The Study Of Xufuzhuyu Decoctions Promoting Angiogenesis Under Hypoxic Condition Through EphB4/EphrinB2 Signaling Pathway

Objective:In the present study, the Human Microvascular Endothelial Cell line (HMEC-1) will be chosen as the object to observe the effect of different concentration s(1.25%?2.5%?5%) of the XuefuZhuyu Decoction containing serum (XFZY-CS) on angiogenesis under hypoxic condition and the underlying mechanisms, and to provide experimental evidence for the clinical application.Methods:1. Preparation of XuefuZhuyu-containing (XFZY-CS) and non-containing (blank) serum of rats.60 SPF SD male rats, weighting from 220 to 240 g, were randomly divided into the XFZY-CS group and the blank control group, with 30 in each group. The XFZY-CS group were given gavage in a dosage of 0.41 g/kg (12 time as human oral dosage) XueFuZhuYu capsules in aqueoussolution. The control group were given a gavage of saline at the same volume. On the 8th day, rats were intraperitoneal injected and anesthetized with sodium pentobarbital 2 hours after gavage, and the abdominal aortic blood were collected, sterilizated subpackaged, and stored in -80? for further use.2. Cell Culture.11.6 g of MCDB-131 powder medium and 1.18 g of sodium bicarbonate were dissolved completely in ultrapure water, then diluted to 1000 ml, and adjusted the pH value from 7.2 to 7.2, then keep under 4? overnight, and used microporous membrane filter for sterilization, subpackaged for further use. The MCDB-131 medium was added with EGF,hydrocortisone and 10% fetal bovine serum to prepare the complete medium.3. Tube formation in vitro angiogenesis assay. Cells treated with same serum levels of the XFZY-CS group and the blank group were incubated under hypoxia for 24h,48h and 72h, then harvested and seeded into ECMatix gel coated 96-well plate and incubated for 5 h. The series of tube-like structures were photographed randomly using a phase-contrast microscope at a magnification of 200×. The number of the formed tubes were calculated and compared between the 2 groups.4.Quantitative Real-time PCR to detect EphB4 and EphrinB2 gene expression. Used trizol reagent to extracte total RNA, reverse transcribed into cDNA. According to gene sequence released by Genbank, the software designed the primers of target genes and ?-actin. Then detected the gene expression in different time points, with 2-??CT quantitative method to evaluate relative.5.To detect the expression of EphB4/EphrinB2 and phosphorylation EphrinB2 by western blot. Extracted the total protein and detected the contents of protein to determine the sample amount by a bicinchoninic acid (BCA) assay kit. Samples were separated by electrophoresis in SDS-PAGE gels and blotted onto 0.45?m polyvinylidene difluoride (PVDF) membranes. The membranes were treated with 5% non-fat milk or BSA in room temperature depending on weather they were used for phosphor-antibody or not. The primary antibodis were incubated at 4? overnight and the secondary antibodies were incubated at room temperature.Result:1.In vitro angiogenesis assay, compared with the same concentration of serum to the blank group,2.5% and 5% of XFZY-CS promoted tube formation after the intervention of XFZY-CS for 24h, only 5% of medicated serum group played a role in the formation of the tube after treatment for 48h. Three concentration of XFZY-CS showed no statistical differences in promoting angiogenesis compared with the blank group in 72h.2.Results of real-time PCR showed that under the 2.5% serum concentration, XFZYD up-regulates the expression of EphrinB2-mRNA in 6h and 12h (P<0.01)and down-regulates the expressionEphB4-mRNA in 6h. Continue being intervened to 24h and 48h, the gene expression of the drug group compared with the same concentration of the blank group, no difference.3.Western Blot analysis of EphrinB2 showed that protein expression appeared in 9h and 24h with the treatment of 2.5% serum concentration(P<0.01). XFZYD induced changes of EphrinB2 phosphorylation in24 h, which can not be tested in other time period. Analysis of EphB4 showed that protein expression with the treatment of 2.5% serum concentration below to the blank serum group in 12h (P<0.05).Conclusion:XFZYD has significant effect on promoting angiogenesis under the condition of hypoxia, which was related to the EphB4/EphrinB2 reverse signaling pathway.