MiR-124 Reduced Neuroinflammation after Traumatic Brain Injury by Inhibiting TRAF6

Introduction: Neuroinflammation contributes to secondary injury after traumatic brain injury (TBI), which has been mainly mediated by the microglia. MiR-124 was reported to play an important role in the polarization of microglia by targeting TLR4 signaling pathway. However, the role and mechanism of miR-124 in neuroinflammation mediated by microglia after TBI is unclear. To clarify this, we performed this research. Methods: The expression of miR-124 was first measured by RT-PCR in the injured brain at 1/3/7 days post-TBI. Then, miR-124 mimics or inhibitors administration was used to interfere the expression of miR-124 at 24 h post-TBI. Subsequently, the microglia polarization markers were detected by RT-PCR, the expression of inflammatory cytokines was detected by ELISA, the expression of TLR4/MyD88/IRAK1/TRAF6/NF-κB was measured by WB, and the neurological deficit was evaluated by NSS and MWM test. At last, in vitro experiments were performed to explore the exact target molecule of miR-124 on TLR4 signaling pathway. Results: Animal research indicated that the expression of miR-124 was downregulated after TBI. Upregulation of miR-124 promoted the M2 polarization of microglia and inhibited the activity of TLR4 pathway, as well as reduced neuroinflammation and neurological deficit after TBI. In vitro experiments indicated that miR-124 promoted the M2 polarization of microglia and reduced neuroinflammation by inhibiting TRAF6. Conclusion: This study demonstrated that upregulation of miR-124 promoted the M2 polarization of microglia and reduced neuroinflammation after TBI by inhibiting TRAF6.

© 2023 The Author(s). Published by S. Karger AG, Basel

Introduction

Traumatic brain injury (TBI) is one of the leading causes that results in acute central nerve injury and chronic long-term disability worldwide, especially in the children and young adults [1]. After TBI, there are mainly two successive phases of pathologic alteration in the injured brain. One is the instant primary brain insult which causes axonal shearing, parenchymal damage, or intracerebral hemorrhage. Another one is the subsequent secondary brain damage induced within hours to days which includes neuroinflammation, oxidative stress, or cell death [2]. Among these events, the neuroinflammation plays an important role in the pathologic process of TBI because the secondary brain damage will be aggravated and the neurologic prognosis will be worse if it cannot be properly controlled [3]. As a pathologic characteristic, the neuroinflammation takes effects in both acute and chronic phases of TBI and even might last for years in patients who have a history of brain injury and lead to chronic long-term disorders, such as neurodegenerative disease or traumatic encephalopathy at last [4, 5]. Although neuroinflammation is a natural reaction of TBI and has protective effects on the injured brain in a way, the excessive inflammatory response often becomes a significant driving force that may lead to worse brain damage and neurological dysfunction. Thus, exploring effective strategy to inhibit the excessive neuroinflammation has great significance for the improvement of neurologic outcome after TBI [6].

Acting as a similar role of macrophage in the central nervous system, microglia is the essential cell in activating and maintaining neuroinflammation response in the injured brain [7]. After TBI, quiescent microglia are instantly activated and migrated into brain regions surrounding the injury sites, and the inflammatory mediators are generated and released as well [7]. In addition, the activated microglia and inflammatory mediators recruit peripheral immune cells such as lymphocytes and neutrophils into the brain to further amplify neuroinflammation [8]. Specifically, the activated microglia present as M1 phenotype release pro-inflammatory cytokines IL-1β, IL-6, and TNF-α and M2 phenotype release anti-inflammatory cytokines IL-4, IL-10, and TGF-β [7, 9]. Thus, microglia exert double-edged effects on the neuroinflammation reaction after TBI, and its polarization from M1 to M2 phenotype could suppress excessive neuroinflammation and improve the neurologic outcome after TBI. To achieve this aim, the concrete mechanism of microglia activation and polarization after TBI should be explored at first. Recent studies have demonstrated that the absence of TLR4 induced microglia polarization toward M2 phenotype and alleviated the development of neuroinflammation, indicating potential neuroprotective effects after TBI [10, 11]. Therefore, exploring novel strategy to inhibit the TLR4 and its downstream molecular pathway including myeloid differentiation primary-response protein 88 (MyD88), IL-1R-associated kinase 1(IRAK1), TNF receptor-associated factor 6 (TRAF6), and nuclear factor-κB (NF-κB) would be useful to reduce neuroinflammation and improve neurologic outcome after TBI [1215].

