PI3K/Akt途径在MMC诱导的肝干细胞凋亡中的作用

Role of PI3K/Akt Pathway in MMC Induced Apoptosis of Liver Stem Cells

目的探讨PI3K/Akt途径在丝裂霉素(mitomycin,MMC)诱导的肝干细胞凋亡中的作用。方法用MMC刺激大鼠肝干细胞,观察MMC刺激不同时间(6、12、24 h)对WB-F334细胞凋亡率的影响,以及不同浓度(0.2、0.4、0.6、0.8、1.0 mg/mL)的MMC对WB-F334细胞的细胞毒作用,加入磷脂酰肌醇-3激酶/蛋白激酶B(PI3K/Akt)抑制剂曲西立滨(triciribine,API-2)和p38丝裂原活化蛋白激酶(p38 MAPK)抑制剂SB203580后观察MMC对WB-F334细胞凋亡率的影响,探讨MMC调节WB-F334细胞凋亡的信号途径。结果 MMC对WB-F334细胞凋亡率的影响随作用时间延长而上升(P<0.05);与对照组比较,不同浓度的MMC对肝干细胞有明显细胞毒作用,且随着MMC浓度的升高而上升;Western blot检测结果显示:MMC刺激WB-F334细胞15 min后MAPK、Akt均发生磷酸化,且随着时间延长而减弱,60 min后磷酸化消失,说明p38 MAPK及PI3K/Akt途径能够被MMC激活;当MMC处理的细胞加入了p38MAPK抑制剂后,抑制剂处理组中细胞的凋亡率与MMC处理组并无显著差异(P>0.05),说明MAPK途径对MMC诱导WB-F334细胞凋亡并没有明显的作用;而当MMC处理的细胞加入了Akt抑制剂后,Akt抑制剂处理组中细胞的凋亡率与MMC处理组相比显著降低(P<0.05),表明PI3K/Akt途径对MMC诱导WB-F334细胞凋亡有明显的作用。结论 MMC能够刺激肝干细胞WB-F334发生凋亡,而其诱导凋亡的作用是通过激活PI3K/Akt信号通路实现的。

Objective To investigate the role of PI3K/Akt pathway in mitomycin（MMC） induced apoptosis of liver stem cells.Methods Rat liver stem cells were stimulated with MMC,and the effect of MMC on the apoptosis rate of WB-F334 cells at different time points（6,12,24 h）,as well as the effects of different concentrations of（0.2,0.4,0.6,0.8,1.0 mg/mL） MMC on the cytotoxicity of WB-F334 cells were evaluated;moreover,cells were treated with p38 MAPK inhibitor and PI3K/Akt inhibitor,and the roles of MAPK and PI3K/Akt signaling pathway in MMC mediated apoptosis of WB-F334 cells were explored.Results The apoptosis rate of the MMC-treated WB-F334 increased with time（P〈0.05）.Compared with the un-treated control group,different concentrations of MMC had obvious cytotoxicity on liver stem cells,and the cytotoxicity increased with concentration.Western blotting results showed that Akt and MAPK in WB-F334 cells were significantly phosphorylated 15 min after MMC stimulation;the degree of phosphorylation decreased with time,and phosphorylation disappeared after 60 min,suggesting that the p38 MAPK,PI3K/Akt pathway can be activated by MMC;furthermore,when p38 MAPK inhibitor was added to MMC treated cells,the apoptosis rate of p38 MAPK inhibitor treated cells showed no significant difference compared to the un-treated cells（P〈0.05）,indicating that the MAPK pathway had no significant effect on MMC induced WB-F334 cell death;however,when the PI3K/Akt inhibitor（API-2） was added,the apoptosis rate of API-2 treated cells was significantly decreased compared to the un-treated cells（P〉0.05）,indicating that the PI3K/Akt pathway had a significant effect on MMC induced apoptosis of WB-F334 cells（P〈0.05）.Conclusion Stimulation of MMC can induce apoptosis of hepatic stem cells WB-F334 through the activation of the PI3K/Akt signaling pathway.