A total of 30 SPF-class male Wistar rats (age, 8 weeks; weight, 200±10 g) were purchased from Beijing HFK Bioscience Co., Ltd. The rats were housed (temperature: 22±2°C; humidity: 50±5%) in Tianjin Chest Hospital Cardiovascular Institute (Tianjin, China) under a constant temperature of 25±1°C and ad libitum access to food and water, in accordance with the requirements of the Animal Research: Reporting of In Vivo Experiments guidelines (version 2.0) (22). All animal experiments were approved by the Animal Ethics Committee of Tianjin Chest Hospital (approval no. TJCH-2021-007).

H9C2 cardiomyocytes (cat. no. 3101RATGNR5; National Collection of Authenticated Cell Cultures) were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Beyotime Institute of Biotechnology), penicillin (100 U/ml) and streptomycin (100 µg/ml) at 37°C and 5% CO₂. The culture medium was changed at every 48 h. Cells (2×10⁵/well) were inoculated into 6-well plates and pretreated with liriodendrin (20, 50, 100 µM) for 24 h at 37°C. The cells were treated with hypoxia for 4 h at 37°C in glucose-free DMEM (Gibco; Thermo Fisher Scientific, Inc.) and placed in an anaerobic incubator (95% N₂ and 5% CO₂). Following hypoxia, the cells were reoxygenated in DMEM containing 4.5 mm glucose and placed in an incubator (95% air and 5% CO₂) at 37 °C for 24 h. Dmethyl sulphoxide (DMSO; cat. no. ST038; Beyotime Institute of Biotechnology) was used as control with a final concentration of 0.1%.

Wistar rats (matched for age, sex and body weight) were randomly divided into the sham operation, I/R and liriodendrin groups (n=10/group). Liriodendrin powder (cat. no. HY-N3377; MedChemExpress; purity >97%) was diluted with PBS to obtain 10 g/l solution. The liriodendrin group was treated with liriodendrin solution (10 ml/kg body weight, equivalent to 100 mg/kg body weight) by intragastric administration daily, from 5 days before surgery to 3 days after surgery. I/R and sham operation groups were intragastrically administered normal saline (10 ml/kg/body weight) daily. After 5 days, animals were anesthetized through inhalation of 3% sevoflurane (~1.3 minimum alveolar concentration [MAC]) and endotracheal intubation was used to assist respiration, with a tidal volume of 28-32 ml/kg, respiratory rate of 75 breaths/min and an inspiratory to expiratory ratio of 1:2. A thoracotomy was performed between the 3rd and 4th intercostal space on the left side of the sternum to expose the heart. The anterior descending coronary artery was ligated at the lower edge of the left atrial appendage. Pale myocardium in the anterior wall of the left ventricle and 0.2 mV ST-elevation in electrocardiograms I, II and augmented voltage left (aVL) were considered markers of successful ligation. Following 30 min assisted ventilation, the ligature was cut to restore the blood supply. In the sham group, the suture was threaded without ligation. The animals were anesthetized with 3% sevoflurane (~1.3 MAC) prior to blood collection or echocardiography. The humane endpoints for animal experiments followed the guidelines outlined in recommended industry standard (RB/T) 173-2018 (rbtest. cnca.cn/portal/stdDetail/500828). The humane endpoints were critical respiratory infections, respiratory distress and incapacity to autonomously ingest food and water. No rats have reached any of the humane endpoints. Animals were euthanized by intravenous administration of pentobarbital sodium (150 mg/kg).

At 2 h after the operation, 1 ml venous blood was collected from the subclavian vein. Quantitative detection of creatine kinase isoenzymes (CKMB; cat. no. E-EL-R1327) and high-sensitivity cardiac troponin (cTn; cat. no. E-EL-R0151) T was conducted using ELISA kits (both Elabscience Biotechnology Co. Ltd.).

Cardiac function was measured by echocardiography (cat. no. L15-7io; Philips Healthcare) 3 days after surgery. End-diastolic volume (EDV), left ventricular end-diastolic diameter (LVIDd), ejection fraction (EF), left ventricular end-systolic diameter (LVIDs), and end-systolic volume (ESV) were measured in the short axis of the papillary muscle. A total of three cardiac cycles was measured for each index and the mean was calculated.

After rats were euthanized, the hearts were harvested and myocardial tissue was fixed in 10% neutral formalin for 24 h at room temperature. After dehydration, transparency, wax immersion and embedding, paraffin blocks were produced. Tissues were sliced in successive sections parallel to the long axis of the heart at a thickness of 4 µm. H&E staining was performed at room temperature for 1 min and visualized under a light microscope (BX53, Olympus Corporation) at a magnification of ×100 and ×400.

