Animal model
A total of 24 healthy male C57BL/6 mice (8 weeks old, weight 25-30g), were maintained under a 12h/12h dark/light regime (7:00 a.m.-7:00 p.m.) at 21 ± 1℃ and at a humidity of 55%-60% with free access to food and water. After 7 days of adaptive feeding, all animals were randomly divided into four groups: (1) The sham-operated group (sham group, n = 6), mouse left anterior descending (LAD) coronary artery was subjected to suture without ligation; (2) AMI group, n = 6, the suture on needle was passed through mouse LAD coronary artery followed by ligation; (3) AMI + (-)-EPI group (n = 6), mouse received injection of (-)-EPI (1 mg/kg/day) for continuous 10 days followed by ligation of LAD coronary artery; (4) AMI + (-)-EPI + HY-100347A group (n = 6), mice were administrated (-)-EPI (1 mg/kg/day) for continuous 10 days after intraperitoneal injection of HY-100347A (10 mg/kg/day) for 30 min followed by LAD occlusion through ligation.
Hematoxylin and eosin (HE) staining
The sections were dewaxed with xylene Ⅰ for 10 min and xylene Ⅱ, Ⅲ for 5 min, followed by incubation in absolute ethanol and gradient ethanol (95%, 85%) for 1 min, respectively. After rinsing with tap water, sections were stained with hematoxylin for about 1-5 min, differentiated with 1%/0.5%/0.25% hydrochloric acid for 3-5 s, and stained with eosin for 20 s-2 min. After that, the sections were dehydrated with gradient ethanol from 85% to 100%, immersed in xylene Ⅰand Ⅱ, blocked with neutral gum, and finally sealed. hematoxylin stains the cell nuclei are stained with a blue or purplish-blue color, muscle, cytoplasm, red blood cells, and connective tissue with a pink color, and eosinophilic particles in cytoplasm with a bright red color; collagen fibers are stained light pink; elastic fibers are stained bright pink.
Masson staining
Myocardial tissues were cut into sections and deparaffinized in distilled water. The sections were stained with hematoxylin for 5-10 min, differentiated with hydrochloric acid and alcohol, and rinsed in running water. After washing, the sections were stained in acid fuchsin solution for 5-8 min, washed in distilled water, and stained with 1% phosphomolybdic acid for 1-3 min and with aniline blue solution or bright green solution for 5 min. Then the samples were dried in a 60℃ incubator, cleared with xylene, and sealed. Collagen fibers were counterstained with aniline blue (blue) or bright green (green), cytoplasm, muscle fibers and red blood cells were stained red, and nuclei were stained blue-brown.
Fibroblast cell culture
1-2 day-old neonatal mouse hearts were extracted upon disinfection with 70% alcohol and placed on a DMEM petri dish. After removing blood and cutting off the aorta and atrium, the hearts were cut into pieces (2 mm). The sections were digested with 0.25% pancreatin at 37°C for 5 min, 6 to 8 times, and subjected to differential adhesion to to obtain fibroblasts. The cells were seeded in 30 mm culture flasks, 6-well plates or 96-well plates at a density of 106 cells/ml. The flasks and plates were placed in an incubator (37°C, 95% air and 5% CO2) for 48-72 h and then transferred to serum-free DMEM for culturing for 24 h. The fibroblasts then were randomly divided into groups for various experiments.
Cell Counting Kit-8 (CCK-8) assay
Adherent cardiac fibroblasts in the logarithmic growth phase were seeded in a 96-well plate with 100 μl/well (5 × 103 cells), and cultured under normoxia or hypoxia for 24 h. The fibroblasts were incubated with different concentrations of (-)-EPI for 72 h and 10 µM CCK-8 solution was added to the cells every 24 h and cultured for 2 h. A microplate reader was used to detect optical density (OD) at 450 nm for subsequent analysis of viability.
Flow cytometry
Following washing cells in cold PBS buffer, the cells were suspended in Binding Buffer to prepare a suspension of 1 × 106 cells/ml. 100 μl of cell suspension was taken to mix with 5 μg of purified recombinant Annexin V-FITC, and incubated for 15 min at room temperature in the dark. Then the mixture was centrifuged at 1000 rpm for 5 min with the supernatant removed. 200 μl Binding Buffer was added to resuspend the cells with 5 μl PI, and a flow cytometer was used to evaluate the cell apoptosis.
Reverse transcription polymerase chain reaction (RT-qPCR)
Total RNA was extracted from cell and tissues using Trizol reagent (15596026, Invitrogen, USA) and reverse transcribed into cDNA according to the procedure of PrimeScript RT reagent Kit (RR047A, Takara, Japan). Then the cRNA was subjected to RT-qPCR using Fast SYBR Green PCR kit (Applied biosystems) and ABI PRISM 7300 RT-PCR system (Applied biosystems) to quantitatively analyze RNA in triple. With GAPDH as an internal reference, gene expression was calculated using the 2-ΔΔCt method and the experiment was repeated for at least three times. The primer sequences were listed in table 1.
Western blot
Cardiac fibroblasts were incubated with 100 µl RIPA and 1 µl PMSF, and sonicated. Mouse myocardial tissues were grinded in 200 µl cell lysate and lysed on ice for 30 min. The above cell and tissue samples were centrifuged at 4°C, 13000 r/min for 30 min and the supernatant was collected, with protein concentration determined by Bicinchoninic Acid method. 100 µg of protein was mixed with 5 × loading buffer and denatured at 100°C for 5 min. After centrifugation, proteins were separated through SDS-PAGE and transferred to polyvinylidene fluoride membrane, blocked with 5% skim milk. The membrane was then incubated with the corresponding primary and secondary antibodies, and detected by an infrared fluorescence scanning system after incubation. With GAPDH as an internal reference, the samples were photographed, and analyzed the image analysis software odyssey1.2.
Statistical analysis
All statistics were analyzed using the SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). Measurement data was presented as mean ± standard deviation. Comparisons those among three or more groups were made by one-way analysis of variance (ANOVA), with Tukey post-hoc test. All tests were two-sided and P < 0.05 was considered statistically significant.