The Scree And Mechanism Research On Traditional Chinese Medicine Which Having Anti-Angiogenesis Effect

Part One Screen Traditional Chinese Medicine Which Having Anti-angiogenesis EffectObjectives To screen Traditional Chinese Medicine composition of14 alcohol extracts of Traditional Chinese Medicine, paridis and pulsatillae, obviouslydifference cyotoxic on human hepatoma HepG2, SMC7721 cell strain; human colon carcinomaLovo, SW-116 cell strain; human gastric carcinoma AGS, BGC-823 cell strain; humanesophageal carcinoma CaEs-17 cell strain and endothelial cell (EC) of human umbilical veinin vitro. Methods The MTT assay had been developed for quantitative evaluation of theinhibited effect of 14 alcohol extracts of Traditional Chinese Medicine on proliferation ofhuman cell strains of hepatoma、colon carcinoma、gastric carcinoma、esophageal carcinomaand EC of human umbilical vein. Used growth curve assay to detect the variability cell toxicaction to tumour cell and EC which was cultured in vitro by 14 alcohol extracts of TraditionalChinese Medicine. Screened for the most sensitive cell strain to the Traditional ChineseMedicine and for the Traditional Chinese Medicine which had different cytotoxic to EC.Results The most sensitive cell to the medicine was human colon carcinoma Lovo cell.Alcohol extract of paridis, tetrandrine and alcohol extract of pulsatillae could obviously inhibitthe proliferation of EC in deuto-cytotoxic concentration (P=0.003、0.006、0.024). Effect wasmost obviously at 48h, and had fine dose-dependent relationship. Conclusion Alcoholextract of paridis in 15-60μg/ml, tetrandrine in 2-8μg/ml and alcohol extract of pulsatillae in2-8μg/ml had obviously variability cell toxic action to tumour cell and EC, the inhibitory effectto EC proliferation was most obviously at 48h, and had fine dose-dependent relationship. Part Two Alcohol Extract of Paridis, Puisatillae and Tetrandrine Effect to The BiologicalBehaviour of Vascular Endothelial Cell in VitroObjectives To investigate the effect of migration, cell cycle andapoptosis mechanism of alcohol extract of paridis, tetrandrine and alcohol extract of pulsatillaeto vascular endothelial cell. Methods Used transwell cabin test and out-body canaliculizationtest to observe the alcohol extract of paridis impact the migrational and vaso-formed ability ofHUVEC. And used Annexin V fluorescein stain and flow cytometry to detect the developmentof apoptosis and the variation of the cell cycle after drugs used to the EC. Results While thealcohol extract of paridis in 15-60μg/ml, tetrandrine in 2-8μg/ml and alcohol extract ofpulsatillae in 2-8μg/ml effected at 24 hours to the EC, the number of the tubule was reducedand lumina became unintegrated. There was significant difference compared with the controlgroup, and the inhibition was dose-dependent to canaliculization of human umbilical veinendothelial cell, R=-0.934,-0.982,-0.931; P=0.000,0.000,0.000. The number of cell migrationhad obviously different compared with the control group after medicine effected for 12 hours,and the capability of HUVEC migration was dose-dependent, R=-0.933,-0.929,-0.933;P=0.000,0.000,0.000. And when alcohol extract of paridis in 60μg/ml, tetrandrine in 8μg/mland alcohol extract of pulsatillae in 8μg/ml at 48h, it could obviously induce human umbilicalvein EC apoptosis. The alcohol extract of paridis and tetrandrine could block the cell cycle andarrest at G0-G1 after 24 hours, and the alcohol extract of pulsatillae arrest at G2-M after 24hours. Conclusion The alcohol extract of paridis, tetrandrine and alcohol extract of pulsatillaecould actively suppress angiogenesis in vitro and the mechanism may related to block themigration, canaliculization of the EC and induce the EC apoptosis, suppress the endothelial cellcycle. Part Three Research The Mechanism of Alcohol Extract of Paridis, Pulsatillae and Tetrandrine toAnti-angiogenesis of The Chick Embryo in VivoObjectives To further understand the effect of anti-angiogenesis of thealcohol extract of paridis, tetrandrine and alcohol extract of pulsatillae in vivo. MethodsUsed chickchorio allantoic membrane (CAM) model to observe the effect of anti-angiogenesisof the alcohol extract of paridis, tetrandrine and alcohol extract of pulsatillae. ResultsSurrounding the carrying agent of CAM the blood vessel was suppressed after added medicinefor 72h, then there appeares avascular area that inequality of size. There was inhibitory actionto the CAM angiogenesis when the density of the alcohol extract of paridis is between15-60ng/ml, tetrandrine between 2-8ng/ml and alcohol extract of pulsatillae between2-8ng/ml.Conclusion The alcohol extract of paridis, tetrandrine and alcohol extract ofpulsatillae could inhibit CAM angiogenesis in vivo. Part Four The Effect of Alcohol Extract of Paridis, Pulsatillae and Tetrandrine on Bearing H22Tumor MiceObjectives To study the antiangiogenic effect of alcohol extract ofparidis, pulsatillae and tetrandrine in vivo on bearing tumor Mice. Methods We discoveredthat the