Apoptosis was detected by TUNEL staining (cat. no. G1507; Servicebio Technology, Inc.) in three fresh heart tissue samples from each group of animals and the mean proportion of TUNEL-positive cells in five randomly selected fields of view was calculated to determine the apoptosis index of the cardiomyocytes. In brief, heart tissue samples were fixed in 10% neutral formalin for 24 h at room temperature. Terminal deoxynucleotidyl transferase (TdT) incubation buffer (2 µl recombinant TdT enzyme, 5 µl biotin-dUTP labeling mix, 50 µl equilibration buffer) was added at 37°C for 1 h, and sections were washed 3 times with PBS. 0.5% streptavidin-HRP buffer was incubated at 37°C for 30 min and then washed 3 times with PBS, 5% DAB chromogenic solution was added at room temperature for 30 min and then washed 3 times with PBS, and 0.5% hematoxylin staining solution was stained at room temperature for 5 min. TUNEL staining was followed by nuclear staining with DAPI (1 µg/ml; cat. no. D106471-5 mg; Shanghai Aladdin Biochemical Technology Co., Ltd.) and incubated at room temperature for 10 min and then washed 3 times with PBS. The sections use neutral resin for mounting and visualize with light microscope (BX53, Olympus Corporation). The apoptotic index (AI) of the myocardial cell was calculated as the proportion of the apoptotic cells (brown-yellow granules in the nucleus) relative to the total number of the cells (23).

Immunohistochemical staining. Immunohistochemistry was used to stain myocardial tissues to determine the expression of cleaved caspase-3, Bcl-2 and Bax proteins. Myocardial tissue was fixed in 10% neutral formalin for 24 h at room temperature. After dehydration with descending alcohol series (95, 85, and 75%), transparency, wax immersion and embedding, paraffin blocks were produced. Tissues were sliced in successive sections parallel to the long axis of the heart at a thickness of 4 µm. The slices were boiled in an antigen retrieval solution (1.8% 0.1 M citrate buffer and 8.2% 0.1 M sodium citrate buffer) at 95°C for 10 min. The slices were washed in PBS buffer. Subsequently, slices were treated with 1% BSA) (cat. no. ST023; Beyotime Institute of Biotechnology, Haimen) for 15 min at room temperature. Slices were incubated at 4°C overnight with the following primary antibodies: cleaved caspase-3 (cat. no. #9664; dilution: 1:500; Cell Signaling Technology, Inc.), Bcl-2 (cat. no. sc-7382; dilution: 1:200; Santa Cruz Biotechnology, Inc.) and Bax (cat. no. sc-7480; dilution: 1:200; Santa Cruz Biotechnology, Inc.). Subsequently, the corresponding secondary antibody, SignalStain^® Boost IHC Detection Reagent (HRP, Rabbit; cat. no. #9664; 1:1,000; Cell Signaling Technology) for cleaved caspase-3 and m-IgGκ BP-HRP (at. no: sc-516102; dilution: 1:1,000; Santa Cruz Biotechnology, Inc.) for Bcl-2 and Bax, with a streptavidin-HRP conjugate 1 h at room temperature. Moreover, the slices were stained with DAB kit and Haematoxylin. DAPI (cat. no. D106471-5 mg; Shanghai Aladdin Biochemical Technology Co., Ltd.) incubation for 10 min at room temperature was utilized as a nuclear staining. Brown-stained cells were considered positive cells. Finally, images were captured at ×400 magnification using an opticallight microscope (BX53, Olympus Corporation) and measured by Motic med 6.0 software (Motic China Group, Co., Ltd.).

After rats were sacrificed, the hearts were harvested and myocardial tissue from the left ventricular myocardial injury site was obtained. Tissue was homogenized with PRO200 hand-held laboratory homogenizer (Bio-Gen PRO200, Genosys Tech-Trading Co., Ltd.) and centrifuged for 10 min at 3,000 × g and 4°C to obtain supernatant. IL-1β (cat. no. H002-1-1; Nanjing Jiancheng Bioengineering Institute), TNF-α (cat. no. H052-1-1; Nanjing Jiancheng Bioengineering Institute), C-C motif chemokine ligand 2 (MCP-1) (cat. no. H115-1-1; Nanjing Jiancheng Bioengineering Institute) and ROS (cat. no. E004-1-1; Nanjing Jiancheng Bioengineering Institute) levels were detected using ELISA kits according to the manufacturer's instructions. SOD (cat. no. A001-3-2; Nanjing Jiancheng Bioengineering Institute) levels in the peripheral venous blood of rats 2 h after the operation were also measured using an ELISA kit according to the manufacturer's instructions.

