目的 探讨异钩藤碱调节表皮生长因子受体（EGFR）/局部黏着斑激酶（FAK）信号通路对肝癌细胞增殖、迁移和5-氟尿嘧啶（5-FU）耐药性的影响。方法 取对数生长期的 HepG2 细胞，分为对照组，异钩藤碱低、中、高浓度（32.5、65.0、130.0 μmol·L^-1）组，异钩藤碱（130 μmol·L^-1）＋EGFR 激活剂 NSC228155（2 μmol·L^-1）组 ；对数生长期的 5-FU 耐药肝癌细胞系 HepG2/5-FU 分为对照组 、异 钩 藤 碱（ 130 μmol · L^-1 ）组 、5-FU（ 60 μmol · L^-1 ）组 、异 钩 藤 碱（ 130 μmol · L^-1）＋5-FU（60 μmol·L^-1）组、异钩藤碱（130 μmol·L^-1）＋5-FU（60 μmol·L^-1）＋NSC228155（2 μmol·L^-1）组。给药处理 24 h，对照组不给药。EdU 染色、CCK-8 检测 HepG2 或 HepG2/5-FU 细胞增殖；划痕实验检测 HepG2 或 HepG2/5-FU 细胞迁移；Westernblotting 检测细胞中细胞周期蛋白 D1（CyclinD1）、迁移侵袭增强子因子 1（MIEN1）、P-糖蛋白（P-gp）、p-EGFR、p-FAK蛋白表达。结果 与对照组相比，异钩藤碱低、中、高浓度组 EdU 阳性率、吸光度（A₄₅₀）值、划痕愈合率、CyclinD1、MIEN1、p-EGFR、p-FAK 蛋白表达显著降低，且呈浓度相关性（P＜0.05）；与异钩藤碱 130.0 μmol·L^-1组相比，异钩藤碱＋NSC228155 组 HepG2 细胞 EdU 阳性率、A₄₅₀ 值、划 痕 愈 合 率 、 CyclinD1、 MIEN1、 p-EGFR、 p-FAK 蛋 白 表 达 显 著 升高（P＜0.05）。与对照组相比，5-FU 组 HepG2/5-FU 细胞 EdU 阳性率、A₄₅₀值、划痕愈合率、P-gp、p-EGFR、p-FAK 蛋白表达显著降低（P＜0.05）；与异钩藤碱组、5-FU 组相比，异钩藤碱＋5-FU 组 HepG2/5-FU 细胞 EdU 阳性率、A₄₅₀值、划痕愈合率、P-gp、p-EGFR、p-FAK 蛋白表达降低（P＜0.05）；与异钩藤碱＋5-FU 组相比，异钩藤碱＋5-FU+NSC228155 组 HepG2/5-FU 细胞 EdU 阳性率、A₄₅₀值、划痕愈合率、P-gp、p-EGFR、p-FAK 蛋白显著上调（P＜0.05）。结论 异钩藤碱抑制 HepG2细胞增殖、迁移及降低 5-FU 耐药性的机制可能与阻断 EGFR/FAK 通路有关。

Objective To investigate the effects of isorhynchophylline (isorhy) on the proliferation, migration, and 5-FU resistance of liver cancer cells by regulating the epidermal growth factor receptor (EGFR)/focal adhesion kinase (FAK) signaling pathway. Methods Logarithmic phase HepG2 cells were divided into control group, low, medium, and high concentration groups of isorhy (32.5, 65.0, 130.0 μmol·L^-1), and the high concentration group of isorhy (130 μmol·L^-1) + EGFR activator NSC228155 (2 μmol·L^-1). Logarithmic phase 5-FU-resistant hepatocellular carcinoma cell line HepG2/5-FU was divided into control group, isorhy (130 μmol·L^-1) group, 5-FU (60 μmol·L^-1) group, isorhy (130 μmol·L^-1) + 5-FU (60 μmol·L^-1) group, and isorhy (130 μmol·L^-1) + 5-FU (60 μmol·L^-1) + NSC228155 (2 μmol·L^-1) group. The cells were treated with drugs for 24 hours, and the control group was not treated. EdU staining and CCK-8 were applied to detect HepG2 or HepG2/5-FU cell proliferation. Scratch experiment was applied to detect the migration of HepG2 or HepG2/5-FU cells. Western blotting was applied to detect cyclinD1, MIEN1, P-glycoprotein (P-gp), p-EGFR, and pFAK proteins in cells. Results Compared with the control group, the EdU positive rate, A₄₅₀ value, scratch healing rate, CyclinD1, MIEN1, p-EGFR, and p-FAK protein expression of HepG2 cells in isorhy group were reduced, in a concentration-dependent manner (P<0.05). Compared with isorhy (130 μmol·L^-1) group, the EdU positive rate, A₄₅₀ value, scratch healing rate, CyclinD1, MIEN1, pEGFR, and p-FAK protein expression of HepG2 cells in isorhy (130.0 μmol·L^-1) +NSC228155 group were increased (P<0.05). Compared with control group, the EdU positive rate, scratch healing rate, A₄₅₀ value, P-gp, p-EGFR, and p-FAK protein expression of HepG2/5-FU cells in the 5-FU group were reduced (P<0.05). Compared with the isorhy group or 5-FU group, the EdU positive rate, A₄₅₀ value, scratch healing rate, P-gp, p-EGFR, and p-FAK protein expression of HepG2/5-FU cells in the isorhy + 5-FU group were reduced (P<0.05). Compared with the isorhy+5-FU group, the positive rate of EdU, A₄₅₀ value, scratch healing rate, P-gp, pEGFR, and p-FAK protein expression in HepG2/5-FU cells in the isorhy+5-FU+NSC228155 group were increased (P<0.05). Conclusion Isorhy may inhibit proliferation, migration, and reduce 5-FU resistance of HepG2 cells by blocking the EGFR/FAK pathway.

R965

黑龙江省省属高等学校基本科研业务费科研项目（2023-KYYWF-0959）