌絲體熱水萃出物（GL-5mHW）效果最好（樣品濃度以100 μg/mL為比較基礎）；進而將GL-5mHW樣品以流式細胞儀探究其對Hep 3B細胞受到TGF-β1所致之氧化損傷保護情形，則未發現有顯著抑制細胞凋亡的現象。此外，再以Hep 3B細胞模式中效果較佳者利用HSC-T6細胞模式評估，結果發現僅原料（黑豆、黃耆）與赤芝子實體之熱水萃出物（樣品濃度以500 μg/mL為比較基礎）有顯著抑制HSC-T6細胞中α-SMA之表現（p<0.05），而「靈芝-豆科」液態發酵產物卻無效果。

Hepatic fibrosis is a wound-healing response to chronic liver injury, which may lead to cirrhosis and liver failure if persisted. Previous studies have indicated that transforming growth factor-β1 (TGF-β1) plays a crucial role in the pathogenesis of liver fibrosis. It induces the phenotypic transition of hepatic stellate cells to proliferating myofibroblast-like cells, enhances the production of extracellular matrix components, and induces apoptotic cell death in hepatocytes. Previously, studies have suggested reactive oxygen species may contribute to both the onset and the progression of fibrosis. Therefore, exploitation of antioxidants for prevention and treatment of liver fibrosis has opened a new way. Recent scientific studies have suggested that the polysaccharides (glucans) and triterpenes in Ganoderma lucidum and isoflavones in leguminous plants, such as black bean and Astragalus membranaceus, have antioxidative activities. Using black bean and A. membranaceus as part of the fermentation medium, isoflavones might be transformed into the more bioavailable form (aglycone) by microbial enzymatic hydrolysis of the glycosidic linkage. The polysaccharides (glucans) might also undergo enzymatic degradation into smaller molecules with higher hydrophilicity and bioavailability. The objectives of this research are to investigate the antifibrotic activities of the fermentation products by using black bean and A. membranaceus as part of liquid fermentation medium of G. lucidum under various fermentation conditions and to evaluate the relationship between the oxidative stress and antifibrotic activity. Human hepatoma cell line (Hep 3B) and rat hepatic stellate cell line (HSC-T6) treated with TGF-β1 were used as the models. Results showed that the recovery effects of TGF-β1 induced oxidative damage exhibited by most filtrates of broth were better than the filtrates of broth removal of polysaccharides and the hot water extracts of mycelia were all better than the ethanol and methanol extracts in Hep 3B cell model. Among all the fermentation filtrates of broth of G. lucidum, GL-2b, which was the fermentation broth of 5% black bean and 2% A. membranaceus fermented at 24 ℃ for 11 days with agitation speed 50 rpm and aeration rate 0.375 vvm, gave the best recovery effect. GL-5mHW, the hot water extract of mycelium by fermentation with 5% black bean and 2% A. membranaceus in the medium of G. lucidum at 30 ℃with agitation speed 50 rpm and aeration rate 0.75 vvm for 11days, showed the best recovery effect among all the mycelia samples. However, the cell cycle analysis of GL-5mHW detected by flow cytometer did not show apoptotic inhibition. In the HSC-T6 cell model system, only the hot water extracts of the raw materials of fermentation (black bean and A. membranaceus), and the fruiting body of G. lucidum showed significant inhibition in α-smooth muscle actin (α-SMA) expression (p<0.05). None of the fermentation products had any significant inhibitory effect toward α-SMA expression.