Reagents

Oleuropein was obtained from Chengdu Push Bio-tech Co., Ltd (32619-42-4, Chendu, China). Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin/treptomycin (P/S), and 0.25% Trypsin–EDTA (1 ×) were obtained from Gibco (California, USA). Tert-Butyl hydroperoxide (tBHP) and 3-[4, 5]-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) were purchased from Sigma-Aldrich (298-93-1, Taufkirchen, Germany). Chloroquine (CQ) and TAK242 were purchased from MedChemExpress (Shanghai, China). Dimethyl sulfoxide (DMSO) was purchased from Solarbio (D8370, Beijing, China). Trizol Total RNA Extraction Regent (G3013), SweScript RT I First Strand cDNA Synthesis Kit (G3330), and 2 × Fast SYBR Green qPCR MasterMix (G3324) were purchased from Servicebio (Wuhan, China). Further, trichloromethane was purchased from Sinopharm Chemical Regent Co., Ltd. (Shanghai, China). LDH Cytotoxicity Assay Kit was purchased from Beyotime (C0016, Shanghai, China). Finally, 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH2-DA) and JC-1 fluorescent probes were obtained from MedChemExpress (Shanghai, China), and Stub-RFPSens-GFP-LC3 was purchased from Genechem (Shanghai, China).

NRF2 (ab31163) and NQO1 (ab80588) antibodies were purchased from Abcam (Cambridge, UK). Keap1 (4678s), HO-1(70081s), GAPDH (5174s), PARP (9832s), LC3B (2775s), P62(39749s), p-P38(4511s), P38 (9212 s), p-ERK1/2 (4370s), p-JNK (9255s), and JNK (9252s) antibodies and the secondary antibodies including horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (7074) were purchased from Cell Signaling Technology (Danvers, MA, USA). TLR4(48–2300) antibody was purchased from Invitrogen (California, USA). ERK1/2 (AF6248) antibody and goat anti-mouse IgG Fluor® 488 Conjugate (S0017) were purchased from AFFINITY (Jiangsu, China). Pierce™ BCA Protein Assay Kit (23225) was purchased from ThermoFisher (California, USA). QuantiCyto® Rat IL-6 ELISA kit (ERC003) and QuantiCyto® Rat TNF-α ELISA kit (ERC102a) were purchased from NEOBIOSCIENCE (Shenzhen, China).

Cell culture

H9C2 and HEK293T cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and were cultured in DMEM complemented with 10% FBS and 1% P/S. The abovementioned cells were maintained in a 37 °C incubator filled with 5% CO2.

Cell viability assay

H9C2 cells were seeded into a 96-well plate at the density of 6000 cells per well overnight. The OP solution used in cells was first dissolved in DMSO to prepare a 40 mM stock solution, and then diluted with the cell culture medium to the required concentration. After that, OP was added to H9C2 cells at doses from 10 to 80 μM and incubated for 24 h. After that, 100 μL of 1 mg/mL MTT was added followed by incubation for another 4 h at 37 °C. The MTT in each well was replaced with DMSO for 15 min at room temperature and the plate was then evaluated using a microplate reader (SYNERGYH1, Bio Tek, USA) by measuring absorbance of 570 nm.

LDH release assay

H9C2 cells were seeded into a 96-well plate at the density of 6000 cells per well overnight. This was followed by a pretreatment with OP at different concentrations for 2 h followed by treatment with tBHP for another 4 h. The supernatants were collected to detect the LDH release level according to the manufacture’s protocol. The absorbance was measured at 450 nm using a multimode plate reader.

Flow cytometry

H9C2 cells were seeded into a 12-well plate at the density of 1 × 105 cells per well overnight. This was followed by a pretreatment with OP at different concentrations (10, 20, and 40 μM) for 2 h followed by treatment with tBHP for another 2 h. The cells were labeled with DCFH2-DA probe (1 μM) for 30 min and then washed and collected for detection by the FACSMelodyTM Cell Sorter (BD bioscience, USA).

Fluorescence assay

H9C2 cells were seeded into a 96-well plate at the density of 6000 cells per well overnight, followed by pretreatment with OP at the concentration of 40 μM for 2 h followed by treatment with tBHP for another 2 h. The ROS level was measured using DCFH2-DA (1 µM), and the MMP was measured using JC-1 solution (10 µM), following the manufacturer’s instructions. The fluorescence was observed under a fluorescence microscope (Leica, Wetzlar, Germany).

