Objective： To study the angiogenesis modulation mechanism of Xuefu Zhuyu Decoction （血府逐瘀汤） on the endothelial cell line ECV304. Methods： ECV304 cells were treated with 2.5% Xuefu Zhuyu Decoction- containing serum （XFZYD-CS） for 24 h, 48 h or 72 h. Thiazolyl blue tetrazolium bromide （MTT）, fluorescence activating cell sorter （FACS）, migration, adhesion and in vitro tube formation assays were conducted to confirm an angiogenesis effect of XFZYD at 3 time points. An analysis of angiogenesis regulator profiles was performed at 3 times with real-time polymerase chain reaction （RT-PCR） superarray. Results： At 48 h, XFZYD-CS induced ECV304 significantly improved cell viability, number in S phase, migration, adhesion and tube formation. At 24 h and 72 h, only cell migration was elevated. Microarray results showed that the expression of 27 angiogenesis-related genes was changed. Conclusion： XFZYD-CS treatment induced angiogenesis on ECV304 cells with significant cellcular changes occurring at 48 h and genetic changes as early as 24 h.
Objective:To investigate the effect and mechanism of Xuefu Zhuyu Capsule(血府逐瘀胶囊,XZC)on pro-angiogenesis in the hindlimb ischemic model rats.Methods:A total of 100 Sprague Dawley rats were randomly divided into a model group,a regular-dose XZC group(0.48 g·kg^-1·d^-1)and a high-dose XZC group(0.96 g·kg^-1·d^-1)using random number table method.The model of hindlimb ischemic rats were made through femoral artery embolization with Bletilla microsphere age nt.XZC were give n on the first day after embolization surgery and lasted 5 days.Finally 72 models were obtained with 12 in each group for each time point.The lower Ischemic limb was amputated on the third day after embolization surgery.Histopathological characters and the number of blood vessels of granulation tissues were observed at 36 and 48 h after amputation,respectively.The main genes were obtained from microarray analysis and were validated using real-time quarttitative polymerase chain reaction.Results:The vascular number of granulation tissues at both 36 and 48 h were characterized by new and fresh vessels.The number of angiogenesis in the high-dose XZC group at 36 and 48 h was greater compared with that in the regular-dose XZC and model groups(P<0.01),and high-dose XZC at 36 h increased more vessels than that at 48 h(P<0.01).Consequently,granulation tissues from the high-dose XZC group at 36 h were chosen for microarray analysis.In all,2,085 differentially expressed genes(DEGs)were detected and 25 DEGs were determined to be directly related to an giogenesis.Four biological process terms were found including an giogenesis,regulati on of an gioge nesis,positive regulati on of an giogenesis,and positive regulation of vascular end othelial growth factor receptor sign aling pathway(P<0.05).Microarray an alysis also showed 49 pathways including 11 pathways related to an giogenesis.Conclusion:XZC promoted angiogenesis moderately and the mechanism invoIved multiple DEGs and multiple pathways.
Objective： To compare the angiogenesis behaviors of vascular endothelial growth factor（VEGF） and Chinese medicine Xuefu Zhuyu Decoction（血府逐瘀汤,XZD） treatments.Methods： Human microvascular endothelial cells（HMEC-1） were treated with various concentrations of either XZD-containing serum（XZD-CS） or VEGF for 24,48,and 72 h,respectively.Cell viability,proliferation,migration,adhesion,and in vitro tube formation assays were used to assess their angiogenic effects.Results： VEGF promoted all cellular phases involved in angiogenesis including cell viability,proliferation,migration,adhesion,and tube formation（P〈0.05 or P〈0.01）.Unlike the continuous promotion effects of VEGF at the above stages,XZD inhibited cell viability and proliferation（P〈0.05 or P〈0.01） and only promoted tube formation in the early phase of angiogenesis（P〈0.01）.Conclusions： These two medications promote different angiogenesis behaviors,which might be an important reason for their distinct therapeutic profile in clinical usage.
LIN FanCHEN Bin-lingWANG Yi-zhengGAO DongSONG JunT.J.KaptchukCHEN Ke-ji
Objective： To evaluate the effect of Xuefu Zhuyu Capsule（血府逐瘀胶囊）-containing serum（XFZY-CS） on Eph B4/ephrin B2 and its reverse signal in human microvascular endothelial cell-1（HMEC-1）. Methods： 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 Eph B4/ephrin B2 and the reverse signal were determined by Western blot and real-time quantitative polymerase chain reaction, respectively; we also confirmed the results through activating and inhibiting the reverse signal by Eph B4/fc and pyrophosphatase/phosphodiesterase2（PP2）. Results： 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 Eph B4-m RNA at 12 h（P〈0.05）, and down-regulates its expression at 24 h（P〈0.01）. Protein expression of Eph B4 was apparently up-regulated at 12 h and down-regulated at 24 h. The phosphorylation of ephrin B2 increased at 9 h（P〈0.05）. In addition, 2.5% XFZY-CS played a similar role as the reverse signaling activator Eph B4/Fc ranging from 0.5 to 5 μg/m L（P〉0.05）. XFZY-CS also reduced the inhibitive effect of PP2 in limited periods. Conclusion： Eph B4/ephrin B2 was the upstream signal in the process of angiogenesis and its reverse signaling was responsible for XFZY＇s effect on promoting angiogenesis.
