A total of 60 adult C57BL/6 genetic background male mice (aged 7–8 weeks) were purchased from Shanghai Laboratory Animal Center (Shanghai, China) and randomized to construct the MI model. All the mice were subjected to permanent left anterior descending artery (LAD) ligation or sham surgery as described previously.11) Briefly, the mice were anesthetized with 1.5% isoflurane gas using an induced chamber before being mechanically ventilated with a rodent respirator. Subsequently, the chest cavity was opened to expose the heart and a 6–0 silk suture was used for permanent ligation of the LAD. The suture was passed approximately 2 mm below the tip of the left auricle. Complete vessel occlusion was confirmed by the presence of myocardial blanching in the perfusion bed. The thorax was closed with a 5–0 silk suture. Mice that died within 24 hours after surgery were excluded from the experiment. Sham-operated animals underwent the same procedure without LAD. All the animals were maintained and handled in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Wenzhou Medical University (approval number: 200016-0003).

BDB was purchased from Matrix Scientific (catalog number: 056469; Colombia, SC, USA). One hundred mg/kg BDB (dissolved in phosphate-buffered saline [PBS]) were administered via intraperitoneal (i.p.) injection 1 hour before MI surgery and then was administrated every other day. By comparison, the control group received the same dose of vehicle.

Cardiac function was evaluated using a Vevo2100 Ultrasound system (Fujifilm VisualSonics, Bothell, WA, USA) at 7 days after the surgery. The investigator was blinded to group assignment, and each group includes 6–14 mice. Mice were anesthetized by isoflurane inhalation. Two-dimensional parasternal long axis views of the left ventricle (LV) were obtained. The LV internal diameters at end diastole and systole, as well as the interventricular septal wall and posterior wall thicknesses were measured by M-mode tracing.

Two weeks after MI, 8–14 mice per group were anesthetized with 5% isoflurane (Merck, Darmstadt, Germany) and 95% oxygen in a gas chamber. The hearts of MI mice were harvested and fixed for 24 hours in 4% paraformaldehyde at 4°C, and then embedded in paraffin. Paraffin-embedded hearts were cut into 6-μm-thick sections and detected infarcted sizes by H&E staining. Photomicrographs were obtained using an Olympus microscope (Tokyo, Japan), and the areas were measured by the Image J software (National Institutes of Health, Bethesda, MA, USA). The infarct size was calculated as a percentage of the LV area.

For the TUNEL assay, MI heart from mice that were administered vehicle or BDB were embedded in optimal cutting temperature (OCT) compound and frozen, and cutted into 6-μm-thick sections. The heart sections were stained by using a TUNEL fluorescence In Situ Apoptosis Detection kit (Yeasen, Shanghai, China), according to the manufacturer's instructions.

Heart were harvested at indicated times and were embedded in OCT regent, and each group includes 8–10 mice. OCT-embedded hearts were cut into 6-μm-thick frozen sections using the freezing microtome (Leica Biosystems, Wetzlar, Germany). Frozen sections were fixed 10 minutes in cold acetone, washed with PBS, and incubated in PBS containing 3% bovine serum albumin (BSA) for 40–60 minutes at room temperature to prevent non-specific binding of antibodies. Tissue slides were then incubated with the following primary antibodies: anti-CD68 (1:200; AbDSerotec, Kidlington, UK) antibody at 4°C overnight. The slides were then washed with PBS three times before incubation with Alexa Fluor-conjugated secondary antibodies (1:1,000; Invitrogen, Carlsbad, CA, USA) for 2 hours at room temperature. ProLong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen) was applied to counterstain the slides. The slides were then mounted and the stained sections were visualized under a laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) and analyzed using Image-Pro Plus software (version 6.0; Media Cybernetics, Rockville, MD, USA). At least 3 different heart sections were obtained from each mouse and 6 random images of at the risk areas were taken from each section.