Immunofluorescence analysis

H9C2 cells were seeded into a confocal dish (SPL, Pocheon, Korea) at the density of 1.2 × 105 cells per well overnight, followed by pretreatment with OP at the concentration of 40 μM for 2 h and treatment with tBHP for another 2 h. The immunofluorescence levels of Nrf2, LC3B, and P62 were measured using an anti-Nrf2 antibody, anti-LC3B antibody, and anti-P62 antibody overnight at 4 °C, followed by incubation with goat anti-rabbit Alexa Fluor 488 secondary antibody (1:200) for 2 h at room temperature. Subsequently, the cells were additionally stained for 5 min with Hoechst 33,342 (1 μM) before being imaged. The cells were imaged using a confocal laser microscope (Leica, Wetzlar, Germany).

Autophagic flux assay

According to the manufacturer’s protocol, H9C2 cells were infected with lentiviruses for 36 h and the expression of GFP and RFP was observed under a fluorescence microscope (Leica). Afterward, the cells were fixed with 4% PFA and stained with Hochest33342 for 5 min at room temperature away from light. Finally, the cells were imaged using a confocal laser microscope.

Western blot assay

The cells were lysed by RIPA lysis buffer with 1% cocktail and 1% phenylmethylsulfonyl fluoride (PMSF) and separated by centrifugation. Total proteins were collected, and the concentration of proteins was measured by the BCA protein assay kit. The quantified proteins were separated by SDS-PAGE and then transferred onto a PVDF membrane. After blocking with 5% non-fat dry milk solution for 1 h at room temperature, the membranes were incubated with the primary antibody (1:1000 dilutions) at 4 °C over 16 h. Then, the blot was treated with HRP-conjugated secondary antibodies (1:5000 dilutions) and visualized using the ChemiDoc XRS + System (California, USA). The gray-scale values of related proteins were quantified by Image J. The cytoplasmic and nuclear proteins were extracted using a nuclear and cytoplasmic protein extraction kit (Beyond time, Shanghai, China) according to the manufacturer’s instructions and the protein level detection was as described above.

Quantitative real-time PCR (qRT-PCR) assay

H9C2 cells were seeded into a dish (SPL, Pocheon, Korea) at the density of 1.2 × 105 cells per well overnight, followed by pretreatment with OP at the concentration of 40 μM for 2 h and treatment with tBHP for another 2 h. Total RNA was extracted and 1 μg RNA was used for qRT-PCR analysis. Nrf2, HO-1, NQO1, and GAPDH oligonucleotide primers were as follows (Table 1)

Table 1 Primer Sequence (5′-3′)Molecular docking

The 3D structure of OP was downloaded through Pubchem. The PDB structure of TLR4 (PDB number: 3FXI) was downloaded through PDB, and the structure was processed using Autodock software (https://autodocksuite.scripps.edu/adt/) for hydrogenation and dehydration and finally molecular docking.

Cellular thermal shift assay (CETSA)

The biochemical mechanism of HEK293T cells is suitable for expressing most mammalian proteins requiring post-translational modifications and protein folding, as their protein structure closely resembles that of the human body [21]. Therefore, HEK293T cells are often chosen in experiments such as CETSA to detect the binding of drugs to proteins in their native conformations [22]. Total proteins were obtained from HEK293T cell lysate lysed with RIPA lysis buffer (1% PMSF and 1% cocktail). Then, the respective cell lysates were co-incubated with vehicle control (DMSO) or OP (40 μM) for 0.5 h on ice and then centrifuged at 12,000 rpm for 20 min at 4 °C. Following this, the protein concentration was quantified using a BCA kit. The supernatant was divided into six parts on average and heated at different temperatures (40, 44, 48, 52, 56, and 60 ℃) for 3 min followed by cooling for 30 s at room temperature, and detection by Western blotting.

