目的 基于 VEGFA/VEGFR2-PI3K/AKT 信号轴探讨化瘀明目方含药血清抑制高糖及血管内皮生长因子 （VEGF）诱导人视网膜微血管内皮细胞（HRMECs）血管新生的分子机制。方法 制备化瘀明目方含药血清；采 用CCK-8法筛选VEGF诱导HRMECs的最佳浓度及干预时间。分别复制高糖、VEGF诱导的HRMECs功能紊乱 模型，分组及干预：①空白组：ECM 完全培养基+10% 空白血清；②高糖模型组：含糖 25 mmol·L-1的 ECM 完 全培养基+10% 空白血清；③高糖干预组：含糖 25 mmol·L-1的 ECM 完全培养基+10% 含药血清；④VEGF模型 组：ECM完全培养基+10 ng·mL-1 VEGF+10%空白血清；⑤VEGF干预组：ECM完全培养基+10 ng·mL-1 VEGF+ 10%含药血清。采用划痕实验检测细胞迁移能力；管腔形成实验检测细胞成管能力；流式细胞术检测细胞凋亡 情况；免疫细胞化学法、Western Blot 法及 RT-PCR 法检测细胞血管内皮生长因子 A（VEGFA）、血管内皮生长 因子受体2（VEGFR2）、磷脂酰肌醇3-激酶（PI3K）、蛋白激酶B（AKT）、胱天蛋白酶9（Caspase-9）、Bcl-2相关 死亡促进因子（Bad）蛋白及mRNA表达水平。结果 采用10 ng·mL-1 VEGF干预24 h为条件复制HRMECs功能紊 乱模型。与空白组比较，高糖模型组与 VEGF 模型组 HRMECs 细胞迁移距离、血管形成数均显著增加（P < 0.01），VEGFA、VEGFR2、PI3K、AKT蛋白及 mRNA表达水平均显著升高（P < 0.01），Caspase-9、Bad蛋白及 mRNA 表达水平均无明显变化（P > 0.05）。与高糖模型组和 VEGF 模型组比较，化瘀明目方含药血清干预后， HRMECs细胞迁移距离显著缩短（P < 0.01），血管形成数显著减少（P < 0.05，P < 0.01），细胞凋亡率显著上升 （P < 0.05，P < 0.01），VEGFA、VEGFR2、PI3K、AKT 蛋白及 mRNA 表达水平显著降低（P < 0.05，P < 0.01）， Caspase-9、Bad蛋白及mRNA表达水平显著升高（P < 0.05，P < 0.01）。结论 化瘀明目方可能通过抑制VEGFA/ VEGFR2-PI3K/AKT信号轴，上调促凋亡基因Caspase-9、Bad表达，发挥抗高糖及VEGF诱导的促视网膜血管 新生效应。

Objective To explore the molecular mechanism of Huayu Mingmu Recipe-containing serum on the inhibition of high glucose and VEGF-induced angiogenesis of human retinal microvascilar endothelial cells（HRMECs） based on VEGFA/ VEGFR2-PI3K/AKT signal axis. Methods Huayu Mingmu Recipe-containing serum was prepared. CCK-8 method was used to screen the optimal concentration and intervention time of VEGF-induced HRMECs. The HRMECs dysfunction model was replicated by the induction of high glucose and VEGF. The grouping and intervention were as following：①a blank group：ECM complete culture medium+10% blank serum;②a high glucose model group： ECM complete culture medium containing 25 mmol·L-1 sugar+10% blank serum；③a high glucose intervention group： ECM complete culture medium containing 25 mmol·L-1 sugar+10% drug-containing serum；④a VEGF model group： ECM complete culture medium+10 ng·mL-1 VEGF+10% blank serum；⑤a VEGF intervention group：ECM complete culture medium+10 ng·mL-1 VEGF+10% drug-containing serum. The scratch experiment was carried out to detect cell migration ability. Cell lumen formation was investigated through luminal formation assay. Cell apoptosis rate was detected by flow cytometry. Immunocytochemistry， Western Blot， and RT-PCR were used to detect the protein and mRNA expression levels of vascular endothelial growth factor A（VEGFA）， vascular endothelial growth factor receptor 2 （VEGFR2），phosphatidylinositol 3-kinase（PI3K），protein kinase B（AKT），Caspase-9，Bcl-2 associated death promoter（Bad）in cells. Results The HRMECs dysfunction model was replicated by the intervention of 10 ng · mL-1 VEGF for 24 h. Compared with the blank group，the high glucose model group and the VEGF model group showed a significant increase in cell migration distance and lumen formation number （P < 0.01）. The protein and mRNA expression levels of VEGFA， VEGFR2， PI3K， AKT were significantly increased（P < 0.01）， but no significant change was observed in the protein and mRNA expression levels of Caspase-9 and Bad （P > 0.05）. After the intervention of Huayu Mingmu Recipe-containing serum， HRMECs migration distance decreased（P < 0.01）， the number of lumen formation decreased（P < 0.01，P < 0.05），the apoptosis rate of cells obviously increased（P < 0.01， P < 0.05），the protein and mRNA expression levels of VEGFA，VEGFR2，PI3K，and AKT were remarkably reduced （P < 0.05， P < 0.01）， while the protein and mRNA expression levels of Caspase-9 and Bad were significantly increased （P < 0.05， P < 0.01） as compared with the high glucose model group and the VEGF model group. Conclusion Huayu Mingmu Recipe exerted inhibitory effect on retinal angiogenesis induced by high glucose and VEGF， which may be related to the inhibition of VEGFA/VEGFR2-PI3K/AKT signal axis and up-regulation of the expression of Caspase-9 and Bad.

R285.5

国家自然科学基金项目（82104714）；国家中医药管理局中医药循证能力建设项目（2019XZZX-YK008）