目的 探讨穿破石总黄酮（total flavonoids from Maclura cochinchinensis，TFMC）对血管瘤内皮细胞（hemangioma endothelial cells，HemECs）增殖、迁移及血管生成的影响及作用机制。方法 设置对照组、TFMC（40 μg/mL）组、磷脂酰肌醇3-激酶（phosphatidylinositol 3-kinase，PI3K）抑制剂LY294002（20 μmol/L）组、缺氧诱导因子-1α（hypoxia inducible factor-1α，HIF-1α）抑制剂LW6（30 μmol/L）组、TFMC＋LY294002组、TFMC＋LW6组、PI3K激动剂740Y-P（15 μmol/L）组、HIF-1α激动剂DMOG（0.5 μmol/L）、TFMC＋740Y-P组和TFMC＋DMOG组。HemECs给予药物干预24 h后，收集上清液和细胞。采用MTT法检测细胞增殖能力；克隆形成实验检测克隆形成能力；划痕和Transwell实验检测细胞迁移和侵袭；AnnexinV-FITC/PI双染检测细胞凋亡；PI染色检测细胞周期；免疫荧光法检测细胞线粒体膜电位和细胞中活性氧（reactive oxygen species，ROS）水平；比色法和ELISA法检测细胞上清液中乳酸脱氢酶（lactate dehydrogenase，LDH）和细胞中血管内皮生长因子（vascular endothelial growth factor，VEGF）、血管内皮生长因子受体2（vascular endothelial growth factor receptor 2，VEGFR2）、纤连蛋白（fibronectin，FN）、基质金属蛋白酶-2（matrix metalloproteinase-2，MMP-2）及MMP-9含量；血管生成实验检测细胞成管能力；qRT-PCR和Western blotting测定迁移、侵袭、VEGF/VEGFR2和PI3K/蛋白激酶B（protein kinase B，Akt）/HIF-1α信号通路相关基因和蛋白表达。结果 与对照组比较，TFMC、LY294002和LW6可显著抑制HemECs增殖、迁移、侵袭和成管能力，促进细胞凋亡、LDH释放和G₀/G₁期阻滞，升高细胞内ROS水平，降低线粒体膜电位和细胞中VEGF、VEGFR2、FN、MMP-2及MMP-9含量，下调迁移、侵袭、VEGF/VEGFR2和PI3K/Akt/HIF-1α信号通路相关基因和蛋白表达（P＜0.01）。与TFMC组比较，TFMC与LY294002或LW6联用可进一步加重这种改变（P＜0.01）。相反，740Y-P和DMOG则可以恶化上述变化；TFMC与740Y-P或DMOG联用可逆转这种改变（P＜0.01）。结论 TFMC可抑制HemECs增殖、迁移、侵袭及血管生成，其作用机制与抑制VEGF/VEGFR2和PI3K/Akt/HIF-1α通路激活密切相关。

Objective To investigate the effect and mechanism of total flavonoids from Maclura cochinchinensis (TFMC) on proliferation, migration and angiogenesis of hemangioma endothelial cells (HemECs). Methods Control group, TFMC (40 μg/mL) group, phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (20 μmol/L) group, hypoxia inducible factor-1α (HIF-1α) inhibitor LW6 (30 μmol/L) group, TFMC + LY294002 group, TFMC + LW6 group, PI3K agonist 740Y-P (15 μmol/L) group, HIF-1α agonist DMOG (0.5 μmol/L), TFMC + 740Y-P group and TFMC + DMOG group were set up. After 24 h of drug intervention with HemECs, the supernatant and cells were collected. MTT assay was used to test cell viability. Clony formation assay was utilized to detect the colony formation ability. Cell scratch and Transwell was used to test cell migration and invasion. AnnexinV FITC/PI double staining was utilized to detect cell apoptosis. PI staining was utilized to test cell cycle. Immunofluorescences were used to detect mitochondrial membrane potential and reactive oxygen species (ROS) level in HemECs. Colorimetric and ELISA methods were utilized to test the level of lactate dehydrogenase (LDH) in supernatant and levels of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR2), fibronectin (FN), matrix metalloproteinase-2 (MMP-2) and MMP-9 in cells. Angiogenesis assays was used to detect the anti-angiogenic ability of cells. qRT-PCR and Western blotting were used to measure the expressions of migration, invasion, VEGF/VEGFR2 and PI3K/protein kinase B (Akt)/HIF-1α signaling pathways related genes and proteins. Results Compared with control group, TFMC, LY294002 and LW6 significantly inhibited the proliferation, migration, invasion and angiogenesis of HemECs, promoted cell apoptosis, LDH release and G₀/G₁ phase arrest, elevated intracellular ROS level, reduced mitochondrial membrane potential and VEGF, VEGFR2, FN, MMP-2 and MMP-9 contents in HemECs, down-regulated the expressions of migration, invasion, VEGF/VEGFR2 and PI3K/Akt/HIF-1α signaling pathway related genes and proteins (P < 0.01). Compared with TFMC group, the combination of TFMC with LY294002 or LW6 could further exacerbate this change (P < 0.01). On the contrary, 740Y-P and DMOG could worsen the above changes; The combination of TFMC with 740Y-P or DMOG could reverse this change (P < 0.01). Conclusion TFMC could inhibit the proliferation, migration, invasion and angiogenesis of HemECs, and its mechanism is closely related to the inhibition of VEGF/VEGFR2 and PI3K/Akt/HIF-1α pathway activation.

R285.5

宜昌市医疗卫生研究项目（A22-2-059，A23-1-066）；湖北省科技厅重点研发项目（2025BCB067）；湖北省科技厅重点研发大健康计划项目（2022BCE017）；湖北省卫生健康委员会中医药重点项目（ZY2023Z015）；湖北省卫生健康委员会中医药面上项目（ZY2025M038）；湖北省科技厅自然科学基金项目（2025AFB805，2025AFB839，2025AFD301，2025AFD294，2023AFB600，2022CFB357，2022CFB427）；湖北省卫生健康委员会科研资助项目（WJ2023M153）；湖北省功能性消化系统疾病中医临床医学研究中心开放基金（SXZ202303，SXZ202308，SXZ202311）