SHED-Derived Exosomes Regulate Microglial Polarization in the Treatment of Traumatic Brain Injury in Rats via miR-330-5p

Background: Traumatic brain injury (TBI) causes structural damage and impairs motor and cognitive function of the brain. Our previous study suggested that exosomes (EXs) secreted by stem cells from human exfoliated deciduous teeth (SHED) extenuated motor damage in TBI rats by regulating microglia. The molecular mechanism of SHED-EXs was investigated in the present study. Methods: The miRNA array was performed to determine the differential miRNA expression in SHED-EXs treating microglia. The key miRNA was selected. Flow cytometry, immunouorescence, enzyme linked immunosorbent assay (ELISA) and Griess assay were performed to detect the function of key miRNA. Real-time PCR, Western blotting and dual luciferase reporter assay were used to conrm the relationship between key miRNA and the target gene. Chromatin immunoprecipitation (ChIP) was performed to determine the downstream pathway of EXs-miRNA. Traumatic brain injury rat model was established and local injection of EXs-miRNA was performed to evaluate the effect. Results: SHED-EXs delivery of miR-330-5p was the key in the regulation of microglia polarization by inhibiting M1 polarization and promoting M2 polarization. Mechanistically, miR-330-5p had an inhibitory effect on Ehmt2, and miR-330-5p/Ehmt2 promoted the transcription of CXCL14 through H3K9me2. In vivo data showed that SHED-EXs/miR-330-5p reduced neuro-inammation and repaired neurological function of TBI rats. Conclusions: SHED-EXs/miR-330-5p improved the motor function of rats after TBI by inhibiting M1 polarization and promoting M2 polarization of microglia through Ehmt2/H3K9me2/CXCL14 pathway.


Introduction
Traumatic brain injury (TBI) refers to local anatomical and pathological structural damage in the brain due to trauma, which can cause hemiplegia, aphasia, mental retardation, or even coma and death [1][2][3]. Currently, surgery and drugs are commonly used as symptomatic treatments. However, these approaches can only control intracranial pressure and maintain blood pressure in patients, whereas their overall e cacies in altering prognoses are poor [4]. A large volume of experimental studies have shown that transplantation of stem cells from multiple sources can improve the function of damaged neurons in animal models of TBI [5][6][7][8][9][10]. More importantly, stem cells from human exfoliated deciduous teeth (SHED) represent a promising cell type to restore the function of damaged neurons since they express neuronal surface markers, have the ability to differentiate into neurons, and promote neuronal proliferation [11][12][13]. However, the therapeutic effect and potential mechanism remains to be clari ed.
In recent years, more and more studies have shown that paracrine signaling is a key mechanism by which stem cell transplantation carries out its effects. Furthermore, exosomes (EXs) are important components during this process [14]. After secretion by donor cells, they are internalized by recipient cells and biological molecules within are released, thereby improving the biological functions of recipient cells [15,16]. EXs are mixed vesicles that consist of different components. In addition to characteristic proteins and lipids in cellular membranes, it is noteworthy that these vesicles carry a class of nucleic acids, namely microRNAs (miRNAs), that can inhibit target genes in recipient cells to regulate their functions [17,18].
Our previous study showed that SHED can signal to microglia via secretion of EXs to regulate the polarization and repair neuronal damage resulting from TBI in rats [19]. Therefore, in the present study, we employed microarrays, real-time PCR, immuno uorescence, ow cytometry, chromatin immunoprecipitation, and animal experiments to determine the key substances by which SHED exosomes (SHED-EXs) carry out their effects in ameliorating TBI, as well as to explore their downstream effector mechanisms. Collectively, our ndings may provide theoretical and experimental bases for the application of SHED and their secreted exosomes in TBI treatment and other future clinical applications.

