青蒿琥酯联合热疗诱导肺腺癌A549细胞凋亡作用

Study on mitochondria pathway of apoptosis induced by artesunate and thermotherapy in A549 cell line

目的探讨青蒿琥酯联合热疗诱导肺腺癌A549细胞凋亡的线粒体途径作用机制。方法150μmol/L青蒿琥酯、青蒿琥酯联合热疗(43℃,1h)作用A549细胞24h,流式细胞技术检测细胞周期和凋亡率情况。荧光显微镜观察线粒体膜电位变化。免疫组化、western blot检测细胞色素c、Bcl-2和caspase-3表达,CaspACETM检测试剂盒检测活性caspase-3表达。结果青蒿琥酯、青蒿琥酯联合热疗作用A549细胞24h后,G0/G1期细胞数增多,S期和G2/M期细胞数减少(P<0.01);凋亡率分别是16.77%和28.90%(P<0.01)。线粒体膜电位下降率分别为18.05%,50.98%(P<0.01)。细胞色素c蛋白、caspase-3表达增加而Bcl-2蛋白表达减少(P<0.01)。活性caspase-3蛋白水平增加A值分别为0.2641±0.0027,0.3613±0.0019,与对照组相比,差异有统计学意义(P<0.01)。结论青蒿琥酯、青蒿琥酯联合热疗可诱导肺腺癌A549细胞凋亡,激活线粒体途径可能是诱导凋亡重要作用机制。

Objective To understand the mechanism of mitochondria pathway of apoptosis induced by artesunate com- bined thermotherapy in human lung adenocarcinoma A549 cell line. Methods Lung adenocarcinoma A 549 cell line was trea- ted with artesunate（ 150 pxnol/L） and hyperthermia（43 ±C for lh） for 24 h. Cell cycle and the apoptosis rate were measured by flow cytometry assay（FCM）. Mitochondria membrane potential was observed with JC-1 apoptosis detection kit by fluores- cence microscope. The protein express ions of cytochrome c, Bcl 2 and caspase 3 in A 549 cell line were assessed with western blot and immunocytochemical method. Expression of active caspase 3 was assayed by CaspACETM assay system. Results Compared with the control, the number of cell increased in stage G0/G1 and decreased in stage S and G2/M with statistic difference（P 〈0. 01 ）. The apoptosis rate of A549 cell line treated with artesunate, artesunate and thermothempy increased to 16. 77% and 28.90% compared with 2. 08% of the control with statistic difference（ P 〈0. 01 ）. Rates of mitochondria mem- brane potential decrease were 18, 05% and 50. 98% in A 549 cell treated with artesunate,and artesunate plus hyperthermia re- spectively compared to 1.92% in control A549 cell （ P 〈 0. 01 ）. Protein expression of cytochrome c and caspase 3 increased, while expression of Bcl-2 decreased compared to control group （P 〈 0. 01 ）. Expression of active caspase 3 increased to 0. 2641 ±0. 0027 and 0. 3613 ±0. 0019 with statistic difference（ P 〈 0. 01 ）. Conclusion Artesunate and thermotherapy has an effect of inducing apoptosis in human lung adenocarcinoma A549 cell line. The activation of mito chodria pathway may be an impor- tant mechanism for cell apoptosis.