Mmu-miR-27a-5p Promotes the Polarization of Macrophages to the M2 Phenotype at the Site of Spinal Cord Injury in Mice


 BackgroundTo investigate the effect of mmu-miR-27a-5p on macrophage polarization in the injured spinal cord and the recovery of motor function after spinal cord injury (SCI) in mice.MethodsA total of 160 specific-pathogen-free male mice were randomly divided into sham, model, mmu-miR-27a-5p, mmu-miR-27a-5p-negative control (NC) groups, with 40 mice in each group. Hindlimb motor function was assessed using the Basso Mouse scale (BMS) before injury and at 1, 3, 7, and 14 days after surgery. Spinal cord tissue samples were obtained at 1, 3, 7, and 14 days after surgery, and macrophage polarization types were detected by using western blot analysis, immunofluorescence, flow cytometry and RT-qPCR.ResultsThe BMS score in the mmu-miR-27a-5p group was significantly higher than that in the model and mmu-miR-27a-5p-NC groups at 7 and 14 days after SCI (X2=26.45-57.62, P<0.05). No significant changes in the expression of M1 markers IL-1β, TNF-α and M2 markers IL-10, Arginase-1 at each time point in the sham group (P=0.96). The expression of IL-1β and TNF-α was significantly lower, while the expression of IL-10 and Arginase-1 were significantly higher in the mmu-miR-27a-5p group as compared to the model and mmu-miR-27a-5p-NC groups at 7 and 14 days after SCI (P<0.05).Conclusion﻿Administration of mmu-miR-27a-5p can promote the polarization of macrophages to the M2 phenotype in the injured spinal cord, and improve motor function recovery within 14 days after SCI in mice.

aimed to explore the effect of mmu-miR-27a-5p on the polarization of macrophage in the injured spinal cord in mouse model of SCI.

Behavioral scoring results
There was no statistically signi cant difference in BMS score between the four groups before SCI (P = 1.0). The hindlimb function disappeared within 1 day after SCI (BMS score = 0), and gradually recovered thereafter in both model, mmu-miR-27a-5p, and mmu-miR-27a-5p-NC groups. BMS score returned to the preoperative level on day 3 after surgery in the sham group. The BMS scorewas signi cantly higher in the mmu-miR-27a-5p group than in the model and mmu-miR-27a-5p-NC groups at 7 and 14 days after SCI (X2 = 26.45-57.62, P < 0.05), indicating that the mice in the mmu-miR-27a-5p group showed better recovery of hindlimb and trunk motor function.

Expression of macrophage markers detected by western blot analysis
No signi cant changes were observed in the expression of IL-1β, TNF-α, IL-10, and Arginase-1 at each time point in the sham group (P = 0.96). There was no statistically signi cant difference in the expression of IL-1β, TNF-α, IL-10, and Arginase-1 between mmu-miR-27a-5p, model and mmu-miR-27a-5p-NC groups at 1 and 3 days after SCI (P = 0.84). The expression of IL-1β and TNF-α was signi cantly lower in the mmu-miR-27a-5p group as compared to model and mmu-miR-27a-5p-NC groups at 7 and 14 days after SCI. On the contrary, the expression of IL-10 and Arginase-1 were signi cantly higher in the mmu-miR-27a-5p group as compared to the model and mmu-miR-27a-5p-NC groups at 7 and 14 days after SC (P < 0.05, Fig. 2). It is suggested that there were more M2 macrophages in the injury site of mice in the MMU-miR-27a-5p group.
Immuno uorescent staining results for macrophage markers No signi cant change was observed in the expression of IL-1β, TNF-α, IL-10, and Arginase-1 at each time point after SCI in the sham group. There were also no signi cant difference in the expression of IL-1β, TNF-α, IL-10, and Arginase-1 at 1 and 3 days after SCI between the mmu-miR-27a-5p, model and mmu-miR-27a-5p-NC groups. At 7 and 14 days after SCI, there were signi cantly increased numbers and wider distributions of IL-10-and Arginase-l -positive cells, and signi cantly decreased numbers and restricted distributions of IL-1β-and TNF-α-positive cells in the mmu-miR-27a-5p group compared to the model and mmu-miR-27a-5p-NC groups (Fig. 2), indicating that mice in the mmu-miR-27a-5p group had more M2 macrophages in the injured spinal cord.

