益肺逐积方诱导人肺腺癌A549细胞凋亡机制

Mechanism of Yifei Zhuji Prescription on Inducing Human Lung Adenocarcinoma A549 Cells Apoptosis

目的：通过体外实验观察益肺逐积方对人肺腺癌A549细胞活性、细胞凋亡以及表皮生长因子受体（EGFR）,G蛋白偶联受体30（GPR30）蛋白表达的影响,探讨中药复方益肺逐积方抑制肺癌细胞活性,诱导肺癌细胞凋亡的作用机制。方法：将人肺腺癌A549细胞培养至对数生长期,益肺逐积方高、低质量浓度组分别加入2,1.5 g·L^（-1）益肺逐积方培养液,5-氟尿密啶（5-FU）组加入2.5 g·L^（-1）5-FU培养液,溶剂组加入含10%胎牛血清的RPMI 1640培养液,分别作用24,48 h后,采用噻唑蓝（MTT）比色法检测益肺逐积方对人肺腺癌A549细胞活性的影响;吖啶橙/溴乙啶（AO/EB）荧光染色观察益肺逐积方作用不同时间后人肺腺癌A549细胞的凋亡形态学变化;流式细胞术（FCM）测定益肺逐积方作用下人肺腺癌A549细胞的凋亡率;利用倒置显微镜观察益肺逐积方作用24,48 h后A549细胞的形态学改变,蛋白质免疫印迹法（Western blot）测定人肺腺癌A549凋亡细胞EGFR,GPR30蛋白的表达。结果：与溶剂组比较,1.5,2 g·L^（-1）益肺逐积方均能抑制人肺腺癌A549细胞活性（P〈0.05）,诱导A549细胞发生不同程度的凋亡形态学改变;流式细胞术检测显示,益肺逐积方作用24,48 h后,可使人肺腺癌A549细胞其发生晚期凋亡（P〈0.05）;Western blot结果显示,益肺逐积方可以不同程度地下调人肺腺癌A549细胞GPR30,EGFR蛋白的表达（P〈0.05）。结论：益肺逐积方可抑制人肺腺癌A549细胞活性、诱导其发生晚期凋亡,并使其发生凋亡形态学改变,这可能与下调EGFR,GPR30蛋白表达有关。

Objective： To observe the effect of Yifei Zhuji prescription on cells activity,apoptosis,and the protein expression of epidermal growth factor receptor（ EGFR） and G protein-coupled receptor 30（ GPR30） of human lung adenocarcinoma A549 cells,investigate its mechanism on inhibiting the activity of lung cancer cells and inducing apoptosis of lung cancer cells. Method： Human lung adenocarcinoma A549 cells were cultured to a logarithmic phase; 2,1. 5 g·L^（-1） drug culture medium was added in Yifei Zhuji prescription high and low dose groups; 2. 5 g·L^（-1）5-FU drug culture medium was added in blank group; RPMI 1640 culture medium containing10% fetal bovine serum was added in solvent group. After treatment for 24,48 h,3-（ 4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（ MTT） assay was used to detect the effect of Yifei Zhuji prescription on the activity of human lung adenocarcinoma A549 cells. Acridine orange/ethidium bromide（ AO/EB） was used to observe the morphological changes of apoptosis of human lung adenocarcinoma A549 cells. Flow cytometry（ FCM）was used to detect the apoptosis rate of A549 cells,and the morphological changes of A549 cells were observed by inverted microscope. In addition,Western blot was used to determine the apoptosis-related protein expression of EGFR and GPR30. Result： Compared with solvent group,both 1. 5 and 2 g·L^（-1） Yifei Zhuji prescription could inhibit the activity of A549 cells（ P 0. 05）,and induce the apoptosis morphological changes of A549 cells; FCM results showed that A549 cells were induced to have late stage apoptosis after Yifei Zhuji prescription treatment for24 h and 48 h（ P 0. 05）. Western blot results showed that Yifei Zhuji prescription could down-regulate the apoptosis-related protein expression of EGFR and GPR30 to different degrees（ P 0. 05）. Conclusion： Yifei Zhuji prescription could inhibit the activity of A549 cells,induce the apoptosis of A549 cells and induce the apoptosis morphological changes. The mechanism might be associated with down-regulating the protein expression of EGFR and GPR30.