Bai (purity >99%) was obtained from Damas-Beta (Shanghai Titan Scientific Co., Ltd), ISO was purchased from Amylet Scientific Inc. (Michigan, United States), and verapamil (Ver) was provided by Harvest Pharmaceutical Co., Ltd. (Shanghai, China). Nicardipine (Nic) was obtained from Sigma Chemical Co. (St. Louis, Mo). Bai was diluted into the proper concentrations in carboxymethyl cellulose and used immediately. ISO and Ver were dissolved in normal saline. Dulbecco’s modified Eagle’s medium (DMEM, Catalog: 12430047), fetal bovine serum (Catalog: 10093188), trypsin (Catalog: 25200072), and penicillin/streptomycin were purchased from Gibco (Thermo Fisher Scientific, Inc. Missouri, United States). Unless otherwise specified, other laboratory chemicals were obtained from Sigma-Aldrich (St. Louis, MO, United States) and were of analytical purity.

Forty Kunming male mice (20 ± 2 g; 4–5 weeks) and male adult Sprague-Dawley rats (180–220 g; 42–45 days) were obtained from the Experimental Animal Centre, Hebei Medical University. Before the experiments, animals were brought into the laboratory and allowed to acclimate for 1 week. Mice were caged in 22 ± 2°C with 12 h light/dark cycles and supplied with adequate food and water. The experiments were performed according to the International Association of Veterinary Editors guidelines and approved by the Ethics Committee for Animal Experiments of Hebei University of Chinese Medicine (approval number: DWLL2020073).

Forty male Kunming mice were randomly allocated into five groups with eight mice per group. The doses of ISO and Bai are can be obtained from previous references (Haleagrahara et al., 2011; Dinda et al., 2017). Details of the experimental groups are as follows:

Control (Con): Mice were intraperitoneally injected (i.p.) with 0.9% normal saline (0.1 M/kg/day) once each day for 7 days.

ISO: Mice were subcutaneously administered ISO (85 mg/kg, s. c.) on the last two consecutive days of the experiment.

Low-dose Bai (L-Bai): Mice in the L-Bai group were pre-treated with Bai solution (30 mg/kg/day) via gavage for 7 days. Then mice were subcutaneously administered ISO (85 mg/kg, s. c.) on the last two consecutive days of the experiment.

High-dose Bai (H-Bai): Mice were pre-treated with Bai solution (60 mg/kg/day) via gavage for 7 days. Then mice were subcutaneously administered ISO, the same as the L-Bai group.

Ver: Mice were given Ver (2 mg/kg/day, i. p.) for 7 days. Then ISO was subcutaneously administered, the same as L-Bai and H-Bai groups.

Twenty-four h after the last treatment, pentobarbital sodium (50 mg/kg) was injected i. p. to anesthetize the mice (B. Zheng et al., 2021). The electrocardiogram (ECG) was recorded on anesthetized mice. After that, blood was collected and serum was separated by centrifugation. Then hearts were quickly dissected, rinsed with saline, weighed, and photographed. For further detection, the heart tissues were placed into liquid nitrogen.

After anesthesia, the RM6240BD Biological Signal Collection System (Chengdu Manufacture Factory) was used to record the ECG and monitor changes in heart rate and ST-segment elevation in each group of mice. ECG was recorded using three subcutaneous needle electrodes: 1) green electrode connected to the right upper limb; 2) black electrode connected to the right lower limb; and 3) red electrode connected to the left lower limb.

Mice were anesthetized, blood was drawn from the enucleation of the eyeball and collected in heparinized tubes. The serum was separated by centrifugation at 3,500 rpm for 10 min. Creatine serum (CK) in serum (No. A032-1-1) and lactate dehydrogenase (LDH) levels (No. A020-2-2) were measured to estimate myocardial damage via a colorimetric method using standard diagnostic kits (Jian Cheng Biological Engineering Institute, Nanjing, China). A UNICO-UV2000 spectrophotometer (JianCheng, Nanjing, China) was used to measure corresponding absorbances.

