半枝莲提取物逆转上皮间质转化抑制肝癌细胞迁移侵袭研究

Study on Inhibition of Reversal of Epithelial-mesenchymal Transition by Extracts from Scutellaria Barbata for Cell Migration and Invasion of Hepatoma Cells

目的:探讨半枝莲提取物(Extracts from Scutellaria barbata,ESB)对肝癌HepG2细胞迁移、侵袭的抑制作用及分子机制。方法:利用转化生长因子-β(Transforming growth factor-β,TGF-β)诱导肝癌HepG2细胞上皮间质转化模型,通过MTT法、Transwell实验测定细胞增殖、迁移及侵袭能力,Western blot检测各组细胞相关蛋白表达情况。结果:MTT结果显示ESB抑制HepG2细胞增殖,且呈浓度及时间依赖性,24 h及48 h IC50分别为317.3μg/mL,243.8μg/mL。Transwell实验显示,与对照组比较,TGF-β组细胞迁移与侵袭能力增强(P <0.05);与TGF-β组比较,TGF-β+5μg/mL ESB组,TGF-β+10μg/mL ESB组细胞迁移及侵袭能力减弱(P <0.05)。Western blot实验显示,在建立上皮间质转化模型后,与TGF-β组比较,TGF-β+ESB (5μg/mL、10μg/mL)组E钙黏蛋白(E-cadherin)表达上调(P <0.05),N钙黏蛋白(N-cadherin)和波形蛋白(Vimentin)表达下调(P <0.05),同时磷酸化的Smad2蛋白(Phosphorylated Smad2,p-Smad2)、磷酸化的Smad3蛋白(Phosphorylated Smad3,p-Smad3)、磷酸化的c-Jun氨基末端激酶(Phosphorylated c-Jun N-terminal kinase,p-JNk)、磷酸化的p38蛋白(Phosphorylated p-38,pp38)、磷酸化的胞外信号调节激酶(Phosphorylated extracellular regulated protein kinases,p-ERK)、整合素αV亚单位(IntegrinαV)、整合素β3亚单位(Integrinβ3)、基质金属蛋白酶2 (matrix metalloprotein2, MMP2)、基质金属蛋白酶9 (matrix metalloprotein9,MMP9)表达显著下调(P <0.05)。结论:ESB可能通过阻断TGF-β/Smad/AMPK信号通路,下调IntegrinαV、Integrinβ3、MMP2及MMP9逆转TGF-β诱导的上皮间质转化,抑制肝癌HepG2细胞迁移及侵袭。

Objective:To investigate the inhibitory effect and molecular mechanism of extracts from Scutellaria barbata(ESB)on cell migration and invasion of hepatoma cell HepG2.Methods:Used transforming growth factor-β(TGF-β)to induce transformation model of epithelial-mesenchymal transition in hepatoma cell HepG2.Measured cell proliferation,migration and invasion by MTT assay and Transwell experiment,and detected the expression of related proteins in each group by applying Western blot.Results:Results of MTT assay showed that ESB inhibited HepG2 cell proliferation in a concentration-and time-dependent manner,with IC50 of 317.3μg/mL and 243.8μg/mL at 24 h and 48 h,respectively.Transwell experiments showed that compared with the control group,the cell proliferation and migration were increase in the TGF-βgroup(P<0.05),Compared with the TGF-βgroup,the cell proliferation and migration were reduced in the group of TGF-β+5μg/mL ESB and the group of TGF-β+10μg/mL ESB(P<0.05).Western blot analysis showed that after establishing the epithelialmesenchymal transformation models,compared with the TGF-βgroup,the E-cadherin expression was up-regulated in the group of TGF-β+ESB(5μg/mL,10μg/mL)(P<0.05);N-cadherin and vimentin expressions were down-regulated(P<0.05);at the same time,the expressions of phosphorylated Smad2 protein(p-Smad2),phosphorylated Smad3 protein(p-Smad3),phosphorylated c-Jun N-terminal kinase(p-JNk),phosphorylated p-38 protein(p-p38),phosphorylated extracellular signal-regulated kinase(p-ERK),integrinαV unit,integrinβ3 subunit,matrix metalloproteinase 2(MMP2)and matrix metalloproteinase-9(MMP9)were significantly down-regulated(P<0.05).Conclusion:ESB may block TGF-β/Smad/AMPK signaling pathway,down-regulate the reversal of epithelial-mesenchymal transformation of IntegrinαV,Integrinβ3,MMP2 and MMP9,and inhibit cell migration and invasion of hepatoma cell HepG2.