A total of 80 healthy male SPF grade mice aged 6 weeks and weighing 25.1±5.53 g were selected. All the mice were purchased from the animal experiment center of Zhejiang province, with the production license of SCXK (Zhejiang) 2011-0166. The mice were fed in a plastic box with bedding material on a solid bottom with the temperature of 22°C and the relative humidity between 50 and 65%, 12 h day-night rhythm was normal, and they were free to eat and drink.

The study was approved by the Ethics Committee of Central Hospital of Zibo (Zibo, China). The CVB3 virus was purchased from Cell Signaling Inc. at a titer of 100 TCID50 (50% tissue culture infective dose) /0.1 ml. Mg-132 was purchased from Calbiochem Inc. at a concentration of 0.75 mg/kg.

Twenty mice were randomly selected as the blank group, and they were kept in routine feeding without any intervention. The remaining 60 mice were then randomly divided into the VMC, MG-132 and control group, each containing 20 mice. Mice in the control group were intraperitoneally injected with 0.1 ml PBS (phosphate buffersaline) at 0.1 mmol/l, mice in the VMC and MG-132 group were intraperitoneally injected with 0.1 ml diluent of CVB3 at 100 TCID50/0.1 ml, and mice in the MG-132 group were intraperitoneally injected with 0.75 mg/kg MG-132 the day after the injection of the CVB3 virus. They were continuously injected for 7 days. Mortality rates of each group were recorded and compared on day 8 of modeling, and then peripheral blood and heart samples of the remaining mice were taken for subsequent detection.

Hearts of mice were fixed with 10% formaldehyde and dehydrated routinely, then embedded with paraffin and sectioned. After sectioning, the pathological changes of mouse myocardial tissues were observed under light microscope (Olympus Corp.) and the pathological score of the myocardial tissues was evaluated. The judgement scores were as follows: 1 point, when the proportion of inflammatory cell infiltration and myocardial necrosis was <25%, 2 points, when the proportion of inflammatory cell infiltration and myocardial necrosis was between 25 and 50%, 3 points, when the proportion of inflammatory cell infiltration and myocardial necrosis was between 51 and 75%, 4 points, when the proportion of inflammatory cell infiltration and myocardial necrosis was >75%.

First, the TRIzol reagent (purchased from Applide Invitrogen, Inc.) was added into the myocardial tissue of mice to extract the total RNA in the blood, and the concentration and quality of total RNA were detected by ultraviolet spectrophotometer (purchased from Shanghai Yuanxi Instrument Co., Ltd.). Total RNA (2 µl) was used to compound cDNA in strict accordance with the instructions of minScript reverse transcription kit (Takara Bio, Inc.). Synthetic cDNA (2 µl) was used for qPCR (RT-qPCR kit was purchased from Takara). Total RNA (2 μl) was added to a microcentrifuge tube, then 11 μl of DEPC H₂O was added. A total of 12–18 μl of 10 μM Oligo (dT) was added, mixed and heated at 70°C for 10 min. After ice bath for 1 min, 2 μl 10X PCR buffer, 2 μl 25 mM MgCl₂, 1 μl 10 mM dNTP mix and 2 μl 0.1 M DTT were added, mixed and incubated for 3 min at 42°C. A total of 1 μl of Superscript II was added, incubated at 42°C for 30 min, and heated at 70°C for 10 min. Ice bath was followed for 5 min. The qPCR reaction system was: 2 µl reverse transcription product, 0.5 µl upstream primer, 0.5 µl downstream primer, 2X SYBR Green PCR Master Mix 10 µl, and sterilized and deionized water complemented to 20 µl. Reaction conditions were: 95°C for 2 min, 95°C for 50 sec, 60°C for 45 sec, extension at 72°C for 30 sec, and for a total of 40 cycles. The expression levels of TNF-α mRNA and TGF-β1 mRNA were detected with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal reference. The primer sequences are shown in Table I (the primers were synthesized and designed by Shanghai Gemma Company), and the experiment was repeated 3 times. The result was analyzed with 2^ΔΔ−Cq method (12).

