| Angiopoietin (Angiopoietins, Ang), one of blood vessel growth-regulating factors, had been shown to play a significant role in the regulation of physiological and pathological angiogenesis. The Ang/Tie signaling pathway of the endothelial cells (ECs) was essential during embryonic vessel assembly, maturation and functions as a key regulator of adult vascular homeostasis. Roxarsone (Rox), a kind of organic arsine feed additives which can promote ECs growth in vivo, might be potential cancer risk. The angiogenic effect of roxarsone in vivo or in vitro had been researched in our provious studies. In this paper, the effect of Ang-2/Tie-2pathway of roxarsone on endothelial cells promotion was investrated by using MTT method, immunofluorescence test, Elisa assay, Real-time PCR and Western Blot analysis when Ang-2and Tie-2was blocked by antibody in rat ECs culture.The different treatment on rat ECs was divided as9groups:0.10μM,1.00μM,10.00μM Rox;50ng/mL antibody of Ang-2(Anti-A) and Tie-2(Anti-T);1.00μM Rox+50ng/mL antibody of Ang-2(1+Anti-A);1.00μM+50ng/mL Antibody of Tie-2(1+Anti-T) and PBS was as negative control,10ng/mL VEGF as positive control. After24hours or48hours incubation of ECs with above treatment, the MTT method was used to measure the cells viability, the immunofluorescence test was used to detect Ang-2and Tie-2distribution in ECs, and the Elisa method was used to test the Ang-2secretion level of cell culture medium. Western blot and RT-PCR was applied to detect protein and gene expression of Ang-2and Tie-2.The MTT data of ECs24hours incubation showed that the OD values of0.10μM to10.00μM of Rox were significantly higher than PBS group (P<0.05), and the highest was at1.00μM Rox. The cells viability in1+Anti-A group and1+Anti-T group was significantly lower than1.00μM of Rox group (P<0.05). The results of48hours ECs incubation were same as that of24hours treatment. Results indicated that Ang-2and its receptor Tie-2might be associated with the promotion effect of Rox to ECs growth. The immunofluorescence assay showed that Ang-2was distributed in the cytoplasm, and the fluorescence of Tie-2was mainly distributed among the cell membrane.In Elisa analysis of Ang-2content in culture supernatant of ECs, results showed that Ang-2level of1.00μM and10.00μM Rox group was significantly higher than that of PBS group (P<0.05),0.10μM Rox group was higher than PBS group but there was no significant difference (P>0.05). The level of Ang-2of1.00μM Rox group was the highest one. The Ang-2contents were markedly risen up after blocking Tie-2(P<0.05). Results indicated that Rox might stimulate Ang-2secretion in ECs, and1.00μM Rox had a strongest promotion of Ang-2 expression, Ang-2secretion level might be relate with Tie-2status (blocked or not).According to the Western Blot analysis of Ang-2and Tie-2in ECs, the Ang-2expression of0.10μM,1.00μM,10.00μM Rox, VEGF group and Anti-T group was significantly higher than that of PBS group (P<0.05). Ang-2expression of1+Anti-A group was significantly lower than1.00μM Rox group (P<0.05). The Tie-2expression of VEGF group and0.10～10.00μM Rox group was significantly higher than that of PBS group (P<0.05),1+Anti-A group and1+Anti-T group was significantly lower than1.00μM Rox group (P<0.05). The maximum expression of Ang-2and Tie-2was at1.00μM Rox. Results showed that Rox can promote both Ang-2and Tie-2expression in ECs, indicating that Ang-2/Tie-2played a significant role in growth promotion of Rox to ECs based on the Ang-2/Tie-2protein expression level.In the RT-PCR assay, Ang-2gene expression of VEGF group and1.00μM Rox group was significantly higher than PBS group (P<0.05), Rox0.10μM and10.00μM group was higher than PBS group, but there was no significant differences (P>0.05). The transcription of Anti-T group was significantly higher than that of PBS group (P<0.05). The Ang-2mRNA of1+Anti-T group was significantly higher than that of1.00μM Rox group (P<0.05). Tie-2gene expression of different groups included VEGF group,0.10μM～10.00μM Rox groups, Anti-A group and Anti-T group were all significantly higher than that of PBS group (P<0.05). The1.00μM Rox group was the highest one. The Tie-2gene level of1+Anti-A group and1+Anti-T group was significantly lower than that of1.00μM Rox group (P<0.05). Results indicated that Rox can obviously promote Ang-2and Tie-2mRNA expression, implying Ang-2/Tie-2pathway was associated with the promotion effect of Rox in ECs based on the mRNA expression of Ang-2/Tie-2.In summary, the proliferation of ECs and Ang-2and Tie-2expression can be promoted at the range of0.10to10.0μM roxarsone tested, and Ang-2/Tie-2pathway played a significant role of roxarsone to growth promotion of ECs. |
PDF Full Text Request