Pharmacological And Toxicological Bioactive Ingredient Screening Of Cyadox

As a new growth promotion feed additive, cyadox shows lower toxicity compared with other quinoxalines. Cyadox could be metabolited to different chemicals, and the main metabolites of cyadox were cyl, cy4, cy6, cy12. But the bioactive ingredient of cyadox was not defined before now, so it is necessary to compare and screen the bioactive ingredient of cyadox, which made much sense to undstand the pharmacology and toxicology mechanism of cyadox.The choice of pharmacological and toxicological indicator of cyadox were the bottlenecks of this assay, after pre-experiment, we set EGF mRNA expression level as pharmacological indicator and cell viability parameters as toxicological indicator.1The pharmacological bioactive ingredient screening of cyadoxThe EGF mRNAexpression level was detected by RT-qPCR after incubated with cyadox at different concentrations and time points, we found that the EGF expression was highest at2μM,1h compared with control, which were selected as the best incubation condition. At this condition, we found that EGF mRNA level was about2fold higher than control after incubated with cyadox, while cy12was about1.3fold higher than control and cy6, cy4, cyl almost had no effect on EGF expression. The ability of those chemicals to up regulating EGF expression were defined as follows, cyadox>cyl2>cy6>cy4>cyl. Cyadox induced EGF mRNA level was down-regulated when incubated with PI3K and Akt inhibitor, which indicated that EGF was regulated by PI3K-Akt pathway. The FOXO1and p53were mildly up-regulated by cyadox (2μM). When treated with Akt inhibitor, FOXO1was significant up-regulated while p53was down-regulated. When treated with ROS scavenger (NAC), FOXO1and p53expression level showed no difference compared to control. When treated with PI3K inhibitor, no change was found, which indicated that Akt was activated by cyadox but not PI3K and Akt was possibly activated by ROS. Western blot detecting of Akt was carried out, we found that cyadox induced p-Akt was significant higher than its metabolites.Structure-effect analysis indicated that N-0group was important to the growth promotion effect of cyadox, and N-0group was the function group of cyadox. ROS release was elevated by N-0metabolism, ROS release was increased at15min after treated with2μM cyadox and was highest at about60min. Cyclin D1mRNA level was up-regulated at about2h and was highest at about8h, and protein expression level of cyclin D1was significant higher at8h and12h than control.Those results indicated that Akt was activated by ROS which was triggered by cyadox metabolism. EGF and cyclin D1expression level were up-regulated by p-Akt, which promoted cell proliferation and growth.2The toxicological bioactive ingredient screening of cyadoxAs MTT and LDH assay showed that cell toxicity was appeared after treated with cyadox and its metabolites(100μM), and cyadox treated group was significant higher than its metabolites treated group, which indicated that the cell toxicity of cyadox was reduced after metabolic. The cell toxicity was defined as follows, cyadox>cy12>cy4> cy6>cyl. Cyadox induced EGF, FOXO1and p53expression presented a significant does-response effect, EGF and FOXO1were down-regulated as the concentration increase, while p53was up-regulated. When Akt was inhibited, p53was significant down-regulated, while FOXO1appeared no changes compared to control. When PI3K was inhibited, p53and FOXO1expression appeared no changes compared to cyadox treated group, which indicated that Akt was activated but not PI3K by cyadox. p-Akt expression level was detected after incubated with cyadox and Akt inhibitor, and the result showed that p-Akt was higher than control.ROS released detecting result indicated that cyadox significant increased ROS release compared with its metabolites, and ROS release was increased when the incubating dose was increased. ROS release was highest at about90min(100μM), and cell apoptosis was significant increased at this concentration.To sum up, we analyzed the time-response and does-response effect of cyadox and its metabolites when treated with inhibitors, and compared the pharmacological and toxicological effect of cyadox and its metabolites. We concluded that N-0group induced ROS release controlled the pharmacological and toxicological effect of cyadox. When treated with low does cyadox, Akt was mildly activated by ROS and oxidative phosphorylation was activated which resulted in cell division and growth promotion. When treated with high does cyadox, Akt was severe activated by ROS and oxidative phosphorylation was activated excessively which stimulated cell metabolism and more ROS was produced by mitochondria. And on the other hand, p53pathway was activated which promoted cell apoptosis. We could conclude that the concentration of ROS induced by cyadox played an important role in the effect of cyadox.