Total protein was extracted from myocardial tissue of rats or H9C2 cells and centrifuged at 4°C for 20 min at 10,000 × g with lysis buffer (cat. no. P0013; Beyotime Institute of Biotechnology) containing phenylmethanesulfonyl fluoride (cat. no. ST506; Beyotime Institute of Biotechnology) on ice for 5 min. The protein concentration was determined using the BCA method (cat. no. 5000002; Bio-Rad Laboratories, Inc.). The 30 µg protein samples were then separated using SDS-PAGE with gel (8-12%) and a 5% stacking gel, followed by transfer onto PVDF) membranes. The membranes were blocked using 5% BSA (cat. no. ST023; Beyotime Institute of Biotechnology) at room temperature for 2 h. and subsequently incubated overnight at 4°C with primary antibodies. Proteins were incubated with antibodies (1:1,000) as follows: NF-κB (cat. no. ab19870), phosphorylated (p)-NF-κB (cat. no. ab239882), IKKβ (cat. no. ab178870), IκBα (cat. no. ab178847), p-IκBα (cat. no. ab133462) (all Abcam), Bcl-2 (cat. no. sc-7382), Bax (cat. no. sc-7480; both Santa Cruz Biotechnology, Inc.) and cleaved caspase-3 (cat. no. #9664; Cell Signaling Technology). Subsequently, the corresponding secondary antibody, anti-Rabbit IgG H&L (HRP) (cat. no. ab205718; 1:10,000; Abcam) for cleaved caspase-3, NF-κB, p-NF-κB, IKKβ, IκBα, p-IκBα and m-IgGκ BP-HRP (at. no: sc-516102; dilution: 1:10,000; Santa Cruz Biotechnology, Inc.) for Bcl-2 and Bax, was incubated for 2 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence kit (cat. no. 323106; Thermo Fisher Scientific, Inc.). The images were analyzed using ImageJ software (version 1.54; National Institutes of Health) and the relative content of the target protein was expressed as the gray value of the target protein/GAPDH gray value. A total of three biological replicates was performed for each treatment group. Certain target proteins and loading controls were screened from different integral membranes due to the similarities in molecular weight. Experimental conditions were consistent for protein detection. Anti-GAPDH (cat. no. sc-32233; 1:2,000; Santa Cruz Biotechnology, Inc.) was used to as the control antibody.

Total RNA was extracted from rat cardiomyocytes or H9C2 cells using TRIzol (cat. no. 15596026; Thermo Fisher Scientific, Inc.) and the TaKaRa-RNA Quantification and Reverse Transcription kit (Takara Bio, Inc.). RT was performed using the BeyoRT II M-MLV reverse transcriptase (cat. no. D7160L; Beyotime Institute of Biotechnology) according to the manufacturer's protocol. The qPCR procedure was conducted with SYBR Green (cat. no. SY1020; Beijing Solarbio Science & Technology Co., Ltd.) and 2×Taq PCR MasterMix (cat. no. PC1150; Beijing Solarbio Science & Technology Co., Ltd.) using a fluorescent qPCR instrument (Exicycler 96; Bioneer Corporation). The following thermocycling conditions were used: 95°C for 5 min, followed by 95°C for 10 sec, 54~62°C (different genes use different annealing temperatures) for 10 sec and 72°C for 15 s. The 2^−ΔΔCq method was used to analyze the target gene expression (24). The primer sequences were as follows: β-actin forward (F), 5′-TCA GGT CAT CAC TAT CGG CAAT-3′ and reverse (R), 5′-AAA GAA AGG GTG TAA AAC GCA-3′ (annealing temperature, 54°C); IL-1β F, 5′-CCT CTG TGA CTC GTG GGA TGA-3′ and R, 5′-CAC GAG CAT TTT TGT TGT TCA-3′ (annealing temperature, 56°C); TNF-α F, 5′-CTT CTC ATT CCT GCT CGT GG-3′ and R, 5′-TCA GCC ACT CCA GCT GCT C-3′ (annealing temperature, 55°C); Bcl-2 F, 5′-GGA GCG TCA ACA GGG AGA TG-3′ and R, 5′-GCA GGT CTG CTG ACC TCA CTTG-3′ (annealing temperature, 59°C); Bax F, 5′-CCA AGA AGC TGA GAG AGT GTC TC-3′ and R, 5′-AGT TGC CAT CAG CAA ACA TGT CA-3′ (annealing temperature, 58°C) and cleaved caspase-3 F, 5′-AGC ACT GGA ATG TCA GCT CGC-3′ and R, 5′-CAG GTC CAC AGG TCC GTTC-3′ (annealing temperature, 62°C).

