Isolation of MSCs-derived Exosomes
Mesenchymal stem cells (MSCs) were isolated from six adult C57BL/6J mice by the whole bone marrow adherence method as previously described(22). 2×106 MSCs were cultured in 100 mm dishes, MSCs were washed with PBS and kept in fetal bovine serum (FBS)-free L-DMEM medium (Gibco, CA, USA) for 48 h when cells reached nearly 80 ~ 90% confluence. Then the supernatant was collected and subject to sequential centrifugation to obtain the exosomes according to a previous study(23). The precipitated exosomes were stored at − 80 ℃.
Characterization of MSCs-derived Exosomes
For transmission electron microscopy (TEM), isolated exosomes were fixed with 1% glutaraldehyde, then a drop of fixed exosomes was spotted onto a formvar/carbon-coated grid and negatively stained with 3% aqueous phosphotungstic acid for 1 min. Subsequently, MSCs-derived exosomes were observed under TEM (Hitachi, Tokyo, Japan, SU-8010). For flow cytometry analysis of exosomes, MSCs-derived exosomes were mixed with 3 μm aldehyde/sulfate latex beads (Invitrogen, Batch Num: 979383) for 10 min with continuous rotation. 1M glycine in PBS containing 2% BSA was added into the mixture to stop the reaction. Beads coated with exosomes were incubated with antibodies CD63-FITC (Lot: GR320523-9, Abcam), CD81-PE (Cat: MA5-17941, Invitrogen) at 37 ℃ for 25 min. Then a FCM flow cytometry (BD FACSalibur) was used to detect the mesenchymal markers.
Animal model of cerebral ischemia-reperfusion (I/R)
A total of 30 adult C57BL/6J mice (8 weeks old and 250 g in weight) were provided from Anima Center of Third Military Medical University (Chongqing, China). All animal producers were approved by the Institutional Animal Care and Use Committee of Anima Center of Third Military Medical University (Chongqing,China).. Mice with cerebral ischemia was induced by right middle cerebral artery occlusion (MCAO) as described previously with minor modifications(24). Reperfusion was performed by withdrawing the suture 1 h after MCAO. For sham group, mice accepted the same operation except MCAO procedure. 2 h after reperfusion, 200 μL/mice MSCs-exosomes-miR-NC or MSCs-exosomes-miR-26a-5p mimics (RiboBio, Guangzhou, China) were immediately injected through the tail vein. The mice in the control group were given an equal volume of normal saline (n = 6 in each group). All mice were divided into four groups: Sham group, MCAO group, MCAO + MSCs-exosome-miR-NC, and MCAO + MSCs-exosome-miR-26a-5p mimics group.
Determination of infarct size
After intraperitoneal injection of 3% sodium pentobarbital, the brains of mice in different groups were removed, placed in 4% PFA overnight and then fully dehydrated in 30% sucrose for 2 days. The brain tissue was then cut into coronal sections with approximately 2 mm in thickness, and then quickly incubated in 2% TTC solution (Sigma) at 37°C for 15 min. Subsequently, the sections were fixed by 4% paraformaldehyde and photographed under a ×100 optical microscope (Olympus Corporation). The image analysis software Image J 1.43 (National Institutes of Health) was used to evaluate the relative infarct percentage according to the following equation: infarct size = 100% × (infarcted volume/total brain volume).
MSCs of OGD model
MSCs was cultured with in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) supplemented with 10% (v/v) fetal bovine serum (FBS, Biological Industries, USA), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, USA) at 37°C under 5% CO2. To mimics the ischemia condition in vitro, cells were firstly induced by oxygen-glucose deprivation (OGD) by using deoxygenated glucose-free DMEM medium in an incubator with 95% N2, 5% CO2 for 1, 2 and 4 h, then cells were transferred into normal condition for an additional 24 h for re-oxygenation. Cells treated without OGD were used as the control group.
MSCs were transfected with 50 nM miR-26a-5p mimics or miR-NC (RiboBio, Guangzhou, China) by using Lipofectamine® 2000 Transfection Reagent (Gibco Life Technologies) according to the manufacturer’s instructions. The sequences used in this study as follows: miR-26a-5p mimics: 5′-UUCAAGUAAUCCAGGAUAGGCU-3′; miR-NC: 5′-UUCUCCGAACGUGUCACGUTT-3′. When needed, the exosomes were isolated from MSCs transfected with miR-26a-5p mimics or miR-NC and used for the treatment of microglia cell line BV-2 cells.
