Introduction

Myocardial infarction (MI) and related complications comprise the leading cause of death in humans worldwide [1]. In addition to myocardial injury caused by ischemia and hypoxia, excessive local inflammation can aggravate myocardial injury after MI, which promotes cardiomyocyte apoptosis and accelerates the synthesis of extracellular matrix, leading to myocardial fibrosis, adverse ventricular remodeling, and cardiac dysfunction [2]. Early inflammatory activation in the infarcted myocardium is the basis for a later myocardial repair phase, but optimal repair after MI requires appropriate containment and resolution of inflammation [3]. Inflammatory macrophages (M1-type macrophages) enter the infarcted area at an early stage to secrete various cytokines, such as tumor necrosis factor (TNF)-α, leading to cardiomyocyte apoptosis. Several studies have shown that switching inflammatory macrophages to the reparative phenotype (M2-type macrophages) on time is essential for the containment and resolution of inflammation [4,5,6,Full size image

Gene Ontology enrichment analysis was performed to reveal the biological characteristics of 200 intersecting target genes. Detailed network pharmacology data are provided in Additional file 1: Fig. S2–S4. The top 20 terms significantly enriched for biological processes (Fig. 2b) included negative regulation of the apoptotic process, positive regulation of the apoptotic process, positive regulation of cell proliferation, inflammatory response, and response to hypoxia, which were all closely associated with the treatment of MI. Therefore, we speculated that BBR exerted a protective effect on the myocardium through the above biological processes. Because the effects of natural drugs are often multifaceted, we excluded terms that were in conflict and those appeared as outcomes, we concluded that the therapeutic effect of BBR on MI mainly involves the inflammatory response and hypoxia response. The enrichment score of the inflammatory response was much higher than that of the hypoxia response; therefore, we primarily verified the effect of BBR@PLGA@PLT NPs on the inflammatory response.

Ability of BBR@PLGA@PLT NPs to regulate inflammation in vitro and in vivo

Subsequently, we evaluated the in vitro biological effects of BBR@PLGA@PLT NPs. The cytotoxicity of BBR@PLGA@PLT NPs was investigated in macrophages (Fig. 3a). In both the BBR@PLGA group and BBR@PLGA@PLT group, BBR was coated with nanoparticles and entered the macrophage environment in a sustained release manner. Therefore, the BBR concentration in the cell environment was relatively low in the BBR@PLGA and BBR@PLGA@PLT groups compared with that in the BBR aqueous solution group. The cell viability decreased only slightly with increasing BBR concentration. Only the BBR aqueous solution groups that contained 50 μg/mL and 100 μg/mL of BBR showed significant reductions in the viability of RAW 264.7 cells (P = 0.0304, P = 0.0062). These results demonstrated that the sustained release ability of BBR@PLGA and BBR@PLGA@PLT NPs reduced the toxicity of BBR and that PLGA and PLTs are biocompatible.

Fig. 3
figure 3

The in vitro and in vivo effects of BBR@PLGA@PLT NPs on macrophages. a Cell viability of macrophages after incubation with various concentrations of BBR, BBR@PLGA, and BBR@PLGA@PLT NPs in solution for 12 h. b Confocal microscopic images of macrophage markers after incubation in 10 μM BBR solution and saline for 12 h. c Inflammatory cytokines secreted by RAW 264.7 cells. d Serum levels of inflammatory cytokines in rats on day three after MI. e Representative immunofluorescence images of TUNEL, CD206, and CD86 in the border zone of infarcted hearts on day three after MI. TUNEL-stained (green) cells indicate apoptosis-positive cells, and CD86 (red) and CD206 (green) staining shows M1 and M2-type macrophages, respectively. f Statistical analysis of the percentage of TUNEL-, CD206-, and CD86-positive cells. All bars represent as means ± SD (n = 3). *P < 0.05 and **P < 0.01, ***P < 0.001

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As shown in Fig. 3b, the BBR aqueous solution group showed significant up-regulation of ARG1 and CD206 (M2 subtype marker) in RAW 264.7 cells compared with the saline control group. We demonstrated that 10 μM BBR did not affect macrophage viability (Fig. 3a), suggesting that BBR exerts its effect on macrophages mainly by acting on macrophage M2-subtype polarization rather than directly inhibiting macrophage activity.

