Influence Of Hydroxysafflor Yellow A On The Proliferation And Expression Of Thrombospondin-l In Human Aortic Endothelial Cells

Background:Modern research shows that the growth of tumor needs blood vessels to provide nutriment and exclude metabolites. Therefore, how to prevent tumor angiogenesis has become the new hot spot for cancer treatment. Literature reports that a certain concentration of hydroxysafflor yellow A (HSYA)has the functions like anti-platelet aggregation, inhibition of thrombosis, reducing ischemic injury of heart and brain,anti-tumor effect and so on。Its anti-tumor effect may be related to inhibiting the proliferation of endothelial cells.Thrombospondin 1 (TSP-1) and vascular endothelial growth factor (VEGF) are two important factors to regulate angiogenesis. VEGF is a specific factor in the vascular endothelial growth.It can induce proliferation of endothelial cell, inhibit apoptosis, promote angiogenesis in vivo, and plays a central role in the process of angiogenesis. TSP-1 can inhibit the proliferation, migration and tube formation of vascular endothelial cells,and interference with the effect of vascular endothelial growth factor to induce endothelial cells proliferating ,however,it lead to apoptosis of endothelial cells and inhibit angiogenesis. It is extensively studied in recent years, and is considered the most important type of protein to inhibit angiogenesis. Literature reports that the HSYA shows a two-way effect on the proliferation of endothelial cells: different conditions can promote or inhibit the proliferation of endothelial cells.It is proved by experiment that a certain concentration of HSYA acting on ischemic brain tissue can enhanced the expression of VEGF and promote angiogenesis to reduce cerebral ischemia.However,whether the inhibition of proliferation of endothelial cells is related to the change of STP-1 expression is not yet reported at home and abroad.The aim of this paper is to study the influence of STP-1 expression after the acting of HSYA and to further probe into its mechanism of inhibition of endothelial cell proliferation and antitumor effect.Methods:1.Cell experimentCells at exponential phase were digested off and seeded at a density of 5×104cells/well into 96-well plate, Culture media contained HSYA with the concentration of 0.591 3 g/L,0.197 1 g/L,0.065 7 g/L,0.021 9 g/L,0.007 3 g/L, 0.002 4 g/L acted on the treatment group, establishing the blank and the control groups. We used the method of MTT to observe the proliferation of HAEC with the influence of HSYA.RT-PCR and immunocytochemistry were used to detect the level of mRNA and protein of TSP-1 in 48 hours after the treatment of HSYA.We established the blank and the control groups in the experiment. The experimental procedures was conducted by RT-PCR kits and immunohistochemical staining kit instructions. The content of TSP-1 in the culture medium was determined by ELISA in 48 hours after the treatment of HSYA,and the experimental procedures was conducted by manufacturer's instructions.2.Statistical analysisSPSS13.0 was used to analyze the data. All data was analyze by F test,and tukey's test was used to compare the data of two groups. Statistical significance was assumed for P < 0.05.3. Results:3.1 MTT test resultsExperimental results showed 0.065 7 g / L HSYA had no obvious effect on the proliferation of endothelial cells,and there was no significant difference compared with the control group.(P<0.05). Lower concentration (about<0.0657g/L) of HYSA could promote the proliferation of endothelial cells,and 0.0073g/L HSYA had the the strongest promoting effect(P <0.05). Higher concentrations (> 0.065 7 g / L)of HSYA inhibited the proliferation of endothelial cells significantly (P <0.01). Therefore, we selected 0.591 3 g / L, 0.065 7 g / L, 0.007 3 g/L three concentrations to the next step of experiment. 3.2 RT-PCR test resultsThe expression of normal HAEC of TSP-1 mRNA was positive. The relative expression of TSP-1 mRNA of 0.007 3 g / L HYSA acting group was higher compared with the normal control group, but there was no significant difference between the them(P<0.05). The relative expression of TSP-1 mRNA of 0.065 7 g / L HSYA acting group was higher than the previous two groups,and there was significant difference(P<0.05). The 0.591 3 g / L HSYA acting group had a higher expression level of TSP-1 mRNA than the other groups, but there was no significant difference compared with the 0.065 7 g / L HSYA action group(P<0.05). We could see HSYA promoted the expression of TSP-1mRNA and the effect was enhanced as the concentration increased.3.3 ELISA test resultsELISA assay showed the content of TSP-1 in the supernatant of 0.007 3 g / L HSYA acting group was higher compared with the control group, but there was no statistical significance;The 0.591 3 g / L and 0.065 7 g / L HSYA acting groups had higher content of TSP-1 in the supernatant than the control group, and there was significant difference(P<0.05)between each of them. It proved HSYA could promote the release of TSP-1 that the TSP-1 levels was promote as the HSYA increased.3.4 Immunocytochemistry test resultsThere were several yellow granules in the sarcoplasma of the blank control group,and it was weakly positive.The 0.007 3 g / L HSYA acting group also showed several yellow granules in the sarcoplasma,which was weakly positive too. There were more yellow granules in the sarcoplasma of the 0.065 7 g/L HSYA acting group,and it showed positive. The 0.591 3g / L HSYA acting group showed a lot of brown granules in the sarcoplasma,and it was strongly positive. The negative control cells with PBS instead of primary antibody showed light violet blue in the sarcoplasma and there were no yellow granules,which were negative.. So the effect of HSYA on the proliferation of HAEC showed a concentration-dependence,for higher concentration had stronger catalytic effect.Conclusion:HSYA promoted the expression and secretion of TSP-1 in HAEC,and HSYA showed a two-way effect on the proliferation of HAEC. Lower concentration (about <0.065 7 g / L) of HSYA could promote the proliferation of endothelial cells,and 0.007 3 g / L HSYA had the the strongest promoting effect(P <0.05). Higher concentrations (> 0.065 7 g / L)of HSYA inhibited the proliferation of endothelial cells significantly,and the inhibition would be enhanced as the concentration increased. Promoting the expression of VEGF and TSP-1 and involved in acting on signal pathway of VEGF might cause the result. Lower concentration of HSYA could promote the proliferation of endothelial cells since VEGF and HSYA acting on signal pathway of VEGF played the major role probably. Higher concentrations of HSYA inhibited the proliferation of endothelial cells. It might be resulted from that the inhibition of TSP-1 up regulated was stronger than the promoting effect of VEGF and HSYA .