目的 探討異鉤藤堿調(diào)節(jié)表皮生長因子受體（EGFR）/局部黏著斑激酶（FAK）信號通路對肝癌細(xì)胞增殖、遷移和5-氟尿嘧啶（5-FU）耐藥性的影響。方法 取對數(shù)生長期的 HepG2 細(xì)胞，分為對照組，異鉤藤堿低、中、高濃度（32.5、65.0、130.0 μmol·L^-1）組，異鉤藤堿（130 μmol·L^-1）＋EGFR 激活劑 NSC228155（2 μmol·L^-1）組 ；對數(shù)生長期的 5-FU 耐藥肝癌細(xì)胞系 HepG2/5-FU 分為對照組 、異 鉤 藤 堿（ 130 μmol · L^-1 ）組 、5-FU（ 60 μmol · L^-1 ）組 、異 鉤 藤 堿（ 130 μmol · L^-1）＋5-FU（60 μmol·L^-1）組、異鉤藤堿（130 μmol·L^-1）＋5-FU（60 μmol·L^-1）＋NSC228155（2 μmol·L^-1）組。給藥處理 24 h，對照組不給藥。EdU 染色、CCK-8 檢測 HepG2 或 HepG2/5-FU 細(xì)胞增殖；劃痕實(shí)驗(yàn)檢測 HepG2 或 HepG2/5-FU 細(xì)胞遷移；Westernblotting 檢測細(xì)胞中細(xì)胞周期蛋白 D1（CyclinD1）、遷移侵襲增強(qiáng)子因子 1（MIEN1）、P-糖蛋白（P-gp）、p-EGFR、p-FAK蛋白表達(dá)。結(jié)果 與對照組相比，異鉤藤堿低、中、高濃度組 EdU 陽性率、吸光度（A₄₅₀）值、劃痕愈合率、CyclinD1、MIEN1、p-EGFR、p-FAK 蛋白表達(dá)顯著降低，且呈濃度相關(guān)性（P＜0.05）；與異鉤藤堿 130.0 μmol·L^-1組相比，異鉤藤堿＋NSC228155 組 HepG2 細(xì)胞 EdU 陽性率、A₄₅₀ 值、劃 痕 愈 合 率 、 CyclinD1、 MIEN1、 p-EGFR、 p-FAK 蛋 白 表 達(dá) 顯 著 升高（P＜0.05）。與對照組相比，5-FU 組 HepG2/5-FU 細(xì)胞 EdU 陽性率、A₄₅₀值、劃痕愈合率、P-gp、p-EGFR、p-FAK 蛋白表達(dá)顯著降低（P＜0.05）；與異鉤藤堿組、5-FU 組相比，異鉤藤堿＋5-FU 組 HepG2/5-FU 細(xì)胞 EdU 陽性率、A₄₅₀值、劃痕愈合率、P-gp、p-EGFR、p-FAK 蛋白表達(dá)降低（P＜0.05）；與異鉤藤堿＋5-FU 組相比，異鉤藤堿＋5-FU+NSC228155 組 HepG2/5-FU 細(xì)胞 EdU 陽性率、A₄₅₀值、劃痕愈合率、P-gp、p-EGFR、p-FAK 蛋白顯著上調(diào)（P＜0.05）。結(jié)論 異鉤藤堿抑制 HepG2細(xì)胞增殖、遷移及降低 5-FU 耐藥性的機(jī)制可能與阻斷 EGFR/FAK 通路有關(guān)。

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

黑龍江省省屬高等學(xué)?；究蒲袠I(yè)務(wù)費(fèi)科研項(xiàng)目（2023-KYYWF-0959）