Expression of macrophage markers detected by RT-PCR
No signi cant change was observed in the relative expression of each macrophage marker gene in the sham group (P > 0.05). Relative expression of IL-10 and Arginase-1 were signi cantly increased, while relative expression of IL-1β and TNF-α were signi cantly decreased at 7 and 14 days after SCI in the mmu-miR-27a-5p group than those in the model and mmu-miR-27a-5p-NC groups (P < 0.05), indicating that administration of mmu-miR-27a-5p can inhibit the in ammatory responses, affect macrophage polarization types in the injury site by decreasing M1 macrophage polarization and increasing M2 macrophage polarization within 14 days following SCI.

Discussion
In ammatory response mainly affects axonal regeneration and functional recovery after SCI. Changes in local microenvironment occur rapidly after SCI, neutrophil in ltration and microglia activation are rst initiated, followed by accumulation of monocyte-derived macrophages [6,7], macrophages recognize and scavenge substances at the injury site that are not conducive to nerve repair and promote tissue regeneration [8], reduce or prevent the expanded secondary injury. In this study, we found that changes in BMS scores for hindlimb motor function were basically consistent with the changes in the expression of IL-1β and TNF-α at 7 days after injury, as well as the change in the expression levels of Arginase-1 and IL-10 at 14d after SCI, suggesting that macrophages play a positive role in the recovery of spinal cord function after SCI. A study [7] established a mouse model of traumatic brain injury and found that macrophages isolated from the ipsilateral cerebral cortex were activated due to the expression of NADPH oxidase 2 at 7 days post injuy, which can signi cantly inhibit neuronal growth and enhance in ammatory responses. The reuslts indicated that after central nervous system injury, the macrophage-mediated in ammatory response can also hinder neural repair. Therefore, macrophages play a dual role in SCI repair. Findings from our study and the above mentioned previous studies both suggest that the mechanism may be related to different polarization types of macrophages caused by changes in the local microenvironment after SCI.
Due to different stimulating factors and difference in the microenvironment at the injury sites, macrophages can be polarized into M1 and M2 types, which secrete different cytokines and chemokines, and exert different effects [9,10]. mmu-miR-27a-5p plays an important role in macrophage polarization, which can affect the polarization of macrophages by regulating MCPIP1 expression [3]. In this study, highest expression of M1 markers IL-1β and TNF-α was observed at 7 days after SCI, while highest expression of M2 markers Arginase-1, IL-10 was observed at 14 days after SCI in the model and mmu-miR-27a-5p-NC groups. IL-1β and TNF-α expression levels were signi cantly lower, and Arginase-1 and IL-10 expression levels were signi cantly higher at 7 and 14 days after SCI in the mmu-miR-27a-5p group compared with the model and mmu-miR-27a-5p-NC groups. These results indicate that administration of mmu-miR-27a-5p can increase M2 macrophage polarization at the injury sites, which have bene cial effects on the repair of injured spinal cord. Fan et al. [11] believed that after SCI, M1 macrophages induce the apoptosis of reactive astrocytes through the TLR/MyD88 signaling pathway and promote glial scar formation. M2 macrophages can promote the growth of neuronal axons [12], the underlying mechanism may be that M2 macrophages secrete large amounts of brous protein and matrix-related proteins, inhibit macrophage in ltration, the expression of in ammatory cytokines and activation of their receptors following SCI, alleviate oxidative stress and secondary SCI, and promote endothelial cell proliferation, angiogenesis, and neural function recovery after SCI [13][14][15]. Our results also con rmed that at 7d and 14d following SCI, BMS scores in the mmu-miR-27a-5p group was signi cantly higher than that in the model and mmu-miR-27a-5p-NC groups(X2 = 26.45-57.62, P < 0.05). Administration of mmu-miR-27a-5p can promote recovery of hindlimb function in mice after SCI.
In fact, the polarization types of macrophages is far more complex than characterized by the M1/M2 polarization. M1 and M2 macrophages may represent two extreme ends of macrophage polarization, there are intermediate phenotypes. At present, three subtypes of M2 macrophages have been distinguished: M2a, M2b, and M2c, each one has its own characteristics and functions. After SCI, M1 and M2a macrophages appeared at the early stage after SCI, followed by M2b and M2c macrophages, and nally overall decrease in M2 macrophage can be observed. M1 gradually occupied and maintained a dominant position for a long time at 1 week following SCI [16][17][18], this is one of the reason why patient with SCI can not recover in a long time. In this study, we detected the protein and mRNA expression of M1 phenotypic markers (IL-1β and TNF-α), and M2a phenotypic markers (arginase-1 and IL-10), and we observed decreased expression of M1 phenotypic markers, and increased expression of M2a phenotypic markers in the mmu-miR-27a-5p group compared with the mmu-miR-27a-5p-NC group, we conclude that administration of mmu-miR-27a-5p can increase M2a macrophage and decrease M1 macrophage. Since the total number of macrophages is constant, the increase in M2a macrophage at early stage (within 3 d) following SCI may be accompanied by a decrease in other macrophage subtypes. The trends of changes in M2b and M2c macrophages need to be further investigated using more speci c phenotypic markers.
The limitation of this study is that we mainly focused on the changes in the polarization types of macrophages at the site of injury, did not observe the changes in the macrophages derive from blood monocytes, further study are needed to con rm our ndings.
In summary, administration of mmu-miR-27a-5p can promote the polarization of macrophages to M2 phenotype in the site of injury, improve the recovery of motor function in mice following SCI.

