Ethics statement
All animal experiments were in tally with the Guide for the Care and Use of Laboratory Animal by International Committees. The protocol was approved by the Institutional Animal Care Use Committee of The First Affiliated Hospital of Anhui Medical University.
Experimental animals
Male healthy C57BL/6 mice (n = 90), aged 10–12 week and weighed 20–30 g were bought from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). The mice were fed adaptively for 1 w in a clean animal house with 22–25°C, water and food available ad libitum and normal circadian rhythm. Twenty mice were divided into the sham group and I/R group with 10 mice in each group to perform the establishment of I/R model and the identification experiment. And then the rest 70 mice were treated according to the successful modeling method.
Modeling of I/R mice
Fasted for 8 h and anesthetized with 1% pentobarbital sodium solution (60 mg/kg) (Sigma, Santa Clara, CA, USA), mice were placed in a supine position with the skin of the regiones colli anterior cut off after disinfection. The tissues and muscles were separated and trachea was exposed; trachea cannula was inserted from the mouth and connected with a respirator. The mice were placed in the right lateral position. The left side of the epidermis of the fifth costal space was made a 2-cm longitudinal incision. The skin was cut open, then the ectopectoralis and entopectoralis were separated, and the fourth costal space was exposed. The fourth costal space was impaled by mosquito forceps, the mediastinum to the left was penetrated, and then the heart was extruded by squeezing the right chest with hand. At the 0.5 cm of lower edge of left atrial appendage, the left coronary artery was ligated with a 6 − 0 suture, with a slipknot and timing. Left ventricular anterior wall whitening or ST segment elevation in the electrocardiogram (ECG) monitoring indicated successful ligation. After ischemia of 30 min, the slipknot was loosened and then re-clocked. Meanwhile, the ST-segment raised in the ECG (ST segment recovery or a change significantly different from that of the previous waveform) within 5 min was noted as a reperfusion success. The heart was then returned to the chest and the contralateral thoracic cavity was pressed with hand to remove the pneumothorax. The skin was sutured. After the operation, the mice were put back into the animal cage and the vital signs of the mice were observed. The same thoracotomy was used in the sham group, but the left coronary artery was not ligated. Cardiac ultrasound detection and hemodynamic monitoring were used to determine whether the model was successfully established.
Grouping and treatment
Seventy mice were distributed into 7 groups (10 mice/group) and injected with normal saline, si-LINC00461 vector, si-LINC00461 vector negative control (NC), miR-185-3p mimics, miR-185-3p mimics NC, miR-185-3p mimics and overexpression (OE)-LINC00461 vector, or miR-185-3p mimics NC and OE-LINC00461 vector through the tail vein 24 h before modeling. The left coronary artery was ligated, and the ligature line was loosened after 30 min, and re-perfused for 2 h. si-LINC00461, si-NC, miR-185-3p mimics, mimics NC and OE-LINC00461 were bought from GenePharma Co., Ltd. (Pudong District, Shanghai, China).
Cardiac ultrasound detection
The tissues and skins in the chest of mice were sutured layer by layer at the end of reperfusion. Left ventricular end diastolic volume (LVEDV) and left ventricular end systolic volume (LVESV) were gauged; the left ventricular ejection fractions (LVEF) and left ventricular fractional shortening (LVFS) were computed. LVEF (%) = (LVEDV - LVESV)/LVEDV × 100%.
Hemodynamic detection
The abdominal cavity of mice were injected into 1% pentobarbital sodium solution. The neck tissues were bluntly separated to expose the carotid artery, the distal end was ligated and the proximal part was pulled by silk thread, 1.4F Millar catheter (Millar Instruments, Inc. Houston, TX, USA) was inserted into the carotid artery of mice. When the Millar catheter entered to the left ventricle, the maximum rate of rise of left ventricular pressure increase/decrease (± dp/dt max) were recorded. According to the blood pressure waveform, whether the catheter into the left ventricular chamber was determined.
Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Trizol (Invitrogen, Carlsbad, CA, USA) method was utilized for extracting total RNA in myocardial tissues. The concentration and quality of RNA were determined by NanoDrop2000 (Thermo Fisher Scientific, Massachusetts, USA). Reverse transcription reaction was in accordance with the ReverTra Ace qPCR RT Kit (TOYOBO, Osaka, Japan) with 500 ng loading sample. RT-PCR reaction system was prepared based on the Sybr green reagent kit (Takara Co., Ltd., Dalian, Liaoning, China). The primers were composed by BGI Co. (Shenzhen, Guangdong, China) (Table 1). U6 was the endogenous control of miR-185-3p while glyceraldehyde phosphate dehydrogenase (GAPDH), the endogenous control of LINC00461 and Myd88. The product was verified by agarose gel electrophoresis. Data were reckoned by 2−ΔΔCt method.