Flow cytometric experiment was performed as previously described (Temporal dynamics of cardiac immune cell accumulation following acute MI). Eight to 10 infarcted hearts each group were cutted into pieces and digested at 37°C for 1.5 hours in PBS, containing type II collagenase (1.5 mg/mL; Worthington Biochemical Corporation, Lakewood, NJ, USA), elastase (0.25 mg/mL; Worthington Biochemical Corporation), and DNase I (0.5 mg/mL; Worthington Biochemical Corporation). After digestion, the tissues were passed through a 70 μm cell strainer, and leukocyte-enriched fractions were isolated with 37–70% Percoll^® (GE Healthcare Biosciences, Piscataway, NJ, USA) by density gradient centrifugation. Next, the cells were collected from the interface and washed with RPMI-1640 cell culture medium. Isolated cells were then incubated with the following antibodies: CD45-PE (BD Biosciences, San Diego, CA, USA), CD11b-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany), F4/80-BV421 (BioLegend, San Diego, CA, USA), Ly-6G-APC (BD Biosciences), and CD206-PE-cy7 (BD Biosciences) at 4°C for 30 minutes. Flow cytometric analysis and sorting were performed using a FACSAria™ flow cytometer (BD Biosciences), and the data obtained were analysed with FlowJo 7.6.1 software (Tree Star Inc., Ashland, OR, USA).

We grinded the heart tissues (50 mg per heart) from animals in 1 mL of ice-cold homogenization buffer (containing protease inhibitor, calcineurin inhibitors, and phenylmethylsulfonyl fluoride [PMSF]). Homogenates were then centrifuged at 4°C, 9,000 ×g for 30 minutes. Supernatants were collected and centrifuged at 4°C, 14,000 ×g for 30 minutes, and the bicinchoninic acid (BCA) method was used to quantify proteins. Then the same amount protein was mixing with loading buffer and boiled for 10 minutes. We performed sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with the sampling amount per well of 50 μg of protein. Then we transferred the proteins to a nitrocellulose (NC) membrane and washed them with Tris-buffered saline and Tween 20 (TBST, 20 mmol/L Tris-HCl [pH7.5], 0.05% Tween 20, and 0.6% NaCl), which consists of 5% skimmed milk powder, at room temperature, and sealed the membrane for 1–2 hours. The membranes were incubated with primary antibody, anti-nuclear factor (NF)-κB (catalog number: #8242, 1:1,000; Cell Signaling Technologies, Danvers, MA, USA), anti-p-NF-κB p65 (catalog number: #3033, 1:1,000; Cell Signaling Technologies), or anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, catalog number: #5174, 1:1,000; Cell Signaling Technologies) at 4°C overnight. The membranes were washed 3 times in TBST. The samples were then incubated for 2 hours at room temperature with a horseradish peroxidase (HRP)-conjugated secondary antibody. Blots were visualized by an enhanced chemiluminescence detection system (GE Healthcare Biosciences). The results were analyzed by the professional software Image J to quantify gray value.

The levels of TNF-α, IL-1β, MCP-1, and IL-6 in heart were measured with an ELISA kit following the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA). Briefly, each heart tissue was homogenized by PBS in a 1:10 concentration (0.1 g of tissue to 1 mL of PBS buffer). A monoclonal antibody specific for these proteins was pre-coated into wells. The standards and test samples were then pipetted into the wells to allow binding of these proteins to the immobilized antibodies. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for these proteins was added to the wells. Following removal of unbound antibody-enzyme reagent by washing 5 times, a substrate solution was added to the wells and color was developed. The optical density of each well was measured at the wave length of 450 or 510 nm on a microreader (S190; Molecular Devices, San Jose, CA, USA). The concentration of each cytokine or chemokine was accordingly calculated.

Data have been expressed as mean±standard error of measurement (SEM). Two-tailed Student's t-test was used for comparisons between different groups. Data were analyzed using GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA, USA). The p values less than 0.05 were considered as being statistically significant.