drugs were more sensitive to H22 cell line than S180 cell line in vitro and selected H22cell line to establish H22 transplantation tumor mice models, then intragastric administrationthe alcohol extract of paridis, tetrandrine and alcohol extract of pulsatillae. To observe weightmagnitude and microvessel density of the tumor after management. Results The tumorweight of the alcohol extract of paridis in 2g/kg and 4g/kg were lighter than NS control group(P=0.012, 0.001). The microvessel density of the alcohol extract of paridis in 2g/kg and4g/kg were fewer than NS control group (P=0.015, 0.001). The tumor weight of tetrandrinein 80mg/kg were lighter than NS control group (P=0.017). The microvessel density oftetrandrine in 40mg/kg and 80mg/kg were fewer than NS control group(P=0.015,0.001). Thetumor weight of alcohol extract of pulsatillae in 20g/kg wre lighter than NS control group (P=0.01). The microvessel density of alcohol extract of pulsatillae in 10g/kg and 20g/kg werefewer than NS control group(P=0.037,0.010). Conclusion This experiment showed that thealcohol extract of paridis, alcohol extract of pulsatillae and tetrandrine had antitumor ability invivo on H22 transplantation tumor. They also inhibited tumor MVD. So we presume that theantitumor ability was correlate with the antiangiogenic effect. Part Five The Effect of Alcohol Extract of Paridis, Pulsatillae and Tetrandrine on Nude MiceBearing Human Colon Carcinoma (Lovo)Objectives To study the effect of alcohol extract of paridis,pulsatillae and tetrandrine in vivo on nude mice bearing Lovo transplantation tumor. MethodsEstablished Lovo transplantation tumor nude mouse models, then intragastric administrationthe alcohol extract of paridis, tetrandrine and alcohol extract of pulsatillae. To observe grosstumor volume, weight magnitude, microvessel density of the tumor and body weight aftermanagement. Results The tumor weight of the alcohol extract of paridis in 4g/kg was lighterthan NS control group (P=0.017). The microvessel density of the alcohol extract of paridis in2g/kg and 4g/kg were fewer than NS control group (P=0.035, 0.003). The tumor weight ofalcohol extract of pulsatillae in 20g/kg was lighter than NS control group (P=0.036). Themicrovessel density of alcohol extract of pulsatillae in 10g/kg and 20g/kg were fewer than NScontrol group (P=0.043,0.033). The tumor weight of tetrandrine in 80mg/kg was lighter thanNS control group (P=0.040). The microvessel density oftetrandrine in 80mg/kg was fewerthan NS control group (P=0.035).The body weight of bearing Lovo transplantation tumornude mouse post-treatment with alcohol extract of pulsatillae (10g/kg,20g/kg), alcohol extractof paridis (2g/kg,4g/kg) and tetrandrine (80mg/kg) had no significant change. ConclusionThis experiment showed that the alcohol extract of paridis, alcohol extract of pulsatillae andtetrandrine had antitumor ability in vivo on Lovo transplantation tumor. They also inhibitedtumor MVD. So we presume that the antitumor ability was correlate with the antiangiogenic effect. Part Six Differential Angiogenesis Related Genes Expression of Alcohol Extract of Paridis,pulsatillae and Tetrandrine on nude mice bearing Lovo transplantation tumorObjectives To study the differential angiogenesis related genesexpression of alcohol extract of paridis, pulsatillae and tetrandrine in vivo on nude micebearing Lovo transplantation tumor. Methods We used microarray analysis to detect thedifferential genes expression of alcohol extract of paridis, pulsatillae and tetrandrine in vivo onnude mice bearing Lovo transplantation tumor.Then we approached the effective target on thelevel of gene deeply. Results 13 angiogenesis related genes were found regulated above 2folds or more by the arrays on the tissue of nude mice bearing Lovo transplantation tumor, andamong them 3 up and 10 down. 3 genes were found up regulated effected by alcohol extract ofparidis (4g/kg): COL4A3 up regulated above 3 folds, CXCL9 up regulated above 4 folds,THBS1 up regulated above 10 folds. 3 genes were found down regulated effected by alcoholextract of pulsatillae (4g/kg): CXCL3 down regulated above 2.7 folds, HPSE down regulatedabove 11.3 folds, ID1 down regulated above 12 folds. 7 genes were found down regulatedeffected by tetrandrine (80mg/kg): ANGPTL4 down regulated above 3.8 folds, FGF2 downregulated above 5.5 folds, FGF6 down regulated above 2.4 folds, FIGF down regulated above3.8 folds, HPSE down regulated above 4.8 folds, LAMA5 down regulated above 2.9 folds,VEGFB down regulated above 7.9 folds. 3 genes were found down regulated effected byalcohol extract of pulsatillae (20g/kg): ANGPTL4 down regulated above 3.6 folds, CCL2down regulated above 4.5 folds, VEGFB down regulated above 7.7 folds. Conclusion Basedon our nude mice bearing Lovo transplantation tumor model, and by oligonucleotide arrays, wefound paridis, pulsatillae and tetrandrine can ragulate the expression of angiogenesis relatedgenes through different target in vivo on nude mice bearing Lovo transplantation tumor.