A total of 150 purebred inbred Balb/c mice, 3–7 weeks of age, body weight 15–23 g were purchased from Model Organisms [license no. SCXK (Shanghai) 2005-0002; Shanghai, China]. The mice were kept in a clean environment with an indoor temperature of 22–26°C and a humidity of 52–58%. Standard cages were used, with 5 mice in each cage. The mice were maintained in a 12 h light/dark cycle, and were allowed free access to food and water. This animal study was approved by the Yidu Central Hospital of Weifang Ethics Committee (Weifang, China).

The CVB3 Nancy strain (Virus Research Institute of Wuhan University Medical College, Wuhan, China) was passaged in HeLa cells (Shanghai Gefan Biotechnology Co. Ltd., Shanghai, China). After the cells grew to form a confluent monolayer, the culture fluid was discarded and washed twice with D-Hank's solution, and the virus was diluted and added to the cells, and the supernatant was discarded after adsorption at 37°C for 1 h. The supernatant was then supplemented with 2% calf serum cell culture medium, and cultured in an incubator at 37°C to establish a normal cell control to observe the cytopathogenic condition. Diseased and normal cells were harvested at 24–48 h with cell lesions >50% (++), and were frozen at −20°C. Diseased and normal cells were separated by repeated freezing and thawing twice to completely release the virus particles. The samples were centrifuged at 4°C and 1,006.2 × g for 30 min. The supernatant was obtained and stored at −40°C. Selected sensitive cell lines were placed in tissue culture wells for culture, and maintenance solutions were diluted and added to culture wells, incubated at 37°C, and cytopathological conditions were observed by using a microscope (Olympus Corporation, Tokyo, Japan). One week later, Reed and Muench method was used to calculate the concentration of 10⁵ TCID₅₀ CVB3, and the sample was restored for further use.

Of the total 150 Balb/c mice, based on the principle of similar body weight, 50 mice were selected to establish an acute VMC animal model (acute VMC group), 50 mice were selected to establish chronic VMC animal model (chronic VMC group), and the remaining 50 mice were selected as control group. The mice in the acute and chronic VMC groups were sterilized at the injection site, and the asepsis inoculation concentration was 10⁵ TCID₅₀/0.1 ml CVB3 0.1 ml. After being fed for 7 days, an acute VMC animal model was established. In the chronic VMC group, the same CVB3 was inoculated intraperitoneally every month after the first vaccination. The monthly dose was gradually increased by 0.07 ml. After 3 months, a chronic VMC animal model was established. The control group was injected with virus-free EMEM at the same time. The number of the mice that died was counted. The mice were sacrificed by decapitation 7 days and 3 months after the completion of the modeling. A portion of the myocardium was removed and quickly placed in liquid nitrogen, and stored at −80°C until use.

Myocardial tissue was taken to prepare paraffin sections and refer to the staining methods reported by Rittié (9) to perform picrosirius red collagen stain to observe myocardial fibrosis changes. Collagen volume fraction (CVF, %): myocardial tissue microscopy was performed by using a T1-T2-200V-200VS optical microscope (Olympus Corporation). Each section was randomly selected from 10 fields for microscopy. The myocardium was yellow, and the collagen tissue was red. Pictures were taken with the Leica camera system (Leica Microsystems, Ltd., Shanghai, China), and QWin image analysis system [Keyence (China) Co., Ltd., Shanghai, China] was used for calculating the percentage of collagen in each field of view, and the mean number represented CVF (%), which was the severity of myocardial interstitial fibrosis.

