网络药理学技术预测昆仑雪菊总黄酮治疗非小细胞肺癌作用及初步验证研究

Prediction of the effects of total flavonoids of Kunlun chrysanthemum in the treatment of non-small cell lung cancer by network pharmacology and its preliminary verification study

通过高效液相色谱法(HPLC)对昆仑雪菊总黄酮进行成分分析。利用网络药理学方法,构建“成分-靶点-通路”网络。基于预测结果,CCK8法检测不同浓度的昆仑雪菊总黄酮(Kunlun chrysanthemum total flavonoids,KCTF)对A549细胞的增殖抑制活性,流式细胞术检测细胞凋亡情况,细胞划痕实验观察KCTF对A549细胞迁移能力的影响。RT-qPCR检测Bax、Bcl-2和MMP-2基因的表达,Western blot检测相关凋亡蛋白Bax和Bcl-2的表达。结果显示,HPLC分析获得68个KCTF成分,经筛选后得27个活性成分,通过相关数据库平台构建出KCTF干预非小细胞肺癌(non-small cell lung cancer,NSCLC)相关靶点217个,KEGG通路富集分析结果显示,KCTF干预NSCLC的关键节点主要被富集在细胞增殖、凋亡和周期调控、病毒致癌及其他肿瘤通路等76条信号通路。细胞水平验证实验结果表明,KCTF明显抑制了A549细胞的增殖,促进细胞的凋亡,抑制细胞的迁移;RT-qPCR实验验证了KCTF上调Bax的表达以及下调Bcl-2和MMP-2基因的表达;Western blot结果表明KCTF明显上调Bax/Bcl-2蛋白的表达,诱导了细胞的凋亡。综上,KCTF可通过多靶点、多途径实现干预NSCLC的作用,其抗肿瘤活性可能与影响凋亡基因Bax及Bcl-2的表达有关。

The composition analysis of the total flavonoids of Kunlun chrysanthemums was carried out by the high performance liquid chromatography(HPLC).The“component-target-pathway”network was constructed through the method of network pharmacology.Based on the prediction results,the CCK8 assay was used to detect the proliferation inhibitory activity of Kunlun chrysanthemum total flavonoids(KCTF)at different concentrations on A549 cells,flow cytometry was used to detect cell apoptosis,and a cell scratch assay was used to observe the effects of KCTF on migration ability of A549 cells.RT-qPCR was used to detect the expression of Bax,Bcl-2,and MMP-2 genes,and Western blot was used to detect the expression of related apoptotic proteins Bax and Bcl-2.The results showed that 68 KCTF components were obtained by HPLC analysis,27 active components were obtained after screening,and 217 KCTF intervention non-small cell lung cancer(NSCLC)related targets were constructed through the relevant database platform.The results of KEGG pathway enrichment analysis showed that the key nodes of KCTF intervention in NSCLC were mainly enriched into 76 signaling pathways such as cell proliferation,apoptosis and cycle regulation,viral carcinogenesis,and other tumor pathways.The results of cell-level verification experiments showed that KCTF significantly inhibited the proliferation of A549 cells,promoted cell apoptosis,and inhibited cell migration;RT-qPCR experiments verified that KCTF up-regulated the expression of Bax and down-regulated the expression of Bcl-2 and MMP-2 genes;Western blot results showed that KCTF significantly up-regulates the expression of Bax/Bcl-2,and induces cell apoptosis.In summary,KCTF can intervene in NSCLC through multiple targets and multiple pathways,and its anti-tumor activity may be related to affecting the expression of apoptosis-related genes Bax and Bcl-2.