Astrocyte-Derived Exosomal miR-148a-3p Suppresses Neuroinflammation and Restores Neurological Function in Traumatic Brain Injury by Regulating the Microglial Phenotype

Interactions between astrocytes and microglia play an important role in the regeneration and repair of traumatic brain injury (TBI), and exosomes are involved in cell–cell interactions. A TBI model was constructed in rats. Brain extract (Ext) was isolated 1 d after TBI. Astrocyte-derived exosomes were obtained by coculturing Ext with primary astrocytes, and the morphology of exosomes was observed by electron microscopy. The isolated exosomes were cocultured with microglia to observe phenotypic changes in M1 and M2 markers. Aberrant RNA expression was detected in necrotic brain tissue and edematous brain tissue. The role of miR-148a-3p in regulating microglial phenotype was explored by knocking down or overexpressing miR-148a-3p. Finally, the effect of miR-148a-3p on TBI was studied in a rat TBI model. Astrocyte-derived exosomes stimulated by Ext promoted the transition of microglia from the M1 phenotype to the M2 phenotype. MiR-148a-3p was highly expressed in TBI. Transfecting miR-148a-3p promoted the transition of microglia from the M1 phenotype to the M2 phenotype and inhibited the lipopolysaccharide-induced inflammatory response in pre-microglia. In a rat TBI model, miR-148a-3p significantly improved the modified neurological severity score and attenuated brain injury, which promoted the transition of microglia from the M1 phenotype to the M2 phenotype. MiR-148a-3p alleviated TBI by inhibiting the nuclear factor κB pathway. Astrocyte-derived exosomal miR-148a-3p regulates the microglial phenotype, inhibits neuroinflammation, and restores neurological function in TBI. These results provide new potential targets for the treatment of TBI.


Introduction
Traumatic brain injury (TBI) is the leading cause of death and disability among adolescents worldwide.It is an externally induced alteration in brain function that is characterized by the loss or impairment of consciousness, loss of memory before and after injury, neurological deficits (e.g., weakness, loss of balance, and altered vision), or mental disturbances such as confusion, disorientation, and slowed thinking (Menon et al., 2010;Peeters et al., 2015;Eme, 2017).Pathologically, brain injury is divided into primary and secondary injury.The inflammatory response, which is a pathological feature of the injured brain, is present in the chronic and acute stages of TBI, and it plays an important role in the transformation from primary to secondary injury (Shojo et al., 2010;Blennow et al., 2012).The inflammatory response is protective and a natural defense, but various stimuli and disordered immune effectors (including proinflammatory cells and chemokines) typically activate neuroinflammation and exacerbate the pathological progression of tissue damage (Piao et al., 2012;Faden and Loane, 2015;Chiu et al., 2016;Simon et al., 2017).Therefore, suppressing excessive inflammation is essential for improving neurological function after TBI.
Macrophages in the central nervous system and microglia are key cells that regulate immune responses to maintain brain homeostasis, normally exhibiting a neuro-specific phenotype (Schmid et al., 2009) and maintaining a relatively quiescent surveillance phenotype to detect brain parenchyma (Davalos et al., 2005;Nimmerjahn et al., 2005).After TBI, microglia in the physiological state are rapidly activated and migrate to the site of injury.Activated microglia produce and release inflammatory mediators and affect peripheral immune cells, including neutrophils, lymphocytes, and macrophages, and the interaction between cells and inflammatory mediators leads to peripheral neuronal overreaction, which greatly exacerbates TBI (Chiu et al., 2016).Recent studies have shown that microglia exert dual effects on TBI.Microglia exhibit an M1 proinflammatory phenotype or an M2 anti-inflammatory phenotype after TBI and release growth factors that repair injured tissue (Harry, 2013;Loane and Kumar, 2016).Notably, modulating the polarization of microglia, especially by promoting M2 phenotypic conversion after TBI, may be a new therapeutic strategy to suppress the excessive inflammatory response after TBI and improve neurological function.
Exosomes are membrane vesicles (40-150 nm in diameter) secreted by various cell types, and there is growing evidence that exosomes act as intercellular messengers to regulate cellular function through an intercellular transfer of biologically active molecules such as proteins, RNA, and miRNA (Colombo et al., 2014;Sluijter et al., 2014;Quek and Hill, 2017).Human bone marrow mesenchymal stem cell-derived exosomes attenuate ischemic reperfusion injury (Liu et al., 2021) and demyelination in the central nervous system (Zhang et al., 2022) during spinal cord injury (Fan et al., 2021). .The induction of neuroinflammation and apoptosis is achieved by modulating the M1/M2 phenotype of microglia, which can also suppress neuroinflammation.Astrocytes and microglia actively participate in various pathological conditions, including TBI.Activated astrocytes produce many regulatory factors that affect CNS immunity and provide negative feedback to activated microglia (Min et al., 2006;Farina et al., 2007).Astrocyte recruitment was impaired in a TBI mouse model when the Cdc42 gene was deleted, and an increase in the number of microglia was observed in regions with a reduced astrocyte population (Robel et al., 2011).Furthermore, astrocytes may act as controllers to rapidly inhibit microglial activation in the injured brain (Kim et al., 2010).Astrocyte-derived exosomes are powerful mediators of neuronal plasticity, immune responses, and neuronal survival in a variety of pathological conditions (Lafourcade et al., 2016).Astroglial-derived exosomes play an important role in protecting neurons from oxidative stress, including the regeneration and repair of TBI or related diseases, but little is known about the mechanisms by which they activate microglia and their role in mediating neuroinflammation after TBI.
The present study investigated the effect of astrocyte-derived exosomes on microglial phenotypes in an in vitro TBI model and examined exosome-encapsulated functional miRNAs in TBI rats in vivo.The results provide new support for astrocyte-mediated regulation of microglia and a potential therapeutic target for neuroinflammation in brain injury via this regulatory mechanism.

