Effects Of CD40siRNA On The Expression Of IL-37 And P38MAPK In Rats With Experimental Autoimmune Myocarditis

Research backgroundViral myocarditis(VMC)is a viral infection caused disease with diffuse or focal myocardial interstitial inflammatory cells infiltration and myocardium necrosis or degeneration,leading to different degrees of cardiac dysfunction and other system damage disease.It is one of the most common cardiovascular diseases in children and adolescents.The mild patients may have no obvious symptoms,while the severe cases may be led to arrhythmia,congestive heart failure,Adams-Stokes syndrome,cardiogenic shock,and even sudden death.Chronic myocarditis may develop into dilated cardiomyopathy(DCM)that is serious harm to human health.The experimental autoimmune myocarditis(EAM)in rat induced by purified porcine cardiac myosin is very similar to human severe myocarditis in pathological evolution process,which is considered as an ideal model to study VMC at home and abroad.The pathogenesis of myocarditis is not completely elucidated.At present,it is believed that the autoimmunity mediated by T cells plays an important role in the developing process of myocarditis.Interleukin 37(IL-37)is a newly discovered anti-inflammatory cytokine.It participates in the development of many inflammatory and autoimmune diseases.Studies have shown that IL-37 can inhibit the excessive immune response.Mitogen activated protein kinase(MAPK)is an important signal transduction pathway in cells,and p38MAPK protein is at the center in the pathway.P38MAPK is activated by trauma,inflammation and biological factors,and then translocates into the nucleus or corresponding target area of cells,which regulates cell proliferation,necrosis,fibrosis and other pathological processes.Some studies have shown that p38MAPK is involved in the development of myocarditis.CD40/CD40L is an important pair of costimulatory molecule,and the interaction between CD40 and CD40L plays an important role in the activation of T lymphocytes and initiation of immune response.CD40 small interfering RNA(CD40siRNA)has been used to block the CD40/CD40L receptor-ligand axis,which could significantly improve the general status and reduce the myocardial damage of the EAM rats in our previous studies.However,the effects of CD40siRNA on the expression of IL-37 and p38MAPK in EAM rats have not yet been reported.ObjectiveTo investigate the effects of CD40siRNA on the expression of IL-37 in peripheral blood and p38MAPK in the myocardium of EAM rats.MethodsThirty-two healthy adult male Lewis rats(6-8 weeks old,eight per group)were assigned randomly into four groups:control,EAM,EAM treated with CD40siRNA(EAM+CD40siRNA)or EAM treated with scramble siRNA(EAM+siRNA)group.EAM in Lewis rats was induced by immunization with purified porcine cardiac myosin which was injected subcutaneously into the foot pads of rats on days 0 and 7.The rats in the control group were immunized with only sterile phosphate buffer saline(PBS)in the same manner.The rats in EAM+CD40siRNA group and EAM+siRNA group were both injected via the tail vein on day 7 with CD40 and scramble siRNA mediated by lentiviral vector,respectively.All rats were sacrificed on day 21 after anaesthesia.Histopathologic changes were observed and myocardial histopathology scores were calculated.The expression levels of peripheral blood IL-37 were measured using an enzyme-linked immunosorbent assay(ELISA),the expression levels of p38MAPK mRNA in myocardium were confirmed by real-time quantitative polymerase chain reaction(qRT-PCR),and the expression levels of phosphorylated p38MAPK(P-p38MAPK)protein in myocardium were detected with immunohistochemistry and western blot.Results1.The morbidity of rats in EAM,EAM+CD40siRNA and EAM+siRNA group was 100%,and none of them died.2.The weight gain of the EAM,EAM+CD40siRNA and EAM+siRNA group is slower compared to the control group(P<0.05).However the weight gain of rats was higher in EAM+CD40siRNA group compared to the EAM group(P<0.05).And no statistic difference between the EAM and EAM+siRNA group was observed(P>0.05).3.Compared to the EAM group,the EAM+CD40siRNA group got lower myocardial histopathology scores(P<0.05).No statistic difference between the EAM and EAM+siRNA group was observed(P>0.05).4.Compared to the control group,level of peripheral blood IL-37 was decreased in the EAM group(P<0.05).And higher level of serum IL-37 was observed in the EAM+CD40siRNA group compared to the EAM group(P<0.05).No statistic difference between the EAM and EAM+siRNA group was detected(P>0.05).5.The level of IL-37 in peripheral blood was negatively correlated with myocardial histopathology score in the EAM rats(r=-0.921,P<0.01).6.The expression level of p38MAPK mRNA in myocardium was up-regulated in the EAM group compared to the control group(P<0.05),whereas the expression level of p38MAPK mRNA were down-regulated in the EAM+CD40siRNA group compared to the EAM group(P<0.05).No statistic difference was detected between the EAM and EAM+siRNA group(P>0.05).7.Significant deeper brown color and higher positive cells ratio of P-p38MAPK were observed in the myocardium immunohistochemistry section of EAM group compared to the control group(P<0.05),whereas the brown color was lighter and positive cells ratio was lower in the EAM+CD40siRNA group compared to the EAM group(P<0.05).No statistic difference between the EAM and EAM+siRNA group was detected(P>0.05).8.Compared to the control group,the expression level of P-p38MAPK protein in myocardium was up-regulated in the EAM group(P<0.05),whereas the expression level of P-p38MAPK protein were down-regulated in the EAM+CD40siRNA group compared to the EAM group(P<0.05).No statistic difference was observed between the EAM and EAM+siRNA group(P>0.05).Conclusions1.The CD40siRNA could alleviate cardiac inflammation injury of EAM rats.2.The IL-37 is involved in the development of EAM.The level of peripheral blood IL-37 was negatively correlated with the myocardial histopathology score,and it may partly reflect the degree of myocardial injury.It is speculated that the treatment mechanism of CD40siRNA in the EAM rats may be related to up-regulating the expression level of IL-37.3.The p38MAPK is involved in the development of EAM.It is speculated that the CD40siRNA may play a therapeutic role through down-regulating the expression of p38MAPK mRNA and inhibiting the phosphorylation of p38MAPK protein in the EAM rats.