Effect of gene silence of CD40 by small interfering RNA on balance between Th1/Th2 and Th17/Treg in rats with experimental autoimmune myocarditis

Abstract

Objective To investigate how gene silence of CD40 by small interfering RNA(siRNA) influences the balance between Th1/Th2 and Th17/Treg in rats with experimental autoimmune myocarditis(EAM). Methods Lewis rats were divided into a normal-control group, EAM group, CD40siRNA-therapy group and siRNA-control group by using random number table.EAM, CD40siRNA-therapy and siRNA-control groups were immunized on day 0 and day 7 with purified porcine cardiac myosin to establish EAM, and CD40siRNA was administered intravenously on day 7.The basic data of each group were observed, and the rats were executed on day 21 to study the pathology of myocardial tissues and the myocardial histopathology scores were calculated, and than the changes in electrocardiograms (ECG) and echocardiogram were recorded.Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of interferon (IFN) -γ, interleukin(IL) -10, IL-17 and transforming growth factor(TGF)-β in peripheral blood. Results (1) The overall incidence of EAM, CD40siRNA-therapy and siRNA-control group was 100%, and no rats died from day 0 to day 21.(2) There were only 2 premature rats in EAM and siRNA-control groups, and the ECGs of normal-control group and CD40siRNA-therapy group were normal.(3) The echocardiogram of EAM, CD40siRNA-therapy and siRNA-control group, displayed the left ventricular dilatation, hypertrophy of the left ventricle, the weakening of the left ventricular wall motion, and heart failure, and pericardial effusion was discovered.The left ventricular fractional shortening(LVFS) and the left ventricular ejection fraction (LVEF) of rats treated by CD40siRNA were higher than those of rats in the EAM group[(46.70±8.25)% vs (34.28±11.81)%, (80.92±11.76)% vs (64.03±12.60)%, F=0.652, 0.234, all P<0.05]. (4) Pathologic examination of the EAM group, CD40siRNA therapy group and siRNA control groups, showed myocardial fiber fracture, myocardial tissue necrosis and inflammatory cell infiltration.But myocardial tissue pathological scores of the cardiac tissue of CD40siRNA therapy group was lower than those of the EAM group(2.10±1.07 vs 3.40±0.72, F=1.290, P<0.05). (5) ELISA method examination revealed that the levels of IFN-γ, IL-4, IL-17 and TGF-β in EAM group were increased compared with those of normal control group[(1 245.55±244.56) ng/L vs (501.53±18.93) ng/L, (78.03±10.47) ng/L vs (30.77±1.74) ng/L, (80.82±13.33) ng/L vs (27.98±3.01) ng/L, (156.27±12.11) ng/L vs (4.33±0.79) ng/L, t=7.408, 10.764, 9.250, 30.608, all P<0.05]. However, the levels of IFN-γ and IL-17 in CD40 siRNA therapy group were lower than those in the EAM group[(940.62±128.40) ng/L vs (1 245.55±244.56) ng/L, (60.42±12.40) ng/L vs (80.82±13.33) ng/L, t=2.704, 2.745, all P<0.05], while the levels of IL-4 and TGF-β were higher[(97.91±13.62) ng/L vs (78.03±10.47) ng/L, (178.84±12.10) ng/L vs (156.27±12.11) ng/L, t=2.835, 3.229, all P<0.05]. Conclusions The administration of CD40 siRNA markedly reduces myocardial inflammation of EAM rats and improves their cardiac function, which can be explained by Th1-type and Th17-type cytokines inhibited, Th2-type and Treg-type cytokines increased, and so then the imbalance of Th1/Th2 and Th17/Treg corrected. Key words: Autoimmune myocarditis; Rat; CD40 small interfering RNA; Th1/Th2; Th17/Treg