MicroRNAs (miRNAs) are important molecules in the brain that can regulate gene transcription and their associated signaling pathway in the activation and polarization of microglia [1618]. Among the known miRNAs, miR-124 is the most abundant brain-specific miRNA which is highly expressed in microglia and modulate its function. Under physiological conditions, miR-124 is constitutively and highly expressed in microglia and plays a key role in its quiescence [19, 20]. Under pathological conditions, the downregulation of miR-124 has increased neuroinflammation by polarizing activated microglia toward M1 phenotype [21], while the upregulation of miR-124 has reduced neuroinflammation by polarizing activated microglia toward M2 phenotype [22, 23]. Interestingly, one previous study had indicated that the expression of miR-124 was decreased by TLR4 activator LPS, and miR-124 overexpression was shown to inhibit LPS-induced microglia activation as well [16]. One latest study also has demonstrated that the overexpression of miR-124 inhibited microglia activation and reduced neuroinflammation by targeting TLR4 pathway [21]. Taken together, TLR4 pathway may play an important role in regulating the activation and polarization of microglia by miR-124. Thus, this research intended to explore the role of miR-124 in microglia-mediated neuroinflammation and its correlation with TLR4 pathway after TBI.

This study aimed at exploring the role and mechanism of miR-124 in neuroinflammation mediated by microglia after TBI. In vivo experiments indicated that the expression of miR-124 was downregulated after TBI, upregulation of miR-124 promoted the M2 polarization of microglia, inhibited the activity of TLR4 pathway, and reduced neuroinflammation and neurological deficit after TBI. In vitro experiments indicated that miR-124 promoted the M2 polarization of microglia and reduced neuroinflammation by inhibiting TRAF6. In all, miR-124 reduced neuroinflammation after TBI by inhibiting TRAF6.

Materials and MethodsAnimals

Male C57BL/6 mice (10–12 weeks old, weighing 28–32 g) were purchased from the Experimental Animal Center of Air Force Medical University (Fourth Military Medical University) (Xi’an, China). These animals were housed in the environment with constant temperature (21.0 ± 2°C), appropriate humidity (50–55%), and a 12 h light/dark cycle (lights on at 8:00 a.m. and off at 8:00 p.m.). They were provided with enough water and food. The Air Force Medical University (Fourth Military Medical University) Ethics Committee approved the experimental protocols and animal procedures in this study. All the procedures were conducted according to the National Experimental Animals Guidelines.

Establishment of TBI Model

TBI animal model was induced by the controlled cortex injury device (Hatteras Instruments, Cary, NC, USA). Mice were first anesthetized by the intraperitoneal injection of 2% pentobarbital sodium (60 mg/kg) and fastened on the stereotaxic frame (Kopf Instruments, Tujunga, CA, USA). The head was horizontally secured by an incisor bar and two lateral ear pins. Then, the fascia reflection was made to expose the skull after sagittal incision was performed at the midline of scalp. Later, skull flap was removed in the right hemisphere after a 4.0 mm diameter craniotomy was conducted between lambda and bregma and 3.0 mm lateral to the sagittal suture. Subsequently, a perpendicular impact brain contusion was carried out on the exposed dura using a piston rod with 3.0 mm diameter for contact surface, 3.0 m/s for impact velocity, 100.0 ms for contact time, and 1.5 mm for vertical cortical impact depth. At last, the skull flap was restored, the scalp was sutured, and the incision was sterilized. Mice in sham group only underwent craniotomy but without cortical impact contusion. The heating pad was used to maintain the body temperature of mice at 37.0 ± 0.5°C during the surgical and recovery procedure.

Intracerebroventricular Injection

The in vivo transfection of miR-124 mimics or miR-124 inhibitors was conducted at 24 h after the establishment of TBI, according to the following procedures. First, the stereotaxic coordinate was located at 0.5 mm posterior, 1.0 mm lateral to bregma, and 2.5–3.0 mm ventral to the surface of the skull. Second, the miR-124 mimics or miR-124 inhibitors (2 μg/2 μL) were added into 1.25 μL of Entranster™-in vivo transfection reagent. Then, the solution was mixed gently and left for 15 min. At last, the solution was intracerebroventricularly injected using a micro syringe (Hamilton, NV, USA) according to the guidance of the stereotaxic instrument (RWD Life Science).

