Experimental Research On NLRP3-inflammasome Participated In HSV-1Induced Viral Myocarditis

ObjectiveTo observe whether the Nod-like receptor pyrin domain-containing protein3(NLRP3)-inflammasome could participate in the pathophysiologic process of Herpes simplex virus-1(HSV-1) induced viral myocarditis (VMC), HSV-1was untilized to infect isolated cultured neonatal rat ventricular myocytes(NRVM) to establish the viral VMC cell model.Method1) Cultured NRVM were infected with0.01or0.1PFU HSV-1for24hours at the fifth day, named the model group1and the model group2respectively. Simultaneously, the normal control group was added the vero cell culture supernatants without HSV-1. At24h post infection, morphologic changes of NRVM were observed under light microscope and photographic records of which were reserved. Moreover, the supernatant concentrations of Creatine kinase-MB (CK-MB) were measured by the automatic biochemical analyzer. Additionally, comprehensive analysis of both the morphologic changes of NRVM under light microscope and the supernatant concentrations of CK-MB were performed to certify the successful establishment of HSV-1induced VMC cell model.2) The gene expression of NLRP3-inflammasome and its downstream pathways were measured by quantitative real-time PCR (qRT-PCR) based on successful establishment of HSV-1induced VMC cell model. The expression and location of Caspase-1(cysteinyl aspartate-specific proteases-1) were evaluated by immunofluorescent (IF) method. Moreover, the supernatant concentrations of Interleukin-18(IL-18) were measuredResults1, Establishment of HSV-1induced VMC cell model1) Cytopathic effect (CPE) was observed in NRVM infected with HSV-1:the cell body swelled, the pseudopodia shortened and coarsened, and the cell density reduced obviously. While the presentation of the control group was normal, ninety percent of this group beating strongly in sync;2) The supernatant concentrations of CK-MB (10.17±3.99U/L,9.93±3.21U/L) in both model groups, one of the myocardial injury biomarkers, were significantly increased compared with that of the normal control group(6.62±1.62U/L, P<0.05, respectively).2. The expression of NLRP3-inflammasome and its downstream pathways1)The mRNA levels of NLRP3, Caspase-1, IL-1β and IL-18were up-regulated by10times,14times,5times,6times in the model group1,and27times,28times,7times,12times in the model group2. Nevertheless, it was failed to detect the up-regulation of ASC mRNA levels in HSV-1infected NRVM, the expression of which were down-regulated73%and52%that of the normal contol group.2) The expression of pro-Caspase-1and Caspase-1were dyed green fluorescence and gathered in the cytoplasm of HSV-1infected NRVM, while obvious green fluorescence could not be observed in the normal control group, which elucidated that the expression of pro-Caspase-1and Caspase-1showed in IF were elevated dramatically.3) The concentration of supernatant IL-18increased in model group1(62.65Π6.69pg/ml) compared with that of in the control group (26.99Π11.43pg/ml), the difference between which was of statistical significance(P<0.05), while there was no statistically significant difference between IL-18concentration in the model group2 (32.83±9.25pg/ml) and that in the normal control group.Conclusion1. The VMC cell model was successfully established by HSV-1infecting isolated cultured NRVM.2. NLRP3-inflammasome and its downstream pathways were activated in cell model of HSV-1infected VMC. NLRP3-inflammasome might participate in pathophysiologic process of VMC, and became a potential target for VMC therapy.