Effect Of CD40siRNA On The Expression And Significance Of Peripheral Blood IL-21 And IL-35 In Rats With Experimental Autoimmune Myocarditis

Research backgroundMyocarditis is a common cardiovascular disease in children and adolescent. Mild patients without any clinical symptoms,but severe cases can lead to heart failure, malignant arrhythmia, Adams-Stokes syndrome and sudden death,part of the patients can be developed into dilated cardiomyopathy (DCM).The main pathological changes is focal or diffuse myocardial interstitial inflammatory exudation and degeneration or necrosis of myocardial fibers.The researchers used porcine cardiac myosin to immune susceptible mice to establish the model of experimental autoimmune myocarditis (EAM), which was very similar to the human severe giant cell myocarditis.As a ideal model for research of myocarditis, which has been widely adopted.At present autoimmune mechanisms mediated by T cells play an important role in the pathogenesis of myocarditis and the imbalance of Thl/Th2, Th17/Treg is also play very important role in the development of myocarditis.Interleukin 21 (IL-21) is a proinflammatory cytokine, mainly secreted by Th17 cells, but interleukin (IL-35) is a novel anti-inflammatory factor, mainly secreted by Treg cells, both play an important role in the pathogenesis of autoimmune diseases, such as rheumatoid arthritis (RA).experimental autoimmune meningitis (EAE), and system lupus erythematosus (SLE).The IL-21 expression in the myocardium and peripheral blood of mice with viral myocarditis (VMC) increased suggests that IL-21 is involved in the pathogenesis of myocarditis. With restructuring IL-35 intervention the viral myocarditis mice, which can delay the progression of myocarditis.The RNA interference technology is a new technology in the study of gene knock out in recent years, refers to import a period of double-stranded RNA (dsRNA) in cell,and the dsRNA has sequence homology with the endogenous mRNA in coding regions, cause the mRNA specific degradation and inhibit the target gene expression effectively, Which can block the purpose gene expression from the source.Our recent results confirmed the CD40siRNA can alleviate the myocardial inflammatory and up-regulation of CD4 +CD25+Treg expression, decrease the peripheral blood level of IL-17 and IL-23 produced by Th17 cell, up-regulation the level of IL-22 produced by Th22 cell, correct the imbalance between Thl/Th2 and Thl7/Treg. But whether the CD40siRNA has influence on the level of IL-35 and IL-21 has not been reported. This experimental application CD40siRNA in EAM rats, from the source block CD40/CD40L axis, observation and analysis the effect of CD40siRNA on myocardial pathological, echocardiogram change and peripheral blood IL-21 and IL-35 in EAM rats for further to explore its mechanism in EAM.ObjectiveTo explore the effect of CD40siRNA on the expression and significance of peripheral blood IL-21 and IL-35, myocardial pathological and echocardiogram change in rats with EAM.MethodsTwenty 6-8 weeks male Lewis rats were divided into normal group, EAM group, CD40siRNA group and siRNA group by using random number table, with 5 rats in each group. The normal rats were induced with phosphate buffer saline(PBS)in double foot pads on day 0 and day 7, the rest of three groups were induced with cardiac myosin protein to established EAM model. The rats in CD40siRNA group and siRNA group were respectively injected with CD40siRNA and siRNA slow virus expression vector through the tail vein of rats on day 7.The rats were executed on 21 day after echocardiogram examination was made. The histopathologic changes were observed by light microscope and the myocardial histopathology scores were calculated. Enzyme-linked immunosorbent assay (ELISA) was used to determine the level of IL-21 and IL-23 in peripheral blood.Results1. Except the normal group, the total incidence rate of rats in each group was 100%, and no rats died.2. The weight of normal rats increased gradually since day 0, the rest three groups weight gain is slow, especially the weight gain of EAM and siRNA group rats increase the slowest. Compared with EAM group, the weight of rats was higher in CD40siRNA group (P<0.05).3. Compared with normal group, the heart mass/body ratio was higher in EAM group (P<0.05), while the heart mass/body ratio was lower in CD40siRNA group than EAM group (P<0.05).4. The heart gross appearance of normal group rats was normal, EAM group and siRNA group respectively have one rat in the heart of the surface have pale area with focal leakage. There was no significant abnormality in the appearance of CD40siRNA group and normal group. Compared with EAM group, the myocardial histopathology scores were lower in CD40siRNA group (P<0.05).There was no difference between siRNA group and EAM group (P>0.05).5. The echocardiogram showed that EAM group has only one rat with massive pericardial effusion with, without pericardial effusion in CD40siRNA group, EAM group, CD40siRNA group and siRNA group displayed the hypertrophy of the ventricular septum and left ventricular wall, heart cavity narrow and the weakening ventricular wall motion. The left ventricular shortening rate (LVFS) in CD40siRNA group were significantly higher than the EAM group (P<0.05).6. The peripheral blood level of IL-21 and IL-35 in EAM group were higher than these in normal group (P<0.05). The peripheral blood level of IL-21 in CD40siRNA group was lower than that in group EAM (P<0.05), while its level of IL-35 significantly higher than the EAM group (P<0.05).7. The level of IL-21 in peripheral blood was positively correlated with myocardial histopathology scores in EAM group (r=0.69, P<0.05), while IL-35 was negatively correlated with myocardial histopathology scores (r=-0.64, P<0.05).Conclusions1. CD40siRNA can relieve myocardial inflammation and reduce the myocardial injury in EAM rats.2. The IL-21 and IL-35 were increased obviously in EAM group. The level of IL-21 was positively correlated with myocardial histopathology scores, while IL-35 was negatively correlated with myocardial histopathology scores. The level of IL-21 and IL-35 can be partly reflect the degree of myocardial injury.3. The level of IL-21 in CD40siRNA group was lower than that in group EAM, while its level of IL-35 significantly higher than the EAM group. The mechanism of CD40siRNA in the treatment of EAM rats may be related to down-regulation IL-21 and up-regulation the expression of IL-35.