双氢青蒿素对人肺腺癌细胞A549增殖和凋亡的影响

摘要

目的探讨双氢青蒿素（dihydroartemisinin， DHA ）对人肺腺癌细胞株A549的作用及其机制。方法将对数生长期的A549细胞分成对照组（control）和双氢青蒿素组（DHA）。对照组细胞常规培养，双氢青蒿素组细胞培养体系中加入双氢青蒿素（500 nmol／L）。各组细胞培养72 h后，采用四甲基偶氮唑盐（MTT）法检测细胞增殖，实时荧光定量PCR检测细胞p85、 Akt、 Bax、 Bcl－2基因表达，免疫蛋白印迹法（Western blotting）检测细胞中p85、 Akt、 p－p85， p－Akt、 Bax、 Bcl－2蛋白的变化， Caspase3活性试剂盒检测Caspase3活性。结果与对照组比较， DHA组A549细胞增殖率显著下降（P＜0．01）， p85、 Akt mRNA表达无显著差异， Bax mRNA表达水平增高（P＜0．001）， Bcl－2 mRNA表达水平降低（P＜0．05）； p85、 Akt总蛋白表达无显著变化（P＞0．05）， p－p85、 p－Akt、 Bcl－2蛋白表达显著减少， Bax蛋白表达显著增多（均P＜0．01）， Caspase3活性显著增加（P＜0．001）。结论双氢青蒿素能抑制A549细胞增殖，促进A549细胞凋亡，其作用机制可能是通过抑制PI3K／Akt信号传导通路而促进凋亡。%Objective To investigate the effect of the dihydroartemisinin (DHA) on the human low-differentiated lung adenocarcinoma cell line A549 and to explore its mechanism. Methods The A549 cells at logarithmic growth phase were divided into control group and DHA group. The cells in the control group were incubated with conventional reagent, and the cells in DHA group were incubated with 500 nmol/L of DHA. After incubation for 72 hours, methyl thiazolyl tetrazolium (MTT) assay was used to examine the proliferation of A549 cells in the two groups. Gene expression of p85, Akt, Bax and Bcl-2 was detected by real-time fluorescence quantitative polymerase chain reaction (PCR) . The protein expression of p85, Akt, p-p85, p-Akt, Bax and Bcl-2 was detected by Western blotting method. The activity of Caspase3 was measured by Caspase3 colorimetric assay kit. Results Compared with the control group, the proliferation rate of A549 cells in DHA group was significantly decreased ( P<0.01) . The RT-PCR results showed that the mRNA expression levels of p85 and AKT had no obvious difference between the two groups, but the mRNA expressien level of Bax was increased ( P<0.001), and the mRNA expressien level of Bcl-2 was decreased ( P<0.05) . The Western blotting results showed that there was no significant changes of p85 and Akt proteins between the two groups (P>0.05), but p-p85, p-Akt and Bcl-2 protein expression levels were significantly decreased ( P<0.01) , and Bax protein expression was increased ( P<0.01). Moreover, the activity of Caspase3 was also enhanced ( P<0.001). Conclusion DHA can reduce the proliferation of A549 cells and increase the apoptosis of A549 cells, and its mechanism probably has relationship with the inhibition of PI3K/Akt signaling pathway.