| Objective:Clinical viral myocarditis(VMC)mainly occurs in children and infants.It presents with myocardial cell necrosis and inflammatory cell infiltration,and further develops into chronic cardiomyopathy,dilated cardiomyopathy(DCM)and heart failure.It can even lead to acute heart failure and sudden death.Viral infection is the main cause of VMC.specially Coxsackievirus group B type 3(CVB3),is the most common pathogen that causes VMC.At present,the pathogenesis of VMC is still unclear,and there is a lack of targeted treatment methods and safe effective drugs.It is very important to explore the pathogenesis of viral myocardial inflammation and to find safe and effective drugs for clinical treatment.4’-azid-2 ’-deoxy-2’-fluoroarabinoside(FNC)is a novel nucleoside analogent,which has been shown to have good anti-coxsackie virus B3 activity in previous studies.Therefore,based on previous studies,this topic is through simulating VMC model infected by CVB3 in vitro and in vivo to observe the the protect effects of FNC on myocardiocytes and to approach the mechanism of action of anti CVB3.Methods: 1.In vitro study 1.1 Study on TLR7-TRAF6 signaling pathwayThe titer TCID50 of CVB3 was determined by end-point dilution method.VMC cell model was established by infecting He La cells with CVB3 in good growth state.The effect of FNC on the m RNA expression levels of TLR3,TLR4,TLR7,TLR8,TRAF6,IL-1β and IL-10 in VMC cell model was determined by RT-PCR.Western Blot was used to detect the effect of FNC on the protein expression levels of IL-1β,TLR7,TRAF6 and TNF-α in VMC cell model.1.2 Effect of FNC on NIH/3T3 fibroblasts The fibroblasts in good growth state were infected by CVB3,and the appropriate viral stimulation concentration was selected according to the cytopathic condition(cytopathic effect,CPE).The effect of FNC on fibroblast proliferation was detected by MTT method.The effect of FNC on the migration ability of fibroblasts was detected by scratch test and Transwell test.2 In vivo study2.1 Pharmacodynamic experimentCVB3 virus dilution was intraperitoneally injected into male BALB/c mice to establish the VMC animal model.The mice were given continuous intragastric administration for 7 days from the second day after infection.The general survival status of the mice was observed.RT-PCR was used to detect the expression level of virus in heart tissue.The heart function of mice was detected by ultrasonography.Morphological changes of myocardial tissue were detected.2.2 Mechanism StudyThe m RNA expression levels of IL-1β,TLR7,TRAF6,TNF-α,TGF-β,COI-I,COI-Ⅲ,MMP2,MMP9 and TIMP1 in myocardial tissue of mice were detected by RT-PCR.The protein expression levels of TLR7,TRAF6 and IL-1β in heart of mice were detected by Western Blot.Immunohistochemistry and masson trichrome staining were observed.Results: 1 In vitro study 1.1 Mechanism research resultsRT-PCR results showed that,at the gene level,FNC significantly inhibited IL-1β,TLR7,TRAF6 and TNF-α(P<0.05),and promoted IL-10(P<0.05).Western Blot assay showed that the protein expressions of IL-1β,TLR7,TRAF6 and TNF-α were up-regulated after virus infection,while FNC could effectively inhibit their expressions and play an anti-inflammatory role(P<0.05).1.2 The effect of FNC on NIH/3T3 fibroblastsThe IC50 of FNC against fibroblasts was 9.156 μg/m L.FNC effectively inhibited the proliferation of fibroblasts(P<0.05).At 6 μg/m L,FNC showed inhibitory effect on fibroblast migration,and the wound healing percentage was sig nificantly decreased(P<0.01).With the increase of the concentration,the migration of fibroblast had a higher inhibitory effect(P<0.01).FNC significantly inhibited the migration of fibroblasts(P<0.05).2 In vivo study2.1 Experimental results of efficacyVMC mouse model was successfully established,and the results showed that FNC could effectively relieve the symptoms of viral myocarditis.Reduce the activity of serum LDH,and inhibit the replication of virus in myocardial tissue.Echocardiography showed that FNC effectively alleviated cardiac dysfunction in VMC mice.FNC could also reduce inflammatory infiltration and myocardial necrosis in mice myocardium.2.2 Mechanism study results FNC could significantly inhibit the expression of of IL-1β,TLR7,TRAF6 and TNF-α(P<0.05).It also inhibited myocardial fibrosis related genes expression of TGF-β,COI-I,COI-Ⅲ,MMP2 and MMP9(P<0.05).It had a promoting effect on TIMP1(P<0.05).Conclusion: 1.FNC has a good therapeutic effect on viral myocarditis and ca n effectively inhibit viral replication in myocardial tissue.2.FNC can down-regulate the expression of TLR7 and TRAF6 proteins,inhibit the activation of NF-κB signaling pathway,and reduce the inflammatory response.3.The anti-CVB3 mechanism of FNC may be related to the direct inhibition of viral replication and the inhibition of inflammatory response and fibrosis caused by viral infection. |
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