The Methylation Of IL-10Promoters In Peritoneal Macrophage From Rats Of Adjuvant-induced Arthritis

DNA methylation is an epigenetic mechanism. It can regulate several biologicalprocesses, such as gene transcription, X-chromosome inactivation, genomic imprintingand chromatin modification. Methylation is a process that transfers a methyl group fromthe methyl donor S-adenosyl-L-methionin(SAM) to the cytosine ring of deoxycytosinebases in CG pairs. It is maintained by DNA methyltransferases (DNMTs). Accumulatedevidence demonstrates that DNA methylation may lead to genic mutation and genesilence. It plays an important role in carcinogens, tumor development, autoimmunedisorders and others.Rheumatoid arthritis (RA) is a disease of the most common inflammatory reaction,it is a chronic autoimmune disease which attacks the linings of joints, thereby causingdestruction, inflammation and pain in the joints. The pathogenesis of RA is verycomplex. Such researches have shown that DNA hypomethylation contributes to thechronicity of RA and could be responsible for the limitation of current therapies. IL-10is a multifunctional cytokine with a wide range of biologic activities. It is secreted bymany cell types but especially mononuclear phagocyte and T cells. So many studieshave implicated IL-10is a very important factor in the pathogenesis of RA. Levels ofIL-10in patients with RA are significantly lower than healthy people and are correlatedwith disease activity. However, why IL-10express less in RA patients, we have knownonly a little. So we questioned whether the level of DNA methylation in the IL-10 promoter may be relevant to IL-10production and may be a possible contributory factorin the inflammatory pathogenesis of RA. In this study, we choice peritonealmacrophages (PMФ) from adjuvant-induced arthritis (AA) rats and RAW264.7to testours conclusions.The article is divided into three sections as follows:Objective：In this article，we study the methylation status of IL-10on peritonealmacrophages(PMФ) in adjuvant-induced arthritis(AA) rats and mice mononuclearmacrophages,with the purpose of finding the relation between DNA methylation andRheumatoid Arthritis. Methods: Freund′S complete adjuvant(FCA)was used to induceAA in rats; The changes of secondary paw-swelling were observed and scored；Pathological changes in synovium of joint were examined by Hematoxylineosin (HE)stain method. Peritoneal macrophages (PMФ) from healthy controls and AA rats wereisolated. The PMФ from AA rats were stimulated with5-Azacytidone (5-azaC). Theexpressions of IL-10, DNMT1mRNA were detected by reverse transcriptionpolymerase chain reaction (RT-PCR) analysis. Detected the expression of IL-10in PMФculture supernatant by ELISA. The methylation status of IL-10promoter wasdetermined by methylation specific polymerase chain reaction (MSP). The expressionof protein of DNMT1was measured by western blot analysis. Cultivate the micemononuclear macrophages RAW264.7，and stimulated it with lipopolysaccharide (LPS)followed by5-azaC. Detect the targets what are detected in PMФ. Result: Understandard growth conditions, IL-10expression was significantly decreased and itsprompter was methylation with the rise of DNMT1in PMФ from AA rats andRAW264.7which was stimulated by LPS.5-azaC treatment reverse the methylation ofIL-10promoters and up-regulated its expression in these two cells, whichdownregulated mRNA and protein levels of DNMT1. Conclusion: The CpG island inthe IL-10gene promoter was specifically methylated to down-modulate the expressionof IL-10protein in PMФ from AA rats and RAW264.7which was stimulated by LPS, it may play a role in the pathogenesis of RA.