Animals
All animal experiments had been accredited by Animal Use and Care Committee of Air Power Medical College, and had been performed in compliance with moral rules of animal analysis. All mice used had been supplied by the animal heart of Air Power Medical College, which had been males in C57BL/6J background and had been 6–8 weeks previous on the onset of experiments. Mice had been housed beneath managed circumstances of lighting (12 h mild/12 h darkish cycle), temperature (22 ± 1℃), and humidity (60 ± 5%) with free entry to meals and water.
Building of MI mannequin
MI was induced as beforehand described [11]. Briefly, mice had been anesthetized with 2–3% isoflurane and hearts had been uncovered by means of the incision within the chest. Then, left anterior descending (LAD) coronary artery was ligated with 6 − 0 silk roughly 2–3 mm beneath the origin. After ligation, the center was returned and the chest was closed. Mice within the sham group obtained similar operation besides LAD ligation. All mice had been monitored for 3–6 h earlier than returning to their regular animal room. The success of MI mannequin development was decided by histological staining and echocardiography.
Triphenyl tetrazolium chloride (TTC) staining
After anesthetization, the chest of mice was lower open to show the center. Then the center was harvested, washed, and sectioned into roughly 1–2 mm thick sections. All sections had been incubated with 1% TTC (A610558, Sangon Biotech) for 15 min at room temperature after which mounted in paraformaldehyde (A500684, Sangon Biotech). Picture J was used to calculate the share of infarcted myocardium.
Echocardiography
Echocardiography was carried out with VEVO 3100 echocardiography system (VisualSonics Inc., Toronto, Canada). Mice had been anesthetized with pentobarbital sodium (intraperitoneal injection, 60 mg/kg physique weight) or isoflurane (inhalation anesthesia, 2–3%). Anesthetization was adjusted to take care of the center price inside 400–450 bpm. M-mode echocardiography was carried out as beforehand described [12]. Left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-systolic quantity (LVESV), and left ventricular end-diastolic quantity (LVEDV) had been calculated.
Measurement of creatine kinase (CK), creatine kinase MB (CKMB), lactate dehydrogenases (LDH), and LDH1
Mice had been anesthetized with 2–3% isoflurane and the neck pores and skin had been lower open to show carotid artery. Blood pattern had been instantly collected after carotid artery was lower. The collected blood pattern had been saved in a single day at 4℃ after which centrifuged at 3000 rpm for 15 min at 4 °C. The supernatant was collected as serum. The quantity of CK, CKMB, LDH, and LDH1 had been decided utilizing automated biochemical analyzer (Chemray 240).
Measurement of iron stage
The measurement of iron stage in serum, myocardium lysate, cardiomyocyte tradition medium, cardiomyocyte lysate, and resolution of iron ion, was carried out utilizing Whole Iron Colorimetric Assay Equipment (Elabscience, E-BC-K772-M) following producer’s directions. Briefly, the serum and cell tradition medium pattern had been diluted after which examined. The guts and cardiomyocyte pattern had been homogenized, centrifuged, after which examined. The iron stage was calculated primarily based on the usual curve. To detect the iron stage inside stay cardiomyocytes, fluorescent iron probe FerroOrange (MaokangBio) was additionally used following producer’s directions (knowledge proven in Fig. 3a and b).
Western blotting
Ventricular tissue, cells or EVs had been lysed with ice-cold RIPA buffer (Beyotime, P0013B). The protein focus was then decided with Pierce BCA Protein Assay Equipment (Thermo Scientific, 23225). Protein pattern had been separated by SDS-PAGE and transferred to nitrocellulose membrane. Membranes had been blocked with skim milk, incubated with main and secondary antibodies, and detected by chemiluminescent detection system (Picture Lab, Bio-Rad, US). Equal protein loading can be confirmed with antibodies towards β-Actin. Antibodies used on this research had been listed beneath: anti- ferritin heavy chain 1 (FTH1) (Santa Cruz, sc-376594), anti-transferrin (Proteintech, 17435-1-AP), anti- glutathione peroxidase 4 (GPX4) (Santa Cruz, sc-166437), anti-β-Actin (Proteintech, 66009-1), HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (Proteintech, SA00001-2), and HRP-conjugated Affinipure Goat Anti-Mouse IgG(H + L) (Proteintech, SA00001-1).