Table 1
Gene | Sequence (5’→3’) |
LINC00461 | F: 5’-GACATTTACGCCACAACCCACG-3’ |
R: 5’-AGACAGACCCTCAGATTCCCCA-3’ |
miR-185-3p | F: 5’-GGATCCAAAGAACATCAGATCCATGG-3’ |
R: 5’-AAGCTTAGCAGACATAGCCAGCCAGA-3’ |
Myd88 | F: 5’-- CAACCAGCAGAAACAGGAGTCT-3’ |
R: 5’-ATTGGGGCAGTAGCAGATGAAG- 3’ |
U6 | F: 5’-ATTGGAACGATACAGAGAAGATT-3’ |
R: 5’-GGAACGCTTCACGAATTTG-3’ |
GAPDH | F: 5’-ACGGCAAGTTCAACGGCACAG-3’ |
R: 5’-GACGCCAGTAGACTCCACGACA-3’ |
Note: F, forward; R, reverse; LINC00461, long non-coding RNA LINC00461; miR-185-3p, microRNA-185-3p; Myd88, myeloid differentiation primary response gene 88; GAPDH, glyceraldehyde phosphate dehydrogenase. |
Western blot analysis
The total protein in myocardial tissues of mice were abstracted. The tissues were cleaned twice or three times with phosphate buffered saline (PBS), cut into small pieces and placed in the homogenizer. The obtained homogenate was moved into a centrifuge tube, followed by an oscillation and ice bath, during which the tissues were triturated repeatedly to ensure the cells were lysed. The liquid supernatant (total protein solution) was amassed by centrifugation at 12,000 rpm for 5 min. The protein concentration was determined by bicinchoninic acid method (Boster Biological Technology Co. Ltd., Wuhan, Hubei, China). The protein was appended with loading buffer and boiled at 95℃ for 10 min, with 30 µg loaded in each well. The protein was separated with 10% polyacrylamide gel electrophoresis for 45–70 min, and transferred to a polyvinylidene fluoride membrane and sealed with 5% bovine serum albumin. The membrane was hatched with primary antibody Myd88 (1:1000), Bcl-2 (1:2000), Bax (1:2000) and GAPDH (1:3000) (all from Abcam, Cambridge, MA, USA) and with corresponding secondary antibody (Miaotong Biotechnology Co., Ltd., Xuhui District, Shanghai, China). The image was developed by chemiluminescence reagent and Bio-rad Gel Doc EZ imager (Bio-rad, California, USA). The target band was analyzed by ImageJ software to performing gray value analysis.
RNA pull-down assay
Three different biotin-labeled miRNA sequences were designed: wild type (WT) miR-185-3p (Bio-miR-185-3p-WT), mutant type (MUT) miR-185-3p (Bio-miR-185-3p-MUT, the sequence complementary to the LINC00461 was mutated), and a random miRNA (Bio-NC) complementary to LINC00461 as a NC. When reached 80–90 confluence, the above mentioned miRNA (Bio-miR-185-3p-WT, Bio-miR-185-3p-MUT and Bio-NC) was transfected. The cells were lysed at 48 h post transfection to obtain a protein lysate. The lysate was hatched with M-280 streptavidin-coated magnetic beads (Sigma). The magnetic beads were washed twice with cold buffer, three times with hypotonic buffer and once with hyperosmotic buffer in turn. Finally, the protein-nucleic acid complex adsorbed by the magnetic bead was eluted. Trizol was utilized to lyse the protein-nucleic acid complex, the RNA was extracted and the expression of LINC00461 was detected by RT-qPCR.
Dual luciferase reporter gene assay
The targeting relationship between miR-185-3p and Myd88 and the binding site between miR-185-3p and Myd88 3’untranslated region (UTR) were predicted by bioinformatics software https://cm.jefferson.edu/rna22/Precomputed. The Myd88 3’UTR sequence containing miR-185-3p binding site was synthesized and the Myd88 3’UTR WT plasmid (Myd88 3’UTR-WT) was constructed. On the basis of the plasmid, the Myd88 3’UTR MUT plasmid (Myd88 3’UTR-MUT) was constructed. The logarithmic growth 293T cells were cultured into 70% confluence on 96-well plates. Myd88 3’UTR WT plasmid and miR-185-3p mimics plasmid or its NC/Myd88 3’UTR MUT plasmid and miR-185-3p mimics plasmid or its NC were transfected into 293T cells according to Lipofectamine 2000 (Invitrogen) specification. The luciferase activity was detected by Dual-Luciferase@ Reporter Assay System kit (Promega Corporation, Madison, WI, USA) 48 h later.
