Animals and cells

Ninety-six SPF grade male healthy adult Wistar rats (200–240 g, 7 weeks old) were obtained from SiPeiFu (Beijing) Biotechnology Co., Ltd. [Animal certificate number: SCXK (Beijing) 2016-0002]. These rats were given chow and water-adapted feeding for 3 days and were randomly divided into 6 groups with 16 rats in each group, namely Sham group, I/R group, SYI high-dose group (32 mg·kg− 1), SYI medium-dose group (16 mg·kg− 1), SYI low-dose group (8 mg·kg− 1) and Hebeishuang group (1 mg·kg− 1). Drug doses were determined by reference to previous literature [23,24,25]. Before the operation, the medicine was formulated into the required concentration with physiological saline. After the left anterior descending coronary artery was threaded, the rats in each group were injected medicine (3 mL·kg− 1) once through the tail vein immediately, except the Sham group and the I/R group were injected with the physiological saline. The ethics committee of Xiyuan hospital approved the study (No.2021XLC037, March 29 2021).

Human umbilical vein endothelial cells (HUVECs) were purchased from Procell Life Science&Technology Co., Ltd. (Lot: CL-0122). The experiment was divided into the normal control group (NC), Oxygen-Glucose Deprivation/Reoxygenation group (OGD/R), OGD/R + SYI (35 μg·mL− 1) group (OGD/R + SYI), OGD/R + SYI + chloroquine (50 μmol·L− 1) group (OGD/R + SYI + CQ). The dose of chloroquine was determined by reference to previous literature [26,27,28].

Establishment of animal model

The establishment of the animal model was based on previous literature and refined according to the pre-testing and previous experimental experience of the research group [29,30,31,32]. Wistar rats were anaesthetized with 1% sodium pentobarbital (50 mg/kg) by intraperitoneal anesthesia and connected to the small-animal ventilator for artificial respiration and the small-animal electrocardiograph. Anesthesia was complete when muscle tension, corneal reflexes and response to skin pinching disappeared in the rats. The chest was then opened and the pericardium exposed by breaking the 3–4 ribs. Once the heart was exposed, a suture was placed approximately 2 mm below the bifurcation of the left anterior descending coronary artery and the drug/physiological saline was immediately injected through the tail vein. After stabilization for 10 min, the silk thread was threaded with a small polyethylene tube, tightening the silk thread, causing myocardial ischemia by pushing the tube method. The judgment criteria for ischemia success were adopted by using the electrocardiogram QRS amplitude to increase, ST-segment elevation and T wave towering or inversion. After 45 min of the chest was closed, the silk thread was cut to realize the reperfusion process. Meanwhile, the chest wall was sutured and the animals resumed spontaneous breathing. After 150 min of reperfusion, the rats were anesthetized, and blood and myocardial tissue samples were collected and stored for examination. Notably, the sham operation group only threading without ligation, and other operations were the same as the model group.

Establishment of cell model

The HUVECs were inoculated in a 96-well plate or a clear sterile plastic petri dish with Lids then put in the cell incubator with a 37 °C, 5% CO2 for 24 h. After discarding the old medium and washing with PBS for 2–3 times, in the NC group, the old medium was replaced with DMEM complete medium, and put the 96-well plate or the clear sterile plastic petri dish with Lids back in the incubator for 6 h waiting for testing. Meanwhile, after washing the old medium in the OGD/R group and each administration group, the old medium was replaced with an N2-saturated sugar-free medium in the OGD/R group, and N2-saturated drug-free sugar-free medium in each administration group. In an airtight box, continuously flowing 950 mL·L− 1 N2 + 50 mL·L− 1 CO2 mixed gas for 30 min to fully exhaust the residual oxygen, consequently, the cells were cultivated in the cell incubator for 4 h. After the end of 4 h, the airtight box was opened and the 96-well plate or the clear sterile plastic petri dish with Lids was taken out, moreover, the sterile glucose solution was added to make the glucose concentration in the culture medium of 4.5 g/L. Cell viability was detected by cell-counting-kit-8 after the 96-well plate or the clear sterile plastic petri dish with Lids was placed back in the incubator for 2 h.