The treatment of microglia
To simulate an in vivo environment of MCAO in microglia, BV-2 cells (Bioleaf, Shanghai, China) were induced by OGD/R treatment as similarly as the MSCs of OGD model above. Then 200 μg/mL MSCs-derived exosomes with overexpression of miR-26a-5p mimics or miR-NC were added into the culture medium to explore the effect of MSCs-Exo on microglia function.
Cell viability was evaluated by using a Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Gaithersburg, MD). In brief, MSCs were seeded into 96-well plates overnight. After the treatment, 10 ul of CCK-8 reagent was added to each well at 24, 48, 72 and 96 h and then incubated for another 4 h. The absorbance at 450 nm was detected with a microplate reader. The absorbance was detected at 450 nm with a microplate reader.
Luciferase reporter assay
The putative binding sites between miR-26a-5p and 3’-UTR of CDK6 were predicted by starBase (http://starbase.sysu.edu.cn/). The mutant-type (MUT) of 3’-UTR (CDK6- MUT) and wild type (WT) of 3’-UTR (CDK6-WT) were amplified and cloned into the pmirGLO dual luciferase reporter vector (Promega, Madison, WI, USA). Then the luciferase reporter plasmids were co-transfected with miR-26a-5p mimics or miR-NC into 293T cells by using Lipofectamine® 2000 Transfection Reagent. 48 h fater transfection, cells were lysed and the relative luciferase activity was detected by the dual-luciferase reporter gene assay (Promega).
RNA extraction and qRT-PCR analysis
Total RNA was extracted from cultured cells or mice brains by using Trizol reagent (Invitrogen). Single-strand cDNA was synthesized using a universal cDNA synthesis kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The expression of targets was tested with a fast real-time PCR system (7900 HT, ABI, Foster City, CA) by using a SYBR Green master mix (Qiagen). The relative expression change of targets was analyzed by the 2-ΔΔCt method with GAPDH and U6 as the internal references. The primers used as follows: miR-26a-5p: forward: 5′-GACGGTACCTTGTCCCTGAATGTAACTCG-3′ reverse: 5′-GTTCTCGAGAAAGCAGTCCCAGCCTAAA-3′; U6: forward: 5′-CTCGCTTCGGCAGCACA-3′, reverse: 5′-AACGCTTCACGAATTTGCGT-3′; GAPDH forward: 5′-CAAGGTCATCCATGACAACTTTG-3′, reverse: 5′-GTCCACCACCCTGTTGCTGTAG-3′.
Total protein of mice brains or cultured cells was isolated by using RIPA lysis buffer (Beyotime Institute of Biotechnology). Approximately equal amounts of protein were separated by 10% SDS-PAGE and transferred onto PVDF membranes (Millipore). After blocking with 5% skim milk, the membranes were incubated with primary antibodies including CD9, CD63, CD81, HSP70, CDK6, cleaved Caspase3 (c-Caspase3), cleaved PARP, and GAPDH antibodies (diluted into 1:1000; Cell Signaling Technology, USA) overnight at 4°C. On the next day, the membranes were incubated with HRP-labeled secondary antibody at room temperature for 1 h. After washing with TBS-T, the protein bands were visualized by ECL reagent and band intensity of targets was quantified by Image-Pro Plus 6.0 software (Media Cybernetic).
Cell apoptosis was analyzed by using a commercialized Annexin V-FITC/PI Apoptosis Detection Kit (YEASEN, Shanghai) according to the manufacturer’s protocols. Briefly, BV-2 cells were washed with PBS, and re-suspended in 100 ul of binding buffer, and 5 ul of Annexin V-FITC and 10 ul of propidium iodide (PI) were gently mixed and added into cell suspension, then incubated in the dark for 15 min. Finally, cell apoptosis was detected by flow cytometry (BD FACS Canto II, USA).
In situ detection of fragmented DNA (TUNEL assay)
Brains tissues were collected and the apoptosis in vivo was evaluated TUNEL staining kit (YEASEN, Shanghai) as the manufacturer’s instructions. TUNEL positive brain cells were counted under a fluorescence microscope. Specially, cell nucleus dyed green were considered to be apoptotic cells, and the rate of apoptosis (%) was calculated as the percentage of TUNEL positive cell nucleus in 5 random fields for each sample.
All data were presented as mean ± SD, and each experiment was repeated three times. Statistical analysis was performed by using GraphPad Prism 6.0 software. Groups comparison was performed with two tailed Student’s t test (two groups) or one-way analysis of variance (ANOVA; multiple groups). P < 0.05 was considered to be significant.