Materials and Methods
TBI animal model and experimental groupings.Male Sprague Dawley (SD) rats (age, 8-10 weeks; body weight, 220-250 g) were purchased from Hunan SJA Laboratory Animal.The animals were housed in a controlled environment (temperature, 20-25°C, 12 h light/dark cycle), and the TBI model was induced according to a previous report (Jassam et al., 2017).Briefly, deformational brain injury was induced via craniotomy in previously anesthetized animals using pneumatic or electromechanically controlled cortical impact (CCI) devices.All rats in the TBI group showed loss of motor ability in the injured hindlimb, unilateral hemiplegia, and decreased muscle tone below the injury plane.The injured hind limbs did not respond to acupuncture, and the animals could not walk in a straight line, which indicated that TBI modeling was successful.The rats were randomly divided into four groups: the sham group (n = 20), TBI group (n = 20), TBI + miR-148a-5p agomir group (n = 20), and TBI + miR-148a-5p antagomir group (n = 20).On Days 1, 3, and 7, the rats were killed (n = 6), tissue around the traumatic area of the injured brain was collected, and similar areas were collected from the sham rats for subsequent experiments.The cortex was collected for immunofluorescence analysis, qRT-PCR, and Western blotting.All animal experimental protocols were approved by the Animal Experiment Ethics Committee of Kunming Medical University, and the animal procedures adhered to the ARRIVE guidelines 2.0.
Primary microglia and astrocyte culture.Primary microglia and astrocytes were obtained and purified from postnatal SD rats (Long et al., 2020).Briefly, newborn SD rats were anesthetized via passive inhalation of 2% isoflurane, and the cerebral cortex was collected and sectioned after the rats were decapitated.The tissue was dissociated in trypsin-EDTA (0.25%; Beyotime), filtered, and centrifuged.The supernatant was discarded, and the cells were added to culture flasks supplemented with a medium containing 10% fetal bovine serum.After 8-10 d of incubation, microglia were removed from the astrocyte monolayer by shaking at 220 rpm for 40 min.The medium was added to 24-well plates and shaken continuously at 220 rpm for 18 h to remove oligodendroglia, and the remaining cells in the flask were astrocytes with >90% purity.Astrocytes and microglia were collected for a subsequent experiment.
Brain extract preparation.Brain extract (Ext) was prepared as previously described (Chen et al., 2002).Briefly, TBI was induced in the rats, and the cortex was collected 1 d later.The collected cortical tissues were placed in glass tubes containing a complete culture medium.Glass grinding rods were used, and the ground tissue was centrifuged (1,000 rpm for 10 min).The supernatant was collected and stored in 1.5 ml electropolished tubes at −80°C.
Isolation of astrocyte-derived exosomes and transmission electron microscopy.Primary astrocytes were incubated with TBI brain extract (10 µg/ml) for 48 h.As described in the literature (Xin et al., 2012), supernatants collected from cultured astrocytes were centrifuged at 4°C (3,000 rpm for 10 min) to remove suspended cells or debris, and the supernatant was stored at 4°C for use as a blank exosome control.Samples were homogenized on a vortex shaker and centrifuged at 100,000 × g for 60 min at 4°C to obtain exosome pellets.The pellets were resuspended in 50 µl of PBS and stored at −80°C for use.Exosome morphology was observed by transmission electron microscopy (JEM-1200EX, JEOL).
Exosome labeling and uptake.Exosomes secreted from astrocytes were fluorescently labeled using the lipophilic dye PKH26 (Sigma-Aldrich).Exosomes were resuspended in diluent solution and PKH26 and incubated at room temperature for 5 min.PKH26-labeled exosomes were diluted with PBS and ultracentrifuged at 150,000 × g at 4°C for 60 min to remove the unbound dye.PKH26-labeled exosomes were incubated with primary microglia for 24 h.Then, the cells were fixed and stained with DAPI.The cells were visualized using laser scanning confocal microscopy (DMi8, Leica), and photographic images were obtained.
Neurological function score.TBI rat neurological severity score [modified neurological severity score (mNSS)] assessments were performed before CCI and on Days 1, 3, and 7 after CCI.The severity scores were graded on a scale from 0 to 18, where 0 represented the normal score and 18 represented the maximum deficit score.mNSS assessments included muscle status; abnormal movements; visual, tactile, and proprioceptive sensations; reflex responses; and balance tests.Higher scores indicated more severe injury.
Determination of brain water content.According to the reference (Wei et al., 2015), the animals were killed 7 d after TBI, and brain tissue was collected and weighed (wet weight).Brain tissues were dried at 80°C for 72 h and weighed again (dry weight).Brain water content (%) was calculated using the following formula: (wet weight − dry weight)/wet weight × 100%.
qRT-PCR.Total RNA was extracted from microglia or tissues using TRIzol reagent (Invitrogen), and the concentration was determined.Real-time PCR was performed to analyze miRNA using kits and instruments according to the manufacturer's protocols.mRNA and miRNA expression were measured using SYBR Premix Ex Taq TM II (Takara) on an ABI-Prism 7500 real-time PCR system (Applied Biosystems).Gene expression was analyzed using the 2 −ΔΔCt method.All primer sequences are shown in Table 1.