Real-Time PCR

The frozen mice brain was homogenized, and total RNA was obtained from the injured hemisphere brain tissue at 1, 3, and 7 days after TBI by the TRIzol reagent (Invitrogen, USA). The concentration of RNA was tested by the spectrophotometer. The reverse transcription was performed by the M-MLV Reverse Transcriptase System (Promega, USA). In order to enlarge and detect the mRNA expression, quantitative real-time PCR was fulfilled with a Light Cycler (Roche Diagnostics, GM) and SYBR Green I in SYBR RT-PCR Kit (TaKaRa, China). The primers were purchased from BioAsia Corp (Shanghai, China). The sequences of the PCR primers included: miR-124 forward: 5′-TAA​GGC​ACG​CGG​TGA​ATG-3′ and reverse: 5′-GTG​CAG​GGT​CCG​AGG​T-3′, CD32 forward: 5′-AAT​CCT​GCC​GTT​CCT​ACT​GAT​C-3′ and reverse: 5′-GTG​TCA​CCG​TGT​CTT​CCT​TGA​G-3′, IL-1β forward: 5′-CAGG CTCCGAGATGAACAAC-3′ and reverse: 5′-GGTGGAGAGCTTTCA GCTCATA-30, CD206 forward: 5′-TCT​TTG​CCT​TTC​CCA​GTC​TCC-3′ and reverse: 5′-TGAC ACCCAGCGGAATTTC-3′, Arginase-1 forward: 5′-GAA​CAC​GGC​AGT​GGC​TTT​AAC-3′ and reverse: 5′-TGC​TTA​GCT​CTG​TCT​GCT​TTG​C-3′, and β-actin forward: 5′-GGCATCGTGATGGACT CCG-3′ and reverse: 5′-GCT​GGA​AGG​TGG​ACA​GCG​A-3′. The miRNA RT Kit (ABI) and TaqMan Universal PCR Master Mix (ABI) were used to detect the expression of miR-124, and U6 was used as an internal control. The comparative cycle threshold method was used to measure the relative quantification value for each target gene. The expression of pro-inflammatory and anti-inflammatory chemokines in BV2 microglia was detected according to the above steps as well. To guarantee the accuracy, each experiment was carried out in triplicate. Data were analyzed by the Light Cycler Software 4.0 (Roche Diagnostics).

ELISA

To measure the expression of pro-inflammatory and anti-inflammatory chemokines in the injured brain, mice were anesthetized by 2% pentobarbital sodium (60 mg/kg) via intraperitoneal injection at 3 days post-trauma. The injured brain was isolated and pulverized in the homogenizer with liquid nitrogen. The lysis buffer including 1% Triton X-100, 1 mm EDTA, 10 mm Tris pH8.0, 1 mm phenylmethylsulfonyl fluoride, 150 mm NaCl, and 5 μL/mL protease inhibitor (Sigma-Aldrich, USA) were added into the lysate and incubated at 4°C for 1 h. The lysate was centrifugalized at 3,000 rpm for 20 min, the supernatant was collected to measure the concentration of pro-inflammatory chemokines IL-1β, IL-6, and TNF-α and anti-inflammatory chemokines IL-4, IL-10, and TGF-β by ELISA kits (Minneapolis, USA), according to manufacturer’s protocols. To guarantee the accuracy, each experiment was performed in triplicate.

Western Blot

The injured brain tissue was isolated from the brain on ice and stored at −80°C. The brain tissue was homogenized and digested in a homogenizer on ice for 15 min with a lysis buffer containing 150 mm NaCl, 1% NP-40, 50 mm Tris (pH = 7.4), 0.5 mm EDTA, 1% Triton X-100, 1 mg/mL aprotinin, 10 mg/mL leupeptin, 1% deoxycholate, and 1 mm phenylmethylsulfonyl fluoride. The lysate was centrifuged at 12,000 rpm at 4°C for 20 min, and the protein concentration was examined with Bicinchoninic Acid Protein Assay Kit (Beyotime, China). Equivalent amount of protein (40 μg) was loaded and separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane at 4°C for 50 min. Membranes were blocked with 5% nonfat milk solution in Tris-buffered saline with 0.1% Triton X-100 (TBST) for 1 h and then incubated overnight at 4°C with appropriate primary antibodies as follows: rabbit anti-mouse TLR4 antibody (Thermo Fisher, USA), rabbit anti-mouse myeloid differentiation factor 88 (MyD88) antibody (Santa Cruz, USA), rabbit anti-mouse TNFR-associated factor (TRAF6) antibody (Novus Biologicals, USA), rabbit anti-mouse NF-κB p65 antibody (GeneTex, USA), and rabbit anti-β-actin antibody (Proteintech, USA). After three washes in Tris-buffered saline with 0.1% Triton X-100, membranes were incubated with the second antibody: horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Cell Signaling Technology, USA) at room temperature for 1 h. Immunoreactivity was detected by Western Bright Enhanced chemiluminescence reagents (Advansta, USA), and optical densities of the band were analyzed by Gel-Pro Analyzer software (version 6.0, Media Cybernetics, Rockville, MD, USA).

Neurological Severity Score Test

NSS test was performed to evaluate the neurological function of mice at 7, 14, 21, and 28 days after TBI. The NSS consisted of 10 individual parameters for the alertness measurement, balancing examination, and motor ability evaluation. The mouse would be assigned for one score point if it lacked a tested reflex or failed to accomplish a task. The total score of NSS was graded on a scale of 0–10, with 0 indicated normal status and 10 suggested maximal neurobehavioral deficits. Thus, the higher the accumulated score, the severer the neurobehavioral dysfunction.