To fully evaluate the effect of BDB on cardiac healing post-MI, BDB (100 mg/kg) or vehicle control was injected into the mice before 1 hour of the LAD ligation and then was administrated every other day (Figure 1A). Cardiac function was markedly improved at days 7 after MI in the mice that received BDB than that in the vehicle group. We found that left ventricular ejection fraction (LVEF, 20.51±1.975% vs. 28.84±2.521%; p<0.05) and left ventricular fractional shortening (LVFS, 11.33±0.765% vs. 14.96±1.012%; p<0.05) after MI significantly improved in the BDB treatment group compared to vehicle treatment (Figure 1B and C). A compensatory myocardial hypertrophy always occurs post-MI, which can be assessed by an index of heart weight to body weight (HW/BW) ratio.12) The HW/BW ratio after treatment with BDB before MI was much lower than that in the mice treated with vehicle (6.1±0.246% vs.7.6±0.442%; p<0.05; Figure 1D). There was no significant difference between the 2 sham groups.

The survival rate was found to be significantly lower in Vehicle group than in BDB-treated group at 2 weeks (Figure 1E). We also observed that the infarct size after MI is significantly decreased in BDB-treated MI group than in vehicle MI group (36.15±3.002% vs. 23.84±2.662%; p<0.05; Figure 1F and G). These findings suggest that treatment with BDB exerts a protective effect against heart failure.

In response to MI, different stress response responses are induced and it is important of the cardiomyocytes apoptosis in the damaged heart. To test whether BDB treatment may affect cardiomyocytes apoptosis after acute MI, we performed TUNEL staining in the infarcted heart. However, the percentage of TUNEL-positive cardiomyocytes was not significantly altered in response to the BDB treatment from days 3 post-MI observed (Figure 2A and B), suggesting that BDB administration did not affect cardiomyocytes apoptosis after acute MI.

The infiltration of macrophages plays an important role in early cardiac repair after ischemia,13) and BDB as an anti-inflammatory agent effective to inhibit allergic diseases in experimental models.10) So we detected the infiltration of macrophages in the infarcted hearts after 3 and 7 days post-MI. As expected, the macrophages (CD68⁺) at 3 days after MI were strongly reduced in number at ischemic border zone (BZ) in the infarcted hearts (Figure 3A and B). Similarly, the infiltration of macrophages in the infarcted hearts was also blunted by BDB administration 7 days post-MI (Figure 3C and D), indicating that the acute inflammatory response to ischemia was suppressed by BDB administration.

It is very important of macrophage polarization in acute MI and distinct macrophage lineages contribute to disparate patterns of cardiac recovery and remodeling in the neonatal and adult heart.14) Therefore, we examined the influence of BDB on M1 and M2 macrophage recruitment in hearts after acute MI (Figure 4A). The total number of infiltrating macrophage was markedly suppressed by BDB treatment (Figure 4B). Indeed, both M1 (Figure 4C) and M2 (Figure 4D) recruited into hearts were suppressed in the BDB treatment group during the acute phase after MI (days 3 and 7). These results indicate that BDB facilitated cardiac repair by suppressing the acute inflammatory responses after MI, including the infiltration of M1 and M2 macrophages.

Macrophages play important roles in the progress of acute MI through secreting a number of cytokines and chemokines.15) We examined the levels of TNF-α, IL-1β, MCP-1, and IL-6 at the areas at-risk of the heart tissues. The expression levels of the 4 pro-inflammatory cytokines were significantly decreased in the BDB treatment group compared to vehicle treatment at day 3 and 7 after MI (Figure 5). These results indicated that BDB inhibited the acute inflammatory responses after MI mainly by reducing pro-inflammatory cytokines secretion.

To further explore the function of macrophages, we detected the phosphorylation of NF-κB, a protein complex that is important for the generation of pro-inflammatory cytokines, including IL-6 and TNF-α.16) The phosphorylation levels of NF-κB were significantly different between BDB treated group and vehicle treated group, BDB administration effectively down-regulated NF-κB phosphorylation in the hearts of MI while had no difference in the hearts of sham operation (Figure 6A and B). Taken together, the results showed that administration of BDB improved cardiac healing possibly via suppression of NF-κB phosphorylation.