青蒿琥酯对人源肝星状细胞中Akt/GSK-3β/β-catenin信号通路的影响研究

Effect of Artesunate on Akt/GSK-3β/β-catenin Signal Pathway in Human Hepatic Stellate Cells

目的:探讨青蒿琥酯(Art)对Akt/GSK-3β/β-catenin信号通路的影响。方法:不同浓度(0,12.5,25,50μg·ml^(-1))Art作用于人源肝星状细胞(LX-2),采用CCK-8法检测细胞增殖情况,并确定给药浓度;给予不同浓度Akt抑制药MK-2206 0~8μmol·L^(-1),Western blot法确定其最佳抑制浓度;给予Art、MK-2206、MK-2206+Art,采用Western blot法检测各组Akt、pAkt、GSK-3β、p-GSK-3β、β-catenin蛋白表达情况。结果:CCK-8法检测细胞存活率,当选用25μg·ml^(-1)Art作用于LX-2细胞24h时细胞存活率约80%,Western blot法确定当MK-2206浓度为6μmol·L^(-1)时,可有效抑制p-Akt的表达;Art组(25μg·ml^(-1))、MK-2206组(6μmol·L^(-1))、MK-2206(6μmol·L^(-1))+Art(25μg·ml^(-1))组与对照组相比,Akt、p-Akt、p-GSK-3β、β-catenin蛋白表达均有显著差异(P<0.05),MK-2206(6μmol·L^(-1))+Art(25μg·ml^(-1))组分别与Art(25μg·ml^(-1))组、MK-2206(6μmol·L^(-1))组比较,GSK-3β、Akt蛋白表达无显著差异(P>0.05),p-Akt、p-GSK-3β、β-catenin蛋白表达显著降低(P<0.01)。结论:Art可通过Akt因子对Wnt/β-catenin信号通路中相关因子产生影响,进而抑制细胞增殖,缓解肝纤维发展进程。

Objective： To explore the effect of artesunate （Art） on Akt/GSK-3β/β-catenin signal pathway. Methods： Art at different concentrations （0, 12.5, 25, 50 μg·ml^-1 ） was used to treat human hepatic stellate cells （ LX-2 ） , and CCK-8 assay was used to detect the cell proliferation to determine the optimal concentration. Art inhibitor （MK-2206） at different concentrations （0 -8 μmol·L^-1） was given to LX-2 cells, and a Western Blot method was applied to determine the optimal inhibition concentration. Art, MK- 2206 and MK-2206 ＋ Art were respectively given to LX-2 cells, and a Western Blot method was used to detect the levels of Akt, p- Akt, GSK-3β, p-GSK-3β and β-catenin proteins. Results ： CCK-8 assay was used to detect the cell survival rate, and the survival rate was 80% after the 24-hour treatment with 25μg·ml^-1 Art. The results of Western Blot showed that MK-2206 at 6 μmol·L^-1 could effectively inhibit the expression of p-Akt. Compared with those of the control group, the levels of Akt, p-Akt, p-GSK-3β and β-eatenin protein were significantly different （P 〈0.05） in Art （25 μg·ml^-1 ） group, MK-2206 （6 μmol·L^-1） group and MK-2206 （6 μmol·L^-1） ＋ Art （25 μg·ml^-1） group. The expression of GSK-3β and Akt in MK-2206 ＋ Art group had no significant difference when compared with that in Art group and MK-2206 group （P 〉 0.05 ） , while the levels of p-Akt, p-GSK-3β and β-catenin were sig- nificantly reduced （P 〈 0.01 ）. Conclusion： Art exhibits the influence on the relative factors in Wnt/β-catenin signal pathway by Akt/β-catenin, subsequently inhibits the cell proliferation and alleviates the liver fibrosis process.