Effect Of C-jun N-terminal Kinase Downregulated By CD40 SiRNA On Autophagy In EAM Rat Cardiomyocytes

Research backgroundMyocarditis is a disease caused by various infections or other causes of m yocardial interstitial cell infiltration and necrosis of adjacent cardiomyocytes,le ading to cardiac dysfunction and other systemic damage.The most common is viral myocarditis(VMC),which is pathologically characterized by necrosis or degeneration of cardiomyocytes and sometimes lesions that can also affect the pericardium or endocardium,and is a common cardiovascular disease during ch ildhood and adolescence and is one of the common diseases responsible for th e death of children and adolescents.Dilated cardiomyopathy is now considered to be a late manifestation of viral myocarditis.Experimental autoimmune myo carditis(EAM)induced by purified porcine cardiac myosin in rats is very simi lar to the process of human myocarditis and is an ideal model for studying V MC at home and abroad.The pathogenesis of myocarditis is not fully understoo d and is related to a variety of factors.In recent years,it has been found that autophagy is closely related to the process of CVB3-related VMC.Autophagy is a process of lysosome-mediated degradation and reuse of cytosolic material.In the case of cell damage,it can effectively remove damaged proteins and o rganelles to maintain cell stability.It mainly includes three(1)macroautophagy:commonly known as autophagy,the product to be degraded first enters the au tophagosome,and then fuses with the lysosome and degrades its product.(2)Microautophagy:A way in which lysosomes directly engulf products to be deg raded.(3)Chaperone-mediated autophagy:Some chaperones help unfolded prot eins enter lysosomes and are selective,the former two do not.Defects in auto phagy are associated with many diseases in humans,including those of cancer,muscle,and neurodegeneration.C-Jun N-terminal kinase(JNK)is an important mitogen-activated protein kinase.It is a key enzyme that transmits extracellula r signals into cells and participates in various physiological processes such as c ell growth,development,division,and death.It is also known as death kinase.It is found that JNK is involved in the development of various diseases relate d to autophagy.CD40-CD40L is a group of important costimulatory factors tha t play an important role in the immune response to T lymphocyte activation,a nd it is also very important for the activation of JNK.However,the effect of CD40-CD40L signaling pathway on autophagy in autoimmune myocarditis throu gh the JNK pathway has not been reported.ObjectiveTo investigate the effect of CD40 siRNA on CD40-CD40L signaling pathw ay via c-Jun N-terminal kinase on autophagy in autoimmune myocarditis.MethodsThirty-two Lewis rats aged 6 to 8 weeks were randomly divided into four groups,namely,CD40 siRNA intervention group(EAM + CD40 siRNA grou p),EAM group,normal control group(NC group),and siRNA intervention gro up(EAM + siRNA group),with 8 rats in each group.The EAM model was i nduced by injecting porcine cardiac immunoglobulin into the hind footpad regio n of EAM + CD40 siRNA group,EAM group,and EAM + siRNA group on days 0 and 7,and the NC group was injected with sterile phosphate buffer(P BS).On day 7,EAM + CD40 siRNA group and EAM + siRNA group were i njected with CD40 siRNA lentiviral vector and siRNA lentiviral vector,respecti vely.Body weight of rats was measured regularly,rats were sacrificed on the 21st day,body weight of rats was measured,myocardial tissue of rats was stai ned with HE,pathological changes of myocardial tissue of rats were observed with microscope at different multiples and pathological scores were calculated.The autophagy-related microtubule-associated protein light chain 3-?/? ratio(LC 3-?/?)and Beclinl were detected by immunohistochemical staining for JNK an d Western blot.Results1.The overall incidence of rats in the EAM + CD40 siRNA,EAM + siR NA,and EAM groups was 100%,as evidenced by body weight and pathologic al findings,and there was no mortality in any group of rats.2.The body weight of rats in the NC group increased rapidly and was sig nificantly higher than that of the EAM + siRNA group and the EAM group,a nd there was statistical significance in the analysis of variance for continuous measurement of body weight in the four groups(F??=4.526,P=0.089;F??=43.809,P<0.001;F??=3.159,P=0.053),There was a significant difference betw een the EAM group and the NC group(P = 0.025),between the EAM + siR NA group and the NC group(P = 0.043),and there was no significant differe nee between the other groups.3.There was significant difference in variance analysis of pathological scor es among the four groups(F = 22.66,P<0.001).EAM + CD40 siRNA gro up was lower than EAM group(P<0.001).There was no significant differe nee between EAM + siRNA group and EAM group(P>0.05).4.The rate of JNK-positive cells in the four groups of data was statisticall y significant by the rank sum test,and there was a significant difference betw een the NC group and the EAM group(Z=-20.500,P<0.001),but it was lower in the EAM + CD40 siRNA group than in the EAM group(Z=-12.500,P<0.001),and there was no difference between the EAM + siRNA group and the EAM group(P>0.05).5.The rank sum test of the relative expression of Beclinl and LC3-? prot eins in the four groups was statistically significant.There was a significant diff erence in the relative expression of Beclinl and LC3-? proteins between NC g roup and EAM group(Z=18.500,P<0.001),(Z=18.812,P<0.001).EAM+CD40si RNA was significantly lower than EAM+siRNA group(Z=-13.500,P<0.001)(Z =-13.188,P<0.001).There was no statistical difference between EAM group and EAM+siRNA group(P>0.05).6.there is a positive correlation between JNK and LC3-? and internal refe rence ratio(r=0.985,p<0.001)?Conclusions1.CD40 siRNA could attenuate myocardial inflammation in EAM rats.2.The CD40-CD40L signaling pathway can affect autophagy in autoimmun e myocarditis through the JNK pathway,and CD40 siRNA can reduce its signa ling pathway and thus play a therapeutic role in EAM rats.