The specific pathogen-free male C57BL/6J mice (6–8 weeks old, weighing 18–22 g) were purchased from the Experimental Animal Center of Bengbu Medical University. All mice were housed in a temperature- and humidity-controlled room with a 12-hour light/dark cycle, free access to standard rodent chow and water. The temperature of the room was maintained ranged from 22-24 °C, with humidity kept at 40-60%.Mice were randomly assigned to different groups using a random number table method, and all analyses were performed in a blinded manner, where the investigators were unaware of the groups.

All procedures were conducted in accordance with the guidelines established by the Ethics Committee of Bengbu Medical University (approval number: BBMC-2024-617). To be operated solely by trained personnel to avoid causing distress to mice through improper handling. The specific operational procedures are as follows:

Anesthesia: Mice were induced under anesthesia using 2% isoflurane (RWD, Shenzhen). During surgery, the R500 Series Universal Small Animal Anaesthesia Machine (RWD, Shenzhen) was maintained via an anesthesia machine, with oxygen flow set at 1 L/min to ensure stable anesthetic depth.

Euthanasia: At the experimental endpoint, mice were subjected to progressive exposure to carbon dioxide (flow rate: 20% of chamber volume per minute) until respiratory arrest occurred. Cervical dislocation was then employed as a secondary confirmation method.

ZOON-BIO BIOTECHNOLOGY synthesized the gene sequence encoding EgAgB subunit 2 (GenBank: ACZ51457) and cloned into E. coli expression vector pPET-28a(+) through homologous recombination with 8His-tag expressed at C-terminus. The correct sequence and reading frame of recombinant EgAgB8/2/pPET-28a(+) was confirmed by double-strand DNA sequencing. The recombinant plasmid was transformed into E. coli BL21(DE3) and rEgAgB8/2 was expressed under induction of 1 mM IPTG at 30 °C for 4 hours. The expressed rEgAg8/2 was soluble and purified through nickel-affinity chromatography. The contaminated endotoxin in the purified protein was removed using the ToxOut Endotoxin Removal Kit (BioVision, USA), and residual endotoxin levels were measured with the ToxinSensor Chromogenic LAL Endotoxin Assay Kit (GenScript Biotech, Nanjing, China). The purified rEgAgB8/2 was stored in PBS buffer without imidazole, and its concentration was determined using a BCA Protein Assay Kit (Biosharp, Hefei). SDS-PAGE was used to confirm the expression and purity of the target protein. The purified rEgAgB8/2 was subsequently stored at -80 °C for future use.

Coronary artery ligation is a well-established method to create a MI model in mice. In the sham group, the same surgical procedure was performed, except the LAD was not ligated. In this study, we used a novel and rapid surgical approach that does not require mechanical ventilation (Gao et al., 2010). Mice were anesthetized with 2% isoflurane via inhalation, and their respiration was closely monitored throughout the procedure. A small skin incision (approximately 5 mm) was made between the 4th and 5th ribs on the left side of the mouse chest, followed by gentle separation of the intercostal muscles to access the thoracic cavity. The left anterior descending (LAD) coronary artery was identified and ligated 3 mm distal to its origin using a 6–0 silk suture. Successful ligation was confirmed by the anterior wall of the left ventricle turning pale, indicating interruption of blood flow. If a vein is mistakenly ligated, typical myocardial blanching does not occur; instead, local congestion (dark purple area) may develop due to obstructed venous return. This characteristic allows direct identification and exclusion of mice with erroneous vein ligation. In addition to cases of incorrect vein ligation, four categories of mice must be excluded from myocardial infarction modelling: those exhibiting severe postoperative complications such as hemorrhage, pneumothorax, arrhythmia, or respiratory distress; those displaying severe infections at the thoracic incision site, including redness, swelling, or suppuration; and those experiencing weight loss exceeding 20% or significant loss of locomotor function within one week post-surgery. One week postoperatively, there was no significant decrease in left ventricular ejection fraction (LVEF) (difference<10% compared to the sham-operated group). Mice that showed no typical signs of myocardial infarction, as verified by TTC staining or HE staining, were also excluded. All animals were monitored for their survival or recovery.

