The Effects Of Cyadox On Expression Of Genes And Proteins In C2C12Myotubes

Cyadox is one of important derivatives which belongs to heterocyclic N-oxide quinoxaline (QdNO). Compared with other quinoxalines, cyadox shows higher safety. Cyadox stimulates growth of many kinds of animals. The previous study reported that cyadox can exert the promoting growth effect by influencing the expression of IGF-1and GH. GH and IGF-1are two members of growth-promoting hormone axis, have direct and indirect impacts on muscle tissue. Whether cyadox can directly regulate the growth of muscle tissue? We need to find out whether and how cyadox influencs the growth of muscle tissue, further explore the molecular mechanism of cyadox growth promoting. So, the myotubes differentiation from the mouse myoblast line C2C12cells were used as an in vitro model in our studies. First, the appropriate concentration and time were determinated; then, differentially expressed genes and proteins were screened by gene chip technology and the coupling technique of two-dimensional electrophoresis and mass spectrometry. Preliminary discussion on the mechanism how cyadox affects muscle cells is also done in our studies.1. The screening of Cyadox treatment concentration and timeIn order to screen the appropriate concentration and time of cyadox, the concentration gradient (1μM,2μM,4μM,8μM and12μM) of cyadox were used to deal with C2C12myotubes, and the concentration of cellular total protein in C2C12myotubes at4h,8h and12h were measured respectively. Results demonstrated that it is the best processing method that use2μM cyadox incubating cells for4h. We analyzed the differentially expressed proteins in C2C12myotubes in this condition.Due to the different expression patterns between the genes and proteins, another target is chosen to screen the appropriate concentration and time of cyadox. In consideration of protein quantification results, we use2μM cyadox to treat with cells. After1h,2h,4h,6h, the mRNA expression level of MyHC gene in cells was detected through RT-PCR. When C2C12myotubes incubated with cyadox for4h, MyHC mRNA was2.4times than the blank control. Therefore, the appropriate concentration and time are2μM and4h.2. Analysis of differentially expressed genes of C2C12myotube induced by cyadoxCells were incubated with2μM cyadox for4h, then the gene chip was adopted to identify differentially expressed genes between cotrol group and treatment group. Compared with the blank control,68known genes were selected including32up-regulation genes and36down-regulation genes. According to the molecular function, these genes were divided into the following five categories:structural components, binding activity, enzyme activity and receptor activity and transcription activity. Based on the biological process, these genes were divided into the following seven categories: protein modification, metabolism, biological synthesis and regulation, transcription, stress, cell growth. Through the analysis of C2C12myotubes’ growth related gene, the possible mechanisms that cyadox affect the growth of muscle cells were conclude: on the one hand, the increasing of Tceal7mRNA expression indused by cyadox may prompt muscle differentiation and further fusion; on the other hand, cyadox affected the level of protein phosphorylation in cells. In addition, cyadox still can enhance the transcriptional regulation of mRNA in cells, affect differential expression of the related metabolic genes, and provide material and energy for cells.3. Analysis of differentially expressed proteins of C2C12myotube after dealing with CyadoxC2C12myotubes were treated with2μM cyadox for4h. Compared with control group, there were20differentially expressed proteins identified successfully by Two-dimensional gel electrophoresis and Time-of-Flight Mass Spectrometry, two were down regulation and the others were up regulation. The function of these proteins were analyzed, we found that these proteins have the binding activity and enzyme activity. And we could classify the biological processes which these proteins participated in as material and energy metabolism, biological synthesis, cell differentiation, signal transduction, and oxidative stress.Enolization enzyme Enol is significantly up-regulation expression in C2C12myotubes after dealing with cyadox, cytoskeleton-associated proteins and cells differentiation related proteins also are up-regulation, predicting cyadox makes C2C12myotubes remodeling and form more massive muscle fibers. In our research, the protein expression level of superoxide dismutase Sodl and peroxidase Prdxl increased, which can remove free radicals and H2O2, and protect cells from damage. The above results show that cyadox may generate the free radicals, forming H2O2in the metabolic process. Studies indicated that low concentration of H2O2can activate PI3K/Akt and other growth related signaling pathway, playing a important role on regulating glucose transport in muscle cells. So, we speculate that cyadox regulates growth and physiological functions of muscle cells through this mechanism.In conclusion, the main effects and mechanisms of cyadox affecting muscle cells were expounded at the molecular leve in this study. Based on the experiment results and analysis, we found cyadox can affect the reshaping and fusion of the muscle cells, promote the material and energy metabolism of cells, change the level of protein phosphorylation in cells. Additional, it is suggested that cyadox have effect on the content of H2O2in cells, indirect effects on the growth related signaling pathways, such as PI3K/Akt, then promote the growth of muscle cells.