Animals
In this study, we used 150 of 6–8 week old male Sprague–Dawley rats (200-250 g) were used for the experiments, purchased from SPF (Beijing) Biotechnology Co., Ltd. [License No.: SCXK (Beijing) 2019–0010]. The rats were housed in a temperature-controlled environment (21 ± 2 °C) with a 12-h light/dark cycle (lights on at 06:00) and free access to food and water. The facilities where the animals were housed followed the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) guidelines, which were approved at the time of the study. The study protocol was approved by the Ethics Committee of Qingdao University School of Medicine (Qingdao, China).
Type 2 diabetic rat model
The T2DM model was induced as follows: Experimental animals were fed basal chow for 1 week. After 1 week, the diabetic group was fed high-fat and high-sugar chow, and the control group was fed basal chow. At week 5, all animals were fasted for 12 h (without water), and STZ (1%) was dissolved in 0.1 mmol/L citric acid-sodium citrate buffer (pH 4.2; Solarbio, Beijing, China), fully dissolved and stored on ice away from light. A single rapid intraperitoneal injection of 30 mg/kg streptozotocin (MCE, USA) was administered to the diabetic group. An equal volume of 0.1 mM citric acid-sodium citrate buffer was intraperitoneally administered to the control group. Seven days later, blood was drawn from the tail vein to check for glucose levels of the rats in the diabetic group. Rats with blood glucose ≥ 16.7 mmol/L were fed a high-fat, high-sugar diet for another 8 weeks, while rats that did not meet the blood glucose standard were discarded. Blood glucose was retested after 8 weeks, and modeling was successful if blood glucose was still ≥ 16.7 mmol/L [16].
Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT)
OGTT: Rats in the normal group (NG: conventional rat chow feeding group) and the diabetes mellitus group (DMG: high-fat chow feeding group supplemented with low-dose STZ injection group) were fasted overnight 12 h before the test, while ensuring access to drinking water. On the day of the test, a 50% glucose solution was prepared, and the volume of glucose solution required for each rat to be tested was calculated based on body weight (2 g glucose/kg body weight) after weighing the rats to be tested. After the gavage operation, blood was taken through the tail vein to test the blood glucose of rats at 0 min, 30 min, 60 min, 90 min and 120 min, and a line graph was made and the area under the curve (AUC) was calculated to compare the difference in blood glucose between the two groups. ITT: Two groups of rats, NG and DMG, were taken and fasted 5 h before the test, while ensuring access to drinking water. And the insulin aspart was diluted in 0.9% saline to prepare a 1:1000 solution (NovoRapid: 100 U/mL; working concentration 0.1 U/mL). After fasting, the rats to be tested were weighed and the volume of solution required for each rat to be tested was calculated based on body weight (0.5 U of menadione insulin/kg body weight). After intraperitoneal injection of insulin, blood was collected through the tail vein to test the blood glucose of rats at 0 min, 30 min, 60 min, 90 min and 120 min, and a line graph was made and the area under the curve was calculated to compare the difference in blood glucose between the two groups.
Myocardial ischaemia‒reperfusion model
The rats were weighed, fasted, and dehydrated for 12 h before surgery. Sodium pentobarbital (50 mg/kg) was injected intraperitoneally (Item No. P3761, Sigma, USA). Each rat was fixed in the supine position and connected to a Powerlab data acquisition and analysis system. A standard II-lead ECG was recorded, and any abnormalities were excluded. The cervical trachea was incised, and a small animal ventilator (Rivard, USA) was connected for assisted breathing (tidal volume, 5 mL/100 g, frequency 60–80 breaths/min, respiratory ratio 2:1, continuous positive end-expiratory pressure). The skin was cut longitudinally 0.5 cm on the left side of the sternum, using the 3rd and 4th ribs as the upper and lower borders. The subcutaneous tissue, pectoralis major muscle, and intercostal muscle were bluntly separated with forceps and a scalpel. Then, 6–0 ophthalmic sutures were passed under the left atrium and 2–3 mm below the intersection of the cone of the pulmonary artery. The left anterior descending branch of the coronary artery was ligated for 30 min, after which the sutures were cut. After successful ligation, the anterior wall of the left ventricle was bruised or pale, the pulsation was reduced, and the ECG showed ST-segment elevation (≥ 0.25 mV), which is a sign of myocardial ischemia. Thirty minutes after ligation, the ligature was cut with scissors to form a reperfusion, and the ECG showed a gradual decrease in the ST segment by approximately 50% and the pale or cyanotic myocardium gradually turned red when blood flow was restored. Reperfusion was allowed to occur for 2.5 h [17]. After the end of reperfusion, rats were euthanized by a single intraperitoneal injection of an overdose (150 mg/kg) of sodium pentobarbital (Item No. P3761, Sigma, USA).
Nicorandil administration route and dosage
In this study, Nicorandil for injection (trade name: Ricoxyl; specification: 12 mg; State Drug Administration H20120069) was used, and the dried drug powder was prepared into a solution of 100 μg/ml with 0.9% saline before the experiment and stored away from light. We selected the effective dose in the clinical study, a loading dose of 200 μg/kg of nicorandil was given via femoral vein 20 min before reperfusion, and a maintenance dose of 20 μg/kg/min was given for 60 min after reperfusion [18].
Experimental groups
A total of 10 groups were included in this study, with 15 animals in each group. (Flowchart of experimental protocol is shown in the Additional file 1: Figure S1).
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(1)
Sham group: The sutures were threaded, but the descending artery was not ligated. The rats were sacrificed 3 h later.
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I/R group: Rats were treated as described above.
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I-Post group: After ligation, the rats were treated for myocardial ischemia (30 s ischemia/30 s reperfusion given 3 times within 3 min of the start of reperfusion). Then, reperfusion was allowed to occur for 2.5 h.
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Nic group: A loading dose of nicorandil (200 μg/kg) was administered 20 min before reperfusion. Then, a maintenance dose (20 μg/kg/min) was administered for 60 min during reperfusion. Afterwards, reperfusion was permitted for another 1.5 h (2.5 h total reperfusion time).
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I-Post + Nic group: Rats were administered a loading dose of 200 μg/kg of nicorandil 20 min before reperfusion and a maintenance dose of 20 μg/kg/min for the first 60 min of reperfusion. At the same time, the rats were post-treated for myocardial ischemia (30 s ischemia/30 s reperfusion administered 3 times within 3 min of the start of reperfusion). Reperfusion was allowed to occur for a total of 2.5 h.
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DM Sham group: Diabetic rats were used for this group. Sutures were threaded without ligation.
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DM I/R group: Diabetic rats were used for this group. I/R injury was induced as described in Myocardial ischaemia‒reperfusion model.
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DM I-Post group: Diabetic rats were treated for myocardial ischemia (30 s ischemia/30 s reperfusion given 3 times within 3 min of the start of reperfusion). Each rat was reperfused for 2.5 h.
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DM Nic group: Diabetic rats were administered a loading dose of 200 μg/kg nicorandil 20 min before reperfusion and a maintenance dose (20 μg/kg/min) was administered during the first 60 min of reperfusion. Then, the rats were reperfused for another 1.5 h (2.5 h total reperfusion time).
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DM I-Post + Nic group: Diabetic rats were administered a loading dose of 200 μg/kg of nicorandil 20 min before reperfusion and a maintenance dose of 20 μg/kg/min for the first 60 min of reperfusion. At the same time, the rats were post-treated for myocardial ischemia (30 s ischemia/30 s reperfusion administered 3 times within 3 min of the start of reperfusion). Reperfusion was allowed to occur for a total of 2.5 h.
Serum assay
After reperfusion, blood was collected from the rat abdominal aorta. The serum was obtained by centrifugation (4 °C, 1000 g, 10 min) and frozen at − 20 °C until further analysis. Serum creatine kinase-MB (CK-MB) levels were measured using the rat creatine kinase isozyme MB (CK-MB) ELISA kit (Elabscience, Wuhan, China). The detection range of the kit is 31.25-2000 pg/ml, the intra- and inter-batch coefficient of variation is less than 10%, and the minimum detection concentration is less than 18.75 pg/ml. Serum nitric oxide (NO) levels were measured using a rat nitric oxide ELISA kit (Elabscience, Wuhan, China). The detection range of the kit is 0.16-100 μmol/L, with intra- and inter-batch coefficients of variation less than 2.4% and 3.7%, respectively, and the minimum detection concentration less than 0.16 μmol/L.
Reactive oxygen species (ROS) assay
At the end of reperfusion, rats were sacrificed. The heart was lavaged with 100 mL 0.9% saline and stored at 4 °C. Then, 50 mg of heart tissue was isolated from the below the ligation site and was assayed using a Rat Reactive Oxygen Species ELISA kit (Joln, Nanjing, China). The detection range of the kit is 1.0U/ml-80U/ml, with intra- and inter-batch coefficients of variation less than 9% and 11%, respectively, and the minimum detection concentration less than 1.0U/ml. Protein concentration (µg/µL) were determined using a BCA assay kit (Thermo, USA) to derive the mean level of ROS release (U/mg).