Hematoxylin-eosin (HE) staining
Preparation of myocardial tissue sections: after 2 h of cardiac I/R in mice, 4 mice were selected for thoracotomy again. The myocardial tissues were flushed in 4℃ pre-cooled PBS and fastened with paraformaldehyde for 24 h. The tissues were embedded with paraffin and sliced to 5 µm using a paraffin slicer (Leica, Stockholm, Sweden), and the slice was baked in an oven at 60℃ for 20 min. The tissue wax block was sliced with a paraffin slicing machine (Leica), and the tissues were baked at 60℃ in an oven for another 20 min.
The slices were dyed with coaggulating reagent TOⅠ and TOⅡ (Guangzhou Jetway Biotech Co., Ltd, Guangzhou, China) for 10 min, respectively. The tissues were dehydrated in 95%, 80% and 75% ethanol for 2 min in turn. Then, the tissues were cleaned with running water, dyed with hematoxylin, returned to blue by tap water and differentiated with hydrochloric acid alcohol. The slices were rinsed under the running water for 5 min when the color was turned red from blue, dyed with eosin for 1 min, hydrated with inverse concentration gradient ethanol, and placed into TOⅠ for 10 min and finally blocked with neutral gum. The tissues were examined with a light microscope for histopathology and photographed.
Picric acid-Sirius red staining
The myocardial tissue sections of mice were dewaxed in turpentine Ӏ, Ⅱ solution for 15 min, respectively, then step-by-step hydrated in 100% ethanol Ӏ, Ⅱ, 95%, 90%, 80% and 75% ethanol and distilled water for 5 min, respectively, placed in hematoxylin solution for 2–3 min, rinsed with clear water for 30 min, dehydrated with 75%, 80%, 95% and 100% for 3 min, respectively. The sections were cleared with xylene and sealed with neutral gum. The degree of myocardial fibrosis was observed under a light microscope.
Terminal deoxynu-cleotidyl transerase-mediated dUTP-biotin nick end-labeling (TUNEL) staining
Preparation of the frozen section of the mice heart: the remaining 3 mice were euthanized at the end of the experiment, and the heart was perfused with physiological saline to remove the residual blood from the heart, and 10% muriate was injected. The mouse heart was immediately buried in the optimal cutting temperature (OCT) (the bottom of the heart was down, the tip of the heart was up). The embedded tissue can be stored in a -4℃ refrigerator. A continuous frozen section was prepared into 4 µm and the tissue sections were placed on a polylysine coated slide after exposure of the papillary muscle.
The frozen sections were fastened with 4% paraformaldehyde, cleaned with PBS, added with 50 µL DNA labeling solution avoiding light for 1 h, reacted with 100 µL antibody solution for 0.5 h avoiding light and dyed with 100 µL 7-AAD/RNase solution for 0.5 h. Nucleus was stained with 4'-6-diamidino-2-phenylindole solution. The sections were sealed and observed by a microscope.
Serum index detection
All the mice at the end of the reperfusion were taken 4 mL blood by a disposable venous blood sample collection container separation glue or coagulant promoter from the abdominal aorta. The blood was centrifuged at 3000 r/min for 10 min to take the supernatant. According to the instructions of the lactate dehydrogenase (LDH) kit, the creatine kinase-MB (CK-MB) kit, the cardiac troponinI (cTn-I) kit and the nitric oxide (NO) kit (all from NanJing JianCheng Bioengineering Institute, Nanjing, China), corresponding sample and reagent was added for detection. With the optical density (OD) values determined by a microplate reader, the standard curve was drawn and the activity or content of each index in the sample was calculated.
Detection of myocardial tissue index in I/R mice
At the end of reperfusion, all the apical tissues of the mouse heart were removed, shredded and put into the 10 mL homogenate tube. The tissues were homogenized in the precooled 0.9 NaCl solution (1 mL/100 mg) at the weight to volume ratio of 1 : 9, and then the homogenate was placed in precooled homogenate medium (0.01M Tris-HCl, 0.0001M ethylene diamine tetraacetic acid-2Na, 0.01M sucrose, 0.8% NaCl solution, pH 7.4) to prepare 10% homogenate. Reactive oxygen species (ROS) and malondialdeyde (MDA) content, as well as superoxide dismutase (SOD) activity were determined according to ROS, MDA and SOD kits (JianCheng Bioengineering Institute).
Statistical analysis
All date were processed by SPSS 21.0 software (IBM Corp. Armonk, NY, USA). Measurement data were interpreted as mean ± standard deviation. Comparisons between two groups were conducted by t-test in data in normal distribution. Comparisons among multiple groups were assessed by one-way analysis of variance (ANOVA) and the pairwise comparisons after ANOVA were analyzed by Fisher’s least significant difference t test (LSD-t). P value < 0.05 was indicative of statistically significant difference.