Zhang Jing-Si, Wang Yi-Zheng, Hu Ya-Qiong, Lin Fan, Gao Dong, Song Jun, Kaptchuk Ted J, Chen Ke-Ji
Zhongshan Hospital, Fudan University, Shanghai, 200032, China.
College of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, 350108, China.
Chin J Integr Med. 2016 Aug;22(8):605-10. doi: 10.1007/s11655-016-2470-9. Epub 2016 Jun 14.
To evaluate the effect of Xuefu Zhuyu Capsule ()-containing serum (XFZY-CS) on EphB4/ephrinB2 and its reverse signal in human microvascular endothelial cell-1 (HMEC-1).
XFZY-CS and the blank control serum were collected. HMEC-1 cells were randomly assigned to 6 groups including the concentration 1.25%, 2.5%, and 5% XFZY-CS groups and their blank serum control ones. The angiogenesis effect of XFZY-CS was tested with an in vitro tube formation assay and the best condition of pro-angiogenesis was determined. The effect of XFZY-CS on EphB4/ephrinB2 and the reverse signal were determined by Western blot and real-time quantitative polymerase chain reaction, respectively; we also confifirmed the results through activating and inhibiting the reverse signal by EphB4/fc and pyrophosphatase/ phosphodiesterase2 (PP2).
XFZY-CS promoted angiogenesis at the concentration of 2.5% corresponding serum after being cultured for 48 h, while inhibited angiogenesis at the concentration of 5% after culturing for 48 and 72 h. Under the 2.5% serum concentration, XFZY up-regulated the expression of EphB4-mRNA at 12 h (P<0.05), and down-regulates its expression at 24 h (P<0.01). Protein expression of EphB4 was apparently up-regulated at 12 h and down-regulated at 24 h. The phosphorylation of ephrinB2 increased at 9 h (P<0.05). In addition, 2.5% XFZY-CS played a similar role as the reverse signaling activator EphB4/Fc ranging from 0.5 to 5 μg/mL (P>0.05). XFZY-CS also reduced the inhibitive effect of PP2 in limited periods.
EphB4/ephrinB2 was the upstream signal in the process of angiogenesis and its reverse signaling was responsible for XFZY's effect on promoting angiogenesis.
评价血府逐瘀胶囊含药血清（XFZY-CS）对人微血管内皮细胞-1（HMEC-1）中EphB4/ephrinB2及其反向信号的影响。
收集XFZY-CS和空白对照血清。将HMEC-1细胞随机分为6组，包括1.25%、2.5%和5%浓度的XFZY-CS组及其空白血清对照组。采用体外管腔形成试验检测XFZY-CS的血管生成作用，并确定促血管生成的最佳条件。分别通过蛋白质印迹法和实时定量聚合酶链反应检测XFZY-CS对EphB4/ephrinB2及其反向信号的影响；我们还通过EphB4/fc和焦磷酸酶/磷酸二酯酶2（PP2）激活和抑制反向信号来证实结果。
培养48 h后，2.5%浓度的XFZY-CS相应血清促进血管生成，而培养48 h和72 h后，5%浓度的XFZY-CS抑制血管生成。在2.5%血清浓度下，XFZY在12 h上调EphB4-mRNA的表达（P<0.05），在24 h下调其表达（P<0.01）。EphB4的蛋白表达在12 h明显上调，在24 h下调。ephrinB2的磷酸化在9 h增加（P<0.05）。此外，2.5%的XFZY-CS在0.5至5μg/mL范围内与反向信号激活剂EphB4/Fc发挥相似作用（P>0.05）。XFZY-CS在有限时间内也降低了PP2的抑制作用。
EphB4/ephrinB2是血管生成过程中的上游信号，其反向信号传导是XFZY促进血管生成作用的原因。
