Establishment of a rat model of MIRI under CPB
Adult male SD rats, 300-350g, were anesthetized by intraperitoneal injection of 2% pentobarbital (50 mg/kg), fixed in supine position, and shaved for skin preparation. Hydroxyethyl starch, mannitol and other pre-filled CPB pipelines are reserved. Endotracheal intubation, connected to a ventilator, adjusted parameters according to the respiratory rate of 60 times/min, tidal volume of 3ml/100g, and respiratory ratio of 1:1.5. The tail vein was punctured and fixed with a 22G trocar to establish an intravenous administration route. 0.5cm above the right midclavicular line of the rat, the neck skin was carefully incised, the right jugular vein was bluntly dissociated, punctured with a 16G trocar, and connected to a three-way tube prefilled with heparin to establish a CPB venous drainage channel. The rat bilateral inguinal skin was incised, the left and right femoral arteries were bluntly dissociated, punctured with a 20G trocar, connected to a three-way tube prefilled with heparin, and one side was connected to a monitor and a bio-signal acquisition system for real-time monitoring of intraoperative heart rate, blood pressure and other indicators, and the other side is used as a CPB oxygenated blood return channel. After the arteriovenous cannulation was completed, the skin was longitudinally incised from the middle of the sternum at the level of the second rib, and the surrounding subcutaneous tissue and muscles were bluntly separated, and the bleeding site was sutured to stop the bleeding at any time. The sternum was then held up with a knife handle and cut open, the heart was fully exposed with a chest expander, and the aortic root was freed and threaded for clamping. The venous drainage access was connected to the blood reservoir, peristaltic pump and rat membrane oxygenator, and the rat was systemically heparinized by injection of heparin sodium (4 mg/kg). When waiting for heparinization, the air bubbles in the CPB pipeline can be discharged with the priming solution. When ACT>480s, turn on the constant flow peristaltic pump to perform CPB. At this time, the depth of the jugular vein cannulation can be adjusted to keep the flow rate at 100ml/kg·min-1 and ensure that the mean arterial pressure (MAP) ≥ 60mmHg, the rat CPB modeling was successful at this time. During the entire operation, a heating blanket was used to keep the rats warm to prevent the death of the rats caused by hypothermia. After the rat CPB model was established, the aortic root was clamped to cause global ischemia, and the heating blanket was turned off. After 30 minutes of complete cardiac arrest, the heating blanket was turned on for rewarming, the aorta was opened, and the CPB flow was maintained for 120 minutes, and the modeling was completed
Experiment grouping and processing strategy
160 adult male SD rats were randomly divided into 8 groups: normal group (Nor), dimethyl malonate control group (dm+ Nor), ischemia-reperfusion group (I/R), dm + ischemia-reperfusion group (dm+ I/R), ischemia postconditioning group (IPO), dm+ ischemic postconditioning group (dm+ IPO), 5-hydroxydecanoic acid+ ischemia postcondition group (5-HD+IPO), dm+5-HD +IPO group (dm+5-HD+IPO), 20 cases in each group. Nor group: The experiment was terminated after maintaining the flow rate of 100ml/kg·min-1 for 160min. dm+ Nor group: The transfer procedure was the same as that of the Nor group. In the corresponding period of 10 min before the whole cardiac arrest in the I/R group, dm 4 mg/kg·min-1 was pumped from the tail vein for 40 min. I/R group: After maintaining the flow rate of 100ml/kg·min-1 for 10 minutes, the whole heart stopped beating for 30 minutes, the aorta was opened, and the experiment was terminated after continuing the transfer for 120 minutes. dm+ I/R group: The transfer process is the same as the I/R group, and the dm pump injection is the same as the dm+ Nor group. IPO group: After maintaining the flow rate of 100ml/kg·min-1 for 10 minutes, whole-heart arrest was performed for 30 minutes, followed by 30s reperfusion and 30s ischemia. After a total of 3 cycles, the aorta was opened, and the experiment was terminated after 117 minutes of transfer. dm+ IPO group: The transfer process is the same as the IPO group, and the dm pump injection is the same as the dm+ Nor group. 5-HD+IPO group: The transfer process was the same as that of the IPO group. 5-HD 5mg/kg was injected from the tail vein 5 minutes before IPO. dm+5-HD+IPO group: The transfer process was the same as the IPO group, the dm pump injection was the same as the dm+ Nor group, and the 5-HD injection was the same as the 5-HD+IPO group. For the experimental group that did not receive dm and/or 5-HD treatment, the same volume of normal saline was pumped/injected at the corresponding time (as shown in Figure 1).
