Cells, media, and reagents
RAW264.7, a macrophage cell line, was cultured in Dulbecco’s minimal important medium (DMEM; HyClone, Logan, USA) added with 10% fetal bovine serum (FBS; Gibco, New York, USA), 100 U/ml penicillin (Gibco, New York, USA), and 100 μg/ml streptomycin (Gibco, New York, USA). ATDC5, a chondrogenic cell line, was cultured in DMEM: nutrient combination F12 (DMEM: F12; HyClone, Logan, USA) supplemented with 5% FBS. The cells have been positioned at 37 °C underneath humidified circumstances with 5% CO2. The bacterial LPS of Escherichia coli and the recombinant mouse IFN-γ and IL-4 have been bought from PeproTech (Rocky Hill, USA). Exosome-depleted FBS was purchased from System Biosciences (California, USA).
Induction of macrophage polarization
RAW264.7 cells have been seeded on tradition plates, incubated at 37 °C in a single day to stick to the underside of the plates. Cells have been stimulated with an entire medium containing 100 ng/ml LPS plus 20 ng/ml IFN-γ 24 h to induce M1 polarization and 20 ng/ml IL-4 24 h to stimulate M2 polarization [6].
Exosome isolation and identification
RAW264.7 cells have been induced to an M1/M2 kind for twenty-four h and cultured in an exosome-depleted FBS-containing full medium. The supernatant was collected, and exosomes have been obtained by ultracentrifugation [35]. Briefly, the supernatant was subjected to a sequence of differential centrifugation steps (300×g for 10 min, 2000×g for 10 min, and 10,000×g for 30 min) to take away intact cells and cell particles. Subsequently, the supernatant was centrifuged at 100,000×g at 4 °C for 70 min to isolate the proteins-containing exosomes. The exosomes have been then purified by washing them with PBS and subjected to a further centrifugation step at 100,000×g for 70 min.
The protein content material of the exosomes was measured with a bicinchoninic acid (BCA) protein assay equipment (Beyotime Biotechnology, Shanghai, China) referring to the producer’s directions. CD9, CD63, CD81, TSG101 and Calnexin (Proteintech, Chicago, USA), which have been exosomal marker proteins, have been assessed by western blotting as beforehand described [51]. M2 macrophage-derived exosomes have been scanned utilizing a TEM (JEM1400, Tokyo, Japan). The scale and focus of exosomes have been analyzed by NTA (Nanosight NS300, Malvern, UK).
Exosome labeling and mobile uptake
M2 macrophage-derived exosomes have been labeled with fluorescent dyes referring to a earlier research [52]. They have been labeled utilizing a PKH26 purple fluorescent cell linker equipment (Sigma-Aldrich, St. Louis, USA) in accordance with the producer’s directions. The labeled exosomes have been washed and centrifuged at 100,000×g for 60 min. Then, the resuspended exosomes have been deployed to RAW264.7 and ATDC5 cells for six h. After the supernatant was eliminated, FITC-conjugated phalloidin (Sigma-Aldrich, USA, St. Louis, USA) and 4,6-diamidino-2-phenylindole (DAPI; Beyotime Biotechnology, Shanghai, China) have been used to stain the cytoskeleton and nuclei, respectively. The cells have been washed with PBS twice and photographed with a confocal microscope (Leica TCS-SP5, Germany).
Exosomal miRNA sequencing and evaluation
Excessive-throughput miRNA sequencing and subsequent bioinformatic evaluation have been carried out by CloudSeq Biotech Co., Ltd. (Shanghai, China). The full RNA of exosomes was ready and quantified utilizing a NanoDrop ND-100 (Thermo Fisher Scientific, USA). Then, complete RNA of every pattern was used to organize the miRNA sequencing library. The libraries have been denatured as single-stranded DNA molecules, captured on Illumina circulation cells, amplified in situ as clusters, and sequenced for 50 cycles on an Illumina HiSeq4000 sequencer (Illumina, CA, USA). Uncooked knowledge have been generated by sequencing, picture evaluation, base calling, and high quality filtering by utilizing an Illumina sequencer.
RNA extraction, reverse transcription-PCR and real-time qPCR
The full RNA of cells was extracted utilizing TRIzol reagent (Invitrogen, CA, USA), and NanoDrop ND-100 was used to measure the standard and concentrations of RNA. cDNA was synthesized with TaqMan reverse transcription reagents (Utilized Biosystems, CA, USA). Actual-time qPCR was carried out utilizing an ABI 7500 system (Utilized Biosystems, CA, USA) in accordance with beforehand described strategies [53]. The reverse transcription primers of miRNAs have been ready utilizing the stem-loop methodology. The transcript ranges of mRNA and miRNA have been normalized to β-actin and U6, respectively. Gene expression ranges have been analyzed utilizing 2−ΔΔCt methodology. The primers used for these analyses are listed in Further file 1: Desk S1.
