Healthy male SD rats were selected and fasted 12 hours before the animal experiment. The rats were anesthetized by pentobarbital sodium according to their body weight. The ECG were monitored and recorded by electrocardiograph.
Cut the skin of the rat neck with aseptic scissors, fully expose the trachea, intubate the trachea with an indwelling needle, connect the small animal ventilator, give artificial ventilation (pressure 3kpa, frequency 70 times / min) to assist breathing, open the chest in the left fifth auxiliary room beside the sternum, separate the pericardium, fully expose the heart, the left coronary artery is located between the left atrial appendage and the pulmonary artery, and use a small curved needle at 1-2 mm under the left atrial appendage. The left anterior descending branch of the coronary artery was punctured at the upper 1/3 of the left anterior descending branch (note that the needle depth was not more than 1 mm, and the width was not more than 3 mm). The color of the anterior wall of the distal end of the coronary artery was observed to become purplish red. ECG monitoring showed that the ST segment in lead I, II, avL of ECG elevated 0.2mV as a sign of successful operation. 40uM curcumin or cur-hydrogel was injected to ventricular wall at the bilateral 1mm of the ligation site with an insulin injection needle (30 G), and then the chest was closed and sutured.
2.2 Cardiac hemodynamic measurement
A small latex balloon filled with water was inserted into the left atrium by polyethylene intubation and entered into the left ventricle. The pressure transducer was connected to measure the left ventricular diastolic pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), and the maximum rate of rise and fall of left ventricular pressure. The data were input into the computer to analyze and record them.
2.3 Cardiac ultrasound examination of cardiac function.
Five mice in each group were randomly selected for cardiac ultrasound 24 hours after reperfusion. After intraperitoneal injection of pentobarbital sodium, the mice were fixed on a constant temperature plate at 37 ℃. The chest was evenly smeared with ultrasonic coupling agent and detected by Mindray DP-50 ultrasound system. Each mouse was measured for 10 consecutive cardiac cycles and the left ventricular ejection fraction (LVEF) and left ventricular short axis shortening rate (LVFS), LVEF=[(LVEDV-LVESV)/LVEDV]×100%,LVFS=[(LVEDD-LVESD)/LVEDD]×100% .
2.4 Evans blue staining.
The coronary artery was ligated in situ, and retrograde perfusion was performed through the aorta with 2-3 ml Evans Blue (1%) under a certain pressure (80mmHg). Under the action of staining, the non-ischemic heart showed blue, which supported the display of ischemic AAR (risk zone) myocardium.
2.5 HE/MASSON staining
The experimental animals were euthanized by injecting excessive pentobarbital sodium. The heart was removed immediately, and part of the myocardial tissue was fixed in paraformaldehyde, and ethanol was used for gradient dehydration, followed by transparent treatment and paraffin embedding. After the embedded myocardial tissue was cooled and solidified, it was cut into thin slices with a thickness of 5 μ m. The myocardial tissue of rats was stained with HE (hematoxylin-eosin staining) and collagen was stained with Masson.
2.6 Determination of biochemical Indexes of Myocardial tissue.
The myocardial tissue was homogenized at 60 Hz for 60 s for 2 times in an automatic sample grinder, and the supernatant was centrifuged at 3500 r / min for 10 min at 4 ℃. The samples were separated and stored in the refrigerator at-80 ℃ for the determination of content and the activities of SOD, CAT and GPS,GR, respectively. After strictly following the instructions of the kit, the OD values were determined by enzyme labeling instrument.
2.7 Enzyme colorimetric method.
The activities of Ca2+-ATPase and Na+-K+ -ATPase in aorta were determined by enzyme colorimetry (Nanjing Jiancheng Institute of Biological Engineering, China). Strictly according to the instructions of ultra-trace Ca2+-ATPase and Na+-K+ -ATPase detection kit, the activities of enzyme in myocardial tissue homogenate were determined.
2.8 Preparation of cur-hydrogel
The peptide RADA16-I (Ac-RADARADARADARADA-CONH2) stored at 4°C and was synthesized by Shanghai Bootech BioScience & Technology (Shanghai, China).