Experimental animals and grouping
A total of 160 speci c-pathogen-free C57BL/6j male mice (45-56 days old) were purchased from Three Gorges University. Mice were randomly divided into sham, model, mmu-miR-27a-5p, mmu-miR-27a-5pnegative control (NC) groups, with 40 mice in each group. The animal study was approved by the ethics committee of our hospital.
Preparation of mmu-miR-27a-5p mimics mmu-miR-27a-5p mimics was synthesized by Wuhan Biofavor Biotech Services Co., Ltd. (Wuhan Biofavor Biotech Services Co., Ltd., Wuhan, China), an unrelated universal sequence was as a negative control (NC). RAW 264.7 macrophages were seeded into 6-well plates and cultured with mmu-miR-27a-5p mimics and mmu-miR-27a-5p-NC, respectively, at 37 °C overnight. Lentivirus vector solution (1 × 109TU/ml) were mixed and diluted with medium at a multiplicity of infection of 100, 10, 1. The old medium was aspirated from the 6-well plate, and replaced with the virus gradient dilutions. After 24 h of culture at 37 °C, culture medium was replaced with fresh media. After 48 hours of culture, transfection e ciency was observed under a uorescent microscope. The optimal transfection condition was chosen and used for subsequent experiments.

SCI modeling
After 3 days of acclimation, all mice were anesthetized intraperitoneally with 4% chloral hydrate. After hair removal and skin disinfection in the surgical eld, a longitudinal incision was made centered on T10 spinous process, then the T9, T10 spinous processes and lamina were removed to expose the dura mater. In the model, mmu-miR-27a-5p, and mmu-miR-27a-5p-NC groups, SCI at T9-10 levels was produced by freely dropping a 10 g weight from a height of 5 cm using a weight drop device onto the T9-10 spinal segment [17]. The signs of successful model establishment included spastic swinging of the tail and retraction-like utter and accid paralysis of the hind limbs immediately after injury. In the sham group, T9-10 spinous processes and lamina were removed without damaging the spinal cord. After surgery, the mice were kept warm. And after recovery from anaesthesia, mice were moved to the animal room. All mice were fed normally. 20,000 units of penicillin were injected intraperitoneally into mice once a day within 3 days after surgery, and the bladder of the mice was squeezed manually twice daily. If mice died during surgery, new mice was added and animal model was established again.

Treatment
All mice in each group were treated at day 3 after SCI model was successfully established. Mice in sham group received no treatment, mice in model group were treated with 10 µl saline. Mice in mmu-miR-27a-5p group and mmu-miR-27a-5p-NC group received 10 µl of mmu-miR-27a-5p suspension and mmu-miR-27a-5p-NC suspension, respectively. Before administration, mmu-miR-27a-5p suspension and mmu-miR-27a-5p-NC suspension were co-cultured with BrdU (10 µmol/L) for 72 hours. Multi-point injections were slowly given for 5 minutes at the injured spinal cord, the needle was left in place for additional 5 min before removal, then feeding was continued.

Behavioral testing
Before surgery and at 1, 3, 7, and 14 days after surgery, open eld test was perfromed [5], mice were placed in an open eld, allowed to freely explore fo for 5 min. Hindlimb and trunk motor function were assessed using Basso Mouse scale (BMS) by a examiner who did not participate in the study design.

Spinal cord tissue acquisition
After successful established of SCI model, 10 mice of each group were sacri ced at 1, 3, 7, and 14 days after SCI to collect their spinal cord samples. Mice were deeply anesthetized with ketamine/xylazine, and perfused through the heart with saline, followed by 40 g/L paraformaldehyde. Approximately 1.5 cm length of spinal cord tissue centered at the injury site was taken, placed in paraformaldehyde overnight at 4 °C, dehydrated in 30% sucrose solution at 4 °C until sinking. Then the tissue was embedded in an optimal cutting temperature, 20-µm sections were cut, and frozen at − 80 °C.