Triphenyl tetrazolium chloride and Evens blue staining to determine the area of myocardial infarction
After reperfusion, the rat LAD vessels were again ligated and 1 ml of 1% Evans Blue staining solution was injected rapidly from the left ventricle to show the ischemic risk area (AAR). After clipping the heart (Heart samples shown in Additional file 1: Figure S2), the hearts were lavaged with 100 mL 0.9% saline at 4 °C and placed on ice at − 80 °C for 10–15 min. After freezing, the hearts were cut into 2-mm thick transverse sections. The transverse sections were placed in 1% 2,3,5-triphenyltetrazolium chloride (TTC; Sigma, USA) in 0.1 mM phosphate (1X PBS) buffer (pH 7.4) at 37 °C for 15–30 min. Then, the tissues were washed with 1X PBS buffer (3 times for 10 min each), and a bluish-purple color was seen in non-infarcted myocardium and no coloration in infarcted myocardium. It was then placed in 4% paraformaldehyde at 4 °C overnight. Images were analyzed using ImageJ data acquisition software (National Institutes of Health, Bethesda, MD, USA). Area measurement method: expressed as infarct area (IS) as a percentage of AAR (IS/AAR) and AAR as a percentage of total area (AAR/LV).
TUNEL assay
After reperfusion, the hearts were then successively lavaged with 100 mL 0.9% saline and 50 mL 4% paraformaldehyde at 4 °C. After lavage, a cross Sect. 2 mm thick was cut perpendicular to the sagittal plane of the heart 2 mm below the ligature site. Fixed in 4% paraformaldehyde 3 days. Paraffin sections were prepared by dehydration and paraffin embedding. The prepared paraffin sections were analyzed using a TUNEL assay kit (Roche). In each slide, color images of five different fields were randomly captured and digitized. Cells that stained blue were normal cardiomyocytes and cells that stained brown were defined as TUNEL-positive cells. The apoptosis index (AI) was calculated as the number of TUNEL-positive cells/total number of cardiomyocytes × 100.
Hematoxylin–eosin (HE) staining
After taking the paraffin sections prepared in the previous step of the experiment and staining them with HE staining kit (Roche), pathological sections were evaluated by a double-blinded pathologist. Lesions consisting of interstitial oedema, myofiber degeneration (i.e., myofiber swelling and myofibrillar lysis), and the formation of myocardial hypercontraction bands were graded according to their severity (0 = no lesion, 1 = mild, 2 = moderate, 3 = marked) and distribution (0 = no lesion, 1 = focal lesion, 2 = multifocal lesion, 3 = diffuse lesion). The mean score for each variable was calculated for each heart, and the group mean score was calculated [19].
Wheat germ agglutinin (WGA) immunofluorescence staining
Plaster sections were made by taking the previous step. The sections were blotted dry with absorbent paper, the heart sections were circled with a histochemical pen, and WGA staining solution (iFluor 488 wheat germ agglutinin conjugate, ATT-Bioquest, USA) diluted 200 times in PBS was added dropwise to the circles, and then incubated for 30 min at 37 °C in a constant temperature chamber protected from light. After incubation, the slides were washed 3 times with PBS (5 min/time) and fluorescence quenching agent was added dropwise for 5 min. After washing, the slides were washed 3 times with PBS (5 min/time), anti-fluorescence quenching sealer was added dropwise in the circle, and the slides were covered with coverslips and sealed with nail polish and stored in a wet box at 4 °C. The digital section scanning system of the Department of Pathology, The Affiliated Hospital of Qingdao University was used to take pictures for observation and analyze the cross-sectional area of cells and the degree of tissue lesions.
Western blot
The rats were sacrificed 15 min after the resuscitation. Then, the heart was lavaged with 100 mL 0.9% saline at 4 °C. Heart tissue from the anterior wall of the left ventricle was isolated from the AAR and used for western blot. The extracted heart tissue was homogenized with RIPA lysis buffer (Elabscience, Wuhan, China). The tissue was then centrifuged at 15,000 × g for 10 min at 4 °C. The supernatant was collected, and the protein concentration was determined using a BCA assay kit (Thermo, USA). The samples were separated using 10% SDS-PAGE gels (10 μg/well). The protein bands were transferred onto nitrocellulose membranes (Merck Millipore, USA). The membranes were then blocked in 5% skim milk for 2 h and incubated overnight at 4 °C with the following primary antibodies: p-PI3K (#4228), PI3K (#4257); p-GSK3β (#5558), GSK3β (#12,456); p-Akt (#4060), Akt (#4691); p-mToR (#5536), mToR (#2983); p-eNOS (#9574), eNOS (#32,027); GAPDH (#5174) (all rabbit, 1:1000, Cell Signaling Technology, USA). horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1; 10,000, Absin, Shanghai, China) was used as the secondary antibody. Target bands were detected using chemiluminescent ECL (Merck Millipore, USA) and visualized using an Amersham Imager 600 (GE Healthcare, Little Chalfont, UK). The images were analyzed using ImageJ data acquisition software.
Statistical analysis
The monitoring data were statistically analyzed using GraphPad Prism9 (La Jolla, CA, USA) and Image J. All data were expressed as mean ± standard error of mean (SEM). T-test was used for statistical analysis of differences between two groups; Differences between multiple groups were analyzed using one-way ANOVA, and Bonferroni multiple comparison test was used for comparison between groups. Statistical significance was set at P < 0.05.
