Experimental Study On Anti-hepatoma Pharmacodynamic And Mechanism Of Action Of Compound Phyllanthus Urinaria ?

Objective1.HepG2cells tumor xenografts model in BALB/C nude mice was used to observe pharmacodynamic effects of Compound phyllanthus urinaria II,by comparing before and after the administration of body weight,tumor volume change,and solid tumor weight size,to evaluate anti-tumor effect of Compound phyllanthus urinaria ? in vivo.2.In vitro experiments,observing the effect of Compound phyllanthus urinaria ?on human hepatoma cell line HepG2 about cell proliferation,colony formation,cell migration and the impact on the cell cycle and apoptosis,was used to evaluate anti-hepatoma effect of Compound phyllanthus urinaria ? in vitro.After CPu ? roled in the HepG2 cells,the gene expression of HNF-4 a and miR-122 and the activation effect of Wnt/?-catenin signaling pathway was studied,in order to explore the role of anti-hepatoma targets and mechanisms from the molecular level.experimental content1.Study of inhibitory effect of CPU ? on HepG2 cells in BALB/C nude mice xenograftsBALB/C nude mice Established subcutaneously implanted tumor of HepG2 cells were randomly divided into five groups:model group,CPU?high,medium and low dose group,5-Fu positive control group.Continuous administration of 6 weeks,the weekly measurement of body weight changes and solid tumor volume size,were sacrificed 24h after the last administration,the tumor weighed stripped and photographed.The results show that compared with the model group group,tumor weight in 5-Fu positive control group,CPU? high,medium and low dose group were significant differences(P<0.01).the inhibition rate of CPU?high,medium and low dose group were 65.8%,59.6%,58.4%;compared with 5-Fu in the control group,the difference was not significant(P>0.05),shows that the intervention of CPU ?has inhibited the growth of solid tumors,liver cancer.2.Study of pharmacodynamic effect and mechanism of anti-hepatoma of CPU?2.1 Effect of CPU?on human hepatoma HepG2 cell proliferation inhibitionHepG2 cells was used a model in vitro,by MTT assay,to measure the inhibitory effect of CPU? on the proliferation of HepG2 cells.The result showed that CPU II could inhibit the proliferation of HepG2 cells,which have a significant dose-response relationship?The 24h,48h,72h half inhibitory concentration(IC50)were 24.4 ± 1.4mg/ml,21.0 ± 1.3mg/ml,14.2 ±1.2mg/ml.,and with time extending,the inhibition increased significantly.2.2 Effect of CPU ?Ion human hepatoma HepG2 cell colony forming abilityWhen CPU?roled in HepG2 cells after 10d,Using Giemsa staining dye,Under the microscope count the number of cell clones than 50,and then calculated as colony-forming efficiency.The results showed that different concentrations of CPU?(5mg/ml,4mg/ml,3mg/ml,2mg/ml,lmg/ml)roling in the HepG2 cells,the cell colony formation inhibition rate was 2.1%,27.9%,38.2%,48.1%,90.2%.This meant that with the dose concentration increasing,inhibition of colony formation rates were increased and when the concentration of CPU?more than 2mg/ml,it have a significant effect on inhibition of colony formation.2.3 Effect of CPU?on human hepatoma HepG2 cell migrationCultured cells after culture plates covered in the 24-well plate painting"-" word,the cells in the control group complete medium,leaves under strain Compound II treatment group,were added to the drug-containing concentration of 20mg/ml,15mg/ml,10mg/ml 1640 complete medium.Respectively,Oh?24h?48h?when 72h,taking pictures.Compared with the control group cells,the concentration of each Phyllanthus Compound II significantly inhibit cell migration effect,and with the extension of time,the inhibition was significantly enhanced.2.4 Effect of CPU?on human hepatoma HepG2 cells cyclereferencing the concentration of IC50 roling in HepG2 cells after 48h,we set up CPU?dose groups(20mg/ml,15mg/ml,10mg/ml),control group,5-Fu group.When CPUII roled in HepG2 cells after 48h,cells were collected,adding PI dye,using the flow cytometry to detecte the changes in cell cycle.Compared with the control group,the proportion of cells in G0/G1 phase of each concentration dose group of CPU? decreased significantly,but the proportion of cells in S phase increase significantly and with the increase of the concentration of CPU II,Cycle arrest significantly increased and the difference was statistically significant(P<0.01).2.5 Effect of CPU ? on human hepatoma HepG,cell apoptosisCPU ?roled in HepG2 cells after 48h.,Flow cytometry was used to detect apoptosis,compared with cells control group,the rate of early apoptotic and total apoptosis cells ratehad a significant difference(P<0.01).When the concentration of CPU ?was more than lOmg/ml,it significantly induced the role of early apoptotic cells,And with concentrations of CPU ? increasing,it induced apoptosis increasing;at a concentration of 20mg/ml,its early apoptosis rate had no significant difference with 5-Fu group.2.6 Regulation of CPU ?on human hepatoma HepG2 cells miR-122 and Wnt/?-catenin signaling pathwayCPU ? roled in HepG2 cells after 48h,using qRT-PCR to detect the relative expression of miR-122,Wntl mRNA,?-catenin mRNA,HNF-4? mRNA.The results showed that compared with the control group,the expression of miR-122,HNF-4 a mRNA significantly increased(P<0.05,0.01)in CPU ? high and low dose groups;the expression of Wnt1mRNA,?-cateninmRNA significantly lower(P<0.05,<0.01).Tip CPU ? can promote the expression of HNF-4? mRNA,thus increasing the expression of miR-122,resulting in the expression of Wnt1mRNA and ?-cateninmRNA declining,making the transduction of Wnt/?-catenin signaling pathway blocked,which ultimately inhibit the growth of liver cancer cells.conclusion1.CPU ? had significantly inhibition on the formation of BALB/C nude mice implanted subcutaneously tumor of HepG2 cells,whhich indicated that CPU II for therapeutic intervention can inhibit the growth of solid tumors,liver cancer.2.CPU ? can significantly inhibited the proliferation of HepG2 cells and significantly block HepG2 cells in S phase.When the S phase DNA synthesis is blocked,the cells can not enter mitosis and ultimately inhibit cancer cell growth.Meanwhile CPU ? can significantly affect cell colony formation,prevent migration of cells,and induced apoptosis.Its anti-hepatoma mechanism may promote the expression of HNF-4? mRNA,thus increase the expression of miR-122.It is related to making transduction of Wntl/?-catenin signaling pathway blocked,further resulting in liver cancer cell cycle arrest,inhibition of proliferation,migration blocked and inducing apoptosis,which played the role of anti-hepatoma.