The concentration of Ca²⁺ (No. C004-2-1) was assessed in the myocardium. Heart tissue was homogenized in 10% deionized water. The homogenate was centrifuged at 2,500 rpm for 10 min and the supernatant was used for the assay. After the samples were processed, the BCA kit was used to detect the protein concentration. The concentration of Ca²⁺ was measured by methylthymol blue staining following the commercially available kit’s instructions (Jiancheng, Nanjing, China). Finally, the OD value of each well was measured at 610 nm using an enzyme marker.

The heart samples were prepared and immediately stocked in 4% paraformaldehyde. After 24 h, samples were routinely processed and embedded in paraffin. Samples were sectioned (5 μm) and stained using hematoxylin and eosin (H&E). Sections were visualized via light microscopy (Leica DM4000B, Solms, Germany).

IL-6 (No. 88-7064) and TNF-α (No. 88-7324) expressions in heart tissues were measured to test the inflammatory status in mice of the five groups using enzyme-linked immunosorbent assay (ELISA) kits according to manufacturer’s instructions (Servicebio, Wuhan, China).

As previously described (Zhang et al., 2016), the cardiac tissues were dissolved in RIPA lysis buffer (No. G2002; Servicebio, Wuhan, China). The supernatant containing total protein extract was used to determine the concentration using the BCA protein quantitative assay kit (No. G2026; Servicebio, Wuhan, China) after centrifugation (12,000 rpm, 10 min, 4°C). Heart tissue lysates were loaded onto a 10% SDS-PAGE gel (No. G2003; Servicebio, Wuhan, China), transferred into polyvinylidene fluoride membranes (No. G6015–0.45; Servicebio, Wuhan, China), and blocked with TBST buffer carrying 5% (w/v) skim milk at 37°C for 30 min. The membranes underwent overnight incubation at 4°C with primary antibodies against Bax (No. GB11690; Servicebio, Wuhan, China; diluted 1:1,000), Bcl-2 (No. PAA778Mu01; Cloud-Clone, Wuhan, China; diluted 1:1,000), Caspase-3 (No. 66470-2-lg; San Ying, Wuhan, China; diluted 1:1,000), TLR4 (No. GB11519; Servicebio, Wuhan, China; diluted 1:1,000), MyD88 (No. 23230-1-AP; San Ying, Wuhan, China; diluted 1:1,000), polyclonal anti-NF-κB (p65) (No. GB11142; Servicebio, Wuhan, China; diluted 1:1,000), p-p38 (no. 4511; Cell Signaling Technology, Massachusetts, United States; diluted 1:1,000), p-extracellular signal-regulated kinase1/2 (p-ERK1/2, No. 4370; Cell Signaling Technology, Massachusetts, United States; diluted 1:1,000), and c-Jun N-terminal kinase (p-JNK, no. 4668; Cell Signaling Technology, Massachusetts, United States; diluted 1:1,000). Anti-β-actin (No. GB12001; Servicebio, Wuhan, China; diluted 1:1,000) was used as the internal standard. The next day, the membrane was washed with PBS-T (No. G0001-2L; Servicebio, Wuhan, China) and incubated with horseradish peroxidase (HRP)-conjugated secondary anti-rabbit or anti-mouse antibodies (No. GB23303 or No. GB23301; Servicebio, Wuhan, China; diluted 1:3,000) for 30 min at 37°C. The bound antibodies were visualized using the enhanced chemiluminescence (ECL) system (No. G2014; Servicebio, Wuhan, China) and quantified by densitometry using an Alpha Ease FC system (Alpha Innotech, Shanghai, China).

Single ventricular myocardia were isolated from normal experimental rats that were anesthetized by injection of i. p. heparin sodium (500 IU/kg) and ethyl carbamate (1.0 g/kg). The hearts were promptly dissected, soaked in frozen Tyrode’s solution, and suspended in Langendorff equipment perfused by oxygenated frozen free Ca²⁺ Tyrode’s solution for 5 min. After that, an enzymatic solution (Collagenase Type Ⅱ, ThermoFisher, No. 17101-015) was applied to digest the heart for 15–20 min. Subsequently, the heart was removed, and the tissue was cleaned thoroughly with Tyrode’s solution. The ventricle was torn into tiny pieces in Kreb’s buffer solution. The freshly dissociated cardiomyocytes were preserved in Kreb’s buffer solution filled with oxygen for up to 1 h at room temperature before subsequent experiments were performed. Solutions used in the perfusion process must be oxygenated with 100% O₂ and prepared as described in Table 1. The pH of normal Tyrode’s solution and the enzyme solution was adjusted to 7.4 using 3 M NaOH. The external/internal solution was adjusted to pH 7.3 with CsOH, and the Kreb’s buffer solution was adjusted to pH 7.2 using KOH.