Detection of macrophage markers using western blot analysis
The collected spinal cord tissues were homogenized in RIPA lysis buffer (Beyotime Biotechnology, China) at 4 °C for 5 min to obtain 10% homogenate. The homogenates were lysed in RIPA buffer at 4 °C for 45 min, centrifuged at 12000r/min for 5 min, then the supernatant were collected. The BCA protein assay kit was used to measure total protein concentration according to the manufacturer's protocol. Protein samples were separated on SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked in 5% non-fat dry milk in TBS and Tween-20 for 1.5 h, incubated with primary antibodies against M1 markers IL-1β, TNF-α and M2 markers IL-10, Arginase-1. Immunoblots were visualized using the ECL western blotting detection system Detection of macrophage markers using immuno uorescence staining Sections were permeabilized and blocked in 0.2% Triton X-100 in PBS, and incubated with primary antibodies against Arginase-1 (1:100, WACO, Osaka, Japan), IL-10 (1:200, WACO, Osaka, Japan), TNF-α (1:30,, WACO, Osaka, Japan), IL-1β (1:200, WACO, Osaka, Japan) overnight at 4 °C. After the sections were rinsed, the sections were incubated for 2 h at room temperature with species-speci c secondary antibodies, including CY3 Goat Anti-Rabbit IgG Antibody (1:100 dilution), Alexa-594 (red)-conjugated TNFαand IL-1βantibodies (1:250, Invitrogen, USA), Alexa Fiuor-488 (green)-conjugated Arginase-1, IL-10 antibodies (1:250, Invitrogen, USA). Finally, sections were washed, mounted with DAPI, and visualized using uorescent microscope (DM3000) or a confocal laser scanning microscope (TCS SP5 II; both Leica Microsystems, Inc., Germany). Three non-overlapping elds at the injury site were randomly selected in each slice at 400 magni cation. Positively stained cells were counted from the nine randomly selected microscopic elds by using the Image-Pro Plus (IPP 6.0) software and the mean values were calculated.
Detection of macrophage markers using ow cytometry 1 ml of PBS containing 0.5% bovine serum albumin and 1 × 10 6 cells were added to each ow tube, and incubated with PE-conjugated CD16/CD32 monoclonal antibody (clone 93, 0.125 µg per test, BD Biosciences, USA), and APC-conjugated Arginase-1 monoclonal antibody (A1exF5, 1.0 µg per test) at 4 °C in the dark for 30 min. After centrifugation at 15000r/mim for 5 min, the supernatant was discarded, then cells were washed once with 1 ml PBS, and centrifuged at 15000r/mim for 3 min. The supernatant was removed and the cells were resuspended in 0.2 ml PBS containing 0.5% BSA. Data were analyzed using ow cytometer (Attune NxT, Thermo Fisher Scienti c, Inc. USA).
Detection of macrophage markers using real-time quantitative PCR (RT-qPCR) Total RNA was extracted using the Trizol reagent (Thermo Fisher Scienti c, Inc. USA). Reverse transcription for DNA was synthesized using PrimeScript™ reverse transcription kit (Takara, Japan). RT-qPCR was performed in a 10 µL reaction system using UltraSYBR premixture (CWbio Co., Ltd., Beijing, China). RT-qPCR conditions included 10 min of predenaturation at 95 °C, followed by 35 cycles of denaturation at 95 °C for 15 s, annealing/ elongation at 60 °C for 1 min. The dissolution curve conditions were 95 °C for 15 S, 60 °C for 1 min, 95 °C for 15 s, 60 °C for 15 s. The relative quanti cation was calculated using the 2−ΔΔCT method with reference genes β-actin.

Statistical analysis
Statistical analysis was performed using GraphPad Prism 6.01 software. Measurement data were expressed as mean ± standard deviation (mean ± SD). Differences in the results from RT-PCR and immuno uorescence staining were analyzed using t test, and differences in the results of behavioral scoring were analyzed using repeated measures Analysis of Variance (ANOVA). A difference with a Pvalue of < 0.05 was considered statistically signi cant. Expression of macrophage markers at 7 days after SCI detected by western blot analysis. A:Sham group; B: Model group; C: mmu-miR-27a-5p group; D: mmu-miR-27a-5p-negative control group Immuno uorescent staining for macrophage polarization-associated markers at 7 days after SCI. Red uorescence indicates the expression of IL-1β and TNF-α, green uorescence indicates the expression of Arginase-1 and IL-10