Cells were harvested and fixed using 2.5% glutaraldehyde solution for 4 h at 4°C. The supernatant was discarded after 12 h and cells were rinsed in phosphate buffer (pH 7.0). Cells were fixed with 1% osmium acid solution for 2 h at 4°C and rinsed with an osmium acid solution. Samples were sliced into 70-90 nm sections after dehydration and embedded. Sections were stained using lead citrate (0.5%) and uranyl acetate (1%) for 30 min at room temperature and imaged using a scanning electron microscope with gold as a sputter coating.

Data from ≥3 independent experiments are presented as the mean ± SD. The t-test was used to analyze statistical differences following Shapiro-Wilk normality test and variance homogeneity testing using Levene's test. Inter-group differences were assessed using one-way ANOVA with Tukey's Honestly Significant Difference post hoc test. P<0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using SPSS (version 23.0; IBM Corp.) or GraphPad Prism software (version 5.02; Dotmatics).

To evaluate the success rate of I/R modeling in rats and the protective effect of liriodendrin on myocardial I/R injury, CKMB (Fig. 1A) and cTnT (Fig. 1B) levels were measured. Compared with the sham group, the CKMB levels in the I/R group were significantly increased, indicating successful establishment of the rat model of myocardial I/R injury. Moreover, the levels of CKMB in the liriodendrin group were significantly lower compared with those in I/R group, which indicated that myocardial injury induced by I/R in rats was alleviated by liriodendrin.

Echocardiography showed significant changes of EDV (Fig. 2A and B) and LVIDd (Fig. 2A and C) in I/R compared to those of the normal rats, while liriodendrin administration significantly reversed I/R-induced the elevation of EDV and LVIDd in I/R rats. However, I/R operation had no obvious changes of EF (Fig. 2A and D), LVIDs (Fig. 2A and E), and ESV (Fig. 2A and F). Interestingly, liriodendrin administration significantly lowered EF (Fig. 2A and D), LVIDs (Fig. 2A and E), and ESV (Fig. 2A and F) in I/R rats compared with I/R operation alone.

In the sham group, the left ventricular myocardial fibers displayed a well-organized and structured arrangement, with normal morphology of myocardial cells. The H&E staining demonstrated that in the I/R group, cardiomyocytes exhibited an indistinct morphology, disorganized arrangement and pyknotic, lysed or absent nuclei, along with notable infiltration of inflammatory cells. Liriodendrin group exhibited markedly less severe pathological alterations compared with the I/R group, including a notable reduction in the degree of inflammatory cell infiltration (Fig. 3A).

The inflammatory response cascade is initiated by IL-1β and TNF-α (25). Increased IL-1β levels were observed in the heart tissue of the I/R group when compared with the sham group (Fig. 3B). Conversely, no significant alteration in TNF-α levels were observed in response to I/R (Fig. 3C). MCP-1 serves a crucial role in the migration and infiltration of macrophages, facilitating their accumulation at the site of inflammation and exacerbating the inflammatory response (26). Increased expression of MCP-1 within the myocardium of the I/R group was observed compared with the sham operation group. Conversely, in rats treated with liriodendrin, there was a significant decrease in MCP-1 expression (Fig. 3D). SOD serves a crucial role in the elimination of ROS (27); there was a significant decrease in SOD levels in the peripheral blood of the I/R group at 2 h post-injury (Fig. 3E). Conversely, there was a notable increase in ROS levels in myocardial tissue of I/R rats, suggesting that I/R injury may intensify the oxidative stress response in cardiomyocytes (Fig. 3F). Compared with the I/R group, the liriodendrin group showed a significant increase in SOD (Fig. 3E) and a significant decrease in ROS levels (Fig. 3F), suggesting that liriodendrin may suppress tissue inflammation following I/R injury and counteract oxidative stress induced by I/R, thereby decreasing cardiomyocyte damage. However, there were no significant differences in the levels of IL-1β and IL-6 in the I/R compared with the liriodendrin group (Fig. 3G and H).