Statistical analysis.
All experiments were performed in triplicate.Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software).The results are expressed as the mean ± standard deviation (SD).Two groups were compared with Student's t test, and three or more treatments or groups were compared with one-way ANOVA followed by Tukey-Kramer post hoc analysis.A p value of <0.05 was defined as statistically significant.

Results
Astrocytes release exosomes after stimulation with brain extracts Astrocyte-derived exosomes can mediate TBI.We examined the expression of exosomes using immunofluorescence staining and Western blotting, and the results showed a significant increase in the expression of CD9 and CD63 after stimulation with TBI brain extract (Fig. 1A,B).Exosomes were isolated from astroglia and observed by using electron microscopy.TEM imaging showed that exosomes derived from astrocytes had a typical cup-shaped morphology and were within the range of 100 nm (Fig. 1C).The exosome markers CD9 and CD63 were also detected (Fig. 1D).

TBI-injured astrocyte-derived exosomes are taken up by primary microglia and promote M2 microglial polarization
To investigate the effect of astrocyte-derived exosomes on microglial phenotype, microglia were treated with TBI-injured astrocyte exosomes.PKH26-labeled exosomes were successfully taken up by microglia (Fig. 2A).We detected Iba-1 (microglia marker) and Arg-1 (M2 marker) by using immunofluorescence; the results showed that exosomes from astrocytes could significantly promote the conversion of microglia to the M2 phenotype, and exosomes from astrocytes could further promote this transformation compared with astrocyte-derived exosomes from normal sources of brain tissue extract (Fig. 2B-F).These data suggested that astrocyte-derived exosomes promoted M2 microglial polarization.

