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 VEGF 诱导人视网膜微血管内皮细胞血管新生的分子机制.

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. [ABSTRACT FROM AUTHOR]