目的 探讨橙皮苷通过靶向β-连环蛋白（β-catenin）对肝癌细胞干性的抑制作用，评估其对索拉非尼敏感性的增强效果。方法 取对数生长期的HepG2细胞，设置对照组和橙皮苷低、高剂量（0.5、1.5 mmol/L）组，进行肿瘤成球实验，采用Western blotting和免疫荧光技术检测干性相关蛋白表达，采用qRT-PCR检测干性相关基因表达。HepG2细胞培养1周后，测定细胞对索拉非尼的半数抑制浓度（half inhibitory concentration，IC₅₀）曲线，观察细胞增殖与凋亡情况。构建稳定过表达β-catenin的HepG2细胞系，设置过表达β-catenin组和过表达β-catenin＋橙皮苷（1.5 mmol/L）组，进行肿瘤成球实验，并检测干性相关蛋白表达；测定细胞对索拉非尼的IC₅₀曲线，观察细胞增殖与凋亡情况。裸鼠注射HepG2细胞后，给予橙皮苷或索拉非尼治疗，定期观察并记录瘤体大小，采用免疫组化与Western blotting检测肿瘤组织Ki67、β-catenin、半胱氨酸天冬氨酸蛋白酶-3（cystein-asparate protease-3，Caspase-3）、CD44、CD133蛋白表达。结果 橙皮苷能有效抑制肿瘤细胞成球能力（P＜0.001），减少细胞球体的体积和数量（P＜0.001），并显著降低干性相关蛋白表达（P＜0.001）。此外，橙皮苷可增强肿瘤细胞对索拉非尼的敏感性，影响β-catenin的定位。体内实验结果显示，橙皮苷具有抑瘤作用，与索拉非尼联用时效果更佳。结论 橙皮苷通过靶向β-catenin，抑制肝癌细胞干性，并显著增强了索拉非尼对肝癌细胞的敏感性。

Objective To explore the inhibitory effect of hesperidin on stemness of hepatoma cells by targeting β-catenin, and evaluate its enhancing effect on sorafenib sensitivity. Methods HepG2 cells in logarithmic growth phase were selected, and control group, hesperidin low-, high-dose (0.5, 1.5 mmol/L) groups were set up for tumor spheroidization experiments, Western blotting and immunofluorescence techniques were used to detect the expressions of stemness related proteins, and qRT PCR was used to detect the expressions of stemness related genes. After one week of HepG2 cells culture, the half inhibitory concentration (IC₅₀) curve of cells against sorafenib was measured to observe cell proliferation and apoptosis. A stable HepG2 cell line overexpressing β-catenin was constructed, overexpressing β-catenin group and overexpressing β-catenin + hesperidin (1.5 mmol/L) group were set up for tumor spheroidization experiments, and the expressions of stemness related proteins were detected; The IC₅₀ curve of cells against sorafenib was measured to observe cell proliferation and apoptosis. After injecting HepG2 cells into nude mice, hesperidin or sorafenib was administered, tumor size was regularly observed and recorded. Immunohistochemistry and Western blotting were used to detect the expressions of Ki67, β-catenin, cysteine aspartate protease-3 (Caspase-3), CD44 and CD133 proteins in tumor tissue. Results Hesperidin could effectively inhibit the spheroidization ability of tumor cells (P < 0.001), reduce the volume and quantity of cell spheroids (P < 0.001), and significantly decrease the expressions of stemness related proteins (P < 0.001). In addition, hesperidin could enhance the sensitivity of tumor cells to sorafenib and affect the localization of β-catenin. The in vivo experimental results showed that hesperidin had anti-tumor effects, and the combination with sorafenib had a better effect. Conclusion Hesperidin inhibits the stemness of hepatoma cells by targeting β-catenin, and significantly enhances the sensitivity of sorafenib to hepatoma cells.

R285.5

山东省中医药科技面上项目（M-2023215）