Experimental Study Of Marsdenia Tenacissima Extract Inhibiting Angiogenesis

Objective Marsdeniae tenacissimae extraction(MTE)can inhibit tumor angiogenesis,but its mechanism is not clear.The present study aimed to evaluate the effect of MTE on Human Umbilical Vein Endothelial Cells(HUVEC)’s proliferation,migration,apoptosis,the impact of new angiogenesis and to explore its mechanism.Methods We cultured HUVEC,used MTS assay to detect the effect of proliferation,wound heating assay detect the effect of migration,Hoechst/PI and FCM detect the apoptosis and the cell arrest,ATP and glucose detection kit detect the cellular metabolite changes,qRT-PCR detect the express of relevant genes and Chick Chorioallantoic Membrane(CAM)assay detect the angiogenesis.Results The effects of MTE on the inhibition of the proliferation of HUVEC was significantly different,and the inhibition rate increased significantly with the increase of concentration(**P<0.01),showing dose-dependent relation.In vitro cell scratch test showed that the migration ability of MTE group was significantly lower than that of the control group,and further verified that MTE could inhibit the migration of HUVEC cells.Hoechst/PI fluorescence staining showed that when the MTE concentration reached 160 μg/mL,the nucleus was dyed bright blue Hoechst,and the PI was characterized by apoptosis morphology.Apoptosis detected by flow cytometry showed that MTE could promote the early apoptosis of HUVEC and block the cell cycle in S phase.24h after MTE treatment of.different concentrations,the ATP metabolism and glucose consumption of MTE treatment group showed a downward trend,these results indicated that MTE could decrease cell proliferation by inhibiting HUVEC metabolism.RT-PCR showed that MTE can increase Caspase-3,PICK1,ICA69,LC3-II,Beclin-1 and PTEN mRNA expression,down-regulate Bcl-2,VEGFR,P2Y6R,CCL-2,AKT,mTOR mRNA expression.These results indicated that MTE could affect the apoptosis and migration of HUVEC cells;qRT-PCR showed that,after the treatment of MTE,Caspase-3 mRNA expression was increased and VEGFR,P2Y6R,CCL-2 mRNA were reduced,these results indicated that MTE could induce apoptosis of HUVEC cells and inhibit cell migration significantly.In vivo CAM experiments showed that,compared with the PBS group,the number of internal vessels was almost invisible in the group of treatment with 16g MTE and the inhibitory effect was obviously.Compared with the control group,there was significant difference between the experimental group and the positive control(**P<0.01),indicating that MTE can effectively inhibit angiogenesis.Conclusion This experiment demonstrates that MTE can inhibit theproliferation and metabolism of Human Umbilical Vein Endothelial Cells,induce the apoptosis of HUVEC,and MTE has anti-angiogenic effect in vivo detected by CAM assay.