Effect of miR-148a-3p on LPS-induced primary microglia
To further examine the function of miR-148a-3p, a miR-148a-3p mimic or miR-148a-3p inhibitor was transfected into primary microglia, which were then stimulated with LPS (1 µg/ml, 24 h).MiR-148a-3p expression was measured using qRT-PCR to show successful transfection of the miR-148a-3p mimic or miR-148a-3p inhibitor (Fig. 4A).Western blot and qRT-PCR were used to detect the expression of IL-1β, IL-6, TNF-α, and iNOS.The results showed that compared with the LPS group, the LPS + miR-873a-5p mimic group had significantly reduced expression of the proinflammatory factors IL-1β, iNOS, TNF-α, and IL-6, while the LPS + miR-873a-5p inhibitor group had significantly increased expression of   these factors (Fig. 4B-F).Taken together, these results suggest that miR-148a-3p inhibits the expression of proinflammatory factors and promotes M2 phenotypic transformation in microglia.

MiR-148a-3p attenuates brain injury in rats after TBI
To identify the effects of miR-873a-5p on TBI, TBI was induced in rats by CCI, after which the miR-873a-5p agomir (mimic) or antagomir (inhibitor) was injected into the lateral ventricle.The expression of miR-148a-3p was detected using qRT-PCR 1, 3, and 7 d after TBI.The expression of miR-148a-3p was increased after TBI compared with that in the sham group and was gradually decreased 7 d after TBI.TBI rats treated with the miR-148a-3p agomir had sustained high miR-148a-3p expression.However, the miR-148a-3p antagomir significantly reduced the expression of miR-148a-3p (Fig. 5A).To investigate the neurorestorative effects of miR-148a-3p, we measured the extent of brain edema on Day 7 after TBI and determined the mNSS.The results showed that the area of brain injury, the degree of brain edema, and the mNSS score increased after TBI.However, on the 7th day after treatment with miR-148a-3p, the area of brain injury, the degree of brain edema, and the mNSS score were significantly decreased after TBI.The miR-148a-3p antagomir significantly aggravated brain injury (Fig. 5B-D).In addition, we evaluated ERK and NF-κB p65 expression to examine the effect of miR-148a-3p on the NF-κB signaling pathway after TBI.Brain tissue was collected 7 d after TBI.Compared with those in the sham group, the levels of phosphorylated ERK and NF-ΚB P65 were substantially increased in brain tissues collected from TBI rats.MiR-148a-3p inhibited the levels of phosphorylated ERK and NF-ΚB P65, and the miR-148a-3p antagomir exerted the opposite effects (Fig. 5E).
MiR-148a-3p suppresses the inflammatory response by promoting M2 polarization in microglia after TBI in rats To assess the effect on microglial polarization after TBI, the expression of M1 phenotypic marker genes (iNOS and CD32) and an M2 phenotypic marker gene (CD206) were examined.The qRT-PCR results showed that the expression of the M1 signature genes CD32 and iNOS and the expression of the M2 signature gene CD206 were significantly increased in the cortex after TBI.MiR-148a-3p agomir treatment significantly decreased CD32 and iNOS expression and increased CD206 expression, and miR-148a-3p antagomir treatment had the opposite effects (Fig. 6A-C).We also detected the expression of a microglial marker (Iba1), an M1 phenotypic marker (iNOS), and an M2 phenotypic marker (Arg1) by immunofluorescence analysis.Compared with that in the sham group, the expression of iNOS was increased in the TBI group.MiR-148a-3p agomir treatment inhibited the expression of iNOS, and miR-148a-3p antagomir treatment further increased the expression of iNOS (Fig. 6D).Compared with that in the sham group, the expression of Arg1 was downregulated in the TBI group.MiR-148a-3p agomir treatment increased the expression of Arg1, and miR-148a-3p antagomir treatment further inhibited the expression of Arg1 (Fig. 6E).These results indicate that treatment with miR-148a-3p promotes the conversion of microglia to the M2 phenotype.