Morris Water Maze Test

Morris water maze (MWM) test was conducted to evaluate the neurocognitive function of mice at 24–28 days after TBI. The instrument used in MWM test was a circular tank (160 cm in diameter and 50 cm in depth) with a black inner wall, which was filled with water of 30 cm in depth and 25°C in temperature. The learning ability and memory function of mouse was measured by the hidden platform trial and probe trial, respectively. In the hidden platform trial, the tank was first divided into four equal quadrants, and then a black circular platform of 12 cm in diameter was hidden 2 cm under water surface in the center of one quadrant. Later on, the mouse was allowed to swim freely in the maze and had a maximum of 120 s to find the platform. If the mouse failed to reach the platform within 120 s, the trial would be terminated, and the mouse would be guided onto the platform and remained on it for 30 s. During this process, the interval time between the mouse placed into the water and reaching the platform was recorded as escape latency, which was used to evaluate the learning ability. Each mouse received four hidden platform trials per day for four consecutive days from 24 days post-trauma. After that, the hidden platform was removed from the quadrant, and the probe test was conducted at 28 days post-trauma. Briefly, the mouse was first put into the water and allowed to swim freely in the maze to find the removed platform. Then the route in target quadrant was identified according to the mouse’s trace in which quadrant the platform was placed. The platform crossing time was calculated based on the time of the mouse swam over the previous platform location. The neurocognitive function of the mouse was evaluated according to the parameters in above trails, which was recorded by the tracking system (Dig Beh-MR, Shanghai Auspicious Software Technology Company Limited, China).

BV2 Microglia Treatment with MiR-124 Mimics/Inhibitors

BV2 microglia were first cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin. Then, cells were seeded onto plates at a density of 1 × 106 cells/well and incubated in the environment of 37°C, 5% CO2, and saturated humidity. To investigate the effect of miR-124 on the expression of TRAF6 and the polarization of microglia, BV2 microglia were grown for 4 days and randomly divided into four groups: control, LPS, LPS + miR-124 mimics, and LPS + miR-124 inhibitors. First, LPS (1 μg/mL) treatment was performed to activate the TLR4 pathway in BV2 microglia for 24 h. Then, miR-124 mimics or miR-124 inhibitors were added into the culture medium and co-cultured for 48 h to explore their effects on the expression of TRAF6 and the polarization of microglia. In detail, the expression of miR-124 in BV2 microglia was measured by RT-PCR, the expression of TRAF6 was measured by WB, the M1/M2 polarization of BV2 microglia was evaluated by flow cytometry, and the expression of pro-/anti-inflammatory cytokines was measured by RT-PCR.

Flow Cytometry

After BV2 microglia were treated with LPS for 24 h and miR-124 mimics or miR-124 inhibitors for 48 h, 3-mL cell suspension from each sample was transferred into a 10-mL centrifuge tube and centrifuged at 500 rpm for 5 min. The supernatant was discarded, and BV2 microglia were reserved and resuspended in 100 μL binding buffer. Then, cells were further incubated with fluorescence-labeled antibodies CD68 and CD206 (Abcam, UK) in the dark at 4°C for 30 min. Later, they were washed in 2.5 mL of 2% FCS (HyClone, USA)/PBS for three times and resuspended in 500 μL of 1% paraformaldehyde/PBS and detected by flow cytometry using FACSCalibur instrument (BD, USA).

Dual-Luciferase Reporter Assay

Bioinformatics analysis showed that the target molecule of miR-124 on TLR4 pathway might be TRAF6. To verify this, online software starBase (http://starbase.sysu.edu.cn/) was used to predict the binding site of miR-124 and TRAF6, and dual-luciferase reporter assay was further performed to confirm the combination of them. Specifically, the wild-type (WT) and mutant-type (MUT) TRAF6 3ʹ untranslated region reporter plasmids (pmiR-TRAF6-wt and pmiR-TRAF6-mut) were first constructed by Promega Corporation (Promega). Then, the two plasmids together with miR-124 mimic/mimic NC were co-transfected into HEK-294 cells using Lipofectamine 3000. After 48 h, those cells were collected and the luciferase activity was measured using Dual-Luciferase Reporter Assay Kit (Promega). Above experiments were conducted according to the manufacturer’s protocols, and three replicates were performed for each group.

