Bone marrow mesenchymal stem cell-derived exosomes shuttling miR-150-5p alleviates mechanical allodynia in rats by targeting NOTCH2 in microglia

Experiment animals

Five male Sprague–Dawley (SD) rats (four weeks old, 80–100 g) for BMSC extraction and 102 healthy male SD rats (eight weeks old) for spinal nerve ligation (SNL) were acquired from Huafukang Biotechnology Co., Ltd. (Beijing, China). Rats were reared in specific pathogen-free rooms at constant room temperature (21–25 °C) and humidity (50–65%) with 12-h light–dark cycles and free access to food and water. All animal experiments abided by the rules and regulations of laboratory animals. The experimentation in the study was ratified by the ethics committee of the Second Xiangya Hospital.

Isolation of primary rat BMSCs

Five four-week-old male SD rats were anesthetized with 2% pentobarbital sodium (50 mg/kg) by intraperitoneal (i.p.) injection. The bilateral femurs and tibiae of the rats were collected and the bone marrow cavity was exposed and washed repeatedly with α-MEM (Gibco, Grand Island, NY, USA) until the bone turned pale. Muscle tissue debris in the fluid was removed with a bacteria-proof filter, and the cell filtrate was centrifuged at 2000 rpm for 10 min. The supernatant was discarded and the cell pellet was resuspended in α-MEM supplemented with 10% fetal bovine serum (FBS). The cells (1 × 106) were then cultured at 37 °C with 5% CO2 and saturated humidity. After cell culture for 3 days, the cells were observed and the medium was replaced and refreshed every 2 ~ 3 days thereafter. Upon reaching 80 ~ 90% confluency, cell passage was carried out at a ratio of 1:3.

Identification of BMSCs

BMSC culture medium was refreshed every three days and the BMSCs were obtained by several times of digestion. The BMSCs at a confluency of 80% were passaged and the third generation (P3) of the BMSCs was obtained to observe their morphology and growth status. The proliferative ability of BMSCs was tested using the MTS method, and flow cytometry detected the expression of stem cell markers (positive markers: CD29, CD44, CD73, CD90, and CD105; negative markers: CD34, and CD45).

BMSCs underwent Alizarin red staining (Solarbio, Beijing, China), Oil red O staining (ab150678, Abcam, Cambridge, MA, USA), and Alcian blue staining (BP-DL241, Nanjing SenBeiJia Biological Technology Co., Ltd., Jiangsu, China) to evaluate their capabilities to differentiate into osteoblasts, adipocytes, and chondrocytes. After identification, only the P3 BMSCs were used in subsequent experiments.

Alizarin red staining

Alizarin red dye was used to detect the osteogenic differentiation potential of BMSCs strictly according to the manufacturer’s instructions. Briefly, after osteogenic differentiation induction for 14 days, osteogenic induction medium was removed and BMSCs were washed twice or thrice in PBS, fixed for 15 min at room temperature, washed with ddH2O, and stained with alizarin red at room temperature for 30 min, after which the BMSCs were rinsed in ddH2O and observed under an IX50 microscope (Olympus, Japan).

Alcian blue staining

After 14 days of chondrogenic differentiation induction, BMSCs were washed twice in phosphate buffer saline (PBS), fixed in 4% paraformaldehyde (PFA) (Sigma-Aldrich, St. Louis, MO, USA) for 30 min, and washed thrice with PBS. Afterwards, the BMSCs were dyed with Alcian blue solution for 30 min, followed by washing thrice with PBS and microscopic observation (IX50, Olympus, Japan).

Oil red O staining

Fourteen days after adipogenic differentiation induction, BMSCs were fixed in 4% PFA for 10 min, washed thrice with distilled water, and stained with Oil red O solution for 30 min. Afterwards, 60% isopropanol was used to remove excess Oil red O solution in culture plates and the cells were gently washed with distilled water until the cleaning solution became clear, and then images were captured with an IX50 microscope (Olympus, Japan).