Detection of myocardial infarction size
Prepare 1% TTC staining solution, incubate at 37°C for 10min, avoid light. At the end of the transfer of the rats in each group, the hearts were cut out, washed with PBS, and frozen at -80°C for 10 min. Make horizontal cuts along the long axis of the heart and place the sections in TTC staining solution. Incubate at 37°C for 30 min in the dark. Heart sections were fixed in 10% paraformaldehyde for 24h. The camera was photographed, and the myocardial infarction area (%) was calculated and analyzed by Image-Pro Plus for statistical analysis.
Observation of cardiac ultrastructure and mitochondrial Flameng score in each group
At the end of the transfer of the rats in each group, the heart was quickly cut off, the ventricular muscle tissue was taken, and the tissue was trimmed into small pieces of about 1 mm × 1 mm × 1 mm with a sharp knife. Immerse the myocardial tissue block in 2.5% glutaraldehyde fixative solution at 4°C for 2h. The tissue blocks were fixed in 1% osmic acid at 4°C for 2 h. The tissue blocks were dehydrated with different concentrations of ethanol and acetone, and embedded in epoxy resin. Trim and prepare ultrathin sections. Heavy metal salts (uranyl acetate and lead citrate) were electronically stained for 1 h, and electron microscope specimens were prepared. The specimen was observed under a transmission electron microscope, and 10 fields of view were taken to evaluate the changes of myocardial ultrastructure.
Detection of plasma CK-MB and cTnI concentrations
At the end of the transfer of the rats in each group, 1 ml of whole blood was drawn from a blood collection tube, and allowed to stand at room temperature. Pre-cool the centrifuge at 4°C, set the parameters to 3000 × g for 20 min, centrifuge the samples, and operate according to the instructions of the CK-MB, cTnI detection kit. Finally, measure the OD value on the preheated microplate reader at a wavelength of 450 nm. The OD value was substituted into the standard curve to calculate the concentration of CK-MB and cTnI, and made statistical analysis.
Determination of ROS content in myocardial tissue
At the end of the transfer of the rats in each group, the hearts were quickly cut off, and the ventricular myocardium was taken out and embedded and fixed with OTC. The frozen microtome was pre-cooled in advance and used to cut the tissue block into thin slices with a thickness of 10um. Adhere the tissue sheet flat to the glass slide, taking care not to fold or fold. The slides with attached samples were washed with PBS pre-warmed at 37°C for 5 min, repeated 3 times. Take a 10um/L DHE probe preheated to 37°C, drop it on the tissue sheet, place the slide in a light-proof box, and incubate at 37°C for 30 min in a drying box. In the dark, the slides with the samples were washed with PBS at 37°C for 5 min, repeated 3 times. After the samples were naturally dried, 1 drop of DAPI was added dropwise, and the slides were mounted. The prepared samples were placed under a laser confocal microscope to observe the ROS fluorescence intensity, and the relative fluorescence value was measured for statistical analysis.
Measurement of SDH activity in myocardial tissue
At the end of the transfer of the rats in each group, the heart was quickly cut off, and 100 mg of ventricular muscle tissue was taken, washed with PBS, and placed in a 1.5 ml centrifuge tube. Add 1 ml of reagent 1 and 10 ul of reagent 2 to fully grind the myocardial tissue. This procedure needs to be performed on crushed ice. Pre-cool the centrifuge at 4°C, set the parameter to 11000g, 10min, centrifuge the sample, take the supernatant and place it in crushed ice for testing. Preheat the microplate reader for 30min, adjust the wavelength to 600nm, and use distilled water to zero. Reagents 3 and 5 were allowed to stand at 37°C for 10 min and then added to a 96-well plate, and the samples were added using the SDH activity detection kit instructions. Preheat the microplate reader for 30min, measure the OD value of the sample at 20s and 80s respectively, denoted as A1 and A2, ΔA=A1-A2, obtain ΔA measurement, ΔA blank. The SDH activity was calculated according to the formula.
