Mice husbandry
The male C57Bl6 mice had been obtained from the experimental animal heart of the Fourth Army Medical College (Xi’an, China). C57Bl/6 mice had been housed at 22 ± 2 °C with a 12-h gentle/darkish cycle. All animal experiments complied with the ARRIVE tips and had been carried out in accordance with Nationwide Institutes of Well being information for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978).
Doxorubicin remedy in mice
To assemble the doxorubicin induced cardiotoxicity mouse mannequin, mice had been injected intraperitoneally with doxorubicin (Yuanye biotechnology, China) diluted in PBS at 10 mg/kg, along with different indicated remedies. On the finish of the experiment, the mice had been subjected to echocardiography examination and/or histology evaluation.
Histology
Hearts from handled mice had been mounted with 4% paraformaldehyde (pH 7.4), embedded in paraffin, after which sliced into 5-µm thickness part. The sections had been stained with Hematoxylin and Eosin (H&E) for routine histological examination. To measure collagen deposits, sections had been stained with Sirius purple. Quantification was carried out utilizing ImageJ software program (Nationwide Institutes of Well being).
Stream cytometry evaluation of lipid peroxidation
Cells with indicated remedies had been incubated in 2 µM C11-BODIPY581/591 (Invitrogen) for 20 min at 37 °C. The entire course of was saved at midnight. Fluorescent sign was analyzed by move cytometry.
DHE and TUNEL staining
Contemporary coronary heart tissues had been sectioned to 10 μm thicknesses and incubated with superoxide anion fluorescent probe dihydroethidium (Cat HY-D0079, MCE) or TUNEL as instructed. The slices had been counterstained with Hoechst after which washed thrice with PBST for five min earlier than examination underneath fluorescence microscope.
Cell tradition and remedy
Mouse cardiomyocyte HL-1 was cultured in DMEM supplemented with 10% FBS, 1% penicillin and streptomycin, and incubated at 37 °C in a humidified environment of 5% CO2 and 95% air. Cells had been incubated with 0.5 µM doxorubicin for 48 h. For Hmox1 knockdown, adverse management or siRNA (20 nM) was transfected into HL-1 cells utilizing Lipofectamine 2000, adopted by doxorubicin remedy someday later.
qPCR evaluation
Whole RNA was remoted utilizing Trizol (Invitrogen) and reverse-transcription was carried out utilizing PrimeScript First-Strand cDNA Synthesis Package (Takara, China). Gene expression was analyzed utilizing PrimeScript RT Grasp Combine (Roche, Switzerland) as instructed. PCR was carried out no less than in triplicate and relative expression was calculated by normalizing to the management samples utilizing 2-ddCt methodology. The primer sequences are listed in Desk S1.
Western blot
Protein samples had been ready utilizing RIPA lysis buffer and separated in 12% SDS-PAGE gel (120 V for stacking gel and 160 V for separation gel). After transferred to nitrocellulose membrane, the membrane was blocked with 5% bovine serum albumin for 1 h, adopted by incubation in a single day with major antibodies anti-GM130 (66662-1-Ig, Proteintech), anti-CD9 (60232-1-Ig, Proteintech), HMOX1 (66743-1-Ig, Proteintech) or anti-GAPDH (60004-1-Ig, Proteintech) at 4 °C. Then, the membrane was washed and incubated with corresponding secondary antibodies at room temperature for 1 h, and visualized by the ECL Prime Western Blotting Detection Reagent.
Measurement of MDA content material
Malondialdehyde (MDA) ranges had been measured utilizing Lipid Peroxidation MDA Assay Package (Cat#S0131, Beyotime) as instructed.
Exosome isolation, loading, labeling and monitoring
Cell tradition supernatant was collected and centrifuged at 13,200 g and 4 °C for 30 min to take away giant microvesicles. The supernatant was then filtered twice via 0.22 μm filters after which exosomes had been pelleted with ultracentrifugation at 120,000 g for 4 h. Pellets had been washed as soon as with PBS and centrifuged at 120,000 g, 4 °C for 1 h once more. The exosome morphology was confirmed by electron microscopy. Briefly, the exosomes had been added onto the grid and stained with 2% uranyl acetate, adopted by imaging with the transmission electron microscope (JEM-2000EX TEM, JEOL Ltd., Tokyo, Japan). Measurement distribution of the remoted exosomes was additionally analyzed by nanoparticle monitoring assay.
To load siRNA (adverse management, siHmox1, or siGapdh) into exosomes, about 10^10 exosomes in 400 µl had been electroporated with 1 OD siRNAs of curiosity (GenePharma, Shanghai) at 700 V/150 mF in 0.4 cm large electroporation cuvettes. Free nucleic acids exterior the exosomes had been eliminated by RNase, adopted by extra exosome isolation with ultracentrifugation as described above. The exosome suspension was dissociated to keep away from attainable aggregates earlier than use.
For in vivo monitoring, purified exosomes had been first incubated with fluorescent dye DiR/DiO/DiI (on the closing focus of 10 mM, Invitrogen, Carlsbad, CA) after which free dye was eliminated by one other spherical of centrifugation. Mice had been injected with labelled exosomes (100 µg at protein focus in 100 µL PBS) with or with out the help of ultrasound focused microbubble destruction. Completely different organs had been harvested and processed for ex vivo picture or microscope evaluation. Cardiomyocytes had been stained with anti-TNNT-2 to visualise the mobile location of exosomes/FAM-labeled siRNA.
Ultrasound focused microbubble destruction and exosome supply into the center
UTMD and UTMD aided exosome in vivo supply had been carried out related as describe earlier than. Briefly, mice had been ready with the irradiated area hair shaved, after which anaesthetized with 2% isoflurane. The combination composed of 100 µL SonoVue™ microbubble (Bracco imaging, SA) and 100 µL exosomes (100 µg at protein focus in 100 µL PBS) was injected slowly by way of tail vein. Two minutes after injection, ultrasound irradiation was carried out within the coronary heart area via the thorax. Ultrasound pulses had been utilized to the focused area for 1 min in whole at an obligation cycle of fifty% by a 0.66 MHz US instrument (Present from Chongqing Medical College) armed with the probe space of 4.5 cm2. For therapeutic intervention, the mice had been handled with exosomes encapsulated with management or Hmox1 siRNA (100 mg at protein focus in 100 µL PBS) with or with out the help of UTMD someday earlier than doxorubicin remedy. Teams handled with doxorubicin, UTMD, or exosome alone served as controls.
Echocardiography
Mice had been anaesthetized with inhaled isoflurane by way of a circuit anesthesia equipment. Visible Sonics Vevo 2100 ultrasound instrument (Visible Sonics Inc., Toronto, ON, Canada) armed with 40 MHz linear transducer was employed for cardiac perform evaluation. Left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) had been obtained from M-mode, and E/A price was obtained by pulsed-wave Doppler echocardiography. The guts price was monitored repeatedly throughout the entire course of [15]. The identities of animals had been saved blinded to the echocardiography investigators.
Statistics
The information had been expressed as imply ± SEM or in any other case indicated. One-way ANOVA evaluation was used for a number of group comparability or t-test for comparability between two teams by GraphPad Prism8. Important variations had been thought of at p < 0.05.