The normalized levels of inflammatory cytokines secreted by RAW 264.7 cells are shown in Fig. 3c (the non-normalized data are shown in Additional file 1: Fig. S5). Only the BBR solution group showed significant differences, with down-regulation of IL-6 (a proinflammatory factor) and TNF-α (a major cytokine that mediates cardiomyocyte apoptosis by inflammation) secretion and up-regulation of IL-10 (an anti-inflammatory factor) secretion. These data again demonstrated that BBR was released from BBR@PLGA and BBR@PLGA@PLT NPs in a sustained manner, and PLGA and PLTs did not produce changes in macrophage secretion in vitro.

In vivo experiments, the serum levels of IL-10, IL-6, and TNF-α were increased on day four after MI, and the IL-10 level was further increased after BBR@PLGA and BBR@PLGA@PLT treatment, while the TNF-α and IL-6 levels were decreased. Moreover, the BBR@PLGA@PLT group had the highest serum IL-10 level and the lowest IL-6 and TNF levels (all P < 0.05) (Fig. 3d, the non-normalized data are shown in Additional file 1: Fig. S5).

Thus, we reasoned that the most likely mechanism of action of BBR@PLGA@PLT NPs on MI involved the cardiac enrichment capacity of the PLT coating, the sustained BBR release capacity of PLGA nanoparticles, and the ability of BBR to promote M2 polarization in macrophages.

We demonstrated that BBR@PLGA@PLT NPs could promote M2 polarization of macrophages in infarcted myocardium by measuring the expression of markers, including TUNEL (apoptotic marker), CD206 (M2 macrophage surface marker), and CD86 (M1 macrophage surface marker) on day four post-MI (Fig. 3e). There was no significant difference in the percentage of TUNEL-positive cells (apoptotic cardiomyocytes) and CD206-positive cells between the saline group and BBR@PLGA group (P = 0.0683, P = 0.2088). The only significant difference between these groups was in the percentage of CD86-positive cells (P = 0.0328). The BBR@PLGA@PLT group showed significant differences in all markers compared with the BBR@PLGA group. Apoptotic cardiomyocytes (P = 0.0018) and CD86-positive cells (P = 0.0025) were significantly reduced, and CD206-positive cells were increased significantly in the BBR@PLGA@PLT group compared with the BBR@PLGA group (P = 0.0062) (Fig. 3f).

Evaluation of cardiac function by echocardiography

Cardiac function is a direct response to the therapeutic effect of drugs on MI, and was measured by echocardiography in rat models on day 28 after MI. Representative echocardiograms are shown in Fig. 4a. Compared with the sham group, the MI rats treated with saline exhibited typical characteristics of heart failure with a significantly increased left ventricular internal diameter at end-diastole (LVIDd), left ventricular inner diameter at end-systole (LVIDs), end-systolic volume (ESV), and end-diastolic volume (EDV), as well as reductions in left ventricle fractional shortening (FS) and left ventricular ejection fraction (LVEF) (all P < 0.05) (Fig. 4b–g). BBR@PLGA NPs demonstrated a limited protective effect on heart function with increased EF and FS and decreased in LVIDd, LVIDs, EDV, and ESV compared with saline (all P < 0.05). The BBR@PLGA@PLT group had the highest EF and FS and the lowest LVIDd, LVIDs, EDV, and ESV among all MI groups (all P < 0.05) (Fig. 4b–g).

Fig. 4
figure 4

Cardiac function was evaluated by echocardiography on day 28 after MI. a Representative echocardiography images of different groups. b LVIDs. c LVIDd. d FS. e ESV. f EDV. g LVEF. All bars represent as means ± SD (n = 6). *P < 0.05 and **P < 0.01, ***P < 0.001

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Evaluation of fibrosis by pathological staining and western blotting

Myocardial fibrosis is an important pathological process that leads to adverse cardiac remodeling and fatal heart failure [36, 37]. The pathological manifestation of fibrosis is that excess collagen deposition replaces normal tissue. Systematic assessment of myocardial fibrosis on day 28 showed that BBR@PLGA@PLT NPs reduced collagen accumulation and improved collagen composition (Fig. 5).

