艾迪注射液对人肺腺癌A549细胞放疗敏感性的增加作用及其机制

The enhancing radiosensitization effect of Aidi injection on human lung adenocarcinoma A549 cells and its mechanism

目的：观察艾迪注射液对人肺腺癌A549细胞放疗敏感性的影响，分析其可能存在的机制。方法①给予不同浓度的艾迪注射液（1.875、3.75、7.5、15、30、60 mg/mL）处理A549细胞24 h，同时设置空白对照组，采用四甲基偶氮唑盐（MTT）比色法检测艾迪注射液对人肺腺癌A549细胞增殖的影响，计算10%细胞生长抑制剂量浓度（IC10），以IC10作为实验药物浓度。②实验分为空白对照组、艾迪对照组、射线处理组和艾迪预处理组。艾迪对照组给予艾迪注射液IC10孵育24 h；射线处理组给予4 Gy X射线照射后孵育24 h；艾迪预处理组给予艾迪注射液IC10孵育24 h后给予4 Gy X射线照射；空白对照组给予等量生理盐水孵育24 h。采用细胞克隆形成实验检测细胞存活分数（SF）；采用蛋白质免疫印迹试验（Western Blot）检测组蛋白H2AX的139号位点丝氨酸磷酸化为γ-H2AX、同源重组修复途径中的关键蛋白Rad51和细胞自噬体标志性蛋白微血管相关蛋白1轻链3（LC3）的蛋白表达情况；用透射电镜观察自噬体形成情况。结果艾迪注射液对人肺腺癌A549细胞增殖具有抑制作用，各剂量艾迪注射液组A549细胞增殖较空白对照组较低，随药物浓度增加，细胞生长抑制率（IR）逐渐增加，呈浓度依赖性，IC10为3.09 mg/mL。与空白对照组比较，艾迪对照组细胞SF无明显变化〔（94.7±3.85）%比（100.0±0.00）%，P＞0.05〕，射线处理组SF下降〔（71.8±5.90）%比（100.0±0.00）%，P＜0.05〕，而艾迪预处理组较放疗组进一步下降〔（51.9±4.73）%比（71.8±5.90）%，P＜0.05〕。3个治疗组γ-H2AX蛋白表达均较空白对照组明显增加，以艾迪预处理组最为显著，而且明显高于射线处理组（灰度值：1.44±0.11比0.93±0.09，P＜0.05）；但Rad51蛋白表达以射线处理组为最高，且高于艾迪预处理组（灰度值：1.37±0.07比0.78±0.04，P＜0.05）。艾迪对照组、射线处理组、艾迪预处理组的LC3Ⅱ/LC3Ⅰ比值较空白对照组有所增加，以艾迪预处理组增加最显著（0.35±0.06、0.37±0.07、0.49±0.06比0.05±0.04，均P＜0.05）。与空白对照组比较，各治疗组自噬体形成均有不同程度的增加，以艾迪预处理组增加最为明显。结论艾迪注射液对A549细胞具有放疗增敏的作用，其机制可能与上调A549细胞自噬水平有关。

Objective To observe the radiosensitization effect of Aidi injection on human lung adenocarcinoma A549 cells, and to analyze its possible mechanism.Methods ① A549 cells were treated with different concentrations of Aidi injection （1.875, 3.75, 7.5, 15, 30, 60 mg/mL） for 24 hours, and in the mean time, a blank control group was set up; the effect of Aidi injection on lung adenocarcinoma A549 cells proliferation was detected by methyl thiazolyl tetrazolium （MTT） assay, and the 10% cell growth inhibitor concentration （IC10） was calculated. ② The experiments were divided into blank control, Aidi control, radiation and Aidi pretreatment groups. The Aidi control group was incubated for 24 hours by Aidi injection IC10; the radiotherapy group was given X-ray irradiation of 4 Gy followed by incubation for 24 hours; the Aidi pretreatment group was incubated for 24 hours by Aidi injection IC10 and then given X-ray irradiation of 4 Gy; the blank control group received equal volume of normal saline and was incubated for 24 hours. The survival fraction （SF） value was detected by cell colony formation assay; the protein levels of the serine phosphorylation at 139 locus of histone （γ-H2AX protein）, the key protein in homologous recombination repair pathway （Rad51 protein） and the cell autophage characteristic protein （LC3 protein） were detected by Western Blot; the formation of autophagosome was observed by transmission electron microscope.Results Aidi injection possessed the suppression of the growth of human lung adenocarcinoma A549 cells, the proliferation of the cells in various Aidi groups was lower than that in the blank control group, with the increase in drug concentration, the A549 cell growth inhibition ratio （IR） was gradually increased, representing a dose dependent manner, and the IC10 was 3.09 mg/mL. Compared with the blank control group, the SF value in Aidi control group was not significantly different [（94.7±3.85）% vs. （100.0±0.00）%,P 〉 0.05], the SF value in radiation group was decreased [（71.8±5.9）% vs. （100.0±0.0）%,P 〈 0.05], and in Aidi pretreatment group, the value was further decreased compared with that in radiation group [（51.9±4.7）% vs. （71.8±5.9）%,P 〈 0.05]. Compared with the blank control group, the expression of γ-H2AX protein in the three treatment groups was significantly increased, the degree of increase in Aidi pretreatment group was the most obvious, and it was significantly higher than that in radiation group （gray value： 1.44±0.11 vs. 0.93±0.09,P 〈 0.05）. But the expression of Rad51 protein was the highest in radiation group, and it was higher than that in Aidi pretreatment group （gray value： 1.37±0.07 vs. 0.78±0.04, P 〈 0.05）. Compared with the blank control group, the LC3Ⅱ/LC3Ⅰ value in Aidi control group, radiation group and Aidi pretreatment group were increased, and the degree of increase in Aidi pretreatment group was the most significant （0.35±0.06, 0.37±0.07, 0.49±0.06 vs. 0.05±0.04, allP 〈 0.05）. Under transmission electron microscope, compared with the blank control group, the autophagosome in all treatment groups was increased to some extent, and the degree of increase in Aidi pretreatment group was the most remarkable.Conclusion Aidi injection has the enhancing effect of radiosensitization on human lung adenocarcinoma A549 cells, and its mechanism is possibly related to the up-regulation of A549 cell autophagy level.