C57BL/6 mice were randomly divided into four groups with 36 mice for each group: (i) Sham operation control treated with PBS (Sham+PBS); (ii) Sham control treated rEgAgB8/2 (Sham+rEgAgB8/2); (iii) MI group treated with PBS (MI+PBS); (iv) MI group treated with rEgAgB8/2 (MI+rEgAgB8/2). Mice were administered intraperitoneally with 5 µg of rEgAgB8/2 in total volume of 100 µl for treatment or the same volume of PBS 30 minutes after surgery. The same amount of rEgAgB8/2 or PBS was given 2, 4, and 6 days post operation. Mice were monitored daily for weight, behavior, and general health status. Cardiac function was evaluated using echocardiography at days 7 to assess myocardial function and progression of MI. Total 18 mice were sacrificed on Day 7 post-surgery, the blood and sera were collected for cytokine measurement and heart tissues collected for biochemical and histological examination including mRNA measurement of some immunological molecules. The rest 18 mice from each group were kept for long-term survival observation till Day 28. The survival rate was calculated using the Kaplan-Meier method. Mice meet the criteria for humane endpoints included a body weight loss for more than 20% or signs of severe distress will be euthanized in accordance with ethical guidelines.

To evaluate infarct size after MI, the hearts collected from 6 sacrificed mice per group on Day 7 were rinsed in saline and quickly frozen at −20 °C for 10–15 minutes to allow for easier slicing without over-freezing. Each heart was then cut into 5 slices, each 1–2 mm thick, from the ligation point to the apex. The heart slices were incubated in 1% 2,3,5-triphenyltetrazolium chloride (TTC) staining solution at 37 °C for 10–20 minutes in the dark. The white area indicates the infarcted tissue, while the red area indicates viable, non-infarcted tissue. The stained slices were photographed, and infarct size was measured using ImageJ software.

The hearts from 6 sacrificed mice per group were perfusion-fixed using 10 mL of ice-cold PBS at a flow rate of 5 mL/min and then stored in 4% paraformaldehyde at 4 °C for 24 hours. After fixation, hearts were embedded in paraffin blocks and sliced into 5-μm-thick sections. The sections were stained with H&E (Servicebio, China) or Masson’s Trichrome (Servicebio, China) as described (Li et al., 2021). Subsequently, histopathological changes were observed under microscope (Nikon, Japan) and the pathological area was analyzed using ImageJ software. Regions of interest were standardized across samples, analyzing 10 random fields per section, with threshold settings adjusted to highlight specific tissue features. Data were statistically analyzed to quantify histopathological differences between groups.

Echocardiography (ECHO) was performed for all mice on Day 7 post surgery using the Vevo 2100 imaging system paired with a probe (VisualSonics, Canada, MS-400 probe). Before echocardiography was performed on mice, the chest area was shaved, and ultrasound coupling gel was applied to the skin to improve contact between the probe and the skin. Mice were positioned supine on a thermostatic platform, and electrodes were attached to the limbs for continuous monitoring of heart rate, respiratory rate, and oxygen saturation. Superficial anesthesia was maintained with 1% isoflurane in oxygen, and heart rate was monitored throughout the procedure to ensure it remained within a controlled range of 400–500 bpm across all animals. Long-axis B-mode imaging was used to acquire left ventricular images, and M-mode tracings of the left ventricular wall were recorded for 4–6 continuous and stable cardiac cycles. The best 3 cycles, defined by their stability and consistency, were selected for analysis. The left ventricular trace method was applied to obtain stroke volume (SV), left ventricular ejection fraction (LVEF), and left ventricular fractional shortening (LVFS). All values were averaged over the selected cardiac cycles. Ultrasound imaging parameters, such as probe frequency and frame rate, were standardized for all mice to ensure consistency in data collection. Data analysis was performed blindly by a separate investigator who was unaware of the experimental groups. Heart function indices were calculated using the following equations:

Measurements were taken using automated tracing algorithms to minimize human error, and heart rate was maintained at a consistent level to avoid confounding effects on the measurements.

