目的 探讨黄芪多糖通过DNA损伤修复发挥抗非小细胞肺癌（NSCLC）活性的作用及机制。方法 通过ip氨基甲酸酯（0.8 mg/g）建立NSCLC小鼠模型，观察肺组织表面结节的数量及形态在体评价黄芪多糖（300 mg/kg）的抗肿瘤活性；CCK8法评价黄芪多糖体外抗人NSCLC细胞系A549（40.00、20.00、10.00、5.00、2.50、1.25 μmol/L）和HCC827（100、50、25 μmol/L）增殖活性；并通过单细胞凝胶电泳测定（彗星试验）评价黄芪多糖对A549（40 μmol/L）和HCC827（50 μmol/L）细胞DNA损伤的影响；通过实时荧光定量PCR（RT-qPCR）、Western blotting、RNA干扰、免疫荧光染色实验研究黄芪多糖对VRK1/P53BP1信号通路的作用。结果 与模型组小鼠比较，黄芪多糖和吉非替尼（阳性药）组的结节数目显著减少（P<0.05、0.01）；黄芪多糖组的肿瘤结节呈近似圆形，与紧密排列的肿瘤结节边界清晰，模型和吉非替尼组的结节呈现不规则结构，细胞排列松散，更具侵略性。黄芪多糖呈剂量相关性地抑制A549和HCC827细胞增殖，显著缩短的彗尾和橄榄炬（P<0.05、0.001），保护DNA完整性；显著增加NSCLC细胞中VRK1 mRNA和蛋白表达水平（P<0.05、0.01），同时免疫荧光分析发现P53BP1和VRK1蛋白表达水平升高，且VRK1蛋白可在细胞核和细胞质之间移位；通过VRK1敲低反向验证上述结果。结论 黄芪多糖通过激活VRK1/P53BP1信号转导途径增强DNA损伤修复，进而抑制NSCLC细胞增殖。

Objective To investigate the effect and mechanism of the anti-non-small cell lung cancer activity of Astragalus polysaccharides (APS) through DNA damage repair. Methods NSCLC mouse model was established by ip carbamate (0.8 mg/g). The number and morphology of pulmonary surface nodules were observed to evaluate the antitumor activity of APS (300 mg/kg) in vivo. CCK8 method was used to evaluate the proliferation activity of APS against human NSCLC cell lines A549 (40.00, 20.00, 10.00, 5.00, 2.50, 1.25 μmol/L) and HCC827 (100, 50, 25 μmol/L) and the effect of APS on DNA damage of NSCLC cells lines A549 (40.00 μmol/L) and HCC827 (50 μmol/L) was evaluated by single cell gel electrophoresis (comet assay). The effects of APS on VRK1/P53BP1 signaling pathway were verified by experiments such as RT-qPCR, western blotting, siRNA, and immunofluorescence staining. Results Compared with the model group, the number of nodules in APS and gefitinib group decreased significantly (P<0.05, 0.01). In Astragalus polysaccharide group, the tumor nodule was round, with clear boundary with closely arranged tumor nodule. In model group and gefitinib group, the nodule presented irregular structure, loose cell arrangement and more aggressive. APS inhibited the proliferation of A549 and HCC827 cells in a dose-dependent manner, significantly shortened coma tail and olive torch (P<0.05, 0.001), and protected DNA integrity. It can significantly increase the expression level of VRK1 mRNA and protein in NSCLC cells (P<0.05, 0.01), and it was found that the expression levels of P53BP1 and VRK1 proteins were increased through immunofluorescence analysis. Besides, VRK1 protein could be shifted between the nucleus and the cytoplasm. Finally, the above results were verified by VRK1 knockdown. Conclusion APS can enhance the DNA damage repair by activating VRK1/P53BP1 signal transduction pathway, and then inhibit the proliferation of NSCLC cells.

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