青藤碱联合索拉非尼对肝癌 HepG2细胞增殖、迁移及凋亡的影响

Effect of sinomenine combined with sorafenib on the proliferation,migration and apoptosis of hepatocellular carcinoma HepG2 cells

目的探究青藤碱与索拉非尼联合应用对肝癌HepG2细胞生物学功能的协同抑制作用,并对其潜在的相关分子机制进行初步探讨。方法通过MTT、平板克隆、细胞划痕、Transwell及Hoechst 33342/PI双染实验分别评价2种药物单独及联合应用后对HepG2细胞增殖、迁移及凋亡能力的影响;通过ROS试剂盒分别检测HepG2细胞经各组药物处理后ROS表达的变化,利用分子对接技术预测青藤碱与索拉非尼抗肝癌主要作用靶点结合的潜力,通过Western blot实验检测联合给药组VEGFR2、STAT3、p-STAT3蛋白表达水平的变化以进行验证,并检测下游MMP2、MMP9、BCL-2、BAX、Cleaved-Caspase3的蛋白表达水平。结果青藤碱、索拉非尼均可显著抑制HepG2细胞存活率(P<0.05),并且呈现时间及剂量依赖性;MTT、平板克隆、细胞划痕、Transwell及Hoechst 33342/PI双染实验结果表明两药联合应用可以对HepG2细胞分别产生协同抑制增殖、迁移及促进细胞凋亡的作用;联合用药组较单药给药组显著增强ROS的表达;分子对接及Western blot结果表明联合用药组较单药组可以显著抑制VEGFR2、p-STAT3的表达水平,并降低迁移关键蛋白MMP2、MMP9的表达水平、上调BAX/BCL-2比值及Cleaved-Caspase 3的表达(P<0.05)。结论青藤碱与索拉非尼联合使用在体外能够协同抑制肝癌HepG2细胞增殖、迁移并诱导细胞凋亡,其机制可能与下调VEGFR2/STAT3通路及通路下游相关分子有关。

Objective To investigate the synergistic inhibitory effect and potential mechanism of sinomenine and sorafenib on the biological function of HepG2 cells in hepatocellular carcinoma.Methods The effects of two drugs alone and in combination on the proliferation,migration,and apoptosis of HepG2 cells were evaluated by MTT,plate cloning,cell scratch,Transwell and Hoechst 33342/PI double staining experiments.The changes of ROS expression in HepG2 cells after treatment with the various groups of drugs were detected by the ROS kit.Molecular docking technique was used to predict the potential binding of sinomenine and sorafenib as the main targets of anti-liver cancer.Western blot was used to detect the expression of VEGFR2,STAT3,and p-STAT3 proteins in the combination therapy group,as well as the downstream protein expression of MMP2,MMP9,BCL-2,BAX,and cleared Caspase3.Results Both sinomenine and sorafenib significantly inhibited the survival of HepG2 cells in a time-and dose-dependent manner(P<0.05).The results of MTT,plate cloning,cell scratch,Transwell and Hoechst 33342/PI double staining experiments showed that the combination of the two drugs can synergistically inhibit cell proliferation and migration,and promote cell apoptosis in HepG2 cells.The combination therapy group significantly enhanced the expression of ROS compared to the monotherapy group.The molecular docking and western blot results showed that the combination therapy group inhibited the expression of VEGFR2 and p-STAT3,and reduced the expression of migration key proteins MMP2 and MMP9,upregulated the BAX/BCL-2 ratio,and the expression of cleared Caspase3(P<0.05).Conclusion The combination of sinomenine and sorafenib can synergistically inhibit the proliferation and migration of HepG2 cells and induce cell apoptosis in vitro,which may be related to the downregulation of the VEGFR2/STAT3 pathway and downstream related molecules.