Effect Of Cyadox On Expression Of Genes And Proteins In GH3Cells

Cyadox, a broad spectrum antimicrobial animal-specific drug, shows greatly growth-promoting effect. Cyadox is a new member of the quinoxalines, which is derivative of quinoxaline-1,4-dioxides, it is crucial to reserve antibacterial activity at the same time reduce toxicity. In order to confirm its antibacterial growth-promoting activity and clarify the underlying molecular mechanism, our laboratory has payed attention to the study of basic toxicology, antibacterial activity, basal metabolism and mechanism of growth-promoting effect. In2007, Huiling Zhu studied the effect of cyadox on the endocrine system of swine, found that cyadox could regulate the expression of several important endocrine factors, one of which is growth hormone (GH), it is essential for animals’growth; however, the mechanism underlying the effect of cyadox on GH is still unknown. In this paper, using Affymetrix GeneChip? Rat Gene1.0ST Array chip and two-dimensional electrophoresis (2-DE) and matrix-assisted laser-desorption ionization time of flight mass spectrometry (MALDI-TOF/TOF-MS), we assessed the differently expressed genes and proteins in GH3cells following exposure to cyadox. Comprehensive analysis the results of genomics and proteomics, more information could be joined into investigate the effect of cyadox in GH3cells as well as clarify the molecular mechanism underlying growth promoting.1. Screening the optimum drug dose and time of gene chip and proteomicsOur objective is to identify the relationship among cyadox and GH synthesis and secretion in GH3cells. In our research, the mRNA expression level of GH was detected by qReal-time PCR after GH3cells were exposed to cyadox (1-8μM) for0.5-24h.The result indicated that GH mRNA expression level showed a significant dose-dependent effect, and the most up-regulation of GH mRNA expression appeared at GH3cells following exposure to2uM cyadox for1h. At the same time, ELISA kits were adopted to search the level of GH release. The result indicated that cyadox have the ability to promote GH release, especially,2μM cyadox incubation GH3cells12h significantly increased GH release. Then we used gene expression microarray to detect the differential expression genes in GH3cells following exposure to2μM cyadox for1hour and12hour, which could real-time reflect the DNA expression under certain situations.Genomics and proteomics are asynchronous, so BCA kits were adopted to detect the total protein concentration in GH3cells inducted by2μM cyadox. As the result showed, cyadox promoted the accumulation of protein in GH3cells from1h to12h, and the most up-regulation of GH protein expression appeared at GH3cells following exposure to2μM cyadox for4h. Combined with the result of ELISA, we used two-dimensional electrophoresis-mass spectrometry to detect the differential expression proteins in GH3cells following exposure to2μM cyadox for4hour and12hour.2. The differentially expressed genes in GH3cells inducted by cyadoxAnalysis the gene chip result, compared with blank,1hour cyadox group had discovered15significant changed genes (p-value≤0.01, folds≥1.5), including13up regulated genes, and2genes down regulated. Furthermore,12h microarray data showed significant alteration in the expression of226genes, including205genes up-regulation and21genes down-regulation following exposure to2μM cyadox. Then we used Gene Ontology enrichment analysis acquired in DAVID Bioinformatics Resources to classify those differentially expressed genes, which were belonging to7categories in terms of their function:cellular signal transduction, nucleic acid transcription regulation, energy metabolism, cell morphology, cell process, ion transport, and immune regulation. Choose Egrl, Dusp8, Jun, Stat2, Prkar1b, Aldoc, Slc30a and Pik3ipl in cDNAmicroarray checked by RT-qPCR to confirm the reliability of gene chip.3. The differentially expressed proteins in GH3cells inducted by cyadoxCyadox induced37differentially expressed proteins on GH3cells identified by the two-dimensional electrophoresis (2-DE) and matrix-assisted laser-desorption ionization time of flight mass spectrometry (MALDI-TOF/MS-MS) compared with control cells.4hour cyadox group had discovered23significant changed proteins,12hour cyadox group had discovered26significant changed proteins, and there were12proteins at the same time appeared in the two group; all of those proteins were associated with molecular chaperone, cell transport protein, protein kinase factor, cell morphology as well as cells metabolism.Comprehensive analysis the results of genomics and proteomics, we can draw the following solution: At the transcriptional level, Cyadox up-regulated the expression of G protein-coupled receptors, the important second messenger cAMP and cAMP response element binding protein CREB, which were involved in AC/cAMP/PKA signal transduction pathway, and they had the ability to stimulate GH mRNA esppression; Second, Pdia3, Ppia, BIP, Rab and Cacn might be some of the main molecular targets of cyadox on GH molecur biosynthesis and secretion. Cyadox increase BIP, PDI and Ppia to influence growth hormone protein molecular synthesis and processing in endoplasmic reticulum and golgi, and cyadox can activate the voltage-gated Ca2+channels in GH3cells, which are capable to activate Ca2+-triggered exocytotic membrane fusion. Beside, Ca2+as an important factor of endoplasmic reticulum and golgi apparatus can greatly influence their structure and biological function. Rab GTPases function as molecular switches, oscillating between GTP-bound (active) and GDP-bound (inactive) conformations with cycles of membrane association/dissociation. Cyadox up-regulated Rab9b, Rab14b, Rabl8which catalyze GDP/GTP exchange; The last, Cyadox can directly improve the energy supply to GH3cells by up-regulated Aldolase, Eno2, Pdg, APDH, IDH and Acly, which are closely related to glycolysis, the pentose phosphate pathway, and the Krebs cycle, and that could provide more favorable conditions for GH synthesis and release.