Effects Of Cyadox On DNA Methylation,Histone Modification And Structure Of Chromatin

Cyadox belongs to the family of quinoxaline,and also acts as growth promoter especially in food producing animals.Though,a large number of studies have been conducted on the pharmacological mechanism of Cyadox,while the molecular mechanism as a growth promoter is still unclear.Previously,researchers have studied the gene expression,protein and hormones secretions,the phenotypic changes,etc,but the mechanism of growth in organism is very complex.Further study is needed to better study about the function of Cyadox as a growth promoter.The change in epigenetics mainly refers to the modification of genetic materials,such as DNA methylation,histone modification and chromatin remodeling.The modification in these materials can regulate the expression of genes,and plays an important role in the growth and development.Can Cyadox cause the changes in body,epigenetics? Can Cyadox affect the growth of the body by influencing the changes in epigenetics? The answer is unknown and still needs further study.In the growth and development of animal body,the endocrine system plays an important role.While IGF-1 is a necessary object for the normal growth and development of the body,and the liver is not only the main secretion organ of IGF-1,but also a place for a variety of different drugs to metabolize and contact with the body.Therefore,in order to know better about the mechanism of growth promoting by Cyadox,the recombinant adenovirus(r GH)was used as the control drug.The gene of IGF-1 was used as the starting point and the rat hepatocytes(BRL cells)were used as experimental materials for the current study.1.To abtain the best time and concentration of Cyadox for the treatment of BRL cell(Screening the most favorable conditions for cell growth)In the present study,the MTT method was used for the detection of succinate dehydrogenises(SDH)which was produced in the mitochondria of the cells,and to determine the best time and concentration of Cyadox and r GH(control drug)for the treatment of BRL cell in combination with cell growth status and IGF-1 m RNA expression.The results showed that the concentration of SDH was higher when BRL cells were treated with 1 ?mol/L of Cyadox and 0.163×2-8 IU/m L r GH for 4 h,and the status of cells was good.The IGF-1 m RNA up-regulated expression was confirmed by RT-PCR.While the up-regulated expression of GHR m RNA and IGF-1R m RNA has also been observed,it was good for the growth and development of cells.So the condition that BRL cells were treated with 1 ?mol/L Cyadox and 0.163×2-8 IU/m L r GH for 4 h could be used for the following studies.2.Influence of Cyadox on DNA methylation in BRL CellsIt was found that DNA hypermethylation occurred in genome of BRL cells after treated with 1 ?mol/L Cyadox and 0.163×2-8 IU/m L of r GH(control drug)for 4 h respectively.It was observed that DNA hypermethylation could inhibit the expression of some genes.While BRL cell genomic DNA methylation level was increased with the increase concentration of Cyadox,but would not be changed with time.However,the methylation level of IGF-1 gene in most promoter region was lower when BRL cells were treated with 1 ?mol/L of Cyadox and 0.163×2-8 IU/m L r GH for 4 h,and it was detected by BSP.The phenomenon was good for the transcription factor to bind to the gene IGF-1 promoter region,it formed a transcriptional initiation complex,which was helpful in the transcription of IGF-1 gene and the expression of IGF-1 m RNA level.The changes of DNA methylation level in cells might be related to DNA methylation-related enzymes,such as DNMT 1,DNMT 3A and DNMT 3B examined by RT-PCR.The DNA methylation could be regulated by DNMTs which might be influenced by the drugs.3.Influence of Cyadox on histone modification in BRL CellsThe levels of H3K4me3 and H3K27 ac in cells were related to the transcription level of the gene,which had an important effect on the growth and development of the cells.It was found that both Cyadox and r GH(control drug)caused a significant decrease in the levels of H3K4me3 and H3K27 ac in BRL cells.H3K4me3 and H3K27 ac levels in BRL cells became lower with the passage of time in the cells treated with drugs from 0~4 h.This phenomenon was not good for the transcription of genes.The results of Ch IP-q PCR displayed that the levels of H3K4me3 and H3K27 ac in IGF-1 promoter region of BRL cells were significantly increased and it could also increase the expression of IGF-1 m RNA level when BRL cells were treated with Cyadox or r GH.By this study it has been found that the levels of H3K4me3 and H3K27 ac might be regulated by enzymes related to H3K4me3 and H3K27 ac.4.Influence of Cyadox on changes of chromatin structure in BRL CellsTo investigate regarding the influence of Cyadox and r GH(control drug)on changes in chromatin structure of BRL cells,Acetyl-Histone H3(Lys)(D5E4)XP Rabbit m Ab and Tri-Methyl-Histone H3(Lys4)(C42D8)Rabbit m Ab were used to show results by laser scanning confocal microscope.The results showed that the spaces were smaller and the gap among the chromatin was larger in the groups that treated with Cyadox and r GH,and the inhibitor 5-aza-2-deoxycytidine could make the contrary results.So it could be inferred that the structure of chromatin in BRL cells was changed in the groups that treated with Cyadox and r GH,and it became more loose,and it was conducive to the formation of transcriptional complexes,which might be more favorable for the transcription and expression of genes,such as IGF-1 gene.To analyze the present results,it was observed that Cyadox and r GH might affect the level of DNA methylation by regulating DNMTs.On one side they could increase genomic DNA methylation level in cells and inhibit the expression of some genes.While it also cause hypomethylation at the Cp G sites in the promoter regions of IGF-1 and could increase the transcription of IGF-1.Secondly,Cyadox and r GH might affect the level of H3K4me3 and H3K27 ac by regulating enzymes that relate to H3K4me3 and H3K27 ac.The level of H3K4me3 and H3K27 ac became lower with the passage of time that BRL cells were treated with Cyadox and r GH increased in 4 h and inhibit the expression of some genes.But high level of H3K4me3 and H3K27 ac in the promoter regions of IGF-1 increased the transcription of IGF-1.Additionaly,the structure of chromatin in BRL cells was changed in the groups that treated with Cyadox and r GH,and became looser,and it was conducive to the formation of transcriptional complexes,which may be more favorable for the transcription and expression of genes,such as IGF-1 gene.It was the first time to study whether Cyadox can make changes of epigenetics,and the relationship between changes of epigenetics,growth and development of body.The r GH was used as the control drug and all the changes in the cells and IGF-1 gene were used as the starting point to conduct this research.This study offers knowledge on theoretical basis to know about the further mechanism of Cyadox to promote growth.And it also provides ideas and references for the study of promoting growth of antibiotic.