Isolation and identification of BMSC-exosomes

BMSC-exosomes were extracted according to the method reported by Yan et al. (2018). Briefly, BMSCs (1 × 105) were plated in six-well plates containing MSC growth medium. After 24 h of cell cultures, the BMSCs were washed thrice with PBS and future cultured for 48 h in exosome-free FBS-contained α-MEM medium (iCell-0650, iCell Bioscience Inc., Shanghai, China). Then the culture (2 mL) was collected and centrifuged at 500 g and 4 °C for 15 min to remove cells, at 2000 g for 15 min to remove cell debris or apoptotic bodies, and at 10,000 g for 20 min to remove large extracellular vesicles. After centrifugation, the supernatant was obtained and filtered with a 0.22 μm filter membrane. The obtained supernatant was centrifuged at 110,000 g for 70 min to avoid protein contamination, and resuspended in 1 × PBS and stored at − 80 °C.

Isolated exosomes were identified using particle size analysis with a NanoSight NS300 instrument (Malvern, UK), transmission electron microscopy, and western blots for exosome marker proteins. In brief, 10–20 μL exosomes were diluted to 1 mL with PBS and analyzed using a NanoSight NS300 instrument at 25 °C and a constant flow rate for particle size analysis; 20 μL exosomes were added onto a copper grid, placed at room temperature for 2 min, negatively stained with 3% phosphotungstic acid solution (12501-23-4, Sigma-Aldrich), washed thrice in PBS, and observed with a Hitachi H7650 transmission electron microscope (Tokyo, Japan); the expression of exosome markers (CD81, CD63, and CD9) and an endoplasmic reticulum marker Calnexin was detected by western blotting (see in western blot part for article numbers of antibodies and detail methods).

Intrathecal catheterization and administration

Intrathecal catheterization was done reference to the methods described by Shih et al. (2012). After anesthesia with i.p. injection of 2% pentobarbital sodium (50 mg/kg), rats were in prone positions, their lumbar region was raised, and their limbs were fixed. Following disinfection, a 1-cm longitudinal incision was made at the L4-5 vertebrae on the back of the rats. The muscles and fascia were bluntly separated, and a 22-gauge puncture needle, with its bevel cephalad and upward, was inserted into the rat spinal dura mater at an angle of approximately 20° to the rat spinal cord. Tail-flicking indicates that the puncture needle is placed in the subarachnoid space. A PE-10 catheter was placed into the needle and gently advanced 3 cm rostrally at the level of the spinal cord lumbar enlargement segments. At this time, there was cerebrospinal fluid (CSF) flowing in the catheter and CSF leakage was observed at the puncture site. The catheter was secured to the fascia and exited from the back of the neck. About 3 ~ 4 cm of the catheter was left outside, fixed, and washed with saline using a microsyringe. The external end of the catheter was heated and blocked to avoid CSF leakage. Finally, the muscle and skin layers were sutured. All the procedures were operated under sterile conditions and penicillin-G sodium (40,000 U/kg) was given post operation by i.p. injection to avoid infection. Twenty-four hours after recovery from anesthesia, 20 μl of 2% lidocaine solution was injected into rats through the catheter. The catheterization was deemed successful if the rats had temporary paralysis of both lower limbs within 30 s and recovered in few minutes.

SNL of the fifth lumbar spinal nerve (L5) in rats

A SNL rat model was established reference to the methods described in a previous study (Jaggi et al. 2011). Rats were anesthetized by 2% pentobarbital sodium (50 mg/kg, i.p. injection) and placed in prone positions. A total of 10 cm2 fur in the back was shaven off along the intercrestal line. After disinfection, a 1.5-cm longitudinal incision was made 0.5 cm to the left side of the intercrestal line. The skin, fascia, and muscles were bluntly dissected to expose the angle between L5 transverse process and sacrum. The L5 transverse process was wiped with a sterile cotton ball and cut off by elbow hemostatic forceps to expose L4-5 spinal nerves. The L5 spinal nerve was separated and ligated, during which the nerve should be prevented from pulling and was ligated twice in case the thread was detached. Sham-operated rats only underwent L5 spinal nerve exposure and other procedures were performed as in SNL rats. The whole operation was completed under sterile conditions and penicillin-G sodium (40,000 U/kg) was administrated (i.p.) post operation to prevent infection.

