Effects of dl-3-n-butylphthalide on angiogenesis of human umbilical vein endothelial cells under ischemia/hypoxia condition:in vitro experiment and its mechanism / 中华神经科杂志

ABSTRACT

Objective To investigate the effects of dl-3-n-butylphthalide ( NBP) on angiogenesis of human umbilical vein endothelial cells ( HUVEC) in vitro under ischemia/hypoxia and the correlation with vascular endothelial growth factor/vascular endothelial growth factor receptor 2 ( VEGF/VEGFR2 )-Notch1/Dll4 signaling pathway .Methods The control group , ischemia/hypoxia group , and ischemia/hypoxia +NBP group ( high dose group and low dose group ) were set up after HUVEC subculture . The cell concentration was adjusted to be 1 ×105/ml, and each group was randomly added 1 ml (1 ×105 cells in each group).Both of the ischemia/hypoxia group and the ischemia/hypoxia +NBP group were cultured under the condition of ischemia and hypoxia .The NBP concentrations of the high and low dose groups were 20 μmol/L and 5 μmol/L respectively.The cell viability of each group was detected by cell counting kit-8, and cell scratch test was used to detect the migration ability of the cells in each group .The cell formation ability of each group was examined by in vitro angiogenesis assay.Western blotting was used to detect the expressions of receptor proteins VEGFR2, Notch1 and Dll4,and mRNA expressions of VEGF, VEGFR2, Notch1 and Dll4 were detected by real-time quantitative PCR.Results NBP increased the survival rates of HUVEC under ischemia/hypoxia condition.In the low dose group, the survival rates of HUVEC at 6, 12, 24, 48 h were 78.6％ ±3.0％, 59.6％ ±5.3％, 44.6％ ±4.2％, 38.2％ ±4.3％, respectively, significantly higher than that in the ischemia/hypoxia group (75.2％±5.8％, 53.2％±4.8％, 36.2％± 7.8％, 22.5％±4.1％;t=4.513, 6.231, 9.322, 9.674; P=0.021, 0.018, 0.026, 0.015).In the high dose group, the survival rates of HUVEC at 6, 12, 24, 48 h were 88.6％±6.3％, 67.5％±5.4％, 53.3％±4.2％, 46.3％±3.9％, respectively , also significantly higher than that in the ischemia/hypoxia group (t=8.123, 11.211, 12.312, 14.154;P=0.001, 0.002, 0.001, 0.001).The mobility of the low dose group was 52.3％+4.2％, with statistically significant difference compared with that in the ischemia /hypoxia group ( 18.5％ ±3.2％) and the control group ( 22.3％ ±4.1％; t=18.324, 15.183; P=0.000, 0.000).The mobility of the high dose group was 87.5％±5.2％, also with statistically significant difference compared with that in the ischemia/hypoxia group and the control group ( t=22.142, 19.341;P=0.000, 0.000).NBP increased the protein and mRNA expression of VEGF , VEGFR2, Notch1 and Dll4.The relative expression of VEGFR2, Notch1 and Dll4 in the low dose group was 1.12 ±0.17, 0.35 ± 0.07 and 0.42 ±0.08, respectively, with statistically significant difference compared with that in the ischemia/hypoxia group (0.82 ±0.05, 0.30 ±0.03, 0.32 ±0.04;t=6.120, 2.123, 4.112;P=0.000, 0.020, 0.003).The relative expression of VEGFR2, Notch1 and Dll4 in the high dose group was 1.30 ± 0.15, 0.41 ±0.10 and 0.48 ±0.11, respectively, also with statistically significant difference compared with that in the ischemia/hypoxia group ( t=8.122, 3.851, 5.130; P=0.000, 0.000, 0.001 ) .The mRNA expressions of VEGF , VEGFR2, Notch1 and Dll4 in the low dose group were 0.43 ±0.08, 0.41 ± 0.05, 0.38 ±0.03 and 0.36 ±0.04, respectively, with statistically significant difference compared with that in the ischemia/hypoxia group (0.28 ±0.03, 0.34 ±0.04, 0.27 ±0.03, 0.19 ±0.04;t=3.122, 3.825, 4.311, 5.211; P=0.000, 0.006, 0.001, 0.000).And the mRNA expressions of VEGF, VEGFR2, Notch1 and Dll4 in the high dose group were 0.58 ±0.05, 0.50 ±0.06, 0.41 ±0.05, 0.52 ±0.06, respectively, also with statistically significant difference compared with that in the ischemia /hypoxia group (t=4.225, 4.872, 5.311, 8.220;P=0.000, 0.000, 0.000, 0.000).Conclusions NBP can promote HUVEC to form blood vessels under ischemia/hypoxia condition , the mechanism of which may be related to the activation of VEGF/VEGFR2-Notch1/Dll4 signaling pathway .It may be one of the mechanisms that NBP improves cerebral microcirculation in acute ischemic stroke .