Determination of succinic acid content in myocardial tissue
At the end of the transfer of the rats in each group, the hearts were cut off, and 50 mg of ventricular myocardium was taken, washed with PBS, placed in a 1.5 ml centrifuge tube and minced, added with 0.45 ml of PBS, homogenized and ground, centrifuged at 3000 g for 10 min, and the supernatant was taken for testing. According to the instructions of the succinic acid content test kit, preheat the microplate reader to measure the OD value at 340nm, and calculate the succinic acid concentration according to the formula.
Determination of fumaric acid content in myocardial tissue
At the end of the transfer of the rats in each group, the hearts were cut off, 40 mg of ventricular myocardium was taken, 100ul of Fumarate Assay Buffer was added to homogenize and ground, centrifuged at 13000 g for 10 min, and the supernatant was collected for testing. Take 10ul of 0.1M Fumarate Standard and add 990ul of Fumarate Assay Buffer to prepare a 1mM standard solution. Add 0, 5, 10, 15, 20 and 25ul of 1mM standard solution to the 96-well plate and make up to 50ul with Fumarate Assay Buffer. Mix 20ul of Fumarate Developer and 20ul of Fumarate Enzyme Mix respectively, and make up the volume with Fumarate Assay Buffer to prepare a reaction mixture. The above reaction mixtures were added to 96-well plates, 100ul per well, and incubated in the dark for 30 min. The OD value at 450 nm was measured using a microplate reader, and the content of fumaric acid was calculated from the standard curve.
Determination of SDHA mRNA expression in myocardial tissue
At the end of the transfer of the rats in each group, the hearts were cut off, and 100 mg of ventricular myocardium was taken, washed with PBS, and then quickly placed in a -80°C refrigerator or liquid nitrogen for cryopreservation. Total RNA was extracted according to the instructions of Trizol kit (Ambion, USA). Put 2ul of the dissolved RNA into a microplate reader, measure OD260 and OD280 respectively, and calculate the OD260/OD280 value. The ratio of 1.8-2.0 means that the total RNA is relatively pure. At the same time, the concentration of total RNA needs to be calculated according to the OD value. According to the HiScript Reverse Transcriptase (VAZYME, China) kit instructions, reverse transcription was used to synthesize cDNA, and RT-qPCR was used to measure the mRNA levels of GADPH and SDHA. The specific primers are as follows:
GAPDH,forward 5¢-ACAGCAACAGGGTGGTGGAC-3¢;
reverse 5¢-TTTGAGGGTGCAGCGAACTT -3¢; 253bp.
SDHA,forward 5¢-TGCCAAGGACCTAGCATCAA -3¢;
reverse 5¢-AGTGGGAAGGACTGGAATGG -3¢; 217bp.
Determination of SDHA protein expression in myocardial tissue
At the end of the transfer of the rats in each group, the hearts were cut off, and part of the ventricular muscle tissue was taken, carefully washed three times with PBS and fully minced. The shredded myocardial tissue, an appropriate amount of grinding steel beads, and 200ul of single-detergent lysis solution containing 2ul PMSF and 2ul phosphatase inhibitor were added to a 2 ml EP tube and homogenized for 3 times, and allowed to stand on crushed ice for 30 min. Pre-cool the centrifuge at 4°C, set the parameter to 12000rpm, 5min, centrifuge the sample, and the supernatant after centrifugation is the total protein. The protein concentration was determined using the instructions of the BCA protein assay kit. After denaturation at 100°C, the protein was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Sinopharm, China). The separated products were electrophoretically transferred onto PVDF membranes and blocked for 2 hours at 37°C. Membranes were then incubated overnight at 4°C with corresponding primary antibodies (1:1,000 dilution) for SDHA protein and internal control protein (GAPDH). The membrane was then washed with TBST buffer and incubated with secondary antibody (1:50,000) for 2hr at room temperature. Use ECL reagent working droplets to add to the washed PVDF membrane, and after the band is clearly colored, absorb the excess liquid and cover it with plastic wrap to make X-ray film. After the film is prepared, input the BandScan software through the scanner to analyze the gray value.
Statistical analysis
In all the data obtained in this study, the measurement data conforming to the normal distribution were expressed as the mean ± standard deviation (`x ±s). The data between groups were analyzed by SPSS 17.0 statistical software for homogeneity of variance test and one-way analysis of variance. For those with homogeneous variance, the least significant difference (LSD) analysis was used, and for those with unequal variance, Dunnett's T3 test was used. Differences were considered statistically significant when P < 0.05.