Serum samples were used directly, while heart tissue extracts were prepared by homogenizing in ice-cold PBS (1 mL per 100 mg tissue) and centrifuging at 10,000 xg. Cytokine levels were measured in the sera and heart extracts using individual ELISA kits: Mouse TNF-α, IL-1β, and IL-10 ELISA Kits (Dakewe Biotech, China); Mouse IL-18, iNOS, and Arg-1 ELISA Kits (RUIXIN Biotech, China); and Mouse TGF-β ELISA Kit (ABclonal, USA). All samples and standards were incubated for two hours at room temperature, plates washed three times with PBS after each incubation. Results were read using a microplate reader at a wavelength of 450 nm. Data were analyzed using the standard curve method to calculate cytokine concentrations in each sample, with statistical analysis performed using one-way analysis of variance (ANOVA).

Total RNA was extracted from 100 mg of cardiac infarction tissue using TRIzol reagent (TransGen Biotech, China), following tissue disruption with a rotor-stator homogenizer at 6,000 rpm for 30 seconds. RNA purity was assessed by spectrophotometry, with A260/A280 ratio between 1.8 and 2.1. For reverse transcription, 2 μg of total RNA was used in a 20 μL reaction using reverse transcription kits (TransGen Biotech, China). RT-qPCR was performed using PerfectStart^®Green qPCR SuperMix (TransGen Biotech, China) on a Roche LightCycler^® 96 system. The specific annealing temperatures and extension times for each primer set (listed in Table 1 ) were optimized based on primer efficiency tests. GAPDH was used as the internal control, validated under experimental conditions to ensure stable expression. The relative mRNA expression of target genes was calculated using the 2−△△Ct method, and average Ct values were used for calculations.

Total protein was extracted from heart tissues using RIPA lysis buffer (100 μL per 10 mg of tissue) containing 0.1% PMSF. Samples were lysed with agitation for 30 minutes at 4°C. Protein concentrations were determined by a BCA Protein Quantitation Kit, using a standard curve prepared from known concentrations of bovine serum albumin. Equal amounts of protein from each mouse were loaded, separated by 12.5% SDS-PAGE gels, and then transferred onto 0.45 µm polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk at room temperature for 3h and then incubated with rabbit anti-NLRP3 antibody (1:1,000) (Wanleibio, Shenyang, China), rabbit anti-Pro-Casepase-1 antibody (1:1,000) (Wanleibio, Shenyang, China), rabbit anti-Cleaved-Casepase-1 antibody (1:1,000) (Wanleibio, Shenyang, China), rabbit anti-IL-1β antibody (1:1,000) (Wanleibio, Shenyang, China) overnight at 4 °C followed by incubation with goat anti-rabbit IgG-HRP secondary antibody (1:7,000) (Abcam, Cambridge, UK) at room temperature for 1h. The same amount of tissue lysate was reacted with rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:1,000) (Wanleibio, Shenyang, China) as baseline control. The specific bands were visualized by chemiluminescent substrate and semi-quantitated by Image Lab System. The results are expressed as ratios of NLRP3, Pro-Casepase-1, Cleaved-Caspase-1 and IL-1β to GAPDH control.

RAW264.7 cells were purchased from Procell (Wuhan, China) and cultured in Dulbecco’s modified Eagle medium (DMEM; HyClone, USA) containing 10% fetal bovine serum (FBS; Gibco, USA) and 1% Penicillin-Streptomycin solution (P/S; Biosharp, China). Cells were maintained at 37 °C in a 5% CO2 humidified incubator.

Harvested RAW264.7 cells were divided into 4 groups with 1×10⁶ cells in each group: (i) RAW264.7 cells incubated with PBS as the control group (PBS+Mφ); (ii) RAW264.7 cells incubated with rEgAgB8/2 (1 μg/mL) (rEgAgB8/2+Mφ); (iii) RAW264.7 cells incubated with LPS (100 ng/mL) (LPS+Mφ); (iv) RAW264.7 cells incubated with LPS (100 ng/mL) in the presence of rEgAgB8/2 (1 μg/mL) (rEgAgB8/2+LPS+Mφ). After 24 hours incubation, the cells and culture supernatants were collected for subsequent experiments.