Intrathecal administration of 1 mg/ml exosomes (10 μl), 1 mg/kg NOTCH2 overexpression plasmids (pcDNA3.1-NOTCH2), negative control of pcDNA3.1-NOTCH2 (pcDNA3.1), 1 mg/kg NOTCH2 interference plasmids (sh-NOTCH2), or negative control of sh-NOTCH2 (sh-NC) was performed on SD rats from days 3–7 after operation (See in Scheme 1 for in vivo experiment design). There were 24 rats in the sham group and 78 rats in the SNL group. The above-mentioned plasmids were provided by Shanghai Genechem Co., Ltd. (Shanghai, China).

Scheme 1scheme 1

In vivo experimental design

The sham-operated rats (n = 24) were divided into the sham (n = 12), PBS (sham-operated rats intrathecally injected with ATP-stimulated 1 × 103 cells/10 μL microglial cells that were not pretreated with exosomes, n = 6), and BMSCs-exo (sham-operated rats intrathecally injected with 1 × 103 cells/10 μL microglial cells pretreated with BMSCs-exosomes and stimulated with ATP, n = 6) groups. Behavioral tests were carried out one day before and 1, 3, 8, and 13 days after intrathecal administration.

Seventy-eight rats in the SNL group were divided into the SNL (n = 18), exo (rats subjected to SNL and injected with BMSCs-exosome, n = 18; exosome localization was carried out in six of them), sh-NC (rats subjected to SNL and injected with sh-NC, n = 6), sh-NOTCH2 (rats subjected to SNL and injected with sh-NOTCH2, n = 6), pcDNA3.1-NC (rats subjected to SNL and injected with pcDNA3.1, n = 6), pcDNA3.1-NOTCH2 (rats subjected to SNL and injected with pcDNA-NOTCH2, n = 6), Exo + pcDNA3.1-NOTCH2 (rats subjected to SNL and injected with BMSCs-exosome and pcDNA3.1-NOTCH2, n = 6), LV-anti-miR-150-5p (SNL rats injected with exosomes derived from BMSCs transfected with miR-150-5p inhibition lentiviral vectors, n = 6), and vector-BMSCs (SNL rats injected with exosomes derived from BMSCs transfected with the negative control of the miR-150-5p inhibition lentivirus, n = 6) groups. The doses of the recombinant lentiviruses were 8 × 103 transduction units (Zhang, Gao, Li, Wen, Yan, Peng and Xiao 2021).

miR-150-5p inhibition in exosomes

BMSC-exosomes expressing miR-150-5p at a low level were obtained by transfection with miR-150-5p inhibition lentivirus (LV-anti-miR-150-5p, 50 nM) or negative control inhibition lentivirus (vector) into BMSCs, and exosomes were extracted 48 h after transfection. Lentiviruses were acquired from Shanghai Genechem Co., Ltd. (Shanghai, China).

Behavioral testing

Paw withdrawal threshold (PWT) was assessed according to the method developed by Chaplan and colleagues (Chaplan et al. 1994). Specifically, in a quiet environment, rats were placed in a Perspex cage with a mental-mesh floor for 30–60 min of acclimatization, and PWT was evaluated with a von Frey 2390 mechanical pain detector after the rats calmed down. The von Frey filament was used to press the plantar surface of hind paws vertically. The stimuli were lasted ≤ 4 s. When the rats presented paw licking, lifting, or latency, the pressure value on the screen was recorded as PWT. Each rat underwent von Frey tests thrice with intervals of 5 min, and the obtained data were averaged.

Paw withdrawal latency (PWL) was measured reference to the methods reported in a previous study (Chen et al. 2015). In a quiet environment, rats were placed in a Perspex cage with a 3 mm-thick glass floor, with acclimatization to the environment for 30–60 min. After the rats calmed down, the plantar surface of the hind paws was stimulated with infrared radiant heat. When the rats had paw licking, lifting, or latency, the timer was stopped and the time (PWL) was automatically recorded. The cutoff time was 20 s to prevent tissue damage. Each rat was tested thrice with intervals of 5 min and the results of three independent tests were averaged.

ELISA

Expression levels of TNF-α, IL-1β, and IL-6 were measured using TNF-α (PRTA00), IL-1β (PRLB00), and IL-6 (PR6000B) ELISA kits (R&D Systems, Minneapolis, Minnesota, USA) strictly following the instructions. First, 100 μl samples or standards were added into plate wells and incubated at 37 °C for 90 min, followed by incubation with specific antibodies and avidin–biotin-peroxidase complex solution for 60 and 30 min, respectively. After 20–25 min of color development with TMB, absorbance values at 450 nm were evaluated using a microplate reader.