Fig. 5
figure 5

Evaluation and quantitative analysis of fibrosis by pathological staining and western blotting on day 28 after MI. a Representative images of heart sections stained with Masson trichrome, collagen is stained blue. b Representative images of heart sections stained with Sirius Red, the red and yellow tissues are considered type I collagen, while the green tissues are type III collagen. Quantitative analysis of the c ventricular wall thickness, d collagen content, and e collagen I/III ratio in different groups was performed by visual analysis. f Western blot results, g quantification of collagen content, and h collagen I/III ratio. All bars represent as means ± SD (n = 3). *P < 0.05 and **P < 0.01, ***P < 0.001

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The saline group had a large amount of collagen deposited in the ventricle, which was stained blue by Masson staining (Fig. 5a). Stiff collagen lacks the contractile capacity of cardiomyocytes and reduces ventricular thickness during the long-term passive stretch (Fig. 5c). Compared with the saline group, the BBR@PLGA group had greater ventricular thickness and less collagen deposition. The ventricles of the BBR@PLGA@PLT group had the thickest ventricular wall and the least collagen deposition among all MI groups (Fig. 5d).

The collagen fibers observed by Masson staining were composed of multiple components. Type III collagen has good elasticity, and type I collagen is stiff [37]. The collagen I/III ratio can reflect the passive expansion ability of collagen fibers in scar tissue. In this study, type I and type III collagen were observed using Sirius Red staining. The visual analysis results showed that the BBR@PLGA group had a lower collagen I/III ratio than the saline group (P = 0.0267), and the BBR@PLGA@PLT group had a significantly lower collagen I/III ratio than the BBR@PLGA group (P = 0.022) (Fig. 5e). To ensure the reliability of this conclusion, we also performed western blotting analysis of collagen in the infarcted myocardium (Fig. 5f). The densitometry results are presented as arbitrary units and normalized ratios. Semi-quantitative analysis showed that treatment with BBR@PLGA and BBR@PLGA@PLT NPs significantly reduced the content of type I and type III collagen in the scar tissue on day 28 (all P < 0.05). The BBR@PLGA@PLT group exhibited a greater reduction in type I collagen than the BBR@PLGA group (P = 0.0003), but there was no significant difference in the type III collagen content between the two groups (P = 0.8078) (Fig. 5g). The BBR@PLGA@PLT group had the lowest type I/III collagen ratio, which was significantly lower than that in the BBR@PLGA group (P = 0.0031) (Fig. 5h). Thus, we again verified the effect of BBR@PLGA@PLT NPs on improving the composition of scar tissue by western blot analysis. The elasticity of collagen fibers in scar tissue plays an important role in the protection of cardiac function.

Fibroblasts secrete collagen in the myocardium and other organs. Studies [37, 38] have shown that the secretion of collagen by fibroblasts in infarcted myocardium is mainly regulated by inflammatory factors, such as IL-10. IL-10 increases collagen synthesis in the early phase of MI, but inhibits collagen synthesis and regulates collagen composition in the later phase of MI, leading to the up-regulation of type I collagen in scar tissue. Type I collagen, which has greater elasticity than type III collagen, can reduce fibrotic myocardial stiffness and directly affect cardiac function. Importantly, cardiac stiffness is one of the leading causes of decompensation in heart failure [39]. Therefore, we suggest that BBR@PLGA@PLT NPs can be enriched in the infarcted myocardium, and the released BBR can regulate the secretory phenotype of macrophages to reduce fibrosis of the infarcted myocardium and stiffness of cardiac scar tissue.

Assessment of cardiac structures

Cardiac structures, including electrical signal transmission networks, vascular networks, and cardiomyocytes [40], were assessed on day 28 by immunofluorescence analysis.

