Taurine、EGCG和genistein联合对体外活化肝星状细胞自噬的影响

目的:探讨牛磺酸(taurine)、表没食子儿茶素没食子酸酯(EGCG)和三羟基异黄酮(genistein) 3种抗肝纤维化药物联合使用对体外活化大鼠肝星状细胞(HSCs)自噬的影响及可能机制。方法:体外培养大鼠肝星状细胞株HSC-T6,设立对照组、联合药物(taurine+EGCG+genistein,TEG)组、自噬抑制剂巴弗洛霉素A1(Baf-A1)组和联合药物+抑制剂(TEG+Baf-A1)组。采用CCK-8法分别检测各组HSCs的存活率,应用透射电镜观察HSCs内部自噬体超微结构变化,通过吖啶橙(AO)染色和荧光显微镜观察各组细胞内自噬溶酶体变化,Western blot分析自噬标志性蛋白LC3-Ⅱ和beclin-1的表达。结果:与对照组相比,TEG组、Baf-A1组和TEG+Baf-A1组细胞的存活率明显降低(P 0. 05);透射电镜观察到细胞内部出现双层或多层膜结构的自噬体,以及单层膜结构的自噬溶酶体。与对照组相比,AO染色结果显示TEG组和Baf-A1组红色荧光区域显著缩小,TEG组中LC3-Ⅱ和beclin-1的表达量显著降低(P 0. 05)。结论:活化的HSCs发生自噬现象,联合药物TEG可阻断HSCs的自噬过程,其作用机制可能与抑制HSCs中自噬体的生成有关。

Taurine,EGCG and genistein inhibit rat hepatic fibrosis by inhibiting autophagy of hepatic stellate cells

AIM: To investigate the effect of combination of three anti-hepatic fibrotic drugs[taurine, epigallocatechin gallate (EGCG) and genistein] on autophagy of activated hepatic stellate cells in vitro and the possible mechanisms. METHODS: Rat hepatic stellate cell line HSC-T6 was cultured in vitro and divided into control group, combination therapy (taurine+EGCG+genistein, TEG) group, autophagy inhibitor bafilomycin A1 (Baf-A1) group and combination therapy+autophagy inhibitor (TEG+Baf-A1) group. The viability of hepatic stellate cells in each group was measured by CCK-8 assay. The ultrastructural changes of autophagosomes in hepatic stellate cells were observed under transmission electron microscope. The changes of autophagy lysosomes in each group were observed by acridine orange (AO) staining and fluorescence microscopy. The protein expression of autophagy markers LC3-Ⅱ and beclin-1 was determined by Western blot. RESULTS: Compared with the control group, the viability of HSC-T6 cells in TEG group,Baf-A1 group and TEG+Baf-A1 group was significantly lower (P<0.05). Transmission electron microscopy revealed the presence of autophagosomes with bilayer and multilamellar structures, and the autophagic lysosomes with monolayer. Compared with control group, the results of AO staining showed that the red fluorescence area in TEG group and Baf-A1 group was significantly reduced, while the expression of LC3-Ⅱ and beclin-1 in TEG group was decreased (P<0.05). CONCLUSION: Autophagy occurs in activated hepatic stellate cells. The TEG combination therapy may inhibit the viability of hepatic stellate cells indirectly by blocking the process of autophagy, thus inhibiting hepatic fibrosis in rats.