RNA-Immunoprecipitation Assay

RNA-immunoprecipitation (RIP) assay was performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit and Ago2 antibody (Millipore, USA), according to the manufacturer’s protocols. Specifically, cells were first washed twice by precooled PBS, centrifuged at 1,500 rpm for 5 min, and lysed with equivoluminal RIP lysis buffer. Then, the magnetic beads were resuspended in 100 μL RIP wash buffer and incubated with 5 μg antibody against Ago2 (Abcam) or IgG (Abcam) at room temperature. Later, the magnetic beads tube was placed on a magnetic separation rack to discard the supernatant, and the magnetic bead-antibody complex was reserved. After that, cell lysates were centrifuged at 14,000 rpm at 4°C for 10 min, and 100 μL supernatant was incubated with the magnetic bead-antibody complex overnight at 4°C. Subsequently, the above complex was centrifuged at 2,500 rpm for 30 min, and the sediment was resuspended in 500 μL RIP buffer and washed for six times. At last, the samples were incubated with 150 μL of proteinase K buffer at 55°C for 30 min and put in the magnetic separation rack to remove the supernatant for further RNA extraction and RT-PCR assay. In order to verify whether the outcomes resulted from RNAs specifically binding to Ago2, RNAs and IgG were measured at the same time.

Data Analysis

Data in this research were expressed as mean values ± standard deviations (M ± SD). The differences among four groups were analyzed by one-way ANOVA test with Bonferroni correction. The differences between two groups were analyzed by unpaired t test with Welch’s correction. SPSS version 13.0 software (SPSS Inc., USA) was used to perform the analysis, and two-tailed p < 0.05 was considered statistically significant. GraphPad Prism 5 software (GraphPad Software, Inc., USA) was used to draw the figures in this manuscript.

ResultsThe Expression of MiR-124 Was Downregulated in the Injured Brain after TBI

To detect the expression of miR-124 in the injured brain, the experimental TBI model was first constructed. Then, the injured cerebral cortex was isolated, and the miR-124 level was measured by real-time PCR at 1/3/7 days post-TBI. Results indicated that the expression of miR-124 in the injured cerebral cortex of TBI mice was much lower than that in the sham group (Fig. 1).

Fig. 1.

The relative expression of miR-124 was downregulated in the injured brain after TBI. ac Compared to the sham group, the relative expression of miR-124 was lower at 1/3/7 days after TBI. n = 6 in sham group/each time point, n = 6 in TBI group/each time point. ∗ p < 0.05 versus sham group.

/WebMaterial/ShowPic/1496096Upregulation of MiR-124 Reduced Signature Genes of M1 Microglia and Pro-Inflammatory Chemokines after TBI

To investigate the role of miR-124 in the M1/M2 polarization of microglia after TBI, the intracerebroventricular injection of miR-124 mimics or inhibitors was performed at 24 h post-TBI to upregulate or downregulate miR-124 in the brain. Results indicated that the transduction of miR-124 mimics improved miR-124 expression, while the transduction of miR-124 inhibitors attenuated miR-124 expression at 3 days after TBI (Fig. 2a). Then, the expression of the signature genes of M1 microglia (CD16, CD32) and M2 microglia (CD206, Arginase-1) in the injured cerebral cortex was analyzed by RT-PCR and, the concentration of pro-inflammatory chemokines IL-1β, IL-6, and TNF-α, and anti-inflammatory chemokines IL-4, IL-10, and TGF-β in the injured cerebral cortex was measured by ELISA. Results indicated that miR-124 mimics significantly decreased the expression of M1 signature genes CD16 and CD32 and pro-inflammatory chemokines IL-1β, IL-6, and TNF-α, while miR-124 inhibitors produced the contrary effects (Fig. 2b–f). In addition, miR-124 mimics significantly increased the expression of M2 signature genes CD206 and Arginase-1 and anti-inflammatory chemokines IL-4, IL-10, and TGF-β, while miR-124 inhibitors produced the contrary effects (Fig. 3a–e). These results showed that upregulation of miR-124 inhibited the expression of M1 microglia signature genes and pro-inflammatory chemokines after TBI.

Fig. 2.

Upregulation of miR-124 reduced M1 microglial markers and pro-inflammatory cytokines in the injured brain after TBI. a MiR-124 mimics upregulated the expression of miR-124, and miR-124 inhibitors downregulated the expression of miR-124. bf MiR-124 mimics reduced the mRNA expression of CD32, IL-1β and the concentration of IL-1β, IL-6, and TNF-α, but miR-124 inhibitors produced the contrary effects. n = 12 in sham group, n = 12 in other three groups. ∗ p < 0.05 versus sham group, #p < 0.05 versus TBI group.

/WebMaterial/ShowPic/1496095Fig. 3.

Upregulation of miR-124 promoted the expression of M2 microglia markers in the injured brain after TBI. ae MiR-124 mimics increased the mRNA expression of CD206, Arginase-1 and the concentration of IL-4, IL-10, and TGF-β, but miR-124 inhibitors produced the contrary effects. n = 12 in sham group, n = 12 in other three groups. ∗ p < 0.05 versus sham group, #p < 0.05 versus TBI group.