Myocardial structure damage was detected by measuring α-actinin and CX43 protein levels. α-actinin is a vital protein of the myocardial skeleton, and CX43 mediates electrical signal transmission between cardiomyocytes. As shown in Fig. 6a, MI operation significantly changed the myocardium structure. CX43 and α-actinin were significantly down-regulated in the infarcted myocardium of the saline group. Additionally, the location of CX43 in the tissue was altered; CX43 was absent from viable myocardial tissue and mainly distributed around the nuclei of cardiomyocytes. Compared with the saline group, both the BBR@PLGA and BBR@PLGA@PLT groups had CX43 in the correct location. Furthermore, CX43 and α-actinin protein levels were up-regulated in the BBR@PLGA and BBR@PLGA@PLT groups, with greater up-regulation in the BBR@PLGA@PLT group than that in the BBR@PLGA group (all P < 0.0001) (Fig. 6b).

Fig. 6
figure 6

Assessment of cardiac structures, including electrical signal transmission networks, vascular networks, and cardiomyocytes on day 28 after MI. a Representative immunofluorescence images co-stained for CX43 (green) and α-actinin (red). b Optical density semi-quantitative analysis of CX43 and α-actinin expression. c Representative immunofluorescence images of blood vessels co-stained for CD31 (green) and α-SMA (red). d Quantitative analysis of the neovessels in the infarct regions. e Evaluation of cardiomyocyte apoptosis in the border zone of infarcted hearts by TUNEL staining and f semi-quantitative analysis. g Western blot and h quantification of TNF-α expression in infarcted myocardium. All bars represent as means ± SD (n = 3). *P < 0.05 and **P < 0.01, ***P < 0.001

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In infarcted myocardium, cardiomyocytes are more likely to survive when angiogenesis occurs [https://metascape.org/). The enrichment terms with p < 0.05 were collected, and those with p < 0.01 were considered as the critical value of significant pathways and functions. Finally, the top 20 biological processes (BP), cellular components (CC), and molecular functions (MF) were defined as terms with p < 0.01, and the pathways were identified based on p < 0.05. Then, the drug-target-pathway (D-T-P) was constructed using Cytoscape v3.7.2 software by linking the core active constituents, predicted targets, and pathways. In the network, the nodes represented the active ingredients, signaling pathways, or potential targets, while the edges identified their interactions.

Cell viability assay

Cell viability was evaluated by MTT assay. RAW 264.7 cells were seeded in a 96-well planet and cultured overnight, followed by incubation for 12 h in BBR aqueous solution, BBR@PLGA dispersion, and BBR@PLGA@PLT dispersion containing different BBR concentrations. MTT at a final concentration of 0.5 mg/mL was added into each well and incubated at 37 °C for 120 min. The absorbance of viable cells was recorded at 560 nm by a microplate reader (Bio-Tek Instruments Inc., Winooski, VT, USA), and 750 nm was the reference wavelength. Corrected absorbance values (560–750 nm) were expressed as the percentage of viable cells relative to untreated control cells.

Immunofluorescence staining of RAW 264.7 cells

ARG1 and CD26 immunofluorescence were performed to investigate the effect of BBR on macrophage phenotype. Ten μM BBR aqueous solution and RAW 264.7 cells were co-incubated for 12 h, followed by incubation with Anti-ARG1 Polyclonal Antibody (Solarbio, Bei**g, China) and CD206 antibody (Abcam, USA). The marker expression on RAW 264.7 cells was observed by a 510 Meta-type laser scanning confocal microscopy (Carl Zeiss Microimaging Inc., Thornwood, NY) using the 488 nm and 633 nm lasers for green and red fluorescence; images were obtained at oil immersion magnifications of × 400.

Inflammatory factors measurement

To investigate the effect of BBR and BBR@PLGA@PLT NPs on inflammatory factors secreted by macrophages, RAW 264.7 cells were treated with lipopolysaccharide (LPS) combined with corresponding drugs, and the BBR concentration in each group was 10 μM. The changes in IL-6, TNF-α, and IL-10 secreted by RAW 264.7 cells were measured by ELISA according to the kit instructions (Bioswamp, Wuhan, China).

Rats’ MI model preparation and treatment

Male SD rats weighing 220 ± 15 g were obtained from the Experimental Animal Department of Tongji Medical College, Huazhong University of Science and Technology. The rats were anesthetized and artificially ventilated, followed by thoracotomy, and the left anterior descending coronary artery was ligated with a 6.0 suture. Fifteen minutes after the MI operation, 1.5 mL of BBR@PLGA@PLT dispersion liquid containing 0.8 mg of BBR or BBR@PLGA dispersion containing 1 mg of BBR or saline was administrated via the tail vein.

