白花蛇舌草乙醇提取物联合吉非替尼对TGF-β1诱导的肺腺癌细胞H358上皮间质化的干预作用

Intervention Effect of Ethanol Extract of Hedyotis Diffusa and Gefitinib on the Epithelial Mesenchymal Transition Process of Human Lung Adenocarcinoma Cell H358 Induced By TGF-β1

关键词:  白花蛇舌草  吉非替尼  上皮间质化  H358细胞

Key words:  ethanol extract of Hedyotis diffusa  gefitinib  epithelial mesenchymal transition  H358 cell

摘要:

目的 研究白花蛇舌草乙醇提取物(ethanol extract of Hedyotis diffusa，EEHD)联合吉非替尼对TGF-β₁诱导的肺腺癌细胞H358上皮间质化的干预作用。方法 以上皮表型人肺腺癌细胞H358为研究对象，TGF-β₁诱导构建细胞上皮间质化模型，按EEHD、吉非替尼及两药3种不同顺序联合作用分为6组：A组，EEHD作用48 h；B组，吉非替尼作用48 h；A24B24组，先EEHD作用24 h，后吉非替尼作用24 h；B24A24组，先吉非替尼作用24 h，后EEHD作用24 h；AB48组，同时加入吉非替尼和EEHD作用48 h；对照组。CCK-8法测定各组细胞坏死率，Western-blot检测各组细胞中E-cadherin、Vimentin、EGFR蛋白表达情况，流式细胞仪检测各组细胞凋亡情况，比较组间差异。结果 各组药物作用均能不同程度抑制上皮间质化肺腺癌细胞H358增殖，E-cadherin表达呈上升趋势，Vimentin表达下降。其中EEHD联合吉非替尼组的细胞生长抑制率和细胞凋亡率显著高于吉非替尼单用组，E-cadherin蛋白表达率则显著高于吉非替尼单用组，而EGFR、Vimentin蛋白表达率显著低于吉非替尼单用组(P<0.05)，EEHD与吉非替尼先后顺序作用组间差异无统计学意义。结论 EEHD与吉非替尼对上皮间质化的H358细胞具有联合抑制作用，白花蛇舌草可部分逆转H358细胞上皮间质化状态。

Abstract:

OBJECTIVE To investigate the ethanol extract of Hedyotis diffusa(EEHD) and gefitinib(Gef) used on the human lung adenocarcinoma H358 cell, which has been induced to epithelial mesenchymal transition by transforming growth factor beta 1(TGF-β₁) in vitro. METHODS The H358 cells were successfully induced to the epithelial mesenchymal transformation model by 5 ng·mL^-1 TGF-β₁ for 24 h. After preparation of the EEHD, respectively added EEHD for 48 h(Group A), Gef for 48 h(Group B), EEHD for 24 h and then Gef for 24 h(Group A24B24), Gef for 24 h and then EEHD for 24 h(Group B24A24), EEHD combined with Gef for 48 h(Group AB) and the control group to the model cells. Then the rates of cell growth were detected by CCK-8 test, the protein expression of epithelial marker (E-cadherin) and mesenchymal cell marker (Vimentin) were detected by Western-blot. And the rates of apoptosis were tested by flow cytometry. RESULTS In all other groups, the expression of E-cadherin increased while the expression of Vimentin decreased than the control group. The rates of cell growth inhibition and apoptosis of EEHD combined with gefitinib group were significantly higher than gefitinib monotherapy group’s, and the expressions of EGFR, Vimentin were significantly lower than gefitinib monotherapy group’s, while the expressions of E-cadherin was higher than gefitinib monotherapy group’s, P<0.05. There was no statistically significant difference between the groups of different orders(A24B24 and B24A24). CONCLUSION Hedyotis diffusa and gefitinib have a combined effect for the epithelial mesenchymal transition(EMT) and tumor cell growth process. Hedyotis diffusa have partially reversal effect on H358 which has been induced by TGF-β1 to epithelial mesenchymal transformation model. Hedyotis diffusa can partially reverse the EGFR-TKI resistance problems caused by EMT.