/WebMaterial/ShowPic/1496094Upregulation of MiR-124 Prompted M2 Polarization of BV2 Microglia and Inhibited Neuroinflammation

In order to confirm the effect of miR-124 on neuroinflammation mediated by microglia, in vitro experiments were performed to investigate whether miR-124 can regulate the polarization of microglia and the expression of inflammatory factors. As presented in Figure 4, LPS stimulation boosted the number of M1 (CD68+) and M2 (CD206+) microglial cells, as well as prompted the expression of pro-inflammatory cytokines IL-1β, IL-6, TNF-α and anti-inflammatory cytokines IL-4, IL-10, TGF-β, indicating M1 and M2 microglia were both activated by LPS. Furthermore, transfection with miR-124 mimics declined the number of CD68+ microglial cells but elevated the number of CD206+ microglial cells, while transfection with miR-124 inhibitors achieved the opposite effect. Transfection with miR-124 mimics prompted the expression of anti-inflammatory cytokines IL-4, IL-10, TGF-β but inhibited the expression of pro-inflammatory cytokines IL-1β, IL-6, TNF-α, while transfection with miR-124 inhibitors achieved the opposite effect. In all, these results indicated that upregulation of miR-124 prompted M2 polarization of microglia and inhibited inflammation.

Fig. 4.

Upregulation of miR-124 prompted M2 polarization of BV2 microglia and inhibited neuroinflammation. a, b LPS stimulation boosted the number of M1 (CD68+) and M2 (CD206+) microglial cells; transfection with miR-124 mimics declined the number of CD68+ microglial cells but elevated the number of CD206+ microglial cells, while transfection with miR-124 inhibitors achieved the opposite effect. c LPS stimulation prompted the expression of pro-inflammatory cytokines IL-1β, IL-6, TNF-α and anti-inflammatory cytokines IL-4, IL-10, and TGF-β; transfection with miR-124 mimics prompted the expression of anti-inflammatory cytokines IL-4, IL-10, TGF-β but inhibited the expression of pro-inflammatory cytokines IL-1β, IL-6, TNF-α, while transfection with miR-124 inhibitors achieved the opposite effect. n = 6 in each group, ∗ p < 0.05 versus control group, #p < 0.05 versus LPS group.

/WebMaterial/ShowPic/1496093Upregulation of MiR-124 Inhibited the Activation of TLR4 Pathway after TBI

To explore the mechanism of miR-124 regulating the expression of signature genes of M1/M2 microglia and pro-/anti-inflammatory chemokines after TBI, the miR-124 mimics or inhibitors were administrated to regulate the expression of miR-124 as described above. Then, the expression of TLR4 and its downstream molecules MyD88/IRAK1/TRAF6/NF-κB p65 was measured by WB. Results indicated that TBI induced the activation of TLR4 pathway, as the expression of TLR4, MyD88, IRAK1, TRAF6, and NF-κB p65 was elevated in the injured brain (Fig. 5a–e). MiR-124 mimics significantly decreased the expression of TLR4 pathway molecules, while miR-124 inhibitors significantly increased the expression of these molecules (Fig. 5a–e). These results implied that upregulation of miR-124 promoted the expression of an M2 profile and reduced neuroinflammation after TBI by inhibiting TLR4 pathway.

Fig. 5.

Upregulation of miR-124 inhibited the activation of TLR4 pathway after TBI. ae TBI increased the expression of TLR4 and its downstream molecules MyD88/TRAF6/IRAK1/NF-κB p65. MiR-124 mimics reduced the expression of these proteins, but miR-124 inhibitors produced the contrary effects. n = 12 in sham group, n = 12 in other three groups. ∗ p < 0.05 versus sham group, #p < 0.05 versus TBI group.

/WebMaterial/ShowPic/1496092MiR-124 Targeted TRAF6

Above experiments in vivo indicated that upregulation of miR-124 promoted the expression of an M2 microglia profile and reduced neuroinflammation after TBI by inhibiting TLR4 pathway. Furthermore, in vitro experiments were performed to explore the exact target molecule of miR-124 on TLR4 pathway. First, bioinformatics analysis showed that the target molecule of miR-124 on TLR4 pathway might be TRAF6. And, LPS elevated the expression of TRAF6 in microglial cells and upregulation of miR-124 reduced the expression of TRAF6, while downregulation of miR-124 elevated the expression of TRAF6 (Fig. 6b), indicating that miR-124 might target TRAF6. Second, MiR-124 and TRAF6 had latent binding sites on the website https://starbase.sysu.edu.cn (Fig. 6c). Third, WT-TRAF6 and miR-124 mimics impaired the luciferase activity after cotransfection, while MUT-TRAF6 and miR-124 mimics exerted no influence on the luciferase activity (Fig. 6d). Moreover, RIP assay found the enrichment of TRAF6 and miR-124 after Ago2 treatment (Fig. 6e). In brief, these results indicated that miR-124 could target TRAF6.

Fig. 6.

