Study Of Oxymatrine On Angiogenesis Of Human Umbilical Vein Endothelial Cells Induced By HepG2 Cells Conditioned Medium

Objective: To explore the effect of oxymatrine on angiogenesis of human umbilical vein endothelial cells(HUVEC)induced by conditioned medium of human hepatoma Hep G2 cells and the underlying molecular mechanism.Methods: HUVEC induced by Hep G2 conditioned medium has the characteristics of tumor endothelial cells(TECs).The expression of TEM1 and TEM8 was detected by q PCR to identify TECs.The morphology of HUVEC before and after induction was observed by inverted microscope.(2)The content of VEGF in different cell culture media was detected by ELISA,the expression of VEGFR2 and Ets-1 in HUVEC before and after induction was detected by q PCR and immunofluorescence.To investigate the molecular mechanism of tumor conditioned medium promoting the transformation of normal ECs to TECs.(3)CCK-8assay,cell scratch test and in vitro angiogenesis test were used to detect the influence of oxymatrine at the concentrations of 0.375 mg/m L,0.75 mg/m L,1.5 mg/m L,3 mg/m L and 6mg/m L on the proliferation,migration and tube-forming ability of TECs in different period of time.(4)The expression of VEGFR2 and Ets-1 was detected by q PCR and cellular immunofluorescence before and after treatment with oxymatrine,and to explore the possible molecular mechanism of oxymatrine inhibiting the angiogenesis of TECs.Results: QPCR results showed that the m RNA expressions of TEM1,TEM8,Ets-1 and VEGFR2 in HUVEC induced by Hep G2 cells conditioned medium were higher than those in the negative control group(P < 0.05),indicating the successful construction of TECs.(2)ELISA results showed that compared with endothelial cell medium(ECM),the content of VEGF in Hep G2 cells conditioned medium and mixed medium(ECM containing 50% Hep G2 cells conditioned medium)were increased(P < 0.001).(3)Compared with HUVEC,the average fluorescence intensity of Ets-1 and VEGFR2 in TECs increased(P < 0.05).(4)CCK-8 assay showed that the inhibitory effect of oxymatrine on the proliferation of TECs increased with the increase of concentration(P < 0.001),the inhibitory effect of 0.375 mg/m L,0.75 mg/m L,1.5 mg/m L and 3 mg/m L oxymatrine groups on the proliferation of TECs decreased with the extension of time,in which 3 mg/m L oxymatrine group was the most obvious,and the difference was statistically significant(P < 0.05),the inhibitory effect of 6mg/m L oxymatrine group on the proliferation of TECs increased with time(P < 0.05).0.375mg/m L,0.75 mg/m L,1.5 mg/m L and 3 mg/m L oxymatrine were selected as the follow-up experimental intervention conditions,and 3 mg/m L oxymatrine group was used as the time effect experimental group for the follow-up study of gene level and protein level.The results of cell scratch test showed that there was no significant difference in scratch healing rate between the 0.375 mg/m L oxymatrine group and the control group(P > 0.05),while the scratch healing rate of the other oxymatrine groups decreased with the increase of concentration(P < 0.05),the effect of oxymatrine on scratch healing rate increased with time(P < 0.05).The results of in vitro angiogenesis experiment showed that the cells gradually appeared tube-like structure after 3 hours in the plate,and there was no significant difference in each group(P > 0.05).In comparison with the control,there was no significant difference in the number of tube formation in the 0.375 mg/m L oxymatrine group(P > 0.05),the number of tube formation decreased with the increase of concentration in the other oxymatrine groups(P < 0.05).The number of tube formation decreased with the extension of oxymatrine intervention time(P < 0.05).(5)QPCR results showed that compared with the control group,except 0.375 mg/m L oxymatrine group,the m RNA expression of Ets-1 and VEGFR2 in cells treated with different concentrations of oxymatrine decreased with the increase of oxymatrine concentration(P < 0.05).Compared with the control group,the m RNA expression of Ets-1and VEGFR2 in cells treated with 3 mg/m L oxymatrine increased with the extension of treatment time(P < 0.05).(6)Cell immunofluorescence assay showed that compared with the control group,the expression of Ets-1 protein in cells treated with different concentrations of oxymatrine decreased with the increase of oxymatrine concentration(P < 0.05),except for0.375 mg/m L oxymatrine group.Compared with the control group,except 0.375 mg/m L and0.75 mg/m L oxymatrine groups,the expression of VEGFR2 protein in cells treated with different concentrations of oxymatrine decreased with the increase of oxymatrine concentration(P < 0.05).Compared with the control group,Ets-1 and VEGFR2 protein expression in 3 mg/m L oxymatrine increased with time(P < 0.05).Conclusion:(1)Hep G2 conditioned medium simulates tumor microenvironment to induce normal endothelial cells to possess the characteristics of tumor endothelial cells,which provides a cell model for the study of anti-hepatoma angiogenesis based on tumor endothelial cells.(2)Oxymatrine can inhibit HUVEC angiogenesis induced by Hep G2 cells conditioned medium,and the mechanism may be through inhibiting the expression of Ets-1,thereby inhibiting the VEGF/VEGFR2 signal transduction pathway.