To Explore The Effect Of Huoxuehueyu Fang On The Metastasis Of Hepatocellular Carcinoma Based On VEGF/VEGFR2 And Dll4/Notch Signaling Pathway

Objective:To explore the anti-cancer mechanism of Huoxuehuayufang(HXHYF)on hepatocellular carcinoma(HCC),from the perspective of regulating tumor angiogenesis related pathways,that the effect of HXHYF on H22 cells and transplanted HCC model nude mice was observed.Methods: In vitro experiment: H22 cells were selected by HXHYF,then analyzed by chemiluminescence,transwell migration experiment,real-time fluorescence quantitative PCR,Western Blot(WB),and matrigel tube-forming experiment.We observed the effect of HXHYF on proliferation and migration of HCC cells,the expression levels of VEGF,VEGFR-2,Dll-4 and Notch-4,and angiogenesis.In vivo experiment: Building stable cell line H22-PDS224,the transplanted HCC nude mice model was established.Methods of vivo imaging technology,hematoxylin-eosin staining,immunohistochemistry and WB were used to observe effect of HXHYF on tumor weight and volume,tumor metastasis,microvascular generation,and the protein expression of Dll-4、Notch-4、VEGF、VEGFR-2.Results: In vitro experiment one: HXHYF can inhibit proliferation of H22 cells.In HXHYF group,number of migration cells was less,the expression of Dll-4 and Notch-4 were up-regulated,and the expression of VEGF was down-regulated,while the expression of VEGFR-2 was not significantly regulated.In vitro experiment two: VEGF inhibitor could inhibit proliferation of H22 cells,with 1u M concentration being more appropriate.Notch inhibitor has bidirectional regulation effects,and can inhibit or promote cell proliferation,of which0.01 u M is a more suitable concentration to promote proliferation of HCC cells.Compared with control group,under the condition of VEGF inhibitor,the relative inhibitory rate of HXHYF on cell proliferation,migration and angiogenesis was different.The relative inhibitory rate in the case of Notch inhibitor was significantly different and was significantly weakened.The effect of HXHYF on Notch pathway was more obvious.In vivo experiment: After H22-PDS224 was screened,the transplanted HCC nude mice model was established.In HXHYF group,tumor weight and volume of transplanted HCC nude mice were smaller,the number of lung metastases was less and CD34 positive expression was lower.The protein expression level of Dll-4 and Notch-4 were higher,while the protein expression level of VEGF and VEGFR-2 were lower.Conclusion:1、HXHYF could inhibit proliferation and migration of HCC cells,up-regulate the expression of Dll-4 and Notch-4,and down-regulate the expression of VEGF,while the up-regulation of VEGFR-2 was not obvious.2、HXHYF could activate Notch pathway and negatively regulate VEGF pathway to inhibit proliferation and migration of HCC cells and angiogenesis of HCC.3 、 HXHYF could inhibit tumor growth and lung metastasis.It could reduce microvascular invasion,inhibit angiogenesis and the protein expression of VEGF and VEGFR-2,and promote the protein expression of Notch-4 and Dll-4.