Myocardial ischemia was induced in the rats by subcutaneous injection with ISO (85 mg/kg). The dose and pattern of injection were carried out according to a previous study (Han et al., 2019). After two consecutive days of ischemia were established, and experimental treatments were performed as described above to isolate ventricular cells from normal rats.

Whole-cell patch clamp recordings were performed in ventricular myocytes to measure the L-type Ca²⁺ currents. The pipette puller (Sutter Instrument, Novato, CA, United States) was employed for pulling the borosilicate glass microelectrodes of 3–5 MΩ resistance, which were later filled with a pipetted internal solution. I_Ca-L was recorded using an Axon Patch 200B amplifier with a 2 kHz applied to recordings. Data analyses were done using the Pclamp10.0 software (Axon Instruments, Union City, CA, United States). All experiments were performed in an air-conditioned room (23–25°C).

Ventricular myocyte contractions and Ca²⁺ transients were detected using the IonOptix detection system (Ion Optix, Milton, Massachusetts, United States). Cardiac myocytes placed in the bath were perfused with normal Tyrode’s solution. Myocardial shortening was initiated twice by field stimulation (0.5-Hz frequency, 2-ms duration) and observed under inverted microscopy. Only cardiomyocytes with a distinct vein were suitable for measuring contractions. Cardiomyocytes were dyed in the dark for 15 min with the fluorescent indicator fura-2/AM (1 mmol/L) and illuminated by alternating between 340 and 380 nm filters. The emitted fluorescence was tested at 510 nm, and Ca²⁺ transients were recorded with the Ion Optix (United States) fluorescence system.

Rat H9c2 cardiomyocytes were purchased from Bluefbio (Shanghai, China, No. BFN60804388) and cultured in high-glucose DMEM which contained 10% fetal bovine serum (Gibco, Grand Island, NY, United States, No. 2176377) and 100 U/mL penicillin/streptomycin under 5% CO₂ and 95% air at 37°C. The cell culture reagents were changed once every 2 days. H9c2 cardiomyocytes were digested with 0.25 g/L trypsin (Gibco) at approximately 80% cell confluence to be used for subculturing (Y. Zheng et al., 2019). The cells were seeded in 25-cm² culture flasks and passaged every 3 days. The follow-up experiments were performed after H9c2 cardiomyocytes had reached the logarithmic growth phase. H9c2 cardiomyocytes were divided into four groups (A) Con group; (B) ISO group at 50 μmol/L (Hu et al., 2013; Lin et al., 2017); (C) L-Bai group (10 μmol/L); (D) H-Bai group (30 μmol/L).

After the experiment, H9c2 cardiomyocytes were digested with a moderate amount of trypsin and centrifuged for 10 min at 1,500 rpm. The supernatant was discarded, and 500 μL phosphate-buffered saline (PBS) was added to resuspend the cell precipitate after which it was the mix. After that, cells were ruptured 10 times via ultrasound for 2 s to collect cell homogenate for detection. The concentration of IL-6 (No. H007) and TNF-α (No. H052) were determined by enzyme-linked immunosorbent (ELISA) kits according to the manufacturer’s instructions.

As a nucleus-specific dye, Hoechst 33258 (Beijing Solarbio Science & Technology, Beijing, China, No. IH0060) is used to evaluate the extent of apoptosis. Following the experiment, the cell culture media in 24-well plates from different groups were discarded, and H9c2 cardiomyocytes were washed three times with PBS (No. 003,002). After washing, cells in each group were incubated in 500 μL/well of Hoechst 33258 staining solution (10 μg/ml) for 20 min in the dark at room temperature, and they were then rinsed twice with PBS. The cells were observed and photographed immediately under fluorescence microscopy (Leica, Germany) (Y. Zheng et al., 2019). Nuclei with brightly stained and bright fragments were regarded as apoptotic cells, while cells with blue chromatin and organized structure were considered to be normal.

