The Molecular Mechanism Of Gromwth Hormone MRNA Expression Regulated By Cyadox In GH3 Cells

Recently,more and more antimicrobial growth promoters have been severely restricted throughout the breeding production.The new drug of quinoxaline,cyadox,attracted people's attention,because it not only shows obvious antimicrobial growth promotion effect but also has many advantages:little side-effect,high-security,fast absorption,eliminated rapidly,no remains,and so on.With the purpose of better popularizing the registration and using of cyadox,our labatory made a mount of research about the pharmacological effect.Previous research results have revealed that in vivo and in vitro cyadox can upregulate GH expression level,and that a large amount of genes and proteins were expressed differentially in GH3 cells induced by cyadox.Among a number of genes expressed differentially after the treatment of cyadox for 1h,early growth response gene 1(Egrl)expression level was the highest.Egrl,a zinc finger transcription factor,belongs to immediate early genes(IEGs)family,and plays an important role in cell physiological effect by altering target genes expression.Thus,the study speculated that Egrl took part in the pharmacological action of cyadox.Although many studies have shown that cyadox can upregulate GH synthesis,but we still didn't kown which signaling pathway was involved in.Yet our study couldn't make sure whether Egrl was involved in GH mRNA expression regulated by cyadox.This research aims to resolve these questions.1.Determine the signaling pathway of GH mRNA expression induced by cyadox in GH3 cellsIn order to determine the signaling pathway of GH mRNA expression induced by cyadox in GH3 cells,seven inhibitors were involed in,and GH mRNA expression level was detected by real-time quantitative PCR.All of the inhibitors were diluted with basal medium.Following incubation with 2?M cyadox for 1h,our results show that the blockades of the AC/PKA,Jak/PI3K,and Ras/ERK pathways reversed the cyadox-induced upregulation of GH mRNA expression,while the inhibitor of p38 pathway did not affect GH mRNA expression level induced by cyadox.In addition,it was noteworthy that the inhibitors of AC/PKA and ERK pathways modified cyadox-induced GH mRNA expression most significantly.2.Acquire the time-effect relationship and signaling pathway of Egrl mRNA expression induced by cyadox in GH3 cellsIn the present study,to better understand the upstream of Egrl transcription induced by cyadox,the effect of ERK inhibitor,20?M PD98059 was analyzed.Firstly,we detected Egrl mRNA expression level with the treatment of 2?M cyadox for 0.5h,1h,2h,4h,and 8h.Results revealed the Egrl mRNA level was the highest for 1h incubation.And then,the cells were pretreated with ERK inhibitor,20?M PD98059,for 1h before exposure to a 1h challenge with cyadox.Study data showed PD98059 could counteract the Egrl upregulation effect induced by cyadox.These findings indicate that cyadox induce the up-regulated expression of Egrl gene mediated by ERK signaling pathway.3.Grope for the optimal condition of RNA inference in GH3 cellsTo further study the biological action of Egrl gene,RNA interference(RNAi)was established as a powerful way to silence Egrl gene expression.Lipofectamine 2000 transfection reagent was used to transfect FAM-siRNA,which had been signed with fluorescent tags,into GH3 cells.The transfection efficiency was observed by inversion fluorescence microscope to ascertain the dose of lipofectamine 2000.And then,negative and positive control siRNA were transfected into GH3 cell to optimize the transfection conditions.Lastly,three Egrl-siRNA oligos were transfected into GH3 cells,and detect Egrl gene transcription level and protein level by real-time quantitative PCR and western-blot.Given these transfection experiment results,we choosed 40pmol Oligo 1183siRNA to conduct subsequent RNA interference experiments.4.Functional analysis of Egrl in GH mRNA expression induced by cyadoxTo analyze the function of Egrl in GH mRNA expression induced by cyadox,the study detected GH gene expression level and CREB phosphorylation level,and found that both of the levels were decreased significantly.These results revealed Egrl was involved in GH mRNA expression induced by cyadox through mediating CREB phosphorylation.Furthermore,the relationships between Egrl and some other differential genes(Slc30a1,Dusp6,Mtla,and Nr4al)were also analyzed.Results showed that it was a complicated regulatory system among these differential genes,because those genes might produce physiological effects influenced by the concentration of metal ions.In addition,Nr4al,a member of the IEGs family,might take effect independent of Egrl.In conclusion,cyadox upregulated GH mRNA expression through AC/PKA,Jak/PI3K,and Ras/ERK signaling pathways,and upregulated Egrl gene transcription by ERK1/2 signaling pathway in GH3 cells.Moreover,Egrl was involved in cyadox-induced GH mRNA expression by regulating CREB phosphorilation.Combining the results of previous studies,we suggest that MAPK/ERK played an important role in the pharmacological effects of cyadox.The differential gene,Nr4a1,a zinc finger transcription factor,as well as Egrl,could be transcribed rapidly with many stimulants,while the relationship between the other 3 differential genes and Egrl was more complicated.This study ascertained Egrl could influence the CREB phosphorilation for the first time,improved the molecular mechanism of cyadox-induced GH expression,provided data support for systematically clarifying the growth-promotion mechanism of cyadox,and provided a valuable reference for further development and utilization of cyadox.