白桦脂醇调控 VEGFA/VEGFR2 信号通路抑制 HUVEC 血管新生的作用机制OA

Objective To investigate the mechanism by which betulin inhibits angiogenesis in human umbilical vein endothelial cell(HUVECs)based on the vascular endothelial growth factor A(VEGFA)/vascular endothelial growth factor receptor 2(VEGFR2)signaling pathway.Methods The effect of different concentrations(0-100 μmol·L-1)of betulin on the proliferation of HUVECs was assessed using the CCK-8 assay,and the appropriate intervention concentrations were selected.Subsequently,HUVECs were divided into a control group,a sorafenib group(5 μmol·L-1),and low-,medium-,and high-dose betulin groups(10,20,30 μmol·L-1).Wound healing assay was used to evaluate cell migration ability;Transwell migration and invasion assays were performed to assess cell migration and invasion capabilities,respectively;tube formation assay was conducted to examine capillary-like structure formation;rat aortic ring sprouting assay and chick chorioallantoic membrane(CAM)assay were employed to evaluate the anti-angiogenic activity of betulin.ELISA was used to measure the secretion level of VEGFA;qRT-PCR was performed to detect the mRNA levels of VEGFA and VEGFR2;Western Blot was carried out to determine the protein expression levels of VEGFA,VEGFR2,AKT,ERK1/2,p-VEGFR2,p-AKT,and p-ERK1/2.Results(1)Betulin at concentrations above 40 μmol·L-1 significantly inhibited HUVEC proliferation(P<0.05),but the cell viability at 40 μmol·L-1 was>85％,indicating a relatively weak effect on proliferation.Therefore,betulin at 10-30 μmol·L-1 was selected for subsequent experiments.(2)Compared with the control group,the low-,medium-,and high-dose betulin groups and the sorafenib group showed significantly reduced cell migration rates in the wound healing assay,as well as reduced migration and invasion rates in Transwell assays(P<0.05,P<0.01).Betulin inhibited HUVEC migration and invasion in a dose-dependent manner(P<0.01).(3)The control group exhibited a large number of three-dimensional tubular structures formed by HUVECs,whereas the betulin groups and the sorafenib group showed markedly fewer tubular structures.The tube formation rate,as well as the microvessel density in rat aortic rings and CAM,were significantly reduced in a dose-dependent manner(P<0.01).(4)Compared with the control group,the low-,medium-,and high-dose betulin groups showed significantly decreased VEGFA secretion levels and VEGFA mRNA expression,as well as significantly reduced protein expression levels of VEGFA,p-VEGFR2/VEGFR2,p-AKT/AKT,and p-ERK1/2/ERK1/2(P<0.05,P<0.01),in a dose-dependent manner(P<0.01).However,no statistically significant difference was observed in VEGFR2 mRNA levels among the groups(P>0.05).Conclusion Betulin may inhibit angiogenesis by modulating the VEGFA/VEGFR2 signaling pathway.