Exosomes derived from heat shock preconditioned bone marrow mesenchymal stem cells alleviate cisplatin-induced ototoxicity in mice

BMSCs isolation

BMSCs were isolated as described in the previous report [22]. In brief, bone marrow cells were flushed out from the bone marrow of C57BL/6 mice with 2% heat-inactivated fetal bovine serum (FBS, Gibco) and centrifugated in density gradient to isolate bone marrow mononuclear cells. Then, CD11b+ microbeads (StemCell Technologies) were utilized to select mononuclear granulocytes positively, and the remaining cells were further cultured for 72–96 h. The attached BMSCs were used for subsequent experiments.

FACS analyzing BMSCs

BMSCs were incubated with FITC conjugated CD44, PE-conjugated CD105, PerCP-Cy 5.5-conjugated CD34, and PE-Cy7-conjugated CD45 (10 μg/ml: BD Biosciences) for 20 min at room temperature, which were further fixed with 2% formaldehyde/ phosphate-buffered saline (PBS) solution on ice. Isotype controls were also utilized to confirm the specific staining. At least 50,000 singlet events were detected on a BD Facscanto II Flow Cytometer.

Heat shock-preconditioned BMSCs

The procedure for preparing heat shock-preconditioned BMSCs was performed as previously described [9]. In brief, BMSCs in the 3rd passage were exposed to heat shock conditions (42 °C water bath) for one hour in exosome-depleted FBS (Gibco) and subsequently normally cultured (37 °C, 5% CO2). The control group was normally cultivated without heat shock pretreatment.

Cisplatin-induced ototoxicity in mice

C57BL/6 mice (eight-week-old) were purchased from Vital River Laboratory Animal Ltd. (Beijing, China). In brief, cisplatin (30 mg/kg, 1 mg/ml) was intraperitoneally injected to construct cisplatin-induced ototoxicity as described (Tsai et al., 2021). All the study protocols were approved by the the Second Xiangya Hospital, Central South University.

Exosomes isolation and testification

BMSCs derived exosomes using the previously described protocol [2, 20]. In brief, a low-speed centrifuge (300 × g, 10 min, 4 °C) was utilized to remove cells, and a high-speed centrifuge (10,000 × g, 30 min, 4 °C) was utilized to isolate large vesicles and additional cellular debris. Ultracentrifuge (100,000 × g, 70 min, 4 °C) was further applied to sediment exosomes on Optima MAX-XP ultracentrifuge (Beckman Coulter), which were resuspended in PBS or culture medium by trituration.

The distribution of exosomes size was revealed by nanoparticle tracking analysis with NanoSight NS300 equipment (Malvern Instruments LTD). The single membrane structure was revealed with transmission electron microscopy (TEM) analysis with a JEM-1400Flash Electron Microscope (Jeol). The surface markers of the exosomes (CD63, CD9, and Alix) were detected with Western blots.

Auditory brainstem response

Auditory brainstem response (ABR) was detected with the TDT System III apparatus (Tucker Davies Technologies) to measure the hearing threshold seven days post cisplatin administration. In brief, acoustic stimuli (8, 16, 24, and 32 kHz; 100 ms duration; intensities ranged from 10 to 100 dB) were directly introduced into the ear canal of cisplatin-induced ototoxic mice. The detecting electrodes were separately placed on the vertex, below the right ear, and below the pinna of the left ear. Subdermal needle electrodes were utilized to record the ABR data. After ABR measurement, cochlear tissues were collected for expression detection and immunofluorescence staining analyses.

Reverse-transcription PCR (RT-PCR)

Total RNA was extracted from BMSCs with Trizol (Invitrogen), and one microgram RNA was reverse transcribed with Applied Biosystems High-Capacity cDNA Reverse Transcription kits. The reverse-transcription PCR (RT-PCR) reaction was constructed in a 15-µl volume (0.375 μM primer, 0.5 μl cDNA) with 2 × FastStart Universal SYBR Green Master Mix (Roche Ltd.). An initial denaturation of 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 1 min to detect the amplification on an ABI STEPONE System (Applied Biosystems). The relative expression was quantified with the comparative ΔCT method, and β-actin was utilized as internal genomic control. The primers utilized: HSP70, forward, 5’-CAACGTGCTCATCTTCGACC-3’, reverse, 5’-GGCTGATGTCCTTCTTGTGC-3’; β-actin, forward, 5’-CACGATGGAGGGGCCGGACTCATC-3’, reverse, 5’-TAAAGACCTCTATGCCAACACAGT-3’.

Western blotting

The lysates derived from BMSCs, BMSC-Exo, HS-BMSC-Exo, and the middle turns of the cochlear tissues were separated by 10% SDS-PAGE and transferred to PVDF membranes, which were further blocked with 5% non-fat milk and incubated with primary antibodies against HSP70, CD63, CD9, Alix, pro-caspase-1, cleaved caspase-1, NLRP3, ASC, and GAPDH (Santa Cruz). Peroxidase-conjugated secondary antibody (Sigma-Aldrich, 1:2000 dilution) was incubated for 2 h at room temperature, and a Pierce™ Enhanced Chemiluminescence (Thermo Scientific) was applied to obtain the chemiluminescence signal. GAPDH was utilized as an internal control to normalize the relative expression of interest genes with NIH-Image J1.51p 22.

Myosin staining

Cochlea tissues were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100 for two hours at room temperature. The permeabilized tissues were further blocked with 5% goat serum, incubated with anti-myosin 7a antibody (Santa Cruz), and subsequently incubated with FITC-conjugated secondary antibody (Abcam). Images were observed under a Nikon 80i microscope.

Enzyme-linked immunosorbent assay (ELISA)

The content of interleukin (IL)-1β, tumor necrosis factor α (TNF-α), and IL-6 in the cochlea was measured with corresponding ELISA kits (eBioscience) according to the manufacturer’s instructions. All the samples and standards were measured with a SpectraMax M5 microplate reader (Molecular Devices) at the wavelength of 450 nm.

Statistical analysis

Quantitative parameters between different groups were assessed with one-way ANOVA followed Tukey’s multiple comparisons test or Mann Whitney test. The significance level was set as p-value < 0.05. All statistical analyses were performed with GraphPad Prism.

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