Study On The Angiogenesis Of Roxasone And The Expression Of VEGF Induced

Roxarone, one of the organic arsine feed additives, was recently shown that it can promote the growth of human vascular endothelial cells, imaging the angiogenesis promotion and potential risk of cancer. Vascular endothelial growth factor (VEGF) is now known to be the most important blood vessel growth-regulating factor. Little information was focused on the study on angiogenesis of roxarsone and its regulation of VEGF at home and abroad. The murine Matrigel plug assay in vivo and the endothelial vascular cells growth model were used to evaluated the angiogenesis promotion of roxarsone and the VEGF regulation effect which is benefit to the knowledge of specifical toxicoloty and potential risk of carcinogenesis of roxarsone, and is useful for the safety of roxarsone used as an additive in animal production.In the Matrige plug assay model, the doses of roxarone were divided into0.10μM,1.00μM,10.00μM, and10ng/mL VEGF was as positive control, PBS as negative control. Endothelial cells from rat thoracal aorta after different treatment were inject to the subcutaneous of ICR mice. Ten days later, the volum size, weight and content of hemoglobin of the Matrige plugs were detected. Results indicated that the volum size, weight and content of hemoglobin of the stoppers in different treatments of roxarsone were significantly higher than that of PBS control group (P <0.05). At the range of0.10μM to10.00μM of roxarsone doses, the size, weight and hemoglobin content of plugs was increased with the roxarsone dose increase but decreased at10.00μM, with the maximum peak appeared at1.00μM treatment. The content of hemoglob in1.00μM roxarsone group was similar with the VEGF control.In the cultures of endothelial cells from rat thorax aorta, the test groups were designed as10.00μM,1.00μM,0.10μM and0.01μM roxarsone,1.00μM roxarsone+antibody of FLT (1.00μM+Anti-FLT),1.00μM roxarsone+antibody of KDR (1.00μM+Anti-KDR), Anti-FLT、 Anti-KDR, Anti-VEGF and PBS. After6h incubation of endothelial cell trains, the MTT method was used to measured the cell viability, the ELISA method to exam the VEGF content, the Werstern Blot to decete the expression of VEGF, and the tube formation assay was to evaluated the potential formation of tube-like of roxarsone.In MTT assay of endothelial cells from rat thorax aorta, the OD values measured in0.10～10.00μM roxarsone and VEGF group cell were significantly higher than that of PBS control group (P<0.05), and the highest OD value was at1.00μM group within0.10～10.00μM roxarsone. The cells viability in1.00μM+Anti-KDR group was obviously lower than that of1.00μM groups (P<0.05), whereas there was not significant differences between1.00 μM+Anti-FLT groups and1.00μM group (P>0.05). The OD of Anti-KDR and Anti-VEGF was significantly lower than that of PBS control (P<0.05), but there was no significant difference between Anti-FLT and PBS (P>0.05). Results showed the angiogenesis effect of roxarsone on the endothelial cells from rat aorta, and the maximum was at1.00μM in the range of0.10-10.00μM dose. The VEGF and its receptor KDR was clearly associated with the promotion effect of roxarsone to endothelial cell growth.In Matrigel-induced tube formation of endothelial cells, the total number of tubes of0.01μM,0.10μM,1.00μM and10.00μM roxarsone group was substantially higher than that of PBS control group. The total number of tubes were increased with the dosage increase of roxarsone at0.01～1.00μM range with maximum peak at1.00μgroups, whereas decreased at10.00μM groups.The VEGF quantity in culture supernatant of endothelial cell treated showed that VEGF content of0.10～10.00μM roxarsone and VEGF group was significantly higher than that of PBS control group(P<0.05). The level of VEGF of1.00μM+Ant-KDR group was significantly higher than1.00μM groups (P<0.05) whereas there was no significant differences between1.00μM+Ant-FLT group and1.00μM roxarsone (P>0.05). But Anti-KDR and Anti-FLT was lower than1.00μM+Anti-KDR and1.00μM+Anti-FLT groups separately. Results indicated that roxarsone may stimulate VEGF secrete in endothelial cell from rat. The VEGF level was not affected by the specifical block of its receptor1(FLT) but markedly risen after blocking receptor2(KDR). In the Western Blot analysis of VEGF in endothelial cells, the VEGF expression of0.10μM,1.00μM,10.00μM roxarsone group was all significantly higher than that of PBS, with the maximum at1.00μM.In summary, the angiogenical promotion of roxarsone was emerged in the0.1～10.00μM dose exposure, the maximum effect was at1.00μM dosage, in which VEGF and KDR was found take part in the regulation.