Lentivirus preparation and development of stably transfected cell strains
pHBLV-zsgreen-puro (miR-ctrl) and pHBLV-miR-26b-5p-zsgreen-puro (miR-26b-5p overexpression) plasmids have been ready by Hanbio Biotechnology Co., Ltd. (Shanghai, China). The ready plasmids have been transfected into HEK293T cells with LipoFiter™ 3.0 (liposomal transfection reagent). The virus supernatant was collected and filtered utilizing a 0.45 μm cell strainer 48 h after transfection. The supernatant was additional centrifuged to 72,000 g at 4 °C for 120 min and resuspended with recent media for the next experiments. The titer of the concentrated lentivirus was measured reaching 2 × 108 TU/ml.
RAW264.7 and ATDC5 cells have been seeded in six-well plates (5 × 105/nicely) and incubated in a single day. Then, 6 μg/ml polybrene (Sigma-Aldrich, St. Louis, USA) was utilized earlier than transfection. The lentivirus-containing medium was modified with full media 24 h after transfection, and fluorescence sign was noticed utilizing a fluorescent microscope 48 h after transfection. Inexperienced fluorescence indicated profitable transfection in cells. Then, 4 μg/ml puromycin was added to the tradition medium to kill untransfected cells. After about 7 days, the efficiently transfected cells survived, they usually have been used for the next evaluation.
Twin-luciferase development and reporter assay
pmirGLO dual-luciferase miRNA goal expression vectors have been ready by GenePharma (Suzhou, China) to evaluate the miRNA exercise. The 3ʹ UTR sequences of TLR3 and COL10A1 (wild kind or mutant) have been cloned within the vectors. The firefly luciferase reporter gene was managed by an SV40 promoter, and Renilla luciferase was used as a management reporter for normalization. HEK293T cells with overexpressed miR-ctrl or miR-26b-5p have been co‐transfected with numerous pmirGLO vectors for twenty-four h. Then, the luciferase exercise within the lysates was assessed with a dual-luciferase reporter gene assay equipment (Beyotime Biotechnology, Shanghai, China) in accordance with the producer’s instruction.
Cell co-culture experiments
RAW264.7 cells have been polarized to an M1 kind by stimulating with LPS and IFN-γ for twenty-four h. M1 macrophage supernatants have been centrifugated at 1000×g for five min and diluted with a serum-free medium (1:1) to organize M1-CM for the next experiments. The obtained M1-CM was deployed to RAW264.7 to guage the consequences on macrophage polarization and added with insulin, transferrin, and selenous acid (ITS; Sigma-Aldrich, St. Louis, USA) to guage the chondrogenesis of ATDC5 cells [39]. M1-CM was analyzed with a Luminex liquid chip for multi-cytokine detection to evaluate the consequences of miR-26b-5p on macrophage repolarization and chondrocyte hypertrophy.
Immunofluorescence staining
RAW264.7 cells with or with out miR-26b-5p overexpression have been stimulated with M1-CM for twenty-four h. They have been mounted in 4% paraformaldehyde containing 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, USA), and 1% bovine serum albumin (BSA) was utilized to dam nonspecific binding. Afterward, the cells have been incubated with antibodies, together with CD16/32 (BD, CA, USA), CD206 (BD, CA, USA), and TLR3 (Abcam, Cambridge, MA, USA), in a single day. They have been additional incubated with fluorescent secondary antibodies (Abcam, Alexa Fluor 594, Cambridge, MA, USA) and DAPI have been transferred to cells and noticed utilizing a confocal fluorescence microscope (Leica TCS-SP5, Germany).
Move cytometry evaluation of macrophage subsets
CD16/32 and CD 206 have been chosen to respectively mark M1 and M2 phenotypes and to tell apart M1 and M2 macrophage populations. After being washed thrice with PBS, the polarized macrophages have been digested and resuspended with PBS. Then, 5 μl of Alexa anti-CD206 (Fluor 647-conjugated, BD, CA, USA) and 5 μl of anti-CD16/32 (PE-conjugated, BD, CA, USA) have been utilized to guage the macrophage subsets. Alexa Fluor 647 isotype management (BD, CA, USA) and PE isotype management (BD, CA, USA) have been transferred to exclude the cells with nonspecific staining. M1 macrophages have been thought to be CD16/32-positive and CD206-negative cells, whereas M2 macrophages have been denoted as CD206-positive and CD16/32-negative cells.
Western blotting evaluation
The cells have been washed with PBS and lysed with a radioimmunoprecipitation assay buffer (RIPA; Millipore, MA, USA) containing protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, USA). The protein concentrations of cells have been evaluated utilizing a BCA protein assay equipment. Then, 20 μg of proteins was separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes have been blocked with 5% nonfat milk for two h and incubated with major antibodies in a single day. The membranes have been additional incubated with fluorescent secondary antibodies and washed thrice with Tris-buffered saline with Tween (TBST). Fluorescent indicators have been photographed with LI-COR imaging methods (Lincoln, USA).
