目的 探討黃芪多糖通過DNA損傷修復(fù)發(fā)揮抗非小細(xì)胞肺癌（NSCLC）活性的作用及機(jī)制。方法 通過ip氨基甲酸酯（0.8 mg/g）建立NSCLC小鼠模型，觀察肺組織表面結(jié)節(jié)的數(shù)量及形態(tài)在體評價(jià)黃芪多糖（300 mg/kg）的抗腫瘤活性；CCK8法評價(jià)黃芪多糖體外抗人NSCLC細(xì)胞系A(chǔ)549（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；并通過單細(xì)胞凝膠電泳測定（彗星試驗(yàn)）評價(jià)黃芪多糖對A549（40 μmol/L）和HCC827（50 μmol/L）細(xì)胞DNA損傷的影響；通過實(shí)時熒光定量PCR（RT-qPCR）、Western blotting、RNA干擾、免疫熒光染色實(shí)驗(yàn)研究黃芪多糖對VRK1/P53BP1信號通路的作用。結(jié)果 與模型組小鼠比較，黃芪多糖和吉非替尼（陽性藥）組的結(jié)節(jié)數(shù)目顯著減少（P<0.05、0.01）；黃芪多糖組的腫瘤結(jié)節(jié)呈近似圓形，與緊密排列的腫瘤結(jié)節(jié)邊界清晰，模型和吉非替尼組的結(jié)節(jié)呈現(xiàn)不規(guī)則結(jié)構(gòu)，細(xì)胞排列松散，更具侵略性。黃芪多糖呈劑量相關(guān)性地抑制A549和HCC827細(xì)胞增殖，顯著縮短的彗尾和橄欖炬（P<0.05、0.001），保護(hù)DNA完整性；顯著增加NSCLC細(xì)胞中VRK1 mRNA和蛋白表達(dá)水平（P<0.05、0.01），同時免疫熒光分析發(fā)現(xiàn)P53BP1和VRK1蛋白表達(dá)水平升高，且VRK1蛋白可在細(xì)胞核和細(xì)胞質(zhì)之間移位；通過VRK1敲低反向驗(yàn)證上述結(jié)果。結(jié)論 黃芪多糖通過激活VRK1/P53BP1信號轉(zhuǎn)導(dǎo)途徑增強(qiáng)DNA損傷修復(fù)，進(jìn)而抑制NSCLC細(xì)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.

R285.5