Molecular interaction analysis

Biacore X100 (Cytiva, United States) was used for measuring the interaction of OP with TLR4. HBS-EP (Cytiva, United States) was used as the working buffer to dilute the TLR4 recombinant protein (Sino Biological, China) to a final concentration of 20 μg/mL. Next, the CM5 chip was activated and TLR4 recombinant protein was coupled with the CM5 chip by the amino coupling method. Furthermore, OP was diluted with the HBS-EP buffer to 100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 μM. Kinetic experiments were performed using the kinetic and affinity methods in the template of the Kinetic Analysis Wizard to analyze interactions between the ligand and the receptor. The obtained data were fitted according to the analysis software, with time as the abscissa and the response value as the ordinate to calculate the binding kinetics between OP and TLR4.

Myocardial ischemia–reperfusion animal model

Animal experiments were approved by the Ethics Committee on Laboratory Animal Management of Guangxi University of Chinese Medicine (Approval Document No. SYXK Gui-2019-0001). Healthy male Sprague–Dawley rats (SPF grade, 6–8 weeks old, body weight 220–250 g) were purchased from Hunan SJA Laboratory Animal Co., Ltd (Hunan, China, animal license number: SCXK Xiang-2021-0002) and were housed under specific pathogen–free (SPF) conditions at 23–25 °C with 40–50% humidity and free access to food and water. After three days of adaptive feeding, the rats were randomly divided into five groups (n = 10 per group): the sham group, the I/R group, the OP group (OP-L:10 mg/kg and OP-H:40 mg/kg) and the Verapamil group (20 mg/kg) [23]. OP solution and verapamil solution used in rats were formulated with normal saline at 1 mg/mL, 4 mg/mL, or 2 mg/mL, and OP was then administered to mice via an intraperitoneal injection (i.p.). Verapamil was administered to mice by intragastric administration (i.g.); both solutions were administered once daily for seven consecutive days. The sham and model groups were administered the same amount of normal saline. The needle was inserted 2 mm from the lower border of the left atrial appendage, and the left anterior descending coronary artery was ligated. At this time, it was observed that the color of the rat's heart surface turned purple. After 30 min, the ligation was removed, and the heart blood was reperfused for 2 h. During this period, the color of the heart's surface returned to red. In the sham group, needles were only inserted without ligation. After reperfusion, the rats were sacrificed, and tissues were collected for subsequent experiments.

Blood routine

After the rats were sacrificed, blood was collected from the abdominal aorta for routine blood testing using the Auto Hematology Analyzer (BC-500, Mindray, Shenzhen, China).

Enzyme-linked immunosorbent assay (ELISA)

Rat blood was collected and left at room temperature for 2 h, centrifuged at 3000 rpm for 15 min at low temperature to get the upper serum, and the levels of IL-6 and TNF-α in the serum were detected using ELISA kits according to the kit and instrument instructions.

2,3,5-triphenyltetrazolium chloride (TTC)-evens’ blue double staining

After 2 h of reperfusion, rats were anesthetized again, and the coronary artery was ligated again at the position of ischemia; 0.3 mL of 1% Evans blue was injected from the apex of the heart to turn the non-ischemic myocardium blue, and the rest of the myocardium was regarded as ischemia danger zone. The heart was then immediately resected; the residual blood was washed with pre-cooled PBS buffer, blotted dry with filter paper, and placed in a freezer at − 20 °C for 30 min. Subsequently, 5–6 slices were evenly cut across the direction perpendicular to the long axis of the left ventricle, followed by soaking sequentially in warm 1% TTC solution, and incubation at 37 °C in dark for 10 min. At this time, the infarct area was grayish-white.

Hematoxylin and eosin (HE) staining

Other parts of rat hearts were fixed with 4% paraformaldehyde for 24 h at room temperature and prepared for paraffin sectioning. The fixed tissues were routinely dehydrated and transparent, embedded in paraffin and sliced (5 μm), dried, dewaxed, hydrated, and stained with hematoxylin for 10 min. The tissues were then washed with distilled water, differentiated with 1% hydrochloric acid ethanol for 10 s, washed with distilled water again, and stained with 1% eosin for 3 min. This was followed by washing, and routine dehydration and mounting. The pathological changes in the myocardial tissue were finally observed under a microscope.

Data analysis

Each experiment was performed three times. Student’s unpaired t-test and one-way ANOVA in GraphPad Prism were used for statistical analysis in GraphPad Prism 9 (GraphPad Software, San Diego, CA). P < 0.05 was considered statistically significant.