The Research On Traditional Chinese Medicine Of Paridis, Pulsatillae And Tetrandrine Combined With Endostar Inhibiting Angiogenesis

[Background]The tumor growth divides into the blood vessel time and the blood vessel earlier period.In blood vessel earlier period,tumor cells main dependence dissemination function gain nutrition.When the entity lump achieved when 1～2mm~3,the cell number achieves about 10~7.Continueous growth must have to provide the enough oxygen and the nutrients by the newborn blood vessel.After tumor internal vascularizing,its volume will be rapid increase.The tumor shift many kinds of factor influence,and in which the menstruation shift is the tumor occurs the important way which the far position spreads, therefore the tumor blood vessel production has played the very vital role in the tumor shift. The blood vessel production causes in the tumor growth and the shift vital role to block the blood vessel to produce into one feasible tumor method of treatment.At present the clinical commonly used anti-blood vessel production chemotherapy is the small dosage,the high-frequency for the medicine pattern,changes the high dose periodic tradition chemotherapy the continuous low dose anti-blood vessel production chemotherapy to enhance the curative effect also decreases the toxicity.Moreover in the tumor organization the newborn blood capillary endothelial cell belongs to the normal cell category,the genetics is stable,not easy to appear bears the medicine phenomenon.Endostar is the product of Medgenn Corporation in Yantai city,Shandong province in China.The pre-clinical research showed that it can depress the proliferation of vessel endothelial cells and build of vessels and growth of tumor.TheⅠandⅡclinical research showed it was effective and safe to patient when it used alone.The traditional Chinese medicine has a curative effect on tumor.In recent years,we discovered some traditional Chinese medicine have antiangiogenic effect,as ginseng soap glucoside Rg3,the red element(in Tripterygium wilfordii withdraw an active monomer ingredient),hydroxyl camptotheca acuminata alkali,paclitaxel and so on.[Objectives]This experiment aims to study(1)The cell-proliferation,cell-migration, tube-formation assays,and cell-apoptosis,cell cycle tests of vessel endothelial cells treated with Tetrandrine and alcohol extracts of Paridis and Pulsatillae as single angent and combined with Endostar;(2) Observe influance on proliferation of blood vein endothelial cells and growth of chick embryo choriallantoic membrane vessels after treated with drugs. [Methods]MTT assay was used to observe the inhibited effect of Tetrandrine and alcohol extracts of Paridis and Pulsatillae as single angent and combined with Endostar on human umbilical vein endothelial cells(HUVECs) and Lovo.Transwell cabin test and out-body canaliculization test were used to observe the impact of Tetrandrine and alcohol extracts of Paridis and Pulsatillae as single angent and combined with Endostar on the migration and vaso-formed ability of HUVECs.Annexin V fluorescein stain and cell cycle test solution and flow cytometry were used to detect the development of apoptosis and cell cycle of HUVECs after treated with drugs.Chickchorio allantoic membrane(CAM) model was used to observe the effect of anti-angiogenesis of Tetrandrine and alcohol extracts of Paridis and Pulsatillae as single angent and combined with Endostar.[Results]1.The survival rate of HUVECs is among 65%-18.2%,78.4%-28%,84.3%-46.9%treated with 15～60μg/ml Paridis alcohol extracts,2～8μg/ml Pulsatillae alcohol extracts and 2～8μg/ml Tetrandrine as single angent and 44.5%-8.9%,37.6%-22.5%,55.2%-13.0% treated with 15～60μg/ml Paridis alcohol extract,2～8μg/ml Pulsatillae alcohol extracts and 2～8μg/ml Tetrandrine combined with 10μg/ml Endostar,The survival rate of Lovo within this doses is more than HUVECs.2.The migration of HUVECs is obviously drpressed treated with 15～60μg/ml Paridis alcohol extracts,2～8μg/ml Pulsatillae alcohol extracts and 2～8μg/ml Tetrandrine as single angent,and the migratory cell numbers are decreased when the concentration of drugs step up,correlation coefficients are -0.933,-0.929,-0.933,P values are0.000,0.000,0.000,and 15～60μg/ml Paridis alcohol extracts,2～8μg/ml Pulsatillae alcohol extracts and 2～8μg/ml Tetrandrine combined with 10μg/ml Endostar,the correlation coefficients are -0.904,-0.919,-0.943,P values are0.000,0.000,0.000,and the combination group has more effect,and it has statistics difference.3.The number of tubule is reduced and lumina become unintegrated treated with 15～60μg/ml Paridis alcohol extracts,2～8μg/ml Pulsatillae alcohol extracts and 2～8μg/ml Tetrandrine as single angent and combined with Endostar10μg/ml.And there is statistics difference between combination group and single drug group.4.60μg/ml Paridis alcohol extracts,8μg/ml Pulsatillae alcohol extracts and 8μg/ml Tetrandrine as single angent can obviously induce HUVECs to apoptosis at 48h,and the rates of apoptosis are 25%,37.1%,20.8%,and the rates of apoptosis of drugs combined with 10μg/ml Endostar are 79.3%,58.6%,60.4%,and the rate of combination group is more than the single group.5.The 60μg/ml Paridis alcohol extracts,8μg/ml Tetrandrine as single angent and combined with 10μg/ml Endostar could block the cell cycle and arrest G0-G1 phase after 24h,8μg/ml Pulsatillae alcohol extracts as single angent and combined with Endostar 10μg/ml arrest G2-M phase of cell cycle.6.15～60μg/ml Paridis alcohol extracts,2～8μg/ml Pulsatillae alcohol extracts and 2～8μg/ml Tetrandrine as single angent and combined with 10μg/ml Endostar suppressed angiogenesis of CAM in vivo.The inhibition ratio is correlated with the concentration.And the combination group depress more CAM.[Conclusion]This experiment shows that low dose of Paridis,Pulsatillae and Tetrandrine has anti-angiogenic effect in vivo and in vitro,and it has synergetic effect when combined with Endostar.