Effect Of CD40siRNA On Expression Of IL-22, IL-6 And TNF-α In Peripheral Blood Of Rats With Experimental Autoimmune Myocarditis

Research background:Viral myocarditis is defined as degeneration and necrosis of myocardium, infiltration of inflammatory cells and fibrin, the gold standard for diagnosis based on histopathology from an endomyocardial biopsy. It is the leading cause of sudden death in adolescent. The mechanism of myocarditis is unclear up to now. There is compelling evidence that the autoimmune reaction by cardiac myosin after virus infection is an important pathogenesis of myocarditis. Myocarditis is a T cell-mediated autoimmune disease. Experimental autoimmune myocarditis (EAM) is recognized as an animal model of autoimmune response mechanism in viral myocarditis. Recently, Th22 has been recognized as a novel CD4+T cell subset, which is characterized by production of IL-22. IL-22 plays a role of pro-inflammatory or anti-inflammatory depending on the way they participating in the immune response. IL-22 can increase the MCP-1 in synovial fibroblasts and the inflammatory cytokines in colonic subepithelial myofibroblasts as a pro-inflammatory cytokine. On the contrary, IL-22 can reduce the myocardial tissue inflammation by the down-regulation of immunologic molecules. IL-22 was increased significantly in viral myocarditis and decreased after the use of anti-IL-22 Ab. Anti-IL-22 Ab can reduce the inflammation of myocarditis significantly. IL-22-Ig gene therapy can decrease the inflammation of myocarditis and increase the expression of IL-22. RNA interference (RNAi) participate in the defense mechanisms involved in eukaryotic cells by siRNA, and regulate any target genes expression in the transcription. Our recent results indicated that CD40siRNA can ameliorate myocarditis, but its moleculer mechanism is not clear.Objective:To explore the change rule of IL-22 in EAM group, and the effect of CD40siRNA on expression of IL-22, IL-6 and TNF-α in peripheral blood of rats with EAM.Methods:Forty eight male Lewis rats of 6-8 weeks were randomly divided into normal control group, EAM group, CD40siRNA group and siRNA group. On days 0 and 7, 0.2ml emulsion containing cardiac myosin and completed Freund adjuvant was injected in footpad in rats of EAM group, CD40siRNA group and siRNA group. The same volume of PBS buffer was injected in rats of normal control group. On day 7, CD40siRNA Lentiviral vector (5×107TU) and siRNA Lentiviral vector (5×107TU) were injected in caudal vein in rats of CD40siRNA group and siRNA group respectively. On day 21,6 rats were sacrificed respectively in each group, on day 56, the remaining rats were sacrificed, we observed histopathologic and ultrastructure changes by light and electron microscope, myocardial histopathologic scores were counted, then cTnT, BNP, IL-22, IL-6 and TNF-α were tested by ELISA.Results:1. The total incidence of EAM group, CD40siRNA group and siRNA group was 100%. There was no rats died in normal control group and CD40siRNA group until 56 day, on the contrary, two rats were respectively died in EAM group and siRNA group. The mortality in each group has no statistical difference (P>0.05).2. In the acute and chronic phase, myocardial histopathology scores in CD40siRNA group was significantly lower than that in EAM group (P<0.05), there was no difference between siRNA group and EAM group (P＞0.05). The myocardial histopathology scores in CD40siRNA group of chronic phase was significantly lower than that in acute phase (P<0.05), there was no obvious difference between myocardial histopathology scores in the acute and chronic phase in EAM group (P> 0.05).3. In the acute and chronic phase, the serum level of cTnT and BNP in CD40siRNA group was significantly lower than that in EAM group (P<0.05), there was no difference between siRNA group and EAM group (P＞0.05). The cTnT and BNP of CD40siRNA and EAM group in the chronic phase were obviously lower than that in the acute phase (P<0.05) and the serum level of cTnT and BNP had positive correlation with myocardial histopathology scores (r=0.732, r＝0.869, P<0.05).4. In the acute and chronic phase, the serum level of IL-22 in CD40siRNA group, EAM group and siRNA group was significantly higher than that in normal control group (P<0.05), it’s level in CD40siRNA group was significantly higher than that in EAM group (P<0.05), there was no difference between siRNA group and EAM group (P>0.05). The serum level of IL-22 of CD40siRNA and EAM group in the chronic phase was obviously higher than that in the acute phase (P<0.05).5. In the acute and chronic phase, the serum level of IL-6 and TNF-α in CD40siRNA group, EAM group and siRNA group was significantly higher than that in normal control group (P<0.05), it’s level in CD40siRNA group was significantly lower than that in EAM group (P<0.05), there was no difference between siRNA group and EAM group (P>0.05). The serum level of IL-6 and TNF-α of CD40siRNA and EAM group in the chronic phase was obviously lower than that in the acute phase (P<0.05).6. There has no correlation of IL-22 with myocardial histopathology scores, the serum level of cTnT and BNP in EAM group(r=0.104, r=-0.507, r=-0.039, P>0.05). IL-6 and TNF-α has positive correlation with myocardial histopathology scores, the serum level of cTnT and BNP (IL-6:r=0.764, r=0.962, r=0.810; TNF-α:r=0.697, r=0.946, r=0.750, P<0.05)Conclusion:1. CD40siRNA can relieve the inflammation in EAM, reduce the degree of myocardial injury and improve the function of heart.2. IL-22 was increased obviously in EAM group, it’s level was obviously higher in the chronic phase than that in the acute phase.3. IL-6 and TNF-α was increased obviously in EAM group, it’s level was obviously lower in the chronic phase than that in the acute phase. IL-6 and TNF-α had positive correlations with myocardial histopathology scores, the serum level of cTnT and BNP, we can use them as important indices to evaluate the degree of myocarditis.2. The CD40siRNA mechanism may be related with the up-regulation of IL-22 expression and the down-regulation of IL-6 and TNF-α.