翠云草提取物调控miR-145对肝星状细胞增殖、凋亡的影响

Effects of Selaginellauncinata Extract on the Proliferation and Apoptosis of Hepatic Stellate Cells by MiR-145 Regulation

目的:探讨翠云草提取物对肝星状细胞增殖、凋亡的影响及分子机制。方法:分别用12.5,25.0,50.0μg·ml-1翠云草提取物处理肝星状细胞HSC-T6,分别记为翠云草提取物低、中、高浓度组;未经任何处理的细胞作为对照组。将miR-145转染至HSC-T6细胞中,记为miR-145组;将miR-145转染至HSC-T6细胞后再用50.0μg·ml-1翠云草提取物处理,记为翠云草提取物+miR-145组。细胞计数试剂盒8(CCK-8)检测细胞活性;克隆形成实验检测细胞克隆形成数;蛋白质印迹(Western blot)法检测细胞增殖核抗原Ki67、半胱氨酸天冬氨酸蛋白酶-3(caspase-3)蛋白表达;流式细胞术检测细胞凋亡;实时荧光定量PCR(RT-qPCR)检测miR-145表达水平。结果:与对照组比较,翠云草提取物中、高浓度组肝星状细胞的吸光度(A)值、克隆形成数、Ki67表达水平显著降低,细胞凋亡率、caspase-3和miR-145表达水平显著升高,且呈浓度依赖性(P<0.05)。过表达miR-145可抑制肝星状细胞HSC-T6增殖,促进细胞凋亡,且miR-145增强了翠云草提取物对肝星状细胞增殖、凋亡的影响。结论:翠云草提取物可通过上调miR-145抑制肝星状细胞增殖、促进细胞凋亡。

Objective: To explore the effect of Selaginellauncinata extract on the proliferation and apoptosis of hepatic stellate cells and the molecular mechanism. Methods: Hepatic stellate cells HSC-T6 were treated with 12.5,25.0 and 50.0 μg·ml-1 Selaginellauncinata extract,respectively,which were recorded as Selaginellauncinata extract low,medium and high concentration groups. The cells without any treatment were used as control group. miR-145 was transfected into HSC-T6 cells as miR-145 group,and HSC-T6 cells were transfected with miR-145 and then treated with 50.0 μg·ml-1 Selaginellauncinata extract recorded as Selaginellauncinata extract+ miR-145 group. Cell counting kit 8(CCK-8) was used to detect the cell activity;clone formation experiment was used to detect the number of cell clone formation;Western blot method was used to detect the cell proliferation,and nuclear antigen Ki67 and cysteinylaspartate specific proteinase-3(caspase-3) protein expressions;flow cytometry was used to detect the apoptosis;real-time fluorescence quantitative PCR(RT-qPCR) was used to detect the miR-145 expression. Results: Compared with those in the control group,the absorbance(A) value,number of colonies,and Ki67 expression level of hepatic stellate cells in Selaginellauncinata extract medium and high concentration groups reduced significantly,and the apoptosis rate,caspase-3 and miR-145 expression levels increased significantly,and the effects of Selaginellauncinata extract was concentration-dependent(P<0.05). Overexpression of miR-145 could inhibit the proliferation of hepatic stellate cells HSC-T6 and promote the apoptosis. MiR-145 enhanced the effects of Selaginellauncinata extract on the proliferation and apoptosis of hepatic stellate cells. Conclusion: Selaginellauncinata extract can inhibit the proliferation and promote the apoptosis of hepatic stellate cells by up-regulating miR-145.