Cultured RAW264.7 cells were adjusted to a density of 1×10⁶ cells/mL for staining procedures. The following three markers were primarily selected. F4/80: A pan-macrophage surface marker widely used for specific identification of macrophages in various mouse tissues. CD86: A key co-stimulatory molecule highly expressed on M1- polarized macrophages (pro-inflammatory phenotype). CD206 (mannose receptor): a classic marker for M2-polarized macrophages (anti-inflammatory/regulatory phenotype). Cells were first treated with fixable viability dye efluor 510 (BioLegend, USA) at a concentration of 0.5 µg/mL for 10 minutes in the dark to differentiate dead from live cells, followed by blocking with Fc receptor blocker (0.5 µg/mL) for 15 minutes to minimize non-specific antibody binding. Surface markers were stained with FITC-anti-F4/80 and APC-anti-CD86 antibodies (all at 1:200 dilution, BioLegend, USA) for 25 minutes. To accurately quantify total CD206 (surface + intracellular) as an indicator of M2 macrophage polarization, permeabilization is required to allow the antibody to enter the cell and bind intracellular CD206. Cells were fixed and permeabilized using the Thermo Fixation/Permeabilization Kit (Thermo Fisher Scientific, USA) for 20 minutes at room temperature. Subsequently, cells were stained with PE-anti-CD206 (1:200 dilution, BioLegend, USA) for 30 minutes in the dark. Flow cytometry data were acquired on a DxP Athena™ flow cytometer (Cytek Biosciences Inc., CA, USA), collecting a minimum of 10,000 viable cells per sample. Compensation was adjusted for FITC, BV605, APC, and PE to correct for spectral overlap. Data analysis was performed using FlowJo V10 software, focusing on the quantification of surface expression levels relative to controls.

Heart tissue samples were processed for single-cell RNA sequencing. Approximately 16,000 cells were loaded onto a single channel of the 10X Genomics Chromium controller, aiming to recover 10,000 cells. Single-cell gel bead-in-emulsions (GEMs) were generated using Chromium v2 and v3 single-cell reagent kits, followed by cDNA synthesis and 11 cycles of amplification using a C1000 Touch Thermal Cycler. Quality control and quantification were performed using the Agilent 2100 Bioanalyzer. A total of 50 ng of amplified cDNA was used for constructing 3’ expression libraries, which were then pooled and sequenced on an Illumina HiSeq 4000 with 150 bp paired-end reads. The resulting basecall files were converted to FASTQ format and aligned to the murine genome (mm10) using the CellRanger v3.0.2 pipeline. Initial cell processing was conducted using the Seurat R package (v3.0.2), with data normalization and integration via canonical correlation analysis and mutual nearest neighbors (MNN). UMAP dimensionality reduction was performed with the top 30 calculated dimensions and a resolution of 0.6. Cell type identification was achieved using the SingleR R package, correlating single-cell expression profiles with the Immgen database. Differential gene expression analysis was conducted using the Wilcoxon rank-sum test, with additional analysis across clusters using the Seurat FindAllMarkers function (log-fold change > 0.25, minimum group percentage 10%, pseudocount 0.1). Bonferroni correction was applied for multiple hypothesis testing. Gene set enrichment analysis was performed using the escape R package, and differential enrichment was determined using the getSignificance function based on the limma linear fit model.

All treatments and analyses were performed in a blinded manner, with researchers unaware of group assignments during both treatment administration and outcome assessment. Statistics were analyzed using GraphPad Prism version 7 software (GraphPad Software, Inc., USA). All samples were randomly grouped to minimize bias between groups, and the data were tested for normality and homogeneity of variance using Shapiro-Wilk and Levene’s tests, respectively. Results are presented as means ± SEM. Multiple group comparisons were analyzed using ANOVA, with post-hoc adjustments for multiple comparisons made using Tukey’s test. The primary comparison will be between the Sham+PBS group and the MI+PBS group to demonstrate differences following modelling. The MI+rEgAgB8/2 group will be compared with the MI+PBS group to demonstrate therapeutic efficacy. Kaplan-Meier survival analysis was conducted, and survival rates between groups were compared using the log-rank test. Statistical significance was set at P< 0.05. All experiments were conducted with at least three biological replicates.

rEgAgB8/2 with 8His-tag at C-terminus was successfully expressed as a soluble recombinant protein in E. coli BL21(DE3) and purified through nickel-affinity chromatography. The contaminated endotoxin was removed by running through endotoxin removal resin and the final endotoxin level of rEgAgB8/2 was reduced to less than 0.1 EU/mg. SDS-PAGE showed that the purified rEgAgB8/2 was approximately 10 kDa in size (including 8His-tag), which was consistent with the deduced theoretical molecular weight (9.4 kDa) ( Figure 1 ).