Serum inflammatory factors measurement

The inhibitory effect of BBR@PLGA@PLT NPs on inflammation from the serum inflammatory factor levels was analyzed in rats on day three after MI. The whole blood of rats was collected, and serum was isolated. Serum levels of inflammatory factors were measured by rat IL-6 ELISA kit (Multisciences, Hangzhou, China), rat TNF-α ELISA kit (Solarbio, Bei**g, China), and rat IL-10 ELISA kit (ELK biotechnology, Wuhan, China) according to the kit instructions.

Immunofluorescence staining

All of the antibodies were obtained from Abcam (USA). Immunofluorescence staining was performed in the infarcted myocardium Sections (4 mm) on day 3 (TUNEL, CD206, and CD86) and day 28 (CX43, α-actinin, α-SMA, and CD31) post-MI. The slides were permeabilized with PBS for 35 min and blocked with 3% BSA for 35 min at 25 °C, followed by incubation with primary antibody at 4 °C for 8 h. The secondary antibody was then added and incubated for 1 h at 25 °C. DAPI was used to stain the nucleus after PBS rinsing. The percentage of positive cells was calculated by the ratio of the number of TUNEL, CD206, and CD86 positive cells to the number of nuclei. The average optical density was calculated by ImageJ to evaluate the expression of α-actinin and CX43. Loop areas that were positive for both CD31 and α-SMA were considered vessels, and the area of vascularization was calculated by ImageJ.

Echocardiographic assessment

On the 28th day after MI, echocardiograms and left ventricular function of Rats were obtained by Philips EPIQ5 system under isoflurane anesthesia. Parasternal long-axis views at the level of the papillary muscles were obtained using transthoracic two-dimensional guided m-tracking. LVEF, FS, LVIDd, LVIDs, EDV, and ESV were calculated as the mean of 3 consecutive cardiac cycles.

Masson staining

The collagen volume fraction was evaluated on Paraffin sections using Masson Trichrome Stain Kit (Sigma-Aldrich) according to the instructions. Collagen volume fraction was calculated as the ratio of the total area of fibrosis to the myocardium in the entire specimens and measured by ImageJ.

Sirius red staining

Sirius red and picric acid was used for Sirius Red staining. Under polarized light, different types of collagens in sections were visualized. Red and yellow tissues were identified as collagen types I and III. ImageJ was used to quantify the collagen I/III ratio.

Western blot

The content of TNF-α, collagen type I, and collagen type III in infarcted myocardium was assessed by Western blot. The protein concentrations were measured with BCA protein Assay Kit (Beyotime Biotechnology, China). Equal amounts of protein (20 μg) were added into each lane of a 10% SDS-PAGE (New Cell & Molecular Biotech, China) and separated. Then, the protein was transferred onto PVDF membranes (Millipore, United States). And the membranes were blocked by Protein Free Rapid Blocking Buffer (EpiZyme, China) for two h and incubated with primary antibodies (anti- TNF-α, 1:1000, Abclonal, China; anti-collagen type I, 1:1000, Proteintech, United States; anti-collagen type III, 1:5000, Proteintech, United States; anti-GAPDH, 1:1000, Abclonal, China) at 4 ℃ for 12 h. After incubation with HRP-coupled secondary antibody (goat anti-rabbit or rabbit anti-mouse IgG, 1:10,000, Boster, China), the membranes were treated with chemiluminescence (Solarbio, China) for 3 min and visualized on a Visionwork system.

In vivo toxicity evaluation

On day 28 after MI, whole blood was collected, and serum Cr, BUN, ALT, and AST levels were measured using a biochemical analyzer (Chemray240, China). Major organs (heart, liver, lung, kidney, spleen) were harvested for H&E staining (Hematoxylin and Eosin Staining Kit, Beyotime, Shanghai, China) and TUNEL staining according to instructions.

Statistical analysis

GraphPad Prism 9.1 (GraphPad) was used to process and analyze the data. The mean and standard deviation are presented. Statistical analyses were conducted using one-way analysis of variance analysis. P < 0.05 was considered statistically significant.