MiR-124 targeted TRAF6. a, b LPS elevated the expression of TRAF6 in microglial cells, upregulation of miR-124 declined the expression of TRAF6, while downregulation of miR-124 elevated the expression of TRAF6. c MiR-124 and TRAF6 had the latent binding sites. d The dual-luciferase reporter activity examinations of the targeting of miR-124 with TRAF6. e RNA-immunoprecipitation test of the enrichment of miR-124 with TRAF6 in Ago2 magnetic beads. n = 6 in each group, ∗ p < 0.05 versus control group, #p < 0.05 versus LPS group.

/WebMaterial/ShowPic/1496091Upregulation of MiR-124 Reduced the Neurological Deficit after TBI

Above experiments demonstrated that the upregulation of miR-124 promoted M2 polarization of microglia and reduction of neuroinflammation after TBI. To further investigate the effect of upregulated miR-124 on the neurological deficit after TBI, NSS, and MWM tests were conducted to measure the neurobehavioral and neurocognitive functions after TBI. In neurobehavioral assessment, the severity score of TBI mice was higher than that of Sham mice (Fig. 7a). The miR-124 mimics injection obviously improved this condition in TBI mice, while the miR-124 inhibitors injection produced the contrary effect (Fig. 5a). In neurocognitive evaluation, the escape latency to find the hidden platform of TBI mice was longer than that of Sham mice (Fig. 7b). The miR-124 mimics’ injection significantly reduced the latency in TBI mice, while the miR-124 inhibitors’ injection produced the contrary effect (Fig. 5b). The platform crossing time and target quadrant route of TBI mice was less than that of Sham mice (Fig. 7c, d). The miR-124 mimics’ injection significantly increased the two indexes in TBI mice, while the miR-124 inhibitors’ injection produced the contrary effect (Fig. 7c, d). These results indicated that the upregulation of miR-124 reduced the neurological deficit after TBI.

Fig. 7.

Upregulation of miR-124 reduced the neurological deficit after TBI. a, b The NSS score and escape latency was higher in TBI group than in sham group, miR-124 mimics reduced these two score, but miR-124 inhibitors increased these two score. c, d Rats in the TBI group presented shorter platform crossing times and less target quadrant route than the sham group; miR-124 mimics increased the route and crossing times, but miR-124 inhibitors reduced the route and crossing times. n = 12 in sham group, n = 12 in other three groups. ∗ p < 0.05 versus the sham group, #p < 0.05 versus the TBI group.

/WebMaterial/ShowPic/1496090Discussion

In the present study, the effect and mechanism of miR-124 on the polarization of microglia after TBI was investigated in vivo and in vitro. The addressed issues included: (1) the expression level of miR-124 in the injured brain after TBI, (2) the effect of miR-124 on the M1/M2 polarization of microglia and neuroinflammation after TBI, (3) the effect of miR-124 on the TLR4 signaling pathway after TBI, (4) the exact target molecule of miR-124 on TLR4 pathway, (5) the effect of miR-124 on the neurological deficit after TBI. In vivo experiments indicated that the expression of miR-124 was downregulated after TBI, upregulation of miR-124 promoted the M2 polarization of microglia and inhibited the activity of TLR4 pathway, as well as reduced neuroinflammation and neurological deficit after TBI. In vitro experiments indicated that upregulation of miR-124 promoted M2 polarization of microglia and reduced neuroinflammation by inhibiting TRAF6. This study indicated a new strategy to reduce the neuroinflammation and improve the outcome of TBI.

Accumulating evidences have shown that microglia played an important role in the central nervous system [2426]. In brain injury, cerebral ischemia, and cerebral hemorrhage, microglia could activate, proliferate, and express signaling molecules and cytokines to induce secondary brain damage [27, 28]. Therefore, restrain the excessive activation of microglia and reduce the secretion of cytokine might be a new strategy to treat TBI. Microglia show the dual role of pro-inflammatory and anti-inflammatory, which are characterized by M1/M2 polarization [2931]. M1 microglia produce high level of pro-inflammatory factors, whose effects are not only to eliminate the invasion of pathogenic microorganisms but also to cause the normal cell and tissue damage. M2 microglia secrete high level of anti-inflammatory factors, whose roles are immune suppression, such as the inhibition of M1 reaction, immune regulation, tissue repair, and functional remodeling [32, 33]. Numerous studies have found that the expression of miRNA was altered in different polarization of microglia, and some miRNAs regulated the polarization of microglia. For instance, miR-155 and miR-146a are involved in the M1 polarization of microglia, and their expression is promoted by IFN-γ or LPS stimulation [34]. MiR-124 has been regarded as the most abundant miRNA in the cerebral, which has been reported to decrease in ischemic stroke and intracerebral hemorrhage [19, 23]. However, the expression and the role of miR-124 in microglia polarization after TBI is less studied. Therefore, miR-124 was chosen to study in this experiment.