MI was induced in mice by ligating the LAD artery as described (Gao et al., 2010) ( Figure 2A ), followed by a 7-day consecutive treatment with rEgAgB8/2 via intraperitoneal injection, as previously described (Boutennoune et al., 2012). Kaplan-Meier survival analysis indicated that the post-MI survival rate of mice treated with rEgAgB8/2 (MI+rEgAgB8/2) remained 94.4% (17/18) at Day 28, which is significantly higher than MI group without treatment (66.667%,12/18) (MI+PBS) within the same period ( Figure 2B ). The ratio of heart weight to body weight, as a marker for heart edema and inflammation, was also significantly reduced in rEgAgB8/2 treated MI group (MI+rEgAgB8/2) compared to the untreated group after MI surgery ( Figure 2C ). By comparing the infarct area of mouse heart staining with TTC, the rEgAgB8/2 treated MI group had significantly less infarct area than those without treatment ( Figure 2D ). The echocardiogram in long-axis B-mode revealed that untreated MI mice (MI+PBS) exhibited significantly reduced anterior and posterior wall motion of the left ventricle compared with MI mice treated with rEgAgB8/2 (MI+rEgAgB8/2). Treatment with rEgAgB8/2 also significantly improved LVEF, LVFS, and SV in MI mice than those without treatment even though both groups maintained the similar heart rate (400–500 bpm) without significant difference ( Figure 2E ).

H&E ( Figure 3A ) and Masson’s trichrome staining ( Figure 3B ) on affected heart tissue demonstrated that rEgAgB8/2 treatment significantly increased the thickness of the left ventricular wall ( Figure 3C ) and reduced myocardial fibrosis in mice with MI ( Figure 3D ). To determine if rEgAgB8/2 is able to mitigate myocardial fibrosis and facilitate vascular reconstruction post-MI, α-SMA, a key marker for the activation of fibroblasts into myofibroblasts (Gabbiani, 2021), and VEGF, which is critical for vascular reconstruction and promotes repair of the infarcted myocardium (Laakkonen et al., 2019), were measured in the heart tissue. RT-qPCR results indicated the mRNA expression of α-SMA in the infarcted heart tissue was significantly decreased ( Figure 3E ) and VEGF transcription was increased ( Figure 3F ) in the cardiac tissues of MI mice treated with rEgAgB8/2 compared to the mouse group without treatment (PBS). Together, these findings suggest that treatment with rEgAgB8/2 significantly reduced myocardial fibrosis and improved heart tissue repair in mice following MI.

The uniform manifold approximation and projection (UMAP) plot generated using single-cell RNA sequencing technology shows the distribution of different cell types in the hearts of mice. The results indicate a significant increase in the number of fibroblasts and macrophages in MI mice. The rise in fibroblasts reflects the heart’s attempt to repair the damage, while the increase in macrophages is closely associated with the inflammatory response. After being treated with rEgAgB8/2, the number of fibroblasts slightly decreased, whereas the number of macrophages remained at a high level ( Figure 4A ). However, the expression of Mrc1 gene, a marker for M2 macrophages (Gordon and Martinez, 2010), was significantly increased compared to MI group without treatment, suggesting rEgAgB8/2 induces the transition of macrophages to the M2 phenotype, thereby exerting anti-inflammatory and reparative effects in the heart with MI ( Figure 4B ). The expression of Col1a1, which encodes collagen type I (Myllyharju and Kivirikko, 2004), was also decreased, indicating that rEgAgB8/2 inhibits excessive collagen synthesis, thereby reducing the risk of cardiac fibrosis ( Figure 4C ).

