| Roxarsone, a arsenic feed additive, has being widely used to promote animal production for many years in all the world. The grown increase mechanism is not clear so far, it may be related to enhanced cell growth, or to upregurate immunity and energy metabolism-related genes expression, may be related to promote new blood angiogenesis, or increase vascular permeability. Few research was focused on these aspects. The angiogenesis potentiality of roxarsone were investigated in vitro or in vivo in our provious researches. This paper will explore the mechanism of vascular endothelial growth factor (VEGF) and its receptors in angiogenic promotion of roxarsone on endothelial cells by blocking the VEGF or VEGFR with its antibody, in which determined by MTT method, cell immunofluorescence analysis, Elisa for VEGF excretion detection, Western blot and real-time fluorescent quantitative PCR for VEGF/VEGFR expression. It is useful to knowedge the VEGF mechanism of roxarsone angiogenesis effect, is also beneifit to the safety evaluation of roxarsone as a feed additive and cancerigenic risk of its residue in environment.The endothelial cells were come from rat thorax aorta and cultured with DMEM containg20%fetal bovine serum at12,24,36and48h respectively. The different exposure groups were designed as PBS control group,10ng/mL VEGF positive control,0.1μM,1.0μM,10μM,100μM of roxarsone,10ng/mL antibody blockage groups (Anti FLT-1, Anti KDR, Anti VEGF), and1.0μM roxarsone together with10ng/mL VEGFR antibody blockage (Rox1μM+Anti KDR, Roxl μM+Anti FLT-1). The endothelial cell viability was determinated by MTT assay; the Elisa method was to detect the amount of VEGF in culture supernatants, cell immunofluorescence analysis and Western blot was to detect the expression of VEGF、KDR and FLT-1in cells, and fluorescence quantitative PCR to detect the mRNA level of VEGF and VEGFR.MTT assay results showed cell viability of10ng/mL VEGF, Rox0.10μM～100μM was significantly higher, whereas that of Anti KDR and Anti VEGF group was significantly lower compared with that of PBS control (P<0.05). There was no significant difference between1.0μM Rox group and positive control group, Anti FLT-1group and PBS control was no significant difference. The OD value of Rox1+Anti KDR group was significantly lower than PBS control, and significantly higher than Anti KDR group (P<0.05); Rox1+Anti FLT group was significantly higher than the Anti FLT-1group (P<0.05), no significant differences with Rox1μM group (P>0.05). Results showed that0.10-100μM Rox was appeared promotion effect on endothelial cell grown, mainly regurated by VEGF and KDR. Immunofluorescence of endothelial cells can observed VEGF, KDR and FLT-1on treated cells. In Elisa detection to VEGF leval on culture supernatant treated by different treatment, results showed that the VEGF contents of different dose Rox groups were significantly higher than that of control group, peak value at1.0μM group. Compared with PBS control, VEGF concentration of Anti KDR group was clearly higher, that of Anti VEGF group was significantly lower (P<0.05), while no obvious difference at Anti FLT-1group.The VEGF content of Rox1+Anti KDR group was significantly higher than1.0μM group and Anti KDR group (P<0.05); the Rox1+Anti FLT-1group was significantly higher than that of Anti FLT-1group (P<0.05), but no significant difference with1.0μM Rox group (P>0.05). Results showed that Rox stimulate endothelial cells secrete VEGF and the cell growth promotion of roxarsone may be regulated by VEGF/KDR pathway mainly.In Western blot analysis, the expression levels of VEGF, KDR and FLT-1in different dose of Rox were higher than that of PBS control (P<0.05), peak value at1.0μM Rox group. Blocking VEGF and KDR with related antibody, the expression of VEGF and KDR was clearly lower than that of PBS control (P<0.05), no difference with FLT-1expression. The KDR level at Rox1+anti KDR group was significantly lower than that of1.0μM Rox group and obviously higher than that of Anti KDR group (P<0.05). The FLT-1expression at Rox1+Anti FLT-1was significantly higher than that of Anti FLT-1group, but no difference within1.0μM Rox group (P>0.05).In Fluorescence quantitative PCR to detect the mRNA level of VEGF, KDR and FLT-1, the VEGF and KDR mRNA expression of different dose Rox group was higher than the negative control (P<0.05), and highest at1.0μM Rox group. When blockage of VEGF and KDR, the VEGF and KDR mRNA expression is significantly reduced compared with PBS control. The KDR level of Rox1+Anti KDR group was significantly higher than that of Anti KDR group and significantly lower than that of1.0μM Rox group (P<0.05). The FLT-1mRNA of Rox1+Anti FLT-1was significantly higher than Anti FLT-1group (P<0.05), but no significant difference with1.0μM Rox group (P>0.05).In summary,0.1μM～100.0μM roxarsone appeared growth promoting action on rat endothelial cells significantly and VEGF/KDR pathway was implied major regulation the effect of roxarsone on rat endothelial cells. |
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