Immunohistochemistry
Hearts had been mounted with paraformaldehyde (A500684, Sangon Biotech), dehydrated, embedded with paraffin, and sectioned. Hearts sections had been then blocked with goat serum and incubated with main antibodies in a single day at 4℃. Then sections had been incubated with secondary antibodies and Hoechst. Picture evaluation and quantification had been carried out with ImageJ as beforehand described [13].
Cell tradition
RAW264.7 mouse macrophage cell line was bought from Cell Strains Service. Macrophages had been cultured with Dulbecco’s modified Eagle’s medium (10% fetal bovine serum and 1% penicillin/streptomycin). When the cell confluency reached round 80–90%, the tradition medium was changed with Dulbecco’s modified Eagle’s medium supplemented with EV-depleted serum. 48–72 h later, the tradition medium was collected to isolate macrophages-derived EVs.
Neonatal rat ventricular cardiomyocytes (NRVCs) had been remoted from the hearts of 1–3 days previous Sprague Dawley rat as beforehand described [14]. As for hypoxia therapy, cardiomyocytes had been maintained in hypoxia atmosphere (1% O2, 5% CO2, and 94percentN2) for six h.
EV assortment and isolation
The tradition medium of unpolarized RAW264.7 macrophages was collected to isolate EV. The collected medium was centrifuged at 800 g for 10 min at 4 °C to take away cells and cell particles. The collected supernatant was then centrifuged at 12,000 g for 30 min at 4 °C to take away microvesicles. The supernatant was centrifuged at 150,000 g for 90 min at 4 °C, washed with PBS, and centrifuged at 150,000 g for 90 min at 4 °C once more. The EV was resuspended with PBS. The quantity of PBS was 1% of the quantity of unique tradition medium. The focus of EV resolution was decided with nanoparticle monitoring evaluation (NTA) evaluation and Pierce BCA Protein Assay Equipment (Thermo Scientific, 23225).
Administration of macrophages-derived EVs
The focus of EVs resolution was about (2.36 ± 0.34) ×108particles/µL (decided by NTA evaluation) and 446.37 ± 28.60 ng/µL (decided by Pierce BCA Protein Assay Equipment). For in vivo experiments, the macrophages-derived EVs had been intramyocardially injected to mice of sham group or MI group. Mice had been injected with 60–80µL EVs resolution or PBS. Within the MI group, after LAD ligation, the EVs resolution or PBS was injected in 3–4 factors across the infarcted space (the colour of the infarcted myocardium was paler in comparison with the conventional myocardium). Within the sham group, the center was uncovered by means of the incision on the chest after which equal quantity of EVs resolution or PBS was injected in the identical space. After operation, all mice had been monitored for 3–6 h earlier than returning to their regular animal room, and detection about cardiac perform and iron metabolism had been carried out 24 h submit operation. For in vitro experiments, EVs resolution was diluted 100-fold utilizing cell tradition medium, and the identical quantity of PBS was added to the management group.
Electron microscopy
For transmission electron microscopy (TEM), EV resolution was dropped onto the 200-mesh copper grids. Extra resolution was eliminated after 10 min incubation. The copper grids had been then incubated with 2% phosphotungstic acid hydrate for 0.5–1 min, washed with Milli-Q purified water for thrice and air-dried.
