| BackgroundMyocarditis is inflammation of the myocardium caused by infection or non-infection, and the virus is the main pathogenic factor. Therecent study shows that, myocardial injury in patients with viral myocarditis is mainly caused by the virus infection directly and immune response indused by virus. The immune response plays an important role in the occurrence and development of myocarditis, which is mainly cellular immune response mediated by T lymphocytes. Cardiac myosin-induced experimental autoimmune myocarditis (EAM) is a model of inflammatory heart disease initiated by CD4+ T cells. According to the different cytokines, CD4+T lymphocytes is usually divided into Th1, Th2, Th17 and regulatory T cells (Treg). T cell subsets play an important role in cellular immunity and humoral immunity, and dominate the immunoregulation. From the references which have been mentioned, the balance of Th1/Th2 and Th17/Treg plays an important role in the immune response of infectious diseases(e.g. HIV), autoimmune thyroid disease and autoimmune hepatitis and other diseases, among which Th1 and Th17 showed inflammatory reaction and promote the tissue damage, while Th2 and Treg showed anti-inflammatory effect.The activation of T cells depends on antigen recognition providing a first signal and the costimulatory molecules providing a second signal. CD40/CD40L is one of the most representative costimulatory molecules, playing an important role in in infectious diseases and autoimmune diseases, and blocking which could reduce tissue injury caused by autoimmune reaction. Studies have shown that the application of CD40Ig fusion protein blocking CD40/CD40L costimulatory signal pathway has obvious improvement effect on viral myocarditis.Small interfering RNA (siRNA) is a small molecular,21-25 nucleotides in length, dominating the technology of RNA interference (RNAi), and can stimulates the silence of the target mRNA and so then treat the immune disease. The previous study of our group have proved that, blocking CD40/CD40L costimulatory signal pathway by CD40siRNA for the treatment of autoimmune myocarditis can reduce myocardial inflammation, which molecular mechanism is not yet clear.ObjectiveThis study will use CD40siRNA for the treatment of autoimmune myocarditis in rats, estimate the conditions by detecting ECG, Ultrasound and myocardial tissue pathological changes, and explore the treatment mechanism of CD40siRNA through testing the cytokines secreted by Th1, Th2, Th17 and Treg.MethodsLewis rats were divided into normal control group, EAM group, CD40siRNA therapy group and siRNA control group randomly. Experimental autoimmune myocarditis model was induced by myosin from porcine heart on day 0 and day 7, and CD40siRNA therapy group was given CD40siRNA for treatment on day 7. The basic information of each group were observed, and the rats were executed after the changes of electrocardiograms (ECG) and echocardiogram were recorded on day 21. The pathology of myocardial tissue was observed and the myocardial histopathology scores were calculated. Enzyme linked immunoabsorption assay (ELISA) was used to determine the level of IFN-γ, IL-10, IL-17 and TGF-β in peripheral blood.Results1. The overall incidence of EAM group, CD40siRNA group and siRNA group of rats was 100%, and there was no death. The weight of the rats increased gradually from day 0, and EAM group loss of body weight from the day 6, while the weight of rats of CD40siRNA therapy group increased after day 6.2. There was only one premature in EAM group and siRNA control group respectively, while the ECGs of normal control group and CD40siRNA therapy group were normal.3. The echocardiogram of EAM group, CD40siRNA therapy group and siRNA control group, displayed left ventricular dilatation or decrease, hypertrophy of left ventricle, weaken of left ventricular wall motion, and pericardial effusion discovered. Left ventricular fractional shortening(LVFS) and left ventricular ejection fraction (LVEF) of EAM group and siRNA group were lower than normal control group (p<0.05). LVEF and LVFS of rats treated by CD40siRNA were higher than those of rats of EAM group (p<0.05).4. Pathologic examination of EAM group, CD40siRNA therapy group and siRNA control group, showed myocardial fiber fracture, myocardial tissue necrosis and inflammatory cell infiltration. The pathological score of cardiac tissue of CD40siRNA therapy group was lower than that of EAM group(p<0.05).5. The levels of IFN-γ and IL-4 in EAM group is higher obviously than these of normal control group(p<0.05). The level of IFN-γ in CD40siRNA therapy group was lower than EAM group, while the level of IL-4 is higher than EAM group (p<0.05).6. The levels of IL-17 and TGF-β in EAM group is higher obviously than these of normal control group(p<0.05). The level of IL-17 in CD40siRNA therapy group was lower than EAM group, while the level of TGF-β is higher than EAM group (p<0.05).7. The change of IFN-γ was positively correlated with the pathological score of cardiac tissue(r=0.755, p<0.05), while the change of IL-4 was negatively correlated with the pathological score of cardiac tissue(r=-0.636, p<0.05).8. The change of IL-17 was positively correlated with the pathological score of cardiac tissue(r=0.841, p<0.05), while the change of TGF-β was negatively correlated with the pathological score of cardiac tissue(r=-0.563,p<0.05)9. The change of IFN-γ was negatively correlated with the left ventricular ejection fraction (LVEF) of rats (r=-0.570, p<0.05), while the change of IL-4 was positively correlated with LVEF(r=0.521,p<0.05).10. The change of IL-17 was negatively correlated with LVEF (r=-0.635, p<0.05), while the change of TGF-β was positively correlated with LVEF(r=0.541, p< 0.05).Conclusion1. CD40siRNA can reduce myocardial inflammation and promote the cardiac functional recover.2. There is imbalance of Th1/Th2 and Th17/Treg in acute phase of EAM, mainly the immune response mediated by Th1 and Th17, and immune deviation of Th1/Th2 and Th17/Treg arises.3. CD40siRNA can inhibits the proinflammatory mediated by Th1 and Th17, and enhanced the anti-inflammatory reaction mediated by Th2 and Treg. The mechanism of CD40siRNA in the treatment of autoimmune myocarditis may be associated with correcting of imbalance of Th1/Th2 and Th17/Treg. |
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