TUNEL staining

Rat SDH tissues were fixed overnight in 4% PFA, embedded in paraffin, and cut into paraffin sections. After deparaffinization and hydration, the tissue sections were immersed in 0.25% Triton X-100 at room temperature for 20 min, and fresh TUNEL reaction mixture was added to the tissue sections and incubated at 37 °C for 1 h in the dark. The tissue sections were sealed with anti-fade reagent containing DAPI. After sealing, the sections were visualized under a fluorescence microscope and the apoptotic rate was analyzed using Image J software [apoptotic rate (%) = number of TUNEL positive cells/number of total cells × 100].

Immunofluorescence

Exosomes were labeled green by an Exo-Glow labeling kit (System Bioscience Inc., Palo Alto, CA, USA) before intrathecal administration. On days 5 post operation (days 3 after exosome injection), six rats were randomly selected and euthanatized to collect their L5 SDH tissues for immunofluorescence.

Rat L5 SDH tissues were collected and cut into paraffin sections after dehydration and embedding. Following deparaffinization, the sections were immersed in TBS and boiled in a microwave oven for 10 min of antigen repair. After sealing at 37 °C for 30 min in bovine serum albumin, the sections were incubated with diluted primary antibodies of NOTCH2 (ab118824, 1:100, Abcam) and OX42 (GTX76060, 1:50, GeneTex, USA) at 4 °C overnight. The sections were then incubated with a secondary antibody for 1 h, washed with PBS, sealed with DAPI, and observed under a fluorescence microscope.

Immunohistochemistry (IHC)

Rat L5 SDH tissues were fixed in 4% PFA for 48 h, cut into 5-μm-thick sections, and roasted for 20 min, followed by deparaffinization in conventional xylene. After washing once in distilled water and thrice in PBS, the tissue sections were evenly covered with 3% H2O2 for 10 min at room temperature and washed thrice with PBS. After that, the sections were added with goat serum blocking solution and placed at room temperature for 20 min. The sections were incubated with NOTCH2 antibody (ab118824, 1:100, Abcam) at 4 °C overnight. After being washed thrice with PBS, the sections were incubated with a secondary antibody (ab6728, 1:1000, Abcam) at room temperature for 1 h, followed by another round of washing. The sections underwent color development with DAB for 1–3 min and the nuclei were stained in hematoxylin solution for 3 min before dehydration, transparentization, and sealing. The sections were observed under a microscope (× 200) and three random fields were selected and the images were processed with Image J software for IHC scoring. The semi-quantitative results of the micrographs were evaluated by two experienced pathologists using the double-blind method, and the percentage of positive cells and staining intensity were scored. The score scale for percentage of positive cells was: 0, < 5%; 1, 5–25%; 2, 26–50%; 3, 51–75%; 4, 76–100%, and that for staining intensity was: 0, no staining; 1, light yellow; 2, brownish yellow; 3, tan. The products of the two scores were defined as positive grades: 0, negative; 1–4, weakly-positive; 5–8, positive; 9–12, strongly-positive.

Isolation and culture of microglial cells

MicrogliaL cells were obtained from SD rats (1–3 days old) reference to the method described in a previous study (Fung et al. 2015). Briefly, rat cortex was separated, the meninges was removed, and the cortex was sliced and digested with trypsin for 15 min; cell suspension was cultured and shaken on an orbital shaker for 4–6 h at 37 °C and 200 r/min, after which the culture medium was collected. Since microglial cells did not adhere firmly to the wall, most of the cells in the collected culture medium were microglial cells. The cells were centrifuged at 1000 r/min for 3 min, the supernatant was removed, and the cells were resuspended in complete medium. The collected cells were inoculated into a new culture flask and named as primary microglial cells (P0). Microglial cells were incubated with BMSC-exosomes or PBS at 37 °C for 24 h before the following experiments.

