- 學位論文
人參皂苷Rb1對老鼠肝臟星狀HSC-T6細胞活化與纖維化之影響
Effects of ginsenoside Rb1 on cell activation and liver fibrosis in rat hepatic stellate HSC-T6 cells
摘要
近年來，慢性肝炎及肝硬化已成為台灣十大死因之第七位，而其中肝纖維化的病症目前已被認為是可修復的。肝臟星狀細胞的活化及增生在肝纖維化的過程中扮演重要的角色，而肝臟星狀細胞因活化所產生大量細胞外基質的堆積則為造成肝纖維化的主要原因。人參皂苷Rb1為人參中最具有活性的物質，且被認為在許多生理功能皆具療效。因此本研究之目的在探討於過氧化氫的氧化壓力下，給予老鼠肝臟星狀HSC-T6細胞人參皂苷Rb1 (純度為98.8%)，對於細胞的活化、增生及纖維化指標的影響。將細胞培養於Waymouth MB 752/1含10%胎牛血清之培養液中，並置於37℃、含5% CO2的環境下，24小時後以10 nM之H2O2誘導細胞更活化，再於24小時後更換為添加不同濃度人參皂苷Rb1之新鮮培養液，培養液中含人參皂苷Rb1之最終濃度分別為5、10、20、40及80 μg/mL。此外增設負控制組（添加0.08% dimethyl sulfoxide，為80 μg/mL Rb1組之劑量）及正控制組（添加5 mM之N-acetyl-L-cysteine）。細胞培養24小時後，收集細胞液及培養液進行細胞存活率、細胞活化指標α-平滑肌動蛋白(α-smooth muscle actin; α-SMA)、細胞增生情形及纖維化指標等分析。結果顯示：在給予細胞人參皂苷Rb1於5-40 μg/mL劑量下可降低由H2O2所誘導之α-SMA的表現(p < 0.05)，且於5-80 μg/mL劑量下顯著抑制細胞的增生情形(p < 0.05)。纖維化指標方面，在5-80 μg/mL的劑量下，可顯著降低轉化生長因子-β1及膠原蛋白的表現；而在10-80 μg/mL的劑量下則可顯著抑制基質金屬蛋白酶-2及金屬蛋白酶組織抑制物-1等纖維化指標的蛋白質表現量(p < 0.05)，且分別於10及80 μg/mL的劑量下能顯著抑制纖維化指標TGF-β1、type I與III collagen及TIMP-1之mRNA表現量(p < 0.05)。由結果得知，人參皂苷Rb1可藉由抑制肝臟星狀細胞的活化、增生及降低纖維化指標的表現，而達到抑制肝臟星狀細胞之致肝纖維化功能。
並列摘要
Chronic hepatitis and cirrhosis have been currently become the 7th leading cause of death in Taiwan. Recently, hepatic fibrosis is regarded as a reversible process in liver diseases. The activation and proliferation of hepatic stellate cells (HSCs) play important roles during liver fibrosis. The accumulated extracellular matrix proteins produced by activated HSCs are the major cause of liver fibrosis. Ginsenoside Rb1, the most active compound purified from ginseng, has been considered to be an effective regulator in many physiological functions. Accordingly, the present study investigated the effects of ginsenoside Rb1 (98.8% purity) on cell activation, proliferation, and liver fibrotic markers in rat hepatic stellate HSC-T6 cells under oxidative stress of H2O2. HSC-T6 cells were cultured in Waymouth MB 752/1 medium containing 10% fetal bovine serum at 37℃ in 5% CO2 atmosphere. HSC-T6 cells were induced more active by 10 nM H2O2. After 24 hours, the medium was replaced by fresh medium containing different concentrations of ginsenoside Rb1 (5, 10, 20, 40, and 80 μg/mL). Additionally, the medium containing 0.08% dimethylsulfoxide or 5 mM N-acetyl-L-cysteine was used as a negative or positive control. After 24 hours, cell suspension and medium were collected for cell viability assay, expression of cell activation marker - α-smooth muscle actin (α-SMA), cell proliferation assay, and expression of fibrotic markers. The results showed that ginsenoside Rb1 at 5-40 μg/mL significantly reduced α-SMA expression induced by H2O2 (p < 0.05) and inhibited cell proliferation at 5-80 μg/mL in HSC-T6 cells (p < 0.05). Transforming growth factor-β1 (TGF-β1) and collagen expression were reduced by ginsenoside Rb1 (5-80 μg/mL). Matrix metallo- proteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1) were also suppressed by ginsenoside Rb1 (10-80 μg/mL). In addition, mRNA expression of TGF-β1, type I and III collagen, and TIMP-1 were inhibited by ginsenoside Rb1 (10 and 80 μg/mL). Therefore, ginsenoside Rb1 might be able to reverse fibrogenesis in hepatic stellate cells through reduction of cell activation, proliferation, and expression of fibrotic markers.