Animal models and drug administration

30 SPF-grade C57BL/6J male mice, aged 8 weeks and weighing 22–26 g, were purchased from Guangdong Medical Laboratory Animal Center and housed in the SPF-level Experimental Animal Center of Lingnan Medical Research Center. After 1 week of acclimatization feeding, the mice were used in the following experiments.

A MI mouse model was established by ligating the left anterior descending coronary artery (LAD). Briefly, anesthetized mice were secured in the supine position on a thermostatic operating table following airway ventilation. The ventilator was set with a respiratory rate of 120 breaths/min and a tidal volume of 2.5 mL. The thoracic cavity of the mouse was incised along the left 3rd and 4th intercostal space of the sternum to expose the heart. The LAD was ligated 1–1.5 mm below the left atrium using 8/0 nylon sutures. The SHAM group underwent the same thoracotomy operation without ligating the LAD. Mice were used for experiments 4 weeks after surgery.

NXK used in this study is concentrated NXK powder diluted in sterile water for intragastric administration. The NXK powder comprises only two herbs: Ilex pubescens and radix ginseng Rubra, with a ratio of 23:77. The concentrated NXK powders were manufactured by the Sci-tech Industrial Park, Guangzhou University of Chinese Medicine, in accordance with internationally certified Good Manufacturing Practice Guidelines (Serial Number: TA2017014). The doses gradient from high to low of NXK are 1.65 g/kg/day (NXK-H), 0.83 g/kg/day (NXK-M), and 0.42 g/kg/day (NXK-L) (The gradient setting referred to the Methodology of Pharmacological Research of Traditional Chinese Medicine). Perindopril (ACERTIL, No. 2016837) served as a positive control drug, and the intragastric dose of perindopril in mice was 0.607 mg/kg/day, calculated according to the recommended daily oral dosage for humans and converted using the equivalent dose formula based on the body surface area of humans and mice. Correspondingly, mice in our study were randomly divided into 6 groups: SHAM group, MI group, NXK-L group (the low dose of NXK), NXK-M group (the medium dose of NXK), NXK-H group (the high dose of NXK, also known as NXK group) and Perindopril group. Mice were given NXK and Perindopril in the corresponding group, while the SHAM group and MI group received equivalent volumes of normal saline water. The drug administration in all groups lasted for 4 weeks consecutively.

The Ethic Committee of Guangzhou University of Chinese Medicine has approved the ethical and scientific application of all animal experiments in our study. The study was strictly conducted in accordance with “Guide for the care and use of Laboratory animals” published by the National Institute of Health.

Preparation of NXK-containing rat serum

The serum was obtained from 12 SPF-grade SD rats, with an equal distribution of males and females, each weighing 200 g, sourced from the Animal Experiment Center of Guangzhou University of Chinese Medicine. The drug-containing serum was prepared with reference to a recent study [7]. The drug dosage was 1.15 g dry extract powder/kg, which was diluted with sterile water, thoroughly shaken for full dissolution, and appropriately heated before gavage. The dosage is based on the "conversion of the initial dose to equivalent dose" method in the Experimental Methodology of Traditional Chinese Medicine Pharmacology (60 kg for human adults). SPF-grade SD rats, with 3 rats for each gender, were orally gavaged with 1 mL each time, twice a day for five consecutive days. The remaining 6 rats received an equal volume of saline as the control group. Within 3 h after the last gavage, blood was collected from the abdominal aorta, and the serum of the same group was mixed and inactivated in a water bath at 56 °C, then stored at − 80 °C for backup.

Cell culture and treatment

RAW264.7 cells were cultured in a completed medium composed of 89% 1640 medium (Cat. No. C11875500BT, GIBCO), 10% fetal bovine serum (Cat. No. 10100147, GIBCO) and 1% penicillin/streptomycin (Cat. No.15140122, GIBCO). A pro-inflammatory macrophage (M1) model was established by adding 200 ug/mL lipopolysaccharide (LPS) (Cat.No.L2880-10 mg, Sigma). The specific groupings are as follows: Control group (CTRL): Cells were cultured with medium containing 5% ctrl rat serum + 5% FBS, NXK group (NXK): Cells were cultured with medium containing 5% NXK-containing rat serum + 5% FBS, LPS-induced M1 macrophage model group (CTRL + LPS): Cells were cultured with medium containing 5% ctrl rat serum + 5% FBS with the addition of 200 ug/mL LPS treatment for more than 16 h, NXK treatment of LPS induced M1 macrophage group (NXK + LPS): Cells were cultured with medium containing 5% NXK-containing rat serum and 5% FBS with the addition of 200ug/mL LPS treatment for more than 16 h, DCA (Cat. No. S8615, Selleck) treatment of LPS induced M1 macrophage group (DCA + LPS): Cells were cultured with medium containing 5% ctrl rat serum + 5% FBS with the addition of 200 µg/mL LPS and 100 μΜ DCA treatment for more than 16 h, DMOG (Cat. No. S7483, Selleck) treatment of LPS induced M1 macrophage group (DMOG + LPS): Cells were cultured with medium containing 5% ctrl rat serum + 5% FBS with the addition of 200 µg/mL LPS and 5 mM DMOG treatment for more than 16 h, DMOG and NXK treatment of LPS induced M1 macrophage group (NXK + DMOG + LPS): Cells were cultured with medium containing 5% NXK-containing rat serum and 5% FBS with the addition of 200 ug/mL LPS and 5 mM DMOG treatment for more than 16 h.

