基于PI3K/PTEN/VEGF的菟丝子总黄酮抗肝癌作用机制研究

目的：基于PI3K/PTEN/VEGF通路探讨菟丝子总黄酮（TF）体外抗肿瘤作用机制,考察合适的给药浓度,为菟丝子药材的临床合理应用提供参考。方法：以人肝癌细胞HepG2为实验对象,CCK-8法检测TF给药后的细胞活性,计算其IC 50值;Annexin V-FITC/PI双染法分析细胞凋亡坏死比例;实时荧光定量PCR （qPCR）法检测胞内磷脂酰肌醇激酶（PI3K）、人类第十号染色体缺失的磷酸酶及张力蛋白同源基因（PTEN）、血管内皮细胞生长因子（VEGF） mRNA的相对表达;酶联免疫吸附法（ELISA）检测缺氧诱导因子-1α（HIF-1α）、血管内皮细胞生长因子受体2、3（VEGFR-2、VEGFR-3）表达变化趋势。结果：相对阴性对照组,给药组细胞抑制率和凋亡率均具有良好的量-效正相关;qPCR结果显示TF给药可以显著的上调PTEN mRNA,下调PI3K、VEGF mRNA;ELISA结果显示HIF-1α、VEGFR-2、VEGFR-3蛋白的表达在给药后均有下降（P<0.01）。结论：菟丝子总黄酮有较好的体外抗肝癌药效,其作用可能与上调PTEN的表达对PI3K-Akt通路进行负调控诱导细胞发生凋亡、下调HIF-1α以及VEGF相关基因和受体蛋白的表达等作用机制共同发挥抗肿瘤作用。

Study on anti-tumor effects and potential mechanism of total flavonoids of Cuscuta chinensis Lam. based on PI3K/PTEN/VEGF pathway

OBJECTIVE To explore the potential mechanism of total flavonoids of Cuscuta chinensis Lam. (TF) on human liver cancer HepG2 cells based on PI3K/PTEN/VEGF pathway and find out the proper dosage for clinical treatments.METHODS HepG2 cells were chosen to be the model of this study, CCK-8 method was used to detect cells inhibition ratio after different doses of TF and different treatment durations. Annexin V-FITC/PI double-staining method was used to analyze cell apoptosis ratio after administration by flow cytometry (FCM). The relative expression of phosphoinositide 3-kinase (PI3K), phosphatase and tensin homolog deleted on chromosome ten (PTEN) and vascular endothelial growth factor (VEGF) mRNA were detected by real time quantitative PCR (qPCR). Enzyme linked immunosorbent assay was applied to quantitatively detect the expression of hypoxia inducible factor-1alpha (HIF-1α) and vascular endothelial growth factor receptor 2, 3 (VEGFR-2, VEGFR-3).RESULTS Compared with the negative control group, cell inhibition ratio and apoptosis ratio of each administration groups showed a good positive relation in dose-effect. The relative expression of PTEN mRNA was significantly increased, while PI3K and VEGF mRNA and HIF-1α, VEGFR-2, VEGFR-3 proteins were significantly decreased (P<0.01) based on qPCR and ELISA results.CONCLUSION TF shows a good anti-liver tumor effect in vitro. The potential mechanisms may include followings:up-regulation of the relative expression of PTEN mRNA and down-regulation of the PI3K-Akt signaling pathway to promote the apoptosis rate of HepG2 cells; down-regulation of the expression of HIF-1α and VEGF related genes or protein receptors.