Effect Of IL-17on The Expression Of Monocyte Chemoattractant Protein-1in The Murine Cardiac Myocytes

Background and objectiveMyocarditis is an inflammatory disease characterized by mononuclear cell infiltration, cardiac myocytes degeneration and necrosis. The most common cause of myocarditis is viral infection. In recent years, with more and more clinical cases, viral myocarditis (VMC) has threatened people’s health. The pathogenesis of VMC has not been entirely known. And it’s considered that the virus could damage the myocardium in the early stage and the autoimmunity could mediate the severe chronic myocardiopathy.As a proinflammatory cytokine secreted by Th17, a newly discovered CD4+helper T cell subsets, IL-17can mediate the inflammatory cells to local invasion and tissue damage by inducing the expression of other inflammatory factors and plays an important role in the inflammatory diseases especially the autoimmune diseases. Monocyte chemoattractant protein-1(MCP-1), with strong chemotaxis to monocytes, is the first discovered CC chemokine family member and its role in inflammatory diseases has attracted the people’s attention. Previous studies have shown that MCP-1plays an important role in the VMC pathogenesis by inducing inflammatory cell infiltration. Other studies confirmed that IL-17got involved in inflammation and mediated mononuclear cell migration by activating a variety of cells to produce MCP-1. Th17cells and IL-17have also been shown to be closely related to the occurrence and development of the VMC. But it has not been reported whether IL-17gets involved in the development of VMC through mediating inflammatory cell infiltration to myocardium by inducing the expression of MCP-1.To determine the cardiac myocytes being target cells of IL-17, this study firstly detected the expression of the IL-17R. Then the effect of IL-17on the expression of MCP-1was investigated in the primary cultured cardiac myocytes. The results of this study will provide the experimental evidence for revealing roles of IL-17and MCP-1in the pathogenesis of VMC.Methods(1) The cardiac myocytes were isolated from neonatal mice for primary culture by different adhesion method.(2)IL-17RA mRNA in the cardiac myocytes was detected by reverse transcription-polymerase chain reaction (RT-PCR) and the expression of MCP-1mRNA in the cardiac myocytes was analysed by semiquantitative RT-PCR through grayscale scanning.(3) The concentration of MCP-1in the culture supernatant of the cardiac myocytes was detected by enzyme-linked immunosorbent assay (ELISA).(4) Experimental groups:there were four groups containing IL-17treatment group, anti-IL-17monoclonal antibody group, the isotype-matched control (IgG) group and control group. And there were six groups including IL-17treatment dose-response groups and time-response groups. The experiments were all repeated three times.(5) SPSS17.0statistical software was applied for the data statistics. The experimental data was expressed with the mean±standard deviation, the two groups were compared with single factor analysis of variance and the test results of P<0.05for difference was statistically significant.Results(1) The cardiac myocytes of the neonatal mice were successfully cultured. The purity of the cultured cardiac myocytes was more than95%by observing the morphology and pulsation.(2) The PCR products amplified by specific primers for IL-17RA were detected by electrophoresis and the target bands were found, indicating that there was the expression of IL-17RA in the cardiac myocytes.(3) After stimulated by IL-17with final concentration of50ng/mLfor8h, MCP-1mRNA and protein in the cardiac myocytes were significantly increased compared with control group(P<0.05). And MCP-1mRNA and protein in the cardiac myocytes of the neutralizing anti-IL-17monoclonal antibody (10ug/mL) group were not statistically significant with the control group (P>0.05). MCP-1mRNA and protein in the cardiac myocytes were significantly increased in the isotype-matched control (IgG)(10μg/mL) group with the control group and the anti-IL-17monoclonal antibody (10μg/mL) group (P<0.05), but they were not statistically significant compared with IL-17treatment group (P>0.05). The effect of IL-17inducing the expression of MCP-1in the cardiac myocytes was completely abrogated by a anti-IL-17antibody, indicating that it was specific for IL-17.(4) After stimulated by IL-17with final concentration of1,5,10,50,100ng/mL for24h, MCP-1mRNA and protein in the cardiac myocytes were significantly increased in a dose-dependent manner compared with that of culture medium control(P<0.05). After stimulated by IL-17with final concentration of50ng/mL for2,4,8,12,24h,the amount of MCP-1mRNA in the cardiac myocytes was the highest at4h, and then the amount of MCP-1mRNA in the cardiac myocytes began to descend. The concentrations of MCP-1in the culture supernatant of the cardiac myocytes increased in a time-dependent manner and had significant differences with the control grou(p<0.05).Conclusions(1) The cardiac myocytes could express IL-17RA and was the target cells of IL-17.(2) IL-17could up-regulate the expression of MCP-1in the cardiac myocytes.(3) IL-17could induce the expression of MCP-1in the cardiac myocytes in dose and time dependent manners.(4) IL-17may play an important role in VMC partly through inducing the expression of MCP-1in the cardiac myocytes to mediate inflammatory cell infiltration to myocardium.