SHED-EXs isolation
Immortalized TERT-SHED were used in the present study as in our previous report [19]. Dulbecco's modi ed Eagle's medium (DMEM) containing 1% penicillin/streptomycin (all from Gibco, USA) was used to culture TERT-SHED for 48 hours. Culture medium was collected for EXs isolation. ExoQuick-TC Exosome Precipitation Solution (EXOTC50A-1-SBI, BioCat, Germany) was used according to the instructions of the manufacturer. miRNA microarray 1 µg/ml LPS stimulated BV-2 cells (obtained from Chinese Academy of Sciences) were co-cultured with 100 µg/ml SHED-EXs for 48 hours. LPS stimulated BV-2 and co-cultured BV-2 were obtained for RNA extraction. RNA samples were sent for miRNA microarray analysis (Genechem, Shanghai, China). Fold change of more than 2-fold (up-or downregulated) (p < 0.001) were chosen.

Real-Time PCR
Real-Time PCR were performed to validate array data. 1 µg/ml LPS stimulated BV-2 cells were co-cultured with 100 µg/ml SHED-EXs for 48 hours. LPS stimulated BV-2 and co-cultured BV-2 were obtained for RNA extraction. RNA was extracted with TRIzol regent ( Target prediction of miR-330-5p and luciferase reporter assay Target predication for miR-330-5p was carried out using miRDB, microRNA.ORG and TargetScan.
Intersection of the three database were obtained. Ehmt2 was chosen for its reported regulation of microglia development [20], so we speculated that it may also play an important role in the polarization of mature microglia. A recombinant plasmid containing Emmt2-Luc/Rluc was prepared. The binding site of miR-330-5p was completely mutated. Luciferase reporter assay was performed as previously described [21].
Brie y, a circular window with a diameter of 2 mm was drilled at the cortical motor function area. A 20 gram weight was used to vertically hit the injured area from 15 cm high. For SHED-EXs group, 3 µl SHED-EXs (400 µg/ml) was injected locally into the injury site. For miR-330-5p mimics group, 3 µl miR-330-5p mimics (100 nM) was used. Brain tissues were collected 48 hours later for cytokines and immunostaining studies. The recovery of motor dysfunction was evaluated after 48 hours, 7 days, 14 days and 21 days.

Statistical Analysis
A p value of less than 0.05 was considered statistically signi cant. Student's t test and one-way ANOVA were performed between two groups and among multiple groups.

Differentially expressed miRNAs in microglia after SHED-EXs treatment
Array data (Fig. 1A) showed that 27 miRNAs were upregulated and 39 miRNAs were downregulated after SHED-EXs was added to LPS-stimulated BV-2 cells, for which a two-fold change or more was used as the cutoff criterion for consideration of signi cantly altered miRNAs. We selected the most differentially expressed 10 miRNAs (6 up-regulated and 4 down-regulated miRNAs) and con rmed their expression by Real-time PCR (Fig. 1B, C). We found that changes in miR-330-5p expression were most signi cantly different. We further quantitated miR-330-5p expression after LPS stimulation of BV-2 cells and found that miR-330-5p expression was signi cantly downregulated (Fig. 1D). After addition of the EXs inhibitor, GW4869, no signi cant difference in miR-330-5p expression was shown compared with that of the LPS stimulation group (Fig. 1E). In summary, LPS stimulation signi cantly downregulated miR-330-5p in microglia. Furthermore, SHED-EXs delivery rescued miR-330-5p expression and may play a role as a key regulator in microglial function.

miR-330-5p inhibits microglial M1 polarization and promotes M2 polarization
In order to elucidate the speci c function of miR-330-5p in microglia, miR-330-5p was rst upregulated in BV-2 cells. Transfection e ciency was measured 48 h after transfection with miR-330-5p mimics ( Fig. 2A). LPS stimulation of BV-2 cells increased the secretions of the pro-in ammatory factors IL-6 and TNF-α while miR-330-5p overexpression signi cantly reduced the secretions of these two cytokines. In addition, LPS stimulation decreased the secