目的 探讨白头翁皂苷中有效成分抑制人肺癌NCI-H460细胞增殖作用及其有效成分协同对肿瘤糖酵解机制的作用。方法 体外培养NCI-H460细胞，以MTT法检测白头翁皂苷中白头翁皂苷A3（A3）、白头翁皂苷D（PSD）、白头翁皂苷B4（B4）、常春藤碱C（HC）、常春藤碱D（HD）、常春藤皂苷元（HY）、白头翁皂苷R13（R13）和白头翁皂苷PSA（PSA）的抗肿瘤活性；通过Calcusyn 3.0软件将筛选出的白头翁有效成分进行配伍及协同抗肿瘤作用研究；采用生物化学检测法和Elisa法检测糖酵解相关代谢产物（丙酮酸、乳酸、葡萄糖）和葡萄糖转运蛋白1（GLUT1）、乳酸脱氢酶（LDHA）、M2型丙酮酸激酶（PKM2）和己糖激酶（HK）水平；运用实时荧光定量PCR（qRT-PCR）技术和Western blotting技术检测糖酵解相关41个成员基因和关键蛋白细胞外调节蛋白激酶1/2（ERK1/2）、Ras、GLUT1、单羧酸转运体4（MCT4）的表达。结果 体外抗肿瘤结果显示，白头翁皂苷中以PSD、R13、PSA抑瘤活性最强，其对NCI-H460细胞IC₅₀分别为5.2、4.6、7.9 μg/mL；运用Calcusyn 3.0软件确定了白头翁皂苷中3个有效单体成分配伍比例及判定了配伍后具有协同抗肿瘤效应；生化和Elisa结果显示，与对照组比较，白头翁单体组及联合组的丙酮酸、乳酸、葡萄糖、HK、PKM2、LDHA含量均明显降低，且GLUT1含量明显升高（P<0.05），与各单体组相比，其联合组的丙酮酸、乳酸、葡萄糖、HK、PKM2、LDHA含量均明显的降低，GLUT1含量明显升高（P<0.05）；qRT-PCR结果显示，单体联合组在网络图中节点最多，其作用靶点多于各个单体组；Western blotting结果显示，与对照组比较，白头翁单体联合组可显著性降低细胞ERK1/2、Ras、GLUT1、MCT4蛋白表达（P<0.05）。结论 白头翁皂苷有效成分联用对NCI-H460细胞具有协同抗肿瘤作用，其可能通过调节肿瘤细胞糖酵解发挥抗肿瘤作用。

Objective The study was designed to investigate the antitumor effect of Pulsatilla chinensis saponins components and the synergistic effect of its effect components on tumor glycolysis. Methods NCI-H460 cell lines were cultured in vitro, MTT assay was used to detect the inhibitory rate of various components of Pulsatilla chinensis saponins. The compatibility and synergistic antitumor effect of different effect components were studied by Calcusyn 3.0 software. Biochemical assay and Elisa assay were used to detect the glycolysis related metabolites (pyruvate, lactic acid, and glucose) and enzymes (GLUT1, HK, PKM2, and LDHA). Energy metabolism related gene members from KEGG pathway database and key protein (ERK1/2, Ras, GLUT1, MCT4) were detected by qRT-PCR and Western blotting. Results The antitumor results in vitro showed that PSD, R13 and PSA in Pulsatilla chinensis saponins had the strongest antitumor activity for human lung cancer NCI-H460 cells and the IC₅₀ were 5.2, 4.6, and 7.9 μg/mL, respectively. Using Calcusyn 3.0 software to confirm the distribution ratio of three effective monomers in Pulsatilla chinensis saponins and determine the synergistic antitumor effect after compatibility. The results of biochemical and Elisa assay showed that the content of pyruvate, lacate, glucose, HK, PKM2 and LDHA were significantly decreased and the GLUT1 content was significantly increased compared with the blank group (P < 0.05),; Compared with each monomer group, the content of pyruvic acid, lactic acid, glucose, HK, PKM2 and LDHA in the combined group were significantly decreased and the content of GLUT1 was significantly increased (P < 0.05). Real-time PCR results show that the combinations group has the most nodes in the network diagram, and its target was more than each monomer group. Western blotting results showed that compared with the blank group, the combinations group significantly decreased the expression of ERK1/2, Ras, GLUT1, and MCT4 protein (P < 0.05). Conclusion The combination of effect components of Pulsatilla chinensis saponins has synergistic antitumor effect on NCI-H460 cells and the antitumor mechanism may be associated with the regulation of glycolysis.

国家自然科学基金资助项目（81460618）；国家自然科学基金资助项目（81860720）