Echocardiography

Four weeks after surgery, the mice underwent examination for cardiac morphology and function using the Vevo 2100 imaging system (Vevo TM 2100). The cardiac functional parameters collected included: left ventricular ejection fraction (LVEF), left ventricular fraction Shortening (LVFS), stroke volume (SV), Left ventricular anterior wall systolic thickness (LVAWs), left ventricular anterior wall diastolic thickness (LVAWd), left ventricular posterior wall systolic thickness (LVPWs), left ventricular posterior wall diastolic thickness (LVPWd), left ventricular end-systolic volume (LVs) and left ventricular end-diastolic volume (LVd).

Masson’s trichrome staining

Briefly, mouse hearts were excised, and fixed in 4% paraformaldehyde (Art. No. BL539A, Biosharp), embedded in paraffin, and cut into 5 μm thick serial sections. Masson's trichrome staining was performed on each section, and the stained sections were observed using a Pannoramic 250/MIDI microscope (3D HISTECH, Hungary). Image J software was used to measure the fibrotic area in the sections.

2,3,5-triphenyl tetrazolium chloride (TTC) staining

After the mice were sacrificed, the heart tissues were collected and briefly rinsed in icy PBS solution to remove residual blood from the ventricles. Then the heart tissues were semifreezed in a − 20 ℃ freezer for 30 min and then cut into 5 slices (1–1.5 mm thick) in a perpendicular way to the long axis. The heart tissue was totally soaked in 1% TTC solution, incubated in a water bath at 37 ℃, stained in the dark for 30 min, and finally fixed with 4% paraformaldehyde and photographed.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay

TUNEL detection was performed using the colorimetric TUNEL Apoptosis Detection Kit (Cat.No.C1091, Beyotime) following the provided instructions. Mouse heart tissue underwent a series of steps: incubation in 4% paraformaldehyde for 24 h, dehydration in 30% sucrose for 24 h, embedding in optimal cutting temperature (OTC) liquid, and slicing into 5 μm thick sections. The sections were immersed in 4% paraformaldehyde for 15 min to immobilize the cells, which was added with immuno-staining strong permeation solution for incubation at room temperature for 5 min, followed by PBS washing, followed by the addition of endogenous peroxidase blocking solution for incubation at room temperature for 20 min, PBS washing, the addition of an appropriate amount of biotin labeling solution for incubation at 37 ℃ for 60 min in the dark, PBS washing, the addition of labeling reaction stopping solution for incubation at room temperature for 10 min, and PBS washing. Subsequently, the Streptavidin-HRP working solution and DAB developing solution were added in sequence. Finally, the slides were sealed with an anti-fluorescence quencher containing DAPI and observed, and images were collected under a fluorescence microscope (Model No. IX73, Olympus).

Flow cytometric analysis of cellular reactive oxygen species (ROS) and different types of macrophages

Cellular ROS levels were detected using the MitoSox Red Fluorescent Staining Kit (Cat. No. M36008, Thermo), and the working solution was prepared by adding MitoSox Red stock solution to HBSS (containing calcium and magnesium ions) (Cat. No. H1025, Solarbio) at a ratio of 1:1000. The MitoSox Red working solution was incubated with the cells in the dark at 37 °C for 10 min. After incubation, cells were collected, centrifuged at 1000 rpm for 5 min, and the supernatant was aspirated, and the cells were resuspended in 500 µl HBSS.

