2.1 Grouping and treatment of experimental animals
C57BL/6J male mice, aged 8-10 weeks(20-25 g), were purchased from the Institute of Experimental Animal Science, Hubei Medical University(Shiyan, China). Before the experiments, the animals were kept in a thermostatic environment with regular 12-hour light/dark cycles and provided with standard food and water. Forty-eight mice were randomly divided into 3 groups (6 mice in each group):(1) control group, (2) LPS group, (3) JQ1+ LPS group. Mice in LPS group were intraperitoneally injected with LPS 7.5mg/Kg(from Escherichia coli,serotype 0111: B4,Sigma,dissolved in 0.9% NaCl). Mice in the LPS+JQ1 group were given are protective treatment with 50mg/Kg JQ1(MedChem Express, HY-13030, and dissolved in 10% DMSO) intravenously 1 hour before LPS stimulation. The dose of JQ1 was based on a previous study. Mice in the control group were injected with DMSO in the caudal vein. Six mice in each group were euthanized after 12 h of LPS stimulation. Serum and heart tissue were collected and stored at −20℃for subsequent experiments(Fig. 1). The survival rate of the remaining mice was recorded every 12 h until 72 h after LPS stimulation.
Cardiac function was assessed by echocardiography after 12 hours of LPS stimulation. Mice were anaesthetized with sevoflurane and fixed in the supine position. Echocardiography was collected using a Mindray Resona7 imaging system equipped with a 20 MHz linear transducer. Left ventricular end-diastolic diameter (LVDD) and left ventricular end-systolic diameter (LVSD) of mice in each group were detected, and left ventricular ejection fraction (LVEF) and left ventricular short-axis shorting rate (LVFS) were calculated according to the built-in program. The mean values of the three measurements were recorded.
2.3 Measurement of Creatine Kinase-MB(CK-MB), Lactate Dehydrogenase (LDH), IL-6, IL-18
Serum and cell supernatants were collected and CK-MB, LDH, IL-1βand IL-18 levels in serum and cell supernatants according to instructions. The CK-MB and LDH kits were purchased from Nanjing Jiancheng Bioengineering Institute, China. The IL-1βELISA kit and the IL-18 ELISA kit were purchased from MULTISCIENCES (LIANKE) BIOTECH, CO., LTD。
2.4 Oxidation index determination
The activity of superoxide dismutase (SOD), the activity of glutathione peroxidase (GSH-Px), and the content of maldialaldehyde (MDA) were determined using the corresponding kit (Nanjing Jianchen Bioengineering Institute, China). After various treatments of cardiomyocytes and cardiac tissue, the cells and tissue homogenates were collected and fragmented in 500µl PBS using an ultrasonic crush, followed by centrifugation at 1200 rpm at 4℃for 10 min. The supernatant was collected to determine SOD activity, GSH-PX activity, and MDA content using the corresponding kit according to the manufacturer's instructions.
2.5 Histological Staining
The rats were killed by neck removal, and the cardiac tissue was obtained. The cardiac tissue was fixed with 4%(φ) paraformaldehyde, and then paraffin sections were prepared. Then HE staining was performed (baking slices - xylene dewaxing hydration - hematoxylin staining - acid ethanol solution differentiation - ethanol dehydration and transparency - neutral gum sealing slices). Finally, the changes in cardiac tissue were observed under a microscope. Three hearts were analyzed in each group.
2.6 Tissue immunofluorescence staining
Heart tissue fixed in 10% formalin was dehydrated, transparent, waxed in, embedded, fixed on a slicer, and cut into 5µm thick paraffin sections. Overnight with anti-CD45 antibody (1:1000, Proteintcch) and then labeled with Goat anti-rabbit IgG H&L (1:2000, Abcam) secondary antibody for 30 min and Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min. Immunocellular infiltration within the myocardium was visualized by confocal microscopy.