Discussion
TBI is one of the most common neurological disorders with high mortality and morbidity (Dabin et al., 2022).However, there is still no clinically effective treatment to improve cognition or promote recovery, and it is not clear which factors contribute to cognitive recovery after TBI.This study explored the role of miR-148a-3p in TBI and the mechanism by constructing a rat model of TBI and isolating primary astrocytes and microglia in vitro.
Exosomes are novel diagnostic markers of human diseases and ideal drug carriers.A variety of cell-derived exosomes are involved in brain injury-related diseases (Jiang et al., 2019).Astrocytes and microglia play important roles in TBI.Astrocytes can robustly regulate microglial activation.Astrocyte-derived exosomal miR-92b-3p ameliorates oxygen glucose deprivation-induced apoptosis in neurons (Xu et al., 2019).Astrocyte-derived exosomal miR-7 leads to the downregulation of neuronal/glial 2 and ultimately to synaptic changes after HIV-1 infection (Hu et al., 2020).The present study isolated exosomes from brain extract-stimulated astrocytes in the context of TBI.TEM imaging showed that exosomes derived from astrocytes were mostly within the range of 100 nm.Microglia were converted to the M2 phenotype after treatment with exosomes.
MicroRNAs (miRNAs, approximately 20 nt) are small noncoding single-stranded RNA molecules that regulate target genes by binding to the 3 ′ untranslated region (3 ′ UTR).There is increasing evidence that microRNAs play an important role in TBI.For example, Huang et al. (2018) found that an increase in miR-124-3p in microglial exosomes inhibits neuronal inflammation and contributes to neurite outgrowth.MiR-124-enriched exosomes promoted M2 polarization in microglia and enhanced hippocampal neurogenesis after TBI by inhibiting the TLR4 pathway (Yang et al., 2019).In this study, we found that miR-148a-3p expression was upregulated in the necrotic area in comparison with the edematous brain.MiR-148a-3p was enriched in astrocyte-derived exosomes.Overexpression of miR-148a-3p inhibited the expression of proinflammatory factors and promoted M2 polarization in LPS-induced microglia.In vivo experiments exhibited similar results.
Microglia are resident immune cells in the central nervous system and the first responders to neurological changes (Kwon and Koh, 2020).The activation of microglia in transient focal cerebral ischemia may reflect the severity of injury (Emmrich et al., 2015) and is accompanied by morphological and phenotypic changes (Guruswamy and Elali, 2017).The M1 phenotype exerts proinflammatory effects, and the M1 phenotype-specific marker is ion calcium-binding bridging molecule 1 (Iba1) (Patel et al., 2013).The M2 phenotype exerts anti-inflammatory effects and has the specific markers arginase 1 (Arg-1), IL-10, and IL-4 (Estévez et al., 2006).The present study showed that astrocyte-derived exosomes inhibited microglial M2 phenotypic conversion and suppressed the inflammatory response.In TBI rats, the area of brain injury, the degree of brain edema, and the mNSS score were increased.However, on the 7th day after treatment with miR-148a-3p, the area of brain injury, the degree of brain edema, and the mNSS score were significantly decreased after TBI.In addition, the Western blot results showed that miR-148a-3p attenuated TBI by inhibiting the NF-κB pathway.
In conclusion, our study showed that astrocyte-derived exosomal miR-148a-3p promoted neurological recovery from TBI by suppressing the cellular inflammatory response and promoting M2 polarization in microglia.These findings provide a regulatory mechanism based on the interaction of astrocytes and microglia in the development of TBI, which may be a new target for the treatment of TBI.

Figure 1 .
Figure 1.Astrocytes release exosomes after stimulation with brain extracts.A, Exosomes derived from astrocytes were increased after brain extract stimulation (scale bar = 100 µm).B, The expression of the exosomal markers CD9 and CD63 in the medium of primary cultured astrocytes was detected by Western blotting after 24 h of brain extract treatment (n = 3, t test).C, A representative TEM image of exosomes in the culture medium of stimulated astrocytes after 24 h of brain extract treatment (scale bar = 100 nm).D, The expression of the exosomal markers CD9 and CD63 in astrocyte-derived exosomes was detected by Western blotting.Con, control; Ext, brain extracts.Compared with the Con group, *p < 0.05 and **p < 0.01.

Figure 3 .
Figure 3.The expression of miR-873a-5p in necrotic brain tissue and edematous brain tissue in TBI was measured by qRT-PCR.Brain tissue samples from necrotic and edematous areas were collected 3 d after TBI.Compared with the edema group, **p < 0.01 and ***p < 0.001, n = 3, t test.

Figure 6 .
Figure 6.MiR-148a-3p suppresses the inflammatory response by promoting M2 polarization in microglia after TBI.A-C, The expression of the M1 signature genes iNOS and CD32 and the M2 signature gene CD206 was detected by qRT-PCR in the rat cortex (n = 6, one-way ANOVA).D, Immunofluorescence analysis of the expression of the M1 microglial marker iNOS in the rat cortex (scale bar = 100 µm).E, Immunofluorescence analysis of the expression of the M2 microglial marker Arg1 in the rat cortex (scale bar = 100 µm).Compared with the sham group, ***p < 0.001.Compared with TBI, # p < 0.05 and ### p < 0.001.