Expression Of Toll-like Receptor 2 In The Offspring Of Neonatal Mice Myocardial Tissue After Intrauterine Infection Of HCMV

Objective: To preliminarily create an approach to establishing animal models with myocardial injury due to intrauterine infection induced by human cytomegalovirus(HCMV)in neonatal mice,and investigate the activation of TLR2 signaling pathway in the process of myocardial injury in neonatal mice from intrauterine infection induced by HCMV through detecting the expression of TLR2 gene and the level of downstream signaling molecules,including MyD88,IFN-? and IL-8.Methods: BALB/c mice(SPF grade)aged 8 to 10 weeks were randomly selected by female to male ratio of 2:1.Mice with negative serum HCMV-IgM and HCMV-IgG antibodies were included and randomized into experimental group,normal control group and blank control group.Mice in the experimental group and blank control group were intraperitoneally injected with 0.5 ml HCMV AD169 strain(5.0log TCID50)0.5 ml HELF cell suspension,respectively,and those in the normal control group were free of any treatment.Serum HIMV-IgM was determined in mice in the three groups with colloidal gold method following one week.Then mice with positive serum HCMV-IgM were randomly selected(n=60 females;n=30 males)in the experimental group,and those with negative HCMV-IgM were randomly selected in the normal control group and blank control group(n=12 females;n=6 males,respectively).Those mice were matched and fed in cages by female to male ration of 2:1.Then mice following pregnancy were separated to feed to obtain the neonatal offspring by natural delivery.Living births in each group were subjected to detection of serum and myocardial HCMV-IgM by colloidal gold technique.Immunohistochemistry(HE staining)was used to observe the pathological changes of myocardium,RT-PCR to detect the myocardial TLR2 mRNA expression,ELISA to measure the level of myocardial MyD88,IL-8 and IFN-?,and colorimetry to determine the activity of Caspase 8.Results:(1)The positive rate of serum HCMV-IgM in BALB/c mice in the experimental group was 82.5%(66/80)in females and 87.5%(35/40)in males one week after intraperitoneal induction with HCMV AD169;(2)Positive rate of serum HMCV-IgM in living births in the experimental group was 53.4%(118/221),which the positive rate of myocardial HMCV-IgM was 35.6%(42/118);(3)Myocardial pathological changes in newborns with positive myocardial HMCV-IgM in mice in the experimental group were represented by disordered myocardial fiber arrangement,edema or/and apoptosis of myocardial cells,compared to orderly arranged myocardial fibers with uniformly stained color in mice in the normal controls and blank controls;(4)Myocardial TLR2 mRNA ?Ct in mice with positive myocardial HMCV-IgM was lower than that of the normal controls and blank controls[(10.63±1.06)vs.(12.88±1.55)and(12.75±1.28)].The difference was significant(P < 0.01).Neonatal mice in the experimental group with positive myocardial HMCV-IgM had significantly higher level of MyD88(4.48±0.74),IL-8(29.58±1.98)and IFN-?(16.90±1.78)than those of blank controls and normal controls[(3.79±0.37),(21.24±1.86),(12.96±1.64);(3.68±0.49),(22.40±1.71),(12.21±1.53),respectively](P< 0.05),and MyD88 level was positively correlated with that IL-8 and IFN-?(r=0.837,r=0.857,P<0.05);(5)The activity of Caspase 8 remained no significant difference among experimental group(67.25±34.57),the control group(72.37±36.34)and the normal control group(63.18±33.67)(P>0.05).Conclusions:(1)Myocardial injury models of neonatal mice were successfully established in BALB/c mice with intrauterine infection induced by intraperitoneal injection of HCMV AD169 followed by mated feeding and reproduction;(2)TLR2 signaling pathway may be involved in the pathological process of newborn mice with myocardial injury from intrauterine infection induced by HCMV.The major mechanisms for the pathological changes of myocardium in the neonatal mice are associated with the release of inflammatory cytokines mediated by TLR2/MyD88 through classical pathways;(3)The pathway for cell apoptosis mediated by TLR2/MyD88/caspase 8 may not be involved in the pathophysiological process in the myocardial injury of newborn mice.