To determine whether the improvement in tissue damage in MI mice by rEgAgB8/2 is associated with the modulation of cardiac inflammatory cytokines, we measured the levels of pro-inflammatory cytokines TNF-α and IL-1β, as well as regulatory cytokines IL-10 and TGF-β in mouse serum ( Figure 5A ) and in the MI tissue extracts ( Figure 5B ) using corresponding ELISA kits. The results showed that after treatment with rEgAgB8/2, the levels of pro-inflammatory factors TNF-α and IL-1β was significantly decreased, while the levels of IL-10 and TGF-β significantly increased in both sera and MI tissues of the mice with MI. We also measured the mRNA levels of these pro-inflammatory cytokines and regulatory cytokines in MI tissues and the results were consistent with the protein levels measured by ELISA ( Figure 5C ). To determine the changes in macrophage phenotypes after rEgAgB8/2 treatment, we detected both protein and mRNA expression levels of M1 macrophage marker iNOS and the M2 macrophage marker Arg-1 in the infarcted heart tissues. The results revealed that treatment with rEgAgB8/2 significantly decreased both protein and mRNA levels of iNOS and increased both protein and mRNA levels of Arg-1 in the MI tissue ( Figure 5D ), indicating the reduced heart tissue inflammation and damage is possibly related to the polarization of macrophage from M1 type to M2 type in affected heart tissue.

Since NLRP3 (NOD-like receptor family pyrin domain-containing 3) inflammasome is an important component of the innate immune system that stimulates caspase-1 activation and the secretion of proinflammatory cytokines IL-1β/IL-18 in response to inflammation and cellular damage (Kelley et al., 2019), we measured if NLRP3/caspase-1/IL-1β signal pathway is involved in the therapeutic effect of rEgAgB8/2 in MI mice. Western blot with specific antibodies demonstrated the protein expression of NLRP3, caspase-1, and IL-1β in the cardiac tissue of MI mice were significantly increased compared to the sham-operated group, indicating that NLRP3, caspase-1, and IL-1β were involved in the inflammatory response of MI. After treatment with rEgAgB8/2, the expression levels of NLRP3, caspase-1, and IL-1β were significantly reduced ( Figure 6A ). We also utilized RT-qPCR to measure the transcriptional levels of the NLRP3, caspase-1, and IL-1β genes in the affected cardiac tissues of mice from each group, the RT-qPCR results were consistent with those of the Western blot analysis ( Figure 6B ). These results suggest that the inhibition of the NLRP3/caspase-1/IL-1β signaling pathway plays a key role in the therapeutic process of rEgAgB8/2 in MI mice.

To further determine the effects of rEgAgB8/2 on the differentiation of macrophage, the macrophage cell line RAW264.7 was co-cultured with rEgAgB8/2 in vitro to mimic the inflammatory microenvironment of macrophage in infarct heart tissue (Yan et al., 2013; Frangogiannis, 2014; Prabhu and Frangogiannis, 2016). LPS was added into the cultured RAW264.7 cells to induce the inflammatory responses when rEgAgB8/2 was added (Zhang et al., 2023; Wang et al., 2024). Flow cytometry was used to measure the expression of the M1-related inflammatory marker CD86 and the M2-related marker CD206 in RAW264.7 cells after being co-incubated with rEgAgB8/2. The results showed that LPS significantly induced expression of CD86+ in RAW264.7 cells. Co-incubation with rEgAgB8/2 reduced the macrophages expressed with CD86+ and induced cells expressed with CD206+ ( Figures 7A, B ) compared to the group without rEgArB8/2 treatment, indicating rEgAgB8/2 induces the macrophage polarization from M1 to M2 subtype. ELISA was used to measure the levels of inflammatory cytokines in the culture supernatants of each group after co-incubation. Compared to the PBS group, LPS stimulation increased the secretion of pro-inflammatory cytokines TNF-α and IL-1β in RAW264.7 cells. Co-incubation with rEgAgB8/2 reduced the levels of the pro-inflammatory cytokines and increased the levels of the anti-inflammatory cytokines IL-10 and TGF-β ( Figure 8 ), further confirming rEgAgB8/2 promotes LPS-induced M1 macrophages transformation towards M2 in vitro.