For immunogold staining, EV resolution was dropped onto copper grids as earlier than. Then, EVs had been mounted with paraformaldehyde for 15 min at room temperature, blocked with 5% bovine serum albumin (BSA) resolution for 30 min at 37 °C, and incubated with main antibodies for 1–2 h at 37 °C. After washing with Milli-Q purified water for thrice, the grids had been incubated with secondary antibodies for 1–2 h at room temperature. Then the grids had been stained with 2% phosphotungstic acid hydrate as earlier than. Antibodies used had been listed beneath: anti-TfR (Santa Cruz, sc-65882), and Rabbit IgG management Polyclonal antibody (Proteintech, 30000-0).
All photographs had been obtained utilizing a transmission electron microscope (JEM-1230, JEOL Ltd., Tokyo, Japan).
Measurement of glutathione (GSH) and malondialdehyde (MDA) stage
The measurement of GSH and MDA stage was carried out utilizing GSH assay package (Beyotime, S0053) and MDA assay package (Beyotime, S0131) respectively following producer’s directions.
Measurement of ROS stage
The ROS measurement was carried out utilizing ROS assay package (Beyotime, S0033) following producer’s directions. Briefly, cardiomyocytes had been seeded onto glass-bottomed tradition dishes (NEST, 801002). After therapy, the fluorescent iron probe DCFH-DA was added to the tradition medium and cells had been returned to the identical atmosphere for 30 min. Then cells had been washed with PBS for thrice. The confocal laser-scanning microscope (Nikon A1 plus Confocal Microscope, Nikon, Japan) was used to acquire all fluorescent photographs, and ImageJ was used to research.
Measurement of cell viability
The measurement of cell viability was carried out utilizing Calcein AM cell viability assay package (Beyotime, C2013FT) following producer’s directions. The FACS Calibur circulate cytometer (BD Biosciences) was used to research.
Dihydroxyethidium (DHE) staining
Upon harvested, the hearts had been snap-frozen in liquid nitrogen. Then, the frozen hearts had been embedded with OCT and sectioned. Sections had been then stained with DHE and Hoechst.
Liquid chromatography tandem mass spectrometry (LC-MS/MS) evaluation
EVs pattern had been used for MS evaluation as beforehand described [15]. The evaluation was carried out utilizing EASY-nLC 1200 UHPLC system (Thermo Fisher) and Q Exactive HF-X mass spectrometer (Thermo Fisher).
Plasmids and small interfering RNA (siRNA) development and transfection
The TfR-EGFP and empty-EGFP plasmids had been generated by Genechem. The transferrin-mcheery and empty-mCherry plasmids and siTfR had been generated by Hanbio. The sequence of siTfR is CCAGACCGUUAUGUUGUAGUATT (sense) and UACUACAACAUAACGGUCUGGTT (anti-sense). Macrophages had been transfected with plasmid with Lipofectamine 3000 (Invitrogen, L3000150) and had been transfected with siRNA with Lipofectamine RNAiMAX (Invitrogen, 13778500) following producer’s directions.
Enzyme linked immunosorbent assay (ELISA)
The measurement of transferrin stage was carried out with Transferrin ELISA package (Elabscience, E-EL-M1184c) following producer’s directions.
EV tracing
EVs had been labeled with Dil (Invitrogen, C7001) following producer’s directions. Then, labeled EVs had been intramyocardially injected. Twenty-four hours after injection, the center, liver, spleen, skeletal muscle, lung and kidney had been harvested. The harvested organs had been subjected to imaging for fluorescent sign. In addition to, the center and liver had been harvested, washed and frozen in liquid nitrogen. The frozen tissue was embedded with OCT and sectioned. The cell membrane was labeled with anti-ATPA1 antibody and the macrophages had been labeled with anti-F4/80 antibody.
Statistical evaluation
All knowledge had been expressed as imply ± commonplace error of imply (SEM). T-test, one-way ANOVA or 2-way ANOVA had been used to research steady knowledge. All evaluation was carried out with GraphPad Prism 6.0 software program (GraphPad Software program, La Jolla, USA). P < 0.05 indicated a statistical significance between teams.