2.7 Cell culture and treatment
H9C2 myoblast cells were purchased from ProCell Life Science &Technology Co., Ltd. H9C2 cells were cultured in a DMEM medium(containing 10% serum,100 ug/L penicillin,100 ug/L streptomycins)in an incubator at 37℃ and 5% CO2 saturated humidity. The cells were divided into 4 groups: control group, LPS group, LPS+ JQ1group, and JQ1+LPS + EX527 group. Cells were stimulated by LPS(10µg/ mL) for 12h, and JQ1(0.5µm) and/or EX527(1 mM, Selleck Chemicals, Item No S1541) were added 1h before LPS for pre-protection.
2.8 CCK8 assay
H9C2 cells in the logarithmic growth stage and good growth state were inoculated into 96-well plates at a density of 3×104/ mL.JQ1 with final concentrations of 0.25, 0.5, 1, 2, and 4µM were added into the cells. H9C2 cells supplemented with DMEM medium were set as the negative control group, and DMEM medium was set as the blank group. Each group has 3 duplicate holes. After 48h of culture, the absorbance (OD) of each well was measured at 450 nm using an automatic enzyme plate analyzer. The inhibitory rate of cell growth was calculated, and the inhibitory rate (%)=[1 −(OD value of the experimental group − OD value of the blank group)/(OD value of the control group − OD value of the blank group)]×100%.
2.9 Fluorescent staining of reactive oxygen species (ROS)
H9C2 cells were seeded in 12-well plates, and the media was discarded after 12 h of drug intervention.DCFH-DA probes were diluted with serum-free medium with 4 ′,6-diamine-2-benzene-index (DAPI) blue in 1∶1500, and incubated in the incubator for 30 min. The medium was discarded, and the cells were washed twice with a serum-free medium. The staining level of cells was observed under fluorescence microscope, and the fluorescence luminosity value was detected.
2.10 Western Blotting
The expression levels of BRD4, NLRP3, and SIRT1 in heart tissue and H9C2 cells were determined by Western Blotting. The protein samples were electrophoretic with 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes at 200 mA constant current. Then, after sealing with 5% skim milk powder at room temperature for 1 h, the anti-BRD4(1:1000;Abcam,ab128874),anti-SIRT1(1:1000;Abcam,ab189494), anti-NLRP3(1:1000;Cell Signaling Technology,15101S), anti-Caspase-1(1:1000; Cell Signaling Technology,3866T), anti-Cleaved caspase-1(1:1000;Cell Signaling Technology,4199T),anti-GSDMD(1:1000;Abclonal, A18281), and anti-GAPDH(1:1000,Absin,abs132004)were incubated at 4℃overnight.The membrane was washed three times and then incubated with secondary antibody at room temperature for 1 h. The protein bands were revealed by ECL (Beyotime Institute of Biotechnology, China) chemiluminescence solution. The Image J Image analysis system (NIH, USA) was used to quantify protein expression.GAPDH is used as an internal reference for Western Blotting.
2.11 Real-time PCR analysis
Total RNA was extracted from mouse myocardial tissue and H9C2 cells with Trizol reagent. cDNA was synthesized from total RNA using a reverse transcription kit. Amplification and quantitative RT-PCR analysis were performed using 7300 Real-Time PCR System (A p p l i e d B i o s y s t e m s) on a SYBR Green. The primer sequences used are as follows: 5′- CCATGGACATGAGCACAATC- 3′(forward primer) and5′- TGGAGAACATCAATCGGACA- 3′ (reverse primer) for the mouse BRD4 gene; 5′- CCGTGGCAAACTGGTACTTT- 3′(forward primer) and5′- GACGCCAACATAGACCACCT- 3′ (reverse primer) for the mouse SIRT1 gene; 5′- GACA TGCCGCCTGGAGAAAC - 3′ (forward primer) and 5′- AGCCCAGGA TGCCCTTTAGT- 3′ (reverse primer) for GAPDH (used as an internal reference control). The results were quantified using the 2- ΔΔCT method.
2.12 Statistical Analyses
All data in our study were expressed as mean ±SEM and statistically analyzed using GraphPad Prism software version 8.0. The comparison between the two groups was performed using the Two-tailed Student’s t-test. Others used one-way analysis of variance (One-way ANOVA). P < 0.05 was considered statistically significant.