Receptor Screening And The Mechanism Of EGF Expression Induced By Cyadox In Swine Hepatocyte

Cyadox, a new derivative of Quinoxalines, was synthesized in1990s. Except the significant growth-promoting effect on pigs, chickens, fish, it has been ascertained as an antibiotic with low poison, high security, quickly absorption, rapidly eliminate.8differential expressed genes, include Epidermal Growth Factor (EGF), Complement Component3(C3), Insulin-like Growth Factor-1(IGF-1) and so on, were induced by Cyadox in hepatocyte of swine. Cyadox could up-regulated EGF and IGF-1mRNA in the study of hepatocyte in vitro, on the other hand, EGF mRNA was actived through PI3K and NF--κB signaling pathways. Whether cyadox could bind with a receptor or not and activate some signaling pathways to involved in the expression of the8genes, so we studied the mechanism of the8differential express genes induced by cyadox.1. Study on the binding sites of cyadox in the liver of pigInteractions3H-CYA with fractions of the hepatocyte had been studied in vivo and in vitro. Animals were sacrificed at6h and24h after administration3H-CYA, and then the subcellular fraction were separated useing differential centrifugation. Differences have been obseverd in the uptake of cyadox by various cell fractions at different time. The amount of3H-CYA attached to cytoplasm was much more than that of nucleus and membrane.We studied from two aspects in vitro, first, the subcellular fragments of liver or hepatocytes incubate with tritium labeled cyadox (3H-cyadox); second, the subcellular fragments were treated with different concentration of3H-cyadox, and whether the saturation binding or competing binding existed were observed. Methods for the separation of the cell nucleus, cell membrane, cytoplasm endoplasmic reticulum, ribosome, mitochondria, Golgi apparatus and microsome by ultracentrifugation were established. The contribution of cytoplasm to bind cyadox was relatively large, second were membrane and nucleus, the rest sucellular component binding far less.The radioactivity in the cytoplasm, mitochondria, and microsome increased with dose of3H-CYA, in the contrast, decreased in the nucleus and membrane. Saturation binding of various subcellular fractions to the drug had not been found at the durg concentration of1.48-47.23μM. The metabolite of cyadox, Cyl, Cy10and QCA, were not able to displace3H-CYA from its binding protein at concentration up to10000-folds that of cyadox. The level of second messengers, cAMP, Ca2+, IP3and DAG, were not affected by cyadox at various dose and time, demonstrating that cyadox has no binding GPCR in L02and hepatocytes.2. The studying of the mechanism of action of EGF expression induced by cyadoxThe metabolites of cyadox were identified in the medium and hepatocytes. Cyl, and so on, were detected both in the medium and hepatocytes. This means cyadox can enter the hepatocytes and be metabolized to cyadox-monoxide and bisdesoxycyadox.As for the intracellular ROS production in hepatocytes, the peak was apparent at30min, but, not stable and possibly degraded during the co-incubation with the cells, had a characterization of short and unstable release of intracellular ROS. So, we infer that the ROS was produced in hepatocytes in the process of metabolizing.The EGF mRNA was down-regulaed in the condition of the free radical scavengers of mannitol, tiron, catalase, N-acetylcysteine and procyanidine in hepatocytes, indicating that ROS is a key mediator of cyadox-induced gene.In summary, we first look for the binding protein of cyadox in the liver, and study the relationship between the cyadox metabolism and signaling pathway. The radioactivity of cyadox is the highest in the cytoplasm in vivo and in vitro, showed that the cyadox binding proteins exist in the cytoplasm. Cyadox cannot bind the G protein-coupled receptor in the cell membrance and then impact the second messagers in the intracellular. We speculate the cyadox binding proteins exist in the cytoplasm, the protein might the metabolic enzyme.Cyadox could metabolize and strip the oxygen radicals on quinoxaline ring, producing ROS which could activate some signaling pathways to up-regulate EGF mRNA expression. So we speculated that maybe the target of cyadox in the pig primary liver cells might be metabolic enzyme, the gene expression such as EGF was changed by ROS.