Mice
Male BALB/c (6–8 weeks) mice were obtained from Kavins Laboratory Animal Company (Changzhou, China). All animal experiments were performed in accordance with the guidelines for the Care and Use of Laboratory Animals (Ministry of Health, China, 1998).
CVB3 Infection and Anti-ST2 mAb Treatment
CVB3 virus (Nancy strain) was maintained by passage through Hela cells (ATCC number: CCL-2). Mice were infected with CVB3 via intraperitoneal (i.p.) injections at the dose of 104 50% tissue culture infectious dose (TCID50) of CVB3. Anti-ST2 mAb (CNT03914,Centocor)were given to VMC mice by intraperitoneal (IP) injection the day before and the day after the infection, 100 µg/mouse. Seven days or fifteen days later, the hearts and serum were collected for the experiment.
Cell Culture
MCF were purchased from BNCC (Beijing, China) and were cultured with DMEM medium (Gibco) containing 10% FBS in a 5% CO2 incubator. The 3–6 passages of cells were used for experiments. Cells were stimulated with sST2 (50nM, R&D Systems) for 12h or 24 h. The 100uM H2O2 were added for 2 hours prior to the stimulation with sST2.
Quantitative RT-PCR
Using an RNA extraction kit (Invitrogen), total RNA was extracted from cells and tissues. Purified RNA Was reverse transcribed into cDNA, then amplified by SYBR-Green master mix kit. Real-time PCR primer sequences were as follows: for mouse P53, F:5’-GCGTAAACGCTTCGAGATGTT-3’ (forward) and 5’-TTTTTATGGCGGGAAGTAGACTG-3’ (reverse); mouse Sirt1, 5’-ATGACGCTGTGGCAGATTGTT-3’ (forward) and 5’-CCGCAAGGCGAGCATAGAT-3’ (reverse); mouse P21, 5’-GTGATTGCGATGCGCTCATG-3’ (forward) and 5’-TCTCTTGCAGAAGACCAATC-3’ (reverse); mouse P16, 5’-AGGGCCGTGTGCATGACGTG − 3’ (forward) and 5’-GCACCGGGCGGGAGAAGGTA-3’ (reverse); Mouse GAPDH, 5’-AGGTCGGTGTGAACGGATTTG-3’ (forward) and 5’-GGGGTCGTTGATGGCAACA-3’ (reverse). Relative levels of gene expressions were determined by the relative standard curve method and normalized to GAPDH and β-actin.
Western Blot
RIPA buffer was used for preparing whole cell lysates. Protein was separated by SDS-PAGE and transferred to PVDF membranes (Millipore). In order to block the membranes,1% BSA was added and the membranes were washed three times with TBS-0.1% Tween 20 (TBST). The washed membranes were incubated primary antibodies at 4˚C overnight. Afterwards, the secondary antibody (1:3000; Abcam) was incubated at room temperature for 1 h. developed using an ECL chemiluminescence kit. Finally, the antigen-antibody reactions were visualized by chemiluminescence (ECL) kit, and the intensity of protein bands was quantified by using ImageJ software.
Elisa
Soluble sST2 levels were quantified in serum according to the manufacturer’s instructions. The results were normalized to the control condition. Data were expressed as a fold change relative to the control conditions.
CCK-8 Assay
MCF were seeded into 96-well plates at 5000 cells per well 24 hours before treatment and three replicate experiments were performed in each group of cells. At the indicated time points, 10 ul CCK8 solution was added to each well and incubated at 37°C with 5% CO2 for 3 h. Finally, the absorbance was detected using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) at 450 nm. The higher the absorbance in OD450, the more active the cell.
Examination Of Myocardial Markers
The levels of lactate dehydrogenase (LDH), creatine kinase-MB(CK-MB) and glutamic oxalacetic transaminase(AST) in serum were measured using the available commercial kits (JianCheng, Nanjing, China), according to the manufacturer’s instructions.
Senescence Detection
A β-galactosidase Staining Kit (Solarbio, Beijing, China) was employed to detect the senescence of MCFs after the cells were treated with sST2 and H2O2. The short answer is that cells were seeded in a twelve-well plate. The medium was removed before the experiment and washed with phosphate-buffered saline (PBS; D8537, Sigma) once. Then, 1 mL stationary liquid was added at room temperature. After 15 min, the stationary liquid was removed and the cells were washed with PBS for three times (3 min/time). Subsequently, 1 mL of β-galactosidase staining working solution was added and incubated overnight at 37°C. Finally, the senescence of the cells was examined were calculated using ImageJ software.
Histological Examination of the Heart
The heart tissues were fixed in 4% polyformaldehyde and embedded in paraffin. Then sections were stained with hematoxylin-eosin(H&E).H&E staining was used to analyze the level of inflammation in heart.
Immunofluorescence Staining.
Hearts were resected and fixed in 4% paraformaldehyde for 24h and dehydrated with 30% sucrose for 2 h. Then, the specimens were processed into frozen sections. Sections of heart were permeabilized in 0.5% Triton X-100 for 20 min and sealed with 5% BSA for 2 h. Afterwards, the sections were incubated with anti-mouse P21 (Immunoway) and anti-mouse α-SMA (Proteintech) antibodies at 4℃ overnight. After being washed, second antibodies (Alexa Fluor 488 anti-mouse and 594 anti-rabbit) were applied. Finally, Nuclei were identified with DAPI (Thermo Fisher Scientific) and representative figures were taken by fluorescence microscope.
Statistical Analysis
All data were analyzed using GraphPad Prism 8.0 (GraphPad Software, Inc.). In ImageJ, grayscale scans were performed on Western blot analysis results. The data were expressed as mean ± standard deviation. A t test was used to compare the data between two groups and the differences between multiple groups were analyzed via a one-way analysis of variance. A P value of 0.05 or less was considered statistically significant.