Determination of SYI composition and content

The ingredients indicate that every 150 mg of safflower yellow for injection contains 80 mg of total safflower ketone. However, the specific ingredients and content of SYI are unknown, therefore, we carried out qualitative and quantitative research on the main components of safflower pigment for injection, and carried out the mass spectrometry characteristics of possible compounds by liquid chromatography-mass spectrometry, mainly for quercetin, hesperidin, rutin, luteolin, isorhamnetin, luteolinluco side, isoquercitrin, apigenin-7-O-beta-D-glucoside, apiin, hydroxysafflor A, anhydrosafflor yellow B and Baicalin was analyzed. A high-sensitivity analytical method for the above compounds was constructed, and on this basis, the chromatographic conditions were explored to determine which components they contained, and HPLC fingerprint technology was used to carry out quantitative analysis and research to clarify the content of the main components, to further study the mechanism of SYI.

HE Staining of myocardial tissue

After reperfusion, the rats were anesthetized by intraperitoneal injection of sodium pentobarbital. Subsequent experiments were performed after the rats were put under deep anesthesia. The thickness of 4 mm cardiac tissue was cut from the heart cross-section below the ligation line and fixed with 4% formalin for 24 h. Subsequently, the thick 4 mm cardiac tissue was embedded in paraffin, and HE staining was used to observe the pathological changes in myocardial tissue.

Measurement of myocardial infarct size by TTC staining

After the end of reperfusion, the rats were anesthetized by intraperitoneal injection of sodium pentobarbital. At the same time, the heart was quickly taken out and blotted dry with filter paper, and then placed in a -20 °C refrigerator for quick freezing for about 10 to 15 min. 5 uniform slices with a thickness of about 1 mm were cut from the heart, and then the slices were quickly placed in a 6-well plate containing TTC staining solution and incubated at 37 °C for 15–30 min in the dark. After the color development of the section is completed, the slices were placed in a special solution for some time in 10% formalin to increase the contrast. After that, the stained tissues were taken out and rinsed with normal saline to an off the excess staining solution on the surface of the tissue. Image J was used to measure and analyze the infarct size and total myocardial area (percentage of infarct area = total infarct area/total myocardial area × 100%).

Detection of serum myocardial enzymes and inflammatory factors

After the reperfusion was completed, the rats were anesthetized by intraperitoneal injection of sodium pentobarbital. The blood was collected from the abdominal aorta, left standing at room temperature for 1 h, and centrifuged at 3500 rpm for 10 min in order to separate the serum. Adding and mixing a certain amount of PBS to the cell pellet, after the cells were suspended in PBS, the cell suspension was transferred to a glass homogenization tube in the ice-water mixture. Manually homogenizing for 3 min, the broken cell suspension can be used for measurement. After taking the serum sample and preparing reagents according to the instructions of AST, CK, and LDH kits and mixing the reagents with animal serum samples in proportion, the contents of AST, CK, and LDH were calculated by detecting the absorbance values of the sample at 660 nm, 505 nm, or 450 nm by a microplate reader. Meanwhile, according to the instructions of the ELISA kit, a standard curve was drawn to detect the contents of inflammatory factors TNF-α, IL-6, MCP-1, and IL-1β when the microplate reader was used to detect the absorbance value of the serum sample or cell suspension.

Western blot analysis

To obtain the protein of rat myocardial tissue, after reperfusion, the rats were anesthetized by intraperitoneal injection of sodium pentobarbital. The ischemic and peripheral myocardial tissue were collected under the ligature and cut into pieces with surgical scissors. The tissue cells were lysed with RIPA lysis buffer (containing protease inhibitors and phosphatase inhibitors). In order to obtain the protein of HUVECs, adding and mixing a certain amount of PBS to the cell pellet, after the cells were suspended in PBS, the cell suspension was transferred to a glass homogenization tube in the ice-water mixture. Manually homogenizing for 3 min, the broken cell suspension can be used for measurement. The protein was transferred to the PVDF membrane by determining the concentration by BCA protein detection kit. The membranes were incubated with the primary antibodies against mTOR, PI3K, p62, NLRP3, NF-kB, P-Akt, Caspase-3, Caspase-1, LC3, TLR4, IL-1β, IL-6, MCP-1, TNF-α, Bcl-2, Bax and β-actin overnight at 4 °C. Then, the membranes were washed with TBST buffer and incubated with secondary antibody in TBST buffer at 37 °C for 1 h. Finally, the membranes were washed and visualized using Super ECL Western Blotting Substrate, and the bands were scanned and quantified using the ImageJ system.

Cell viability assay

After each treatment, Cell Counting Kit-8 was used to measure the viability of HUVECs according to the standard protocol of the manufacturer. The absorbance was determined at 450 nm using a spectrophotometer.

