The Sutdy On Molecular Biological Mechanism Of Astragaloside Ⅳ Stimulates Angiogenesis

Purpose:This study aimed at determining the angiogenic effect of AST and its underlying mechanism.Methods:1.Cell Proliferation Assay in vitroViability of cells was assessed by Cell Counting Kit-8 (CCK-8) assay. In brief, EA-hy926 cells were seeded in a 96-well plates according to the density of 2×104 cells for each well. Premixed CCK-8 and medium (10μl) were added into 96-well plates, and cells were then incubated for 0.5-1h at 37℃. The values of A450 were obtained with the 3,550 automatic detector from Beckman.2. Tube Formation Assay in vitroA Matrigel tube-formation assay was also performed to assess in vitro angiogenesis. Growth factor-reduced Matrigel (BD) was placed in 96-well culture plates and allowed to set at 37℃ for 1 h. Then 1×104 EA-hy926 cells were added to each well and incubated in basic medium with 10mg/L AST,30mg/L AST, 100mg/L AST or DMSO,another group add 100mg/L AS-Ⅳ and PD98059 for 4h,8h,12h. Five fields were counted for each well. The length of the tube was measured by Image-Pro Plus 6.0.3. mechanism studyThe total RNA was isolated using Trizol-reagent according to manufacture’s instructions. Real-time PCR was used to measure the expression of VEGF. Real-time PCR was performed with the following PCR primers:GAPDH, forward 5’-CGGGAAACTGTGGCGTGAT-3’and reverse 5’-CAAAGGTGGAGGAGTGGGT-3; VEGF, forward 5’-ATGAACTTTCTGCTGTCTTG-3’and reverse 5’-TGCATGGTGATGTTGGAC- 3. SYBR-Green Universal Master Mix kit (ABI) were empoyed to detect the levels of these genes.Each well, with2×104 cells/well, was treated with AST 10mg/L,30 mg/L,100 mg/L respectively for the indicated duration at 5% CO2/95% room air and 37℃ for 24h. Protein of 20 μg/well was separated on 8-12% SDS-polyacrylamide gels and transferred onto nitro-cellulose membranes. After blocking in 1X TBST,5% nonfat dry milk at room temperature for 30 min, the membranes were incubated with the primary antibodies in blocking buffer (1X TBST,5% nonfat dry milk) overnight at 4℃. The membranes were washed three times for 5 min with 1X TBST and then incubated with secondary antibodies. After the membranes were washed three times for 5 min with 1X TBST, the signals were detected using ECL chemiluminescence reagents.Outcome:Our data revealed that AST promoted cell proliferation, and tube formation. Mechanism studies revealed that AST activated the extracellular signal regulated protein kinases 1/2(ERKl/2) pathway. All angiogenic effects of ASTwere reversed by ERK1/2 inhibitor (PD98059)Collectively AS-IV has a certain role in promoting angiogenesis in EA-hy926 cells, its mechanism of action involve in the activation of ERK1/2 cell signaling pathways.