Babao Dan Inhibits Angiogenesis And Lymphangiogenesis Of Gastric Cancer Via Regulating VEGF/VEGFR Pathway

Objective:To study the effect and mechanism of Babao Dan(BBD)on angiogenesis and lymphangiogenesis of gastric cancer(GC)in vitro and in vivo via VEGF/VEGFR pathway(VEGF-A/VEGFR-2 and VEGF-C/VEGFR-3),and to provide experimental evidence for its clinical treatment of gastric cancer.Methods:In vivo experiment: The nude mice model of MGC80-3 subcutaneous transplanted tumor was established and randomly divided into control group and BBD group according to tumor volume.The BBD group(n=10)was administered by gavage according to 0.25 g/kg,and the control group(n=10)was given the same volume of saline.Each group was intragastric administration 6 days a week and the volume of the tumor was measured every other day.The nude mice were sacrificed after 4 weeks,and the blood was collected and the tumor tissue was stripped for the subsequent experiment.Immunohistochemistry(IHC)was used to detect the microvessel density(CD31)and lymphatic vessel density(LYVE-1)of the tumor tissue.Enzyme-linked immunosorbent assay(ELISA)was used to detect the content of VEGF-A and VEGF-C in serum.And western blot(WB)was used to detect the expression level of VEGF-A,VEGF-C,VEGFR-2,VEGFR-3 of VEGF/VEGFR pathway-related proteins in tumor tissues.In vitro experiment: GC cell lines(AGS,BGC823,MGC80-3)were treated with different concentrations of BBD.Cell viability was detected by MTT assays,and the secretion and expression level of protein VEGF-A and VEGF-C were detected by ELISA and WB.By stimulating HUVEC and HLEC with exogenous factors VEGF-A and VEGF-C respectively to simulated internal environment.Tube formation was used to detect the effect of BBD intervention on angiogenesis and lymphangiogenesis ability.Cell viability was detected by MTT assays,Hochest33258 staining was used to observe cell apoptosis,Transwell and wound healing assays were used to detect the effect of BBD on cell migration and damage repair ability.WB assays was used to detect the expression level of VEGF-A,VEGF-C,MMP-2,MMP-9,VEGFR-2 and VEGFR-3.Results:In vivo experiment: The data showed that BBD had a certain inhibitory effect on the volume and weight of GC transplanted tumor(P<0.05 or P<0.01),and there was no adverse reaction.IHC data showed that BBD could inhibit the angiogenesis and lymphangiogenesis of GC transplanted tumor(P<0.01).ELISA data showed that BBD could reduce the content of VEGF-A and VEGF-C in serum(P<0.05).WB showed that BBD could down-regulate the expression level of VEGF-A,VEGF-C,VEGFR-2 and VEGFR-3(P<0.01).In vitro experiment: The results of MTT assay showed that BBD could inhibit the activity of GC cells(AGS,BGC823,MGC80-3)(P<0.01).ELISA and WB data showed that BBD could inhibit the secretion and expression level of protein VEGF-A and VEGF-C(P<0.01).The results of tube formation showed that BBD could inhibit angiogenesis and lymphangiogenesis(P<0.01).The results of MTT assay showed that BBD could inhibit the activity of HUVEC and HLEC(P<0.01).Hochest33258 staining showed that BBD could promote the apoptosis of HUVEC and HLEC(P<0.05 or P<0.01);Transwell and wound healing assays showed that BBD could inhibit the migration and damage repair ability of HUVEC and HLEC(P<0.01).WB showed that BBD could down-regulate the expression level of VEGF-A,VEGF-C,MMP-2,MMP-9,VEGFR-2 and VEGFR-3(P<0.01).Conclusion:BBD can inhibit the development of GC and significantly inhibit angiogenesis and lymphangiogenesis via regulating VEGF/VEGFR pathway in vivo and in vitro.