Experimental instruments

API4000QTRAP mass spectrometer system, equipped with ESI ion source and Analysis1.5.1 data processing system, purchased from Applied Biosystems, Inc (USA). ICE-CL31R cryogenic centrifuge, Ultra-low temperature refrigerator and Spectrophotometer (NANODROP2000) were purchased from Thermo Fisher Scientific (USA). ALC-V8 small animal ventilator was purchased from Shanghai Alcott Biotechnology Co., Ltd. (China). MPIAS-500 Multimedia Color Pathology Graphic Analysis System was purchased from Jingzheng Hengbocheng Technology Development Co., Ltd. (China). The microplate reader (BioTekSYNERGY4) was purchased from Berten Instrument Co., Ltd. (USA). Biological signal acquisition and processing system (BL-420 F) was purchased from Sichuan Chengdu Taimeng Technology Co., Ltd. (China). Electrophoresis instrument (powerpac200) was purchased from Bio-Rad Laboratories, Inc. (USA). Hitachi Transmission Electron Microscope (H-7500) was purchased from Hitachi Limited (Japan). The Gel imaging system (Tanon1600) was purchased from Shanghai Tianneng Technology Co., Ltd. (China).

Reagents and antibodies

Safflower yellow pigment for injection was purchased from Shanxi Deyuantang Pharmaceutical Co., Ltd., (lot No.713020334391, China). Hebeishuang (Diltiazem) was purchased from Tianjin Tanabe Pharmaceutical Co., Ltd. (lot No.B014, China). Autophagy inhibitor, chloroquine, was purchased from Sigma-Aldrich Co., Ltd. (lot No.095m14016V, USA). Anti-Caspase-3 antibody (Cat No.ab13847), Anti-LC3 (Cat No.ab51520), Anti-P62 (Cat No.ab56416), Anti-TLR4 (Cat No.ab13867), Anti- mTOR (Cat No.ab32028), Anti-PI3K (Cat No.ab151549) and Anti-NLRP3 (Cat No.ab214185) were purchased from Abcam Plc (United Kingdom). Anti-GAPDH antibody (Cat No.5174), Anti-NFκB antibody(p65) (Cat No.8242) and Anti-AKT (Cat No.4060) were purchased from Cell Signaling Technology (USA). Anti-Caspase-3 antibody (Cat No.ab13847), Anti-LC3 (Cat No.ab51520), Anti-P62 (Cat No.AB56416), Anti-TLR4 (Cat No.ab13867), Anti-mTOR (Cat No.ab32028), Anti-PI3K (Cat No.ab151549) and Anti-NLRP3 (Cat No.ab214185) were purchased from Abcam Plc (United Kingdom). Anti-GAPDH antibody (Cat No.5174), Anti-NFκB antibody(p65) (Cat No.8242) and Anti-AKT (Cat No.4060) were purchased from Cell Signaling Technology (USA). DAB detection kit (Polymer) (PV6000-D) was purchased from Thermo Fisher Scientific (USA). DMSO (Cat No.3483C285), DTT (Cat No.0281), TEMED (Cat No.0761), and Tween-20 (Cat No.0777) were purchased from Sigma-Aldrich LLC(USA). BCA Protein Assay Kit (Cat No.02912E) were purchased from Jiangsu Cowin Biotech Co., Ltd. (China). Goat anti-mouse IgG (H + L), HRP (Cat No.115-035-003), goat anti-rabbit IgG (H + L) and HRP (Cat No.111-035-003) were purchased from Jackson Immuno Research laboratories. Inc (USA). ECL (WBKLS0500) was purchased from Sigma-Aldrich Co., Ltd. (USA). Rat IL-1β ELISA Kit (Lot No.1229170721), Rat MCP-1 ELISA Kit (Lot No.0119180705), Rat TNF-α ELISA Kit (Lot No.0105180709) and Rat IL-6 ELISA Kit (Lot No.1229170724) were purchased from RayBiotech Life, Inc. (USA). Aspartate Aminotransferase (Cat No. AST/GOT) Activity Assay Kit (Reitman-Frankel Method) (Cat No.C010-1), Creatine kinase (CK) Activity Assay Kit (Cat No.A032), Lactate Dehydrogenase (LDH) Activity Assay Kit (Cat No.A020-2) were purchased from Nanjing Jiancheng Bioengineering Institute(China). Pentobarbital Sodium (Lot No.2017050505) was purchased from Shanghai Yiji Industrial Co., Ltd. (China)

Statistical analysis

All data are expressed as mean ± standard error of the mean. Statistical software uses SPSS20.0 statistical data. The T-test is used for comparison between two groups, and analysis of variance (ANOVA) is used for comparison of three or more groups. P values less than 0.05 were considered significant. GraphPad Prism 9.0 software was used for statistical data, and the level of significance is indicated in the legend.