To investigate the protective effect of liriodendrin on cardiomyocytes, immunohistochemical staining was conducted. Semi-quantitative analysis of key proteins involved in the apoptosis pathway was performed and the AI was calculated. Compared with the sham group, a significant decrease in the anti-apoptotic protein marker Bcl-2 was observed in the I/R group, suggesting I/R injury may exert a pro-apoptotic influence on rat cardiomyocytes (Fig. 4A and B). Furthermore, a significant increase in Bax (Fig. 4A and C) and cleaved caspase-3 levels was observed in the I/R group (Fig. 4A and D). The liriodendrin group exhibited a significant decrease in expression levels of Bax and cleaved caspase-3 in cardiomyocytes, along with a significant increase in expression of the anti-apoptotic protein Bcl-2. The AI was significantly higher in the I/R compared with the sham group. The liriodendrin group exhibited a marked decrease in the apoptosis index but this was not significant (Fig. 4E). These findings suggested that liriodendrin effectively enhanced the anti-apoptotic capacity of cardiomyocytes subjected to I/R.

To determine the impact of liriodendrin on the apoptosis pathway in rats subjected to I/R, western blotting (Fig. 5A) was used to measure protein expression levels of Bcl-2 (Fig. 5B), Bax (Fig. 5C) and cleaved caspase-3 (Fig. 5D), which are pivotal proteins involved in the apoptosis pathway induced by myocardial injury (28). In accordance with the findings of immunohistochemical staining, the liriodendrin group exhibited increased protein expression of Bcl-2 and decreased protein expression of Bax and cleaved caspase-3 compared with the I/R group. Furthermore, the liriodendrin group demonstrated a significant increase in transcriptional activity of Bcl-2 compared with the I/R group (Fig. 5E). These results suggested that liriodendrin decreased activation of the apoptosis pathway of cardiomyocytes in rats subjected to I/R.

Liriodendrin may impede the NF-κB signaling pathway, which is important in the inflammatory response subsequent to myocardial infarction (19). In the context of myocardial injury induced by I/R, liriodendrin inhibited the release of inflammatory factors. Consequently, the present study evaluated the protein expression of NF-κB pathway components, including NF-κB (Fig. 6A and B), p-NF-κB (Fig. 6C), IκBα (Fig. 6D), p-IκBα (Fig. 6E) and IKKβ (Fig. 6F). These results demonstrated a significant decrease in the expression of p-NF-κB and p-IκBα in the liriodendrin group compared with the I/R group. This suggested that liriodendrin effectively inhibited the NF-κB signaling pathway in myocardial I/R injury in rats, mitigating the release of inflammatory factors.

Mitochondria are key target organelles of I/R injury. ROS induce protein and lipid peroxidation, affect ATP synthesis, activate phospholipase and damage structure of mitochondria (29). To clarify the protective effect of liriodendrin, H9C2 cells were treated with 100 µM liriodendrin for 24 h, hypoxia was induced for 4 h and cells were cultured with normal medium for 24 h after reoxygenation to observe the differences in mitochondria. Compared with normal cells, the mitochondria of cells that underwent hypoxia/reoxygenation were swollen and demonstrated vacuolar degeneration, an absence of mitochondrial cristae and an incomplete outer membrane. A majority of the mitochondria in the liriodendrin-treated group exhibited normal morphology, with a few swollen and vacuolated mitochondria (Fig. 7). These results suggested that liriodendrin may serve a role in preserving the mitochondrial morphology of cells subjected to I/R.

A previous study reported that varying concentrations of liriodendrin exhibit distinct therapeutic effects on cardiomyocytes cultured under hypoxic conditions (19). To elucidate the impact of liriodendrin concentration on the therapeutic efficacy of hypoxia/reoxygenation in H9C2 cells, pretreatment of H9C2 cells with three concentrations of liriodendrin was performed. The protein expression of NF-κB, p-NF-κB, IκBα, p-IκBα, Bax and Bcl-2 was assessed across a range of liriodendrin concentrations (0-100 µM; Fig. 8A and B). All concentrations of liriodendrin caused a significant decrease in protein expression levels of p-NF-κB and p-IκBα (Fig. 8A and B). Furthermore, 100 µM liriodendrin caused a significant decrease in Bax protein expression levels and a significant increase in Bcl-2 protein expression. The transcriptional activity of genes related to apoptosis was measured. Transcriptional activity of Bax and caspase-3 demonstrated a marked decrease, while the ratio of Bcl-2/Bax showed a marked increase in cells treated with 100 µM liriodendrin; however, these results were not significant (Fig. 8C).