Alizarin purple staining
The chondrogenesis of ATDC5 cells was induced for 14 days. Then, the cells have been mounted with 4% paraformaldehyde, incubated with 1% Alizarin purple resolution (Sigma-Aldrich, St. Louis, USA) for 30 min, [39] and washed with PBS twice to take away extra Alizarin purple dye. Afterward, they have been photographed underneath an optical microscope.
Institution of ACLT-induced OA mouse mannequin
Animal experiments have been permitted by the ethics committee of the First Affiliated Hospital of Soochow College. Twenty-four 3-month-old male C57BL/6J mice obtained from Laboratory Animal Middle of Soochow College have been uncovered underneath particular pathogen-free (SPF) circumstances and fed with business meals and water. OA was induced by making use of ACLT, which might trigger irregular mechanical loading, to the best knee in accordance with beforehand described strategies [54, 55]. An anterior drawer check was performed to substantiate full transection, and a sham operation was made by opening the joint capsule and suturing the incision.
The mice have been randomly and averagely cut up into 4 teams and assigned to 4 cages. The mice in group 1 have been assigned within the management group and handled with sham operation. The mice in teams 2–4 have been handled with ACLT to the best knee joints. The mice in group 2, 3, and 4 have been additionally intra-articularly injected with PBS, 5 nmol agomir NC, and 5 nmol miR-26b-5p agomir, respectively. Intra-articular injection was administered as soon as every week for 4 weeks [45, 46].
Gait evaluation
Gait evaluation was carried out utilizing a gait evaluation system as described beforehand [47]. In short, the gait evaluation system comprises a fluorescent gentle tube alongside a glass plate runway. The mice have been positioned individually on the runway and allowed to stroll throughout the runway freely. The gait of every mouse was recorded utilizing a high-speed colour digital camera and analyzed with the VisuGait software program (Xinruan Co., Ltd., Shanghai, China). The swing part length (s) indicated the interval when the paw was not touching the bottom in an entire step cycle. The swing velocity (m/s) was calculated by dividing the stride size by the length of the swing part. The responsibility cycle (%) was outlined because the ratio between stance length and full step cycle length [56]. The ratios of the affected proper hind limb to the contralateral left hind limb (RH/LH) have been calculated to remove particular person variations [50].
Von Frey withdrawal threshold testing
Von Frey filaments have been utilized to evaluate the secondary mechanical allodynia by measuring the withdrawal threshold in accordance with beforehand described strategies [56]. The mice have been positioned in a clear plexiglass chamber with a metallic mesh ground for at the least 10 min to acclimatize them earlier than the check. The filaments have been utilized when the mouse stood nonetheless on all 4 paws and operated thrice with an inter-trial interval of 10 s. The response was thought of constructive when the mice flinched their paw greater than as soon as. Then, the paw withdrawal threshold (PWT) of the mouse was recorded.
Histological statement
The mice have been killed 4 weeks after the injection. The proper knee joints of the mice in numerous teams have been dissected and stuck with 4% paraformaldehyde for 48 h. The samples have been decalcified in 10% EDTA for 3 weeks and embedded in paraffin. The sagittal sections of the medial compartment of the knee joints have been minimize at 4 μm for the next microstructural observations. They have been additional stained with S&F and H&E. The OARSI rating was utilized in accordance with beforehand described strategies [57]. Synovitis was assessed utilizing the Krenn scoring system [58]. Immunohistochemical staining was utilized in accordance with a earlier report [59]. Particularly, major antibodies, together with CD16/32, CD206, COL10, and MMP-13 (Abcam, Cambridge, MA, USA, dilution 1:200) have been utilized and incubated at 4 °C in a single day. A secondary antibody was incubated at 25 °C for 1 h. The samples have been stained with diaminobenzene (Dako, North Sydney, NSW, Australia) and hematoxylin (Sigma-Aldrich, St. Louis, USA). The immunohistochemically stained samples have been then photographed underneath a microscope. The share of positively stained cells within the articular cartilage and synovium was calculated (Further file 1).
Statistical evaluation
Knowledge have been expressed as imply ± customary deviation. Variations between two teams have been in contrast by way of two-sided Scholar’s t-test. Multifactorial comparisons have been carried out by one-way evaluation of variance. Knowledge with p < 0.05 have been thought of to have statistically important variations. On this research, “*”and “**” denoted p < 0.05 and p < 0.01, respectively. The information evaluation was calculated with SPSS 22.0 evaluation software program (SPSS Inc, Chicago, IL, USA).