The method of configuration of curcumin-RADA16-I solution was as follows: appropriate curcumin and RADA16-I were placed in 10 ml vials, and water was added tovials, obtained curcumin solution with concentration of 5.0×103 M and RADA16-I solution with concentration of 5.8×105 M (0.1 mg/ml). The solution is left in a mixing pan for about 5 days.
2.9 Dynamic Light Scattering (DLS)
The particle size distribution of the RAD16-I-PY suspension was measured by dynamic light scattering particle size analyzer (Nano-ZS90, Malvern, UK). The suspension is shaken vigorously before measurement.
2.10 Drug release kinetics in vitro.
Under the simulated environment of physiological temperature in vitro, the release time and rate of curcumin from co-assembled hydrogel were observed. It was detected by high performance liquid chromatography-mass spectrometry (LC-MS).
2.11 Hypoxia-reoxygenation model of cardiomyocytes
H9C2 cardiomyocytes were cultured in DMEM medium containing 10% serum and 1% penicillin-streptomycin at 37 ℃ and 5% CO2 incubator. According to the experimental group, the original complete culture medium was replaced by serum-free low-sugar DMEM medium, and H9c2 cardiomyocytes were placed in an anoxic incubator with 37 ℃, 5%CO2 and 2% O2 for 12 hours. After hypoxia, the culture medium was replaced by complete culture medium and reoxygenated in an incubator of 37 ℃ and 5% CO2 for 12 hours to establish a cell HR model.
2.12 MTT assay
H9C2 cells were plated in 96-well plates and we used MTT assay to detect the cell viability. MTT (0.5 mg/mL; Beyotime Biotechnology, China) was added after curcumin treatment and incubated for 3 h at 37°C. And 150 μL DMSO was added and incubated for 15 min. We measured the absorbance at 490 nm.
2.13 Flow cytometry.
The Annexin V-FITC/PI apoptosis detection kit was purchased from Solebao Company(Beijing, China), and an appropriate amount of logarithmic growth phase cells were washed twice with pre-cooled PBS. The cells were suspended with 500 ul of bound buffer, mixed with 5 ul of annexin V-FITC and PI, respectively, and placed at room temperature for 15 min. The apoptosis rate was determined by flow cytometry.
2.14 Single fluorescent GFP-LC3 plasmid transfection.
After transient transfection of GFP-LC3 plasmid into H9C2 cells, the changes of green bright spots of GFP-LC3 in each group were observed under double fluorescence microscope, and 5 visual fields were randomly selected in each experimental group to take pictures.
2.15 TUNEL staining.
According to the instructions, the cells or tissues were successively added with biotin labeling solution and 3,3-diaminobenzidine carbon tetrachloride (DAB) chromogenic solution and used PBS. Finally, the 3DHISTECH Pannoramic SCAN system was used to scan 5 non-overlapping visual fields in each group. The apoptotic nuclei and the total number of apoptotic nuclei in the visual field were calculated by Image J software. The apoptosis rate of cardiomyocytes = the number of apoptotic nuclei / the total number of nuclei × 100%.
2.16 DCFH-DA probe staining.
DCFH-DA probe (Sigma, USA) is used to detect the formation of reactive oxygen species. The cardiomyocyte culture medium was removed, diluted DCFH-DA (10uM) probe 1ml was added and incubated at 37 ℃ in 5%CO2 incubator for 20 min. Rinse with PBS for 3 times × 1 min to fully remove the DCFH-DA probe that did not enter the cell. Then, the fluorescence intensity was detected by laser confocal microscope.
2.17 Determination of mitochondrial membrane potential.
JC-1 reagent (T3168) was purchased from Semel Fisher Technology Co., Ltd. (China). Strictly follow the instructions. 1 μg/mL JC-1 working solution was prepared and incubated at 37 ℃ for 20 min,PBS. Five visual fields were selected for each group and photographed under confocal microscope.
2.18 Western blot.
Take appropriate amount of cells in logarithmic growth phase, after RIPA cleavage, extract total protein, BCA method. After quantitative denaturation, protein electrophoresis-membrane transfer-closure-Ⅰ anti-incubation-Ⅱ anti-incubation-development exposure were carried out according to the operation steps. The expression of the target protein was expressed by the ratio of the gray value.
2.19 Statistical analysis
All data is presented as a mean ± S.E.M. Statistical analysis was performed using a one-way ANOVA. P-value < 0.01was considered as statistically significant