Total RNA extraction: the myocardial tissue cryopreserved at −80°C was obtained, and TRIzol reagent [Thermo Fisher Scientific (China) Co. Ltd.] was used to lyse the solution after shaking for 30 min at room temperature. The extraction process was strictly in accordance with the manufacturer's protocol. The absorption value of RNA was measured by using an Ultrospec III ultraviolet spectrophotometer (Eppendorf, Hauppauge, NY, USA), and the purity of the total RNA was measured on a 1% agarose gel power supply. RT-qPCR assay was performed by using TGF-β1, ADAMTS-1 fluorescence quantitative PCR kit (Biomiga, Inc., San Diego, CA, USA). RT reaction (reaction volume 18.6 µl): RNA 10 µl and oligo(dT) 1.3 µl was added to thin-walled tubes to mix well, and heated at 65°C for 30 min; 7.3 µl Mix liquid (with 2.3 µl dNTP, 4.0 µl buffer and 1.0 µl M-MLV) was added and mixed, reacted at 37°C for 2.5 h, heated at 65°C for 30 min, and 20 µl cDNA was diluted to 100 µl with ionized water and stored at −20°C. PCR reaction: GAPDH as an internal reference. Primer sequences are shown in Table I. All the primers were synthesized by Suzhou Hongxun Biotechnologies Co., Ltd. (Suzhou, China). PCR reaction conditions: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 sec, annealing at 60°C for 30 min, extension at 72°C for 1 min, elongation at 72°C for 10 min, and a total of 35 cycles of amplification reaction. The manufacturer's software was used for amplification analysis, and GAPDH was used as an internal reference gene. The result was processed using 2^−ΔΔCq (10).

SPSS 17.0 (Softnet Technology Co., Ltd., Tianjin, China) was used for statistical analysis. Measured data were expressed as mean ± standard deviation (means ± SD). The t-test was used to compare measurement data between two groups. Chi-square test was used for comparison of enumeration data between the groups. ANOVA was used to compare multiple groups of means. Dunnetts test was used for the post-hoc pairwise comparisons. Pearson's correlation test was used to analyze the correlation between ADAMTS-1 mRNA and CVF and TGF-β1 mRNA in mice with chronic VMC. P<0.05 for the difference was considered statistically significant.

Nine mice died in the acute VMC group, and the mortality rate was 18.00% (9/50). Thirteen mice died in the chronic VMC group, and the mortality rate was 26.00% (13/50). None of the mice in the control group died. The sex, age, body weight, indoor temperature and indoor humidity of the three groups of mice had no effect on this experiment (p>0.05) (Table II). The myocardial tissue of mice was stained with picrosirius acid red collagen after the calculation. It was observed that compared with the control group, the CVF of the myocardial tissue in the acute VMC group was significantly increased, and the increase of CVF in the myocardial tissue of the chronic VMC group was the most obvious (p<0.001) (Fig. 1).

The relative expression levels of TGF-β1 mRNA in myocardial tissue of the acute and chronic VMC and control groups were 1.364±0.564, 2.158±1.086, and 0.692±0.470, respectively. Compared with the control group, the relative expression of TGF-β1 mRNA in myocardial tissue of mice in the acute VMC group was significantly increased (t=6.201, p<0.001). The relative expression of TGF-β1 mRNA in myocardial tissue of the chronic VMC group was significantly increased (t=8.538, p<0.001); the relative expression of TGF-β1 mRNA in myocardial tissue of the chronic VMC group was significantly higher than that of the acute VMC group (t=4.109, p<0.001). The difference between the three groups was statistically significant (F=43.340, p<0.001) (Fig. 2).

The relative expression levels of ADAMTS-1 mRNA in myocardial tissue of the acute and chronic VMC and control groups were 2.697±1.323, 3.781±1.587, and 1.349±0.757, respectively. Compared with the control group, the relative expression of ADAMTS-1 mRNA in the myocardial tissue of the acute VMC group was significantly increased (t=6.094, p<0.001). The relative expression of ADAMTS-1 mRNA in the myocardial tissue of the chronic VMC group was significantly increased (t=9.488, p<0.001); the relative expression of ADAMTS-1 mRNA in myocardial tissue of the chronic VMC group was significantly higher than that of the acute VMC group (t=3.288, p<0.001). The difference between the three groups was statistically significant (F=42.550, p<0.001) (Fig. 3).

Pearson's correlation test results showed that the relative expression of ADAMTS-1 mRNA was positively correlated with CVF in the myocardial tissue of mice (r=0.351, p<0.01), and positively correlated with TGF-β1 mRNA in the mouse myocardium (r=0.401, p<0.01) (Figs. 4 and 5).