The resulting data are shown as mean ± standard error of the mean (S.E.M.) Statistical analyses were performed using Origin Pro version 9.1 software. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc tests was performed to compare more than two groups. A value of p < 0.05 was considered statistically significant.

Figure 2A shows representative ECG tracings. As shown in Figures 2B,C, the heart rate and ST-segment showed significant elevations in ISO-induced MI mice (p < 0.01). Heart rate and ST-segment in L-Bai, H-Bai, and Ver groups had decreased when compared with ISO group (p < 0.01 or p < 0.05). The results revealed that Bai inhibited heart rate and ST segment in a dose-dependent manner in mice.

As shown in Figure 2D and Table 2, a marked difference between the Con and ISO groups in terms of gross appearances and body and heart weights was found. Compared to other groups, ISO treatment caused an enlargement of the heart and a significant decrease in body weight in mice, while heart weights from mice with cardiac hypertrophy increased. At the same time, the heart coefficient in ISO-treated hearts was markedly elevated (p < 0.01 or p < 0.05). In contrast, L-Bai, H-Bai, and Ver administrations resulted in a significant improvement of pathology in addition to the reduction in heart weight, heart coefficients, and augmentation in body weight (p < 0.01 or p < 0.05). These findings suggested that Bai improved the gross appearance and coefficients of the heart in a dose-dependent manner.

As presented in Figures 3A,B, the mouse serum levels of CK, LDH, and myocardium Ca²⁺ concentrations in the ISO group were higher than those in the Con group (p < 0.01), while L-Bai, H-Bai, and Ver groups showed a significant recovery to varying degrees (p < 0.01).

Figure 3C presented the histopathology change of mouse hearts. Cardiac slices in the Con group showed a normal myofibrillar structure, while the ISO group demonstrated evident degeneration, infiltrating inflammatory cells, and necrosis. The necrosis and inflammatory cells in the Bai and Ver groups were less than those in ISO group. These changes in pathological structures were dramatically increased in the ISO group compared to the Con group (p < 0.01). Conversely, the L-Bai, H-Bai, and Ver groups reduced these pathological structural changes with increasing dose compared with the ISO group (p < 0.01 or p < 0.05).

In contrast with the Con group, the results indicate that IL-6 and TNF-α in the ISO treated group were significantly elevated (Figures 5A,B, p < 0.01). Meanwhile, the L-Bai, H-Bai, and Ver groups were found to cause a decrease in the levels of IL-6 and TNF-α to varying degrees in mice (p < 0.01 or p < 0.05). Our findings suggested that Bai inhibited the levels of inflammatory factors in a dose-dependent manner.

We determined protein levels of the apoptotic markers based on Western blots to observe the expression of Bax, Bcl-2, and Caspase-3 factors that are mainly associated with apoptosis (Figures 5C–F). Compared to the Con group, the ISO group showed a noticeable increase (p < 0.01), while the Bcl-2 level showed a significant decrease. Mice given L-Bai, H-Bai, and Ver showed a reduction in Bax and Caspase-3 levels (p < 0.01 or p < 0.05), while the Bcl-2 level showed enhancement during the same period compared to the ISO group (p < 0.01 or p < 0.05). It was manifested that Bai inhibited the expressions of apoptotic factors in a dose-dependent manner.

Figure 6A–H illustrates that the protein expressions of TLR4, MyD88, NF-κB, p-p38, p-ERK1/2, and p-JNK in ISO were significantly increased compared to the Con group (p < 0.01). In groups that mice were treated with L-Bai, H-Bai, and Ver, these protein expressions were significantly depressed (p < 0.01 or p < 0.05). The results revealed that Bai inhibited the expressions of TLR4/MyD88/MAPKs/NF-κB pathway in a dose-dependent manner.