Flow cytometric analysis of macrophage types in heart tissue: Hearts were immediately excised, flushed with HBSS, and torn into small pieces. Collagenase II (Cat. No. A004174-001, Diamond) was added to HBSS, and heart tissue was digested and ground at 37 °C. The supernatant was collected, erythrocytes were removed using Red Blood Cell Lysis Buffer (Cat. No. BL503B, Biosharp), and then the residue was filtered through a 70 μm filter (Cat. No. 352340, BD). The filtrate was centrifuged (1700 rpm, 5 min) and resuspended in 500 μL of icy HBSS. CD11b (Cat. No. 557672, BD), F4/80 (Cat. No. 565410, BD), Ly6G (Cat. No. 127618, Biolegend), and Ly6C (Cat. No. 128015, Biolegend) antibodies were added to the cell suspension and incubated for 30 min on ice in the dark.

FACS was performed by a MoFlo Astrios EQ (Beckman Coulter) and a FACS Calibur flow cytometer (BD). Data analysis was then performed with FlowJo 10.1 software (Tree Star).

Quantitative real-time PCR (qPCR) analysis

Total RNA was extracted by RNAzol RT (Cat. No. RN190, Molecular Research Center) in accordance with the manufacturer's instructions. Subsequently, the extracted total RNA underwent reverse transcription, following the guidelines outlined in the Fastking RT reverse transcription kit (Cat. No. KR116, TIANGEN). The resulting cDNA was utilized for quantitative PCR on a CFX96 Real-Time System (Bio-Rad, Hercules) with the SYBR Select master mix kit (Cat. No.4472908, Life Technology). The relative mRNA expression leves of specific genes was normalized to GAPDH, and data analysis was performed utilizing the ΔΔCt method. The primers sequences used for qPCR can be found in Table 1.

Table 1 Primer sequences of genesEnzyme-linked immunosorbent assay (ELISA)

The ELISA kits were employed to assess the concentration of IL-1β (Cat. No. JL18222, J&l biological), IL-6 (Cat. No. JL20268, J&l biological), LDH (Cat. No. RD-RX20588, Henghuibio), cTn-I (Cat. No. RD-RX20716, Henghuibio) and CK-MB (Cat. No. RD-RX28610, Henghuibio) in mouse peripheral blood serum and ROS production in mouse heart tissues (Cat. No. E004, Nanjin Jiancheng Bioengineering Institute). The experimental procedures followed the guidelines provided by the respective kit manufacturers.

PDH activity detection

The heart tissue was ground using an ice bath homogenizer, followed by centrifugation at 11,000g for 10 min at 4 °C. The resulting supernatant was collected, and the optical density (OD) of the sample at 605 nm was measured in accordance with the manufacturer's instructions provided with the PDH activity assay kit (Cat. No. YX-W-B103, Sinobestbio). PDH activity was then calculated based on the weight of the heart.

Western Blot (WB) analysis

Heart tissue and cellular proteins were extracted with RIPA lysis buffer (Cat. No. P0013B, Beyotime).Subsequently, proteins of varying molecular weights were separated via SDS-PAGE (4–20%) and transferred onto 0.45 μm PVDF membranes (Cat. No. IPVH00010, EMD Millipore). After blocking the membranes with 5% skimmed milk (Cat. No. 9999, CST) for 1 h at room temperature, they were incubated overnight at 4 °C with specific antibodies: GAPDH (1: 1000, Cat. No. 2118, CST), HIF-1α (1: 500, Cat. No. bs-0737R, Bioss), and PDK1 (1: 1000, Cat. No. DF4365, Affinity Bioscience) on a shaker. The following day, secondary antibodies (Cat. No. 7074S, CST) were applied and incubated for 1 h at room temperature. Immunoblotting was visualized using ECL (Cat. No. WBKLS0500, Millipore), and the density of immune-reactive bands was analyzed using Image J software.

Metabolic analysis

For metabolic analysis, RAW264.7 cells were seeded in XFe 24 well microplates at a density of 2 × 106 cells per well (Cat. No. 102340-100, Agilent Technologies). Oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and real-time ATP rate were respectively measured using the Cell Mito Stress Test kit (Cat. No. 103015-100, Agilent Technologies), Glycolysis Stress Test Kit (Cat. No. 103020-100, Agilent Technologies) and Real-time ATP Rate Assay Kit (Cat. No. 103592-100, Agilent Technologies) following the manufacturer's instructions. The measurements of OCR, ECAR, and real-time ATP rate were conducted using a Seahorse XFe24 Analyzer (Cat. No.S7801B, Agilent Technologies), and the results were analyzed with Wave software version 2.6.0 (Agilent Technologies).

Statistical analysis

SPSS25.0 statistical software was used for data analysis. All data were described as mean standard deviation (X ± SD).T test was adopted to assess the significance of differences between the two groups. For comparisons among multiple groups, one-way ANOVA was employed, with statistical significance set at P < 0.05.