Animals
All animal procedures related to this work proceed after approval by the institutional ethical committees. B6.BKS(D)-Leprdb/J (db/db) mice were applied as diabetes models. The low-fat diet fed mice were used as a lean model, as previously reported [23]. All mice were individually housed and had ad libitum access to food and water. Experimenters were not blinded to group assignment and outcome assessment.
Wound Healing Model And Treatment
The mice were anesthetized with 2.5% of isoflurane. The hair on the back of the mice was removed by an electric shaver and sterilized using 70% alcohol. The 8 mm skin wounds were made using a biopsy punch. 100 µg per 100 µL ATMs-Exos were treated from day 1 to day 5. The diameter of each wound (day 0, 5, 10, and 15) was measured using Photoshop to calculate the percentage of wound closure.
To induce local inhibition of expression of miR-222-3p, 1 nmol miR-222-3p inhibitor or negative control (ThermoFisher Scientific) in a total volume of 100µl for each wound was injected intradermally into the wound edges of mice according to manufacturer's instruction, as previously reported [24].
Histological Analysis And Immunofluorescence
Tissue samples were fixed in 4% paraformaldehyde and 3um sections were cut from paraffin-embedded blocks. Sections were stained with standard Masson’s trichrome. Immunohistochemical staining for the presence of TNF-α was conducted in consecutive sections.
Immunofluorescent staining was performed with paraffin-embedded tissues and 4% PFA fixed macrophages. Following primary antibody incubation, slides were washed with 1X PBS and incubated with secondary antibody for one hour at room temperature. Sections were counterstained with DAPI mounting medium and analyzed. Fluorescence was detected under confocal laser scanning microscopy.
Adipose Tissue Macrophage (Atms) Isolation
ATMs were isolated from the visceral adipose tissues (VATs) of mice. The preparation and flow cytometry sorting of ATMs were performed as described previously [13]. Briefly, mechanically minced VATs were digested with collagenase II (Sigma-Aldrich) for 15 min at 37°C. The collected digests were filtered through 100 µm cell strainers and centrifuged at 1,000 x g for 10 min. Pellets were incubated in RBC lysis buffer. For flow cytometry cell sorting, single-cell suspensions were incubated with fluorescence-tagged antibodies against CD11b or F4/80. CD11b+/F4/80 + macrophages were purified using FACS.
Isolation And Characterization Of Exosomes
ATMs were then cultured in a culture medium containing 10% exosome-free FBS to produce exosomes. Briefly, the supernatants were collected after 48-h culture. The exosome quick extraction solution was added to the filtered solution at a 1:5 ratio and stored at 4°C for at least 12 h.
The morphology of exosomes was observed by transmission electron microscopy (TEM, JEM-1400plus). The size was determined by nanoparticle tracking analysis (NTA). Western blot analysis was used for detecting exosomal markers HSP70, TSG101, and CD63.
Microrna Sequencing
RNA was isolated using TRIzol reagent, and the concentration was quantified using the NanoDrop spectrophotometer, while integrity was evaluated. A total amount of 3 µg total RNA per sample was used as input material for the small RNA library. Following transforming the single-stranded DNA adaptor into a double-stranded DNA molecule, PCR amplification was performed. At last, library quality was assessed on the Agilent Bioanalyzer 2100 system using DNA High Sensitivity Chips. DESeq2 was used to test for differential expression of small RNAs between the samples.
Quantitative Real-time Rt-pcr (Qrt-pcr) Assay
Trizol reagent (Invitrogen, USA) was employed for total RNA extraction from treated cells. Then, the cDNA reverse transcription was carried out. qRT-PCR was carried out using SYBR Green Master (ROX) (Roche, USA). The mRNA levels were calculated relative to the control Gapdh (for mRNA) or U6 (for miRNA) using the 2−ΔΔCq method, as previously reported [25].
Immunoblots
Cells were lysed using RIPA solution, and a BCA assay was used to determine the protein content. Separated by electrophoresis, the protein was transferred to PVDF membranes. After blocking, the membranes were incubated overnight with primary antibodies separately, then incubated with HRP-conjugated secondary antibodies. The stained protein bands were visualized using a Bio-Rad ChemiDoc XRS imaging system and analyzed using Quantity One software.
Dual Luciferase Reporter Assays
The luciferase reporter assay was performed to validate the 3′-UTR binding site of Bim with miR-222–3p, according to previous research [26]. Briefly, the HEK293T cells were seeded into 96-well plates and co-transfected with target plasmids and miR-222-3p mimic/miR control. After 24 h transfection, the luciferase activity was determined and the reporter assay was performed according to the manufacturer's protocol (Promega). Renilla luciferase activity was normalized to firefly luciferase and expressed as a percentage of the control.
Flow Cytometry
Flow analysis was performed to evaluate the expression of cell surface markers for M1 or M2 phenotypes as described [27]. Briefly, after treatment, RAW 264.7 cells were surface-stained with antibodies specific for CD86 (M1 marker) and CD206 (M2 marker) for 30 min at 4°C. After additional washing steps, the cells were analyzed using a BD FACS AriaII flow cytometer and analysis was performed using FlowJo software.
Statistical analysis
Data were expressed as mean ± standard deviation. Statistical significance of differences among groups was tested by one-way ANOVA or repeated measures ANOVA. Comparisons between two groups were done using Student’s t-test or paired t-test. A value of P < 0.05 was considered statistically significant.