Cardiac muscle tissues (100 g) of mice were taken and RIPA was added lysate to extract the total protein. Then the extracted protein was boiled at 100°C for 5 min to denaturate, then separated with 10% SDS-PAGE after being cooled, transferred to the PVDF membrane, and sealed with 5% of skim milk at room temperature for 1 h. Then the primary rabbit anti-mice polyclonal antibodies of TNF-α (1:500), TGF-β1 (1:500) and β-actin (1:1,000) (cat. nos. 17590-1-AP, 21898-1-AP, 14395-1-AP; ProteinTech Group, Inc.) were added at 4°C for incubation overnight, then the secondary goat anti-rabbit polyclonal antibody (dil, 1:500; cat. no. SA00001-2; ProteinTech Group, Inc.) at room temperature for 1 h incubation, finally ECL developer was used to develop color.

i) Eight-day survival rates of mice in each group were recorded and compared. ii) The pathological score in myocardial tissues of mice in each group was evaluated and compared. iii) The expression levels of TNF-α mRNA and TGF-β1 mRNA in myocardial tissues of mice in each group were compared. iv) TNF-α protein and TGF-β1 protein in myocardial tissues of mice in each group were compared. v) Correlation analysis was performed between the pathological score and the protein expression levels of TNF-α and TGF-β1 in myocardial tissues of mice, and the protein expression levels of TNF-α and TGF-β1 in myocardial tissues.

SPSS 20.0 (IBM Corp., Armonk, NY, USA) was used for statistical analysis of the experimental data. The Chi-square test was used to compare the enumeration data. Kaplan-Meier curve was used for survival analysis. Measurement data were expressed as mean ± standard deviation. t-test was used for comparison between the two groups and univariate analysis of variance was used for multigroup comparison. LSD/t-test was used for postoperative comparison. Correlation was analyzed by Pearson's correlation analysis. GraphPad Prism 6 software (Hangzhou Aimeilv Biotechnology Co., Ltd.) was used to draw the experimental images. P<0.05 was considered to indicate a statistically significant difference.

The survival rate of the blank group and the control group at day 8 was 100%. A total of 11 mice in the VMC group died at day 8, with a survival rate of 45%. A total of 5 mice in the MG-132 group died at day 8, with a survival rate of 75%. The 8-day survival rates of the blank group and control group were significantly higher than those of the VMC group and MG-132 group, but the 8-day survival rate of mice in the MG-132 group was significantly higher than that of the VMC group (P<0.05) (Fig. 1).

There was no inflammatory cell infiltration in myocardial tissues of mice in blank or control group. There was a large amount of inflammatory cell infiltration in myocardial tissues of mice in VMC group. The inflammatory cells in the myocardial tissues of mice in MG-132 group were significantly reduced compared with those in VMC group. The myocardial histopathological scores of mice in blank and the control group were respectively 0.33±0.04 and 0.34±0.06, the myocardial histopathological score of mice in the VMC group was 1.85±0.11, and was 1.04±0.10 in the MG-132 group. The myocardial histopathological scores of mice in the blank and the control group were significantly lower than those in VMC and MG-132 group, and myocardial histopathological score of mice in MG-132 was significantly lower than that in VMC group (P<0.05) (Fig. 2).

There was no significant difference in expression levels of TNF-α mRNA and TGF-β1 mRNA between the blank group and the control group (P>0.05), but were significantly lower than those in the VMC group and the MG-132 group. However, expression levels of TNF-α mRNA and TGF-β1 mRNA in myocardial tissues of MG-132 group were significantly lower than those of the VMC group (P<0.05) (Table II).

There was no significant difference in the expression levels of TNF-α protein and TGF-β1 protein between blank and control group (P>0.05), but both were significantly lower than those of VMC group and the MG-132 group. However, expression levels of TNF-α and TGF-β1 protein in myocardial tissues of mice in the MG-132 group were significantly lower than those in the VMC group (P<0.05) (Table III).

The expression levels of TNF-α protein and TGF-β1 protein in myocardial tissues were positively correlated with the pathological score of myocardial tissues (r=0.843, P<0.05; r=0.763, P<0.05), and there was a positive correlation between expression levels of TNF-α protein and TGF-β1 protein of VMC mice in myocardial tissues (r=0.672, P<0.05) (Figs. 3–5).