In this research, the experimental TBI model was first constructed, and the expression of miR-124 in the injured brain was measured. Results showed that the expression of miR-124 in TBI group was much lower than that in control group, indicating TBI induced the downregulation of miR-124. Next, the role of miR-124 in M1/M2 polarization of microglia was investigated by analyzing the signature genes of M1/M2 microglia and the pro-/anti-inflammatory cytokines. In this experiment, miR-124 mimics or inhibitors were injected into the ventricle, and the injured brain was collected and investigated after 3 days. Results showed that miR-124 mimics obviously decreased the expression of signature genes of M1 microglia and pro-inflammatory cytokines, but significantly increased the expression of signature genes of M2 microglia and anti-inflammatory cytokines, while miR-124 inhibitors produced the contrary effects. Moreover, in vitro experiments demonstrated that miR-124 mimics decreased the proportion of M1 microglia and the expression of pro-inflammatory cytokines but increased the proportion of M2 microglia and the expression of anti-inflammatory cytokines, while miR-124 inhibitors produced the contrary effects. These data indicated that miR-124 attenuated M1 polarization and promoted M2 polarization of microglia after TBI. In addition, we also performed experiments to clarify the therapeutic effect of upregulation of miR-124. Results indicated that upregulation of miR-124 effectively improved the neurologic outcome. Thus, upregulating miR-124 might be a new therapeutic approach for TBI.

TLR4 is highly expressed on the microglia, which might be activated after brain injury and plays a key role in regulating the innate immunity and inflammatory response [35]. The downstream mechanisms of TLR4 involve two parallel signaling pathways, the MyD88 pathway and the TRIFs pathway, both which ultimately activate NF-κB [36]. Many studies have reported that TLR4 was critical for TBI-induced inflammation in the MyD88 pathway, which ultimately induced the activation of NF-κB and subsequent production of pro-inflammatory cytokines [37, 38]. MiR-124 has been known to maintain microglia in a quiescent state and promote the transition of microglia from a pro-inflammatory to an anti-inflammatory phenotype [16, 39]. One study has indicated that the expression of miR-124 was decreased by TLR4 activator LPS, and miR-124 overexpression was shown to inhibit LPS-induced microglia activation as well [16]. Another study has also demonstrated that overexpression of miR-124 inhibited the activation of microglia and reduced neuroinflammation by targeting TLR4 pathway [21]. These studies have indicated that TLR4 signaling pathway might play an important role in regulating the activation and polarization of microglia by miR-124. Our study demonstrated that TBI induced the activation of microglia, enhanced NF-κB nuclear translocation, and upregulated inflammatory factors, while upregulation of miR-124 reduced the expression of TLR4/MyD88/TRAF6/IRAK1/NF-κB and the production of inflammatory mediators. These data suggested that miR-124 might attenuate the activation of microglia by inhibiting TLR4 pathway. Furthermore, in vitro experiments were performed to explore the exact target molecule of miR-124 on TLR4 pathway. Dual-luciferase reporter assay and RIP assay indicated that miR-124 could target TRAF6. Bringing the result of in vivo and in vitro together, it can be concluded that miR-124 promoted the M2 polarization of microglia and reduced neuroinflammation after TBI by inhibiting TRAF6.

Conclusion

In conclusion, this study indicated that the expression of miR-124 was downregulated after TBI. Upregulation of miR-124 promoted the M2 polarization of microglia and inhibited the activity of TLR4 pathway, as well as reduced the neuroinflammation and neurological deficit after TBI. Furthermore, miR-124 promoted the M2 polarization of microglia and reduced neuroinflammation by inhibiting TRAF6. This research implicated the role of miR-124 in regulating microglia-mediated neuroinflammation through targeting TRAF6 on the TLR4 pathway, thereby indicating a new strategy to reduce the neuroinflammation and improve the outcome of TBI.

Statement of Ethics

This study protocol was reviewed and approved by the Animal Experimental Ethical Inspection Committee of Fourth Military Medical University, Xi’an, Shanxi, China (2020601).

Conflict of Interest Statement

The authors declare no conflict of interest.

Funding Sources

The present work was supported by grants from the National Natural Science Foundation of China (82101465 to Yongxiang Yang), Natural Science Foundation of Sichuan (2022NSFSC1444 to Yongxiang Yang), Natural Science Basic Research Program of Shaanxi Province (2022JM-437 to Yuqin Ye), China Postdoctoral Science Foundation (2020T130789 to Yuqin Ye), and the Hospital Foundation of General Hospital of Western Theater Command (2021-XZYG-A13 to Yuan Ma).

Author Contributions

Yongjian Yang and Yuan Ma designed the experiments and guided the writing of this article. Yongxiang Yang and Yuqin Ye were responsible for performing a part of the experiments and writing the manuscript. Kexia Fan and Jianing Luo performed a part of the experiments and analyze the data. Authors included in this article agreed with the final manuscript.

Data Availability Statement

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding authors.

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