| Roxarsone (Rox), an arsenic compound, has been widely used in the poultry industry as a feed additive to promote growth, prevent coccidiasis and to improve feed conversion ratio. Now some studies show that low concentration roxarsone may promote endothelial cells growth or vessel formation in vivo, but the effect mechanism is not clear yet. VEGF/VEGFR which is probably the most important factor on growth of vascular endothelial cell and angiogenesis of blood vessels. The ERK/MAPK pathway is the most important signal to regulate cell division and proliferation. The mechanism of ERK/MAPK pathway in the proliferation promotion of roxarsone on rat endothelial cells was investigated by specific inhibiting VEGFR, Ras and MEK in ERK/MAPK signal.In the culture of rat vascular endothelial cells, different treatment was designed as follows. Vascular endothelial cells were exposed to 0.1 μM,1.0μM and 10 μM roxasone, arid PBS as negative control. Co-treatment of 1.0 μM Rox with different inhibitor was as below:1.0 μM Rox+anti-VEGFR (1+anti-V),1.0μM Rox+anti-Ras (1+anti-R),1.0 μM Rox+anti-MEK (1+anti-M),1.0 μM Rox+anti-VEGFR+anti-Ras (1+anti-V+anti-R),1.0 μM Rox+anti-VEGFR +anti-Ras+anti-MEK (1+anti-V+anti-R+anti-M). Every special inhibitor was set as control, the concentration of VEGFR inhibitor was 15μM (anti-V),10μM as Ras inhibitor (anti-R),10μM as MEK inhibitor (anti-M). The multi-inhibitor joined anti-VEGFR+anti-Ras (anti-V+anti-R), anti-VEGFR+anti-Ras+anti-MEK (anti-V+anti-R+anti-M) was also as control. The MTT reduction assay was used to reflect the proliferation of the cells, the Elisa analysis was used to determination the VEGF level in supernatant. The expression of signal molecule was detected by Western-blot analysis and real-time PCR.The MTT data of endothelial cells at 24 hours incubation showed that the OD values of 0.1 μM,1.0 μM and 10 μM of roxarsone were significantly higher than that of negative control (P<0.01), the peak was at 1.0μM Rox group. The OD values in VEGFR, Ras and MEK inhibitor co-treatment with 1.0 μM Rox roxarsone groups were significantly lower than that in 1.0 μM Rox group (P<0.01), but higher than the relevant inhibitor treatment group. The cell viability of co-treatment with tow or more inhibitors was significantly lower than that in single blockage group.The data of VEGF level tested by Elisa method were as followed. The VEGF concentration in culture supernatants treated by 0.1 μM, 1.0μM and 10μM of Rox were significantly higher than that in negative control (P<0.01),1.0 μM Rox group was the most. The VEGF content in groups of every inhibitor treated was significantly lower than the PBS control. In 1.0 μM roxarsone co-treatment with one or more inhibitors, the expression of VEGF were significantly lower than that in 1.0μM Rox but higher than the relevant inhibitor treatment group. Result showed that roxarsone may promote the expression of VEGF, and blocking the VEGFR, Ras and MEK signal molecule can affect VEGF expression of rat endothelial cells exposed to roxarsone.In the Western Blot analysis, there was no significant differences in the Ras, MEK and ERK expression between doses of roxarsone groups and PBS control, but the phosphorylated proteins of Ras, MEK and ERK were significantly increased. The levels of Ras in 1.0 μM Rox co-treatment with VEGFR or Ras inhibitor were not significant different from that of 1.0 μM Rox, but was significant difference in the expression of P-Ras (P<0.01). In the treatment of 1.0 μM roxarsone with VEGFR, Ras or MEK inhibitors, the expression level of P-MEK and P-ERK was significantly lower than those in 1.0LM Rox exposed only (P<0.01). The levels of P-MEK in anti-R and anti-V+anti-R groups were significantly lower than those in 1.0 μM Rox co-exposed to different inhibitors (P<0.05). The level of P-ERK in 1.0 μM Rox co-treated with anti-M, anti-V+anti-R, anti-V+anti-R+anti-M groups were significantly higher than those in their inhibitor control (P<0.01). Result showed roxarsone clearly affected phosphorylation of signal proteins in ERK/MAPK pathway, and VEGFR-ERK/MAPK signal transduction was involved in the promotion of roxarsone on endothelial cell growth.The real-time PCR data were as below. The mRNA of ras in 1.0 μM roxarsone was no significant differences compared with the control group. The mRNA of ras in 1.0 μM Rox co-treated with different inhibitor groups were significantly lower than that in 1.0 μM Rox, and the level in anti-V treatmet group is significantly lower than that in 1+anti-V (P<0.01). The mRNA of mek-1 in 1.0 μM roxarsone was significantly higher than that in PBS control, whereas no difference in mek-2 mRNA. The level of mek-1 and mek-2 in 1.0 μM Rox co-treated with one or more inhibitors were significantly lower than those in 1.0 μM Rox (P<0.01). The expression of mek-1 in anti-V+anti-R+anti-M group was significant lower than that in 1+anti-V+anti-R+anti-M. anti-R treatment group. The mek-2 level was significantly different between 1.0μM Rox plus anti-R treatment and 1.0 μM Rox group (P<0.01). The erk-1 expression was significantly higher than control group, the erk-2 was no significant difference. The erk-1 and erk-2 levels in 1.0 μM Rox co-treatment with one or more inhibitor were significantly lower than those in 1.0 μM Rox treatment group (P<0.01). It can demostrated that the genes of mek-1 and erk-lin ERK/MAPK pathway play a signficant role in roxarsone promoting the growth of endothelial cell.In conclusion, ERK/MAPK pathway was involved in proliferation promotion of roxarsone on rat endothelial cell. |
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