Cell transfection

MicrogliaL cells were transduced with miR-150-5p inhibitor/mimic or their respective negative controls (inhibitor/mimic NC) (transfection dose of 50 nM, all from GenePharma, Shanghai, China). Following experiments were conducted 48 h after transfection.

RT-qPCR

Total RNA was extracted from tissues and cells with the use of TRIzol reagent (Takara, Dalian, China), followed by measurement of RNA concentration and purity using a NanoDrop spectrophotometer. RNA was reverse-transcribed into cDNA with the kit (TaKaRa, Tokyo, Japan). RT-qPCR was performed on a Biosystems 7300 real time PCR system (ABI, Foster City, CA, USA) according to the instructions of a SYBR GreenMix kit (TaKaRa). Each PCR experiment was performed in triplicate, and a PCR system was added with 10 ng cDNA. Gene expression was analyzed by the 2−ΔΔCt method (Soejima and Koda 2008) [ΔΔCt = (Ct target gene − Ct housekeeping gene) experimental group − (Ct target gene − Ct housekeeping gene) control group]. GAPDH and U6 were used as housekeeping genes for NOTCH2 and miR-150-5p, respectively. Each experiment was repeated thrice. Primer design and PCR experiments were carried out by RiboBio (Guangzhou, China) (see in Table 1 for primer sequences).

Western blotting

After the cells or tissues were treated with RIPA lysis buffer on ice for 15 min, the lysate was centrifuged for 5 min at 13,000 g, and the concentration of isolated total protein was measured using a BCA kit. The lysate was added with loading buffer and boiled in a water bath for 10 min of denaturation. The loading volume of protein samples was calculated based on the loading quantity (30 μg protein per well). The protein was loaded and separated by electrophoresis (80 V for 3 min and 120 V for 90 min), and transferred onto a 0.22-μm PVDF membrane at 250 mA for 100 min. The membrane was washed thrice (two minutes per time), immersed in 5% skim milk at room temperature for 1 h, and incubated with antibodies of NOTCH2 (ab118824, 1:100, Abcam), CD81 (ab79559, 1:1000, Abcam), CD63 (ab134045, 1:1000, Abcam), CD9 (ab223052, 1:1000, Abcam), Calnexin (ab22595, 1:1000, Abcam), OX42 (GTX76060, 1:50, GeneTex), and GAPDH (ab181602, 1:1000, Abcam) at 4 °C overnight. The membrane was then washed thrice with TBST for 10 min each. Secondary antibodies (ab6721, ab6728, Abcam) were incubated with the membrane at room temperature for 1 h, followed by membrane washing. Enhanced chemiluminescence reagent was used for color development and Image J software for calculating relative protein expression. The results represented the average of three independent experiments.

Dual-luciferase reporter assay

A binding site between miR-150-5p and NOTCH2 was searched by bioinformatics prediction, and wild sequence of the binding site between miR-150-5p and NOTCH2 or mutated sequence were synthesized and inserted into pGL3-Basic vectors (NOTCH2-wt and NOTCH2-mut). The vectors identified by sequencing were transfected with mimic NC or miR-150-5p mimic into microglial cells. The cells were transfected for 48 h and then lysed. A luciferase assay kit (K801-200, Biovision) and a luciferase reporter gene analysis system (Promega, Madison, WI, USA) were used to calculate luciferase activity, using Renilla luciferase as the internal control. The ratio of Firefly luciferase RLU to Renilla luciferase RLU represented the activation degree of the target reporter gene. Each experiment was repeated thrice.

RNA pull-down

Biotinylated miR-150-5p probe (miR-150-5p probe) and negative control probe (NC probe) were transfected into microglial cells which were collected after transfection for 48 h, lysed, and incubated with streptavidin-conjugated magnetic beads according to the instructions of a Pierce™ Magnetic RNA–Protein Pull-Down kit (Millipore, Billerica, MA, USA). Briefly, the beads were washed and incubated with the cell lysate at room temperature in a rotator. RT-qPCR was used to detect NOTCH mRNA expression in the eluted complexes.

Statistical analysis

Data were processed by GraphPad Prism 7 and presented as mean ± standard deviation. T test was used for comparisons between two groups, and one-way analysis of variance was used for multigroup comparisons with Tukey’s multiple comparisons test for post hoc multiple comparison. P < 0.05 was considered statistically significant.

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