Angiogenesis In The Hypoxia Affected By Xuefu Zhuyu Decoction

ObjectiveTo observe the effect of Xuefu Zhuyu containing serum(XFZY-CS) on angigogenesis under both hypoxic and non-hypoxic conditions in human microvascular endothelial cell-1(HMEC-1).We applied the methods of MTT, cell proliferation, migration and adhesion as well as tube formation to evaluated the relevant mechanism with different concentrations(1.25%、2.5%、5%) of XFZY-CS in three different time points(24h、48h、72h), in order to provide convincing evidence for the clinical application of Xuefu Zhuyu Decoction.Methods1 Preparation of XFZY-CSMale Sprague-Dawley rats weighting 220±lOg were randomly distributed into the XFZY-CS group and the control group. We dissolved the Xuefu Zhuyu capsules (XFZYC) with saline and adjusted the concentration to 0.41g/Kg, almost 12 times of the conventional dose of human. After administrating with XFZYC or saline via gastrogavage,2 times/day for 7 days, we anesthetized the rats with pentobarbital sodium and collected the serum from abdominal aorta. Both the XFZY-CS and the blank control serum were centrifuged at 3000rpm for 30min, inactivated at 56℃ for 30 min and then stored at-20℃ after filtering through 0.22um filters.2 Groups divisionWe then randomly divided the HMEC-1 into hypoxic and non-hypoxic groups. Each group was then divided into 1.25%、2.5% and 5% XFZY-CS groups and their blank serum control ones. The effect of XFZY-CS was detected by MTT assay, Fluorescence Activating Cell Sorter (FACS), cell adhesion, Transwell chamber and in vitro tube formation assays respectively.Results1. Cell activity was reduced in hypoxia, but 1.25% XFZY-CS improved cell activity after cultured for 24h and 48h in hypoxia. No difference was observed under the non-hypoxic condition.2. The proportion of cells in the S phase was improved after intervened for 72h in hypoxia, and no cell proliferation was observed in both hypoxic and non-hypoxic conditions.3. We observed that cell migration increased significantly in 1.25% and 2.5% control groups after cultured for 24h,48h and 72h; while 2.5% XFZY-CS inhibited cell migration at 48h but prompted it at 72h, the 5% XFZY-CS accelerated migration at 48h in hypoxia. Under the normal condition,2.5% XFZY-CS inhibited cell migration at 72h and 5% XFZY-CS played the inhibiting role in all the time points.4. In the hypoxic groups, all blank control groups inhibited cell adhesion at 48h and the effect was continued to 72h in the 1.25% concentration.2.5% XFZY-CS accelerated cell adhesion at 48h and 5% XFZY-CS depressed the process after intervening for 48h and 72h. Bi-directional regulation effect could be observed in the non-hypoxic, the same as in the hypoxic condition.1.25% XFZY-CS inhibited cell adhesion at 72h, while 2.5% and 5% XFZY-CS both up-regulated at 72h, and down-regulated adhesion in different time phase.5. In vitro angiogenesis assay, treatment of XFZY-CS in 1.25% concentration for 24h and 2.5% serum concentration for 48h showed evident effect in promoting angiogenesis in hypoxia, while 2.5% and 5% serum concentration inhibited angiogenesis at 72h or 48h respectively. Interesting enough,2.5% XFZY-CS also promoted angiogenesis at 48h and the suppressive effect of 5% XFZY-CS delayed to 72h in the non-hypoxic.Conclusion1. XFZY-CS significantly promotes angiogenesis under the hypoxic condition.2. The bi-directional regulation effect of XFZY-CS in angiogenesis could be observed in both hypoxic and non-hypoxic.3. Compared with the non-hypoxic groups, XFZY-CS groups in hypoxic condition promote vessel formation earlier and last for a longer time with even lower serum concentration, which may indicate that XFZY-CS has more advantages in hypoxia.4. Our experimental results are quite different in hypoxia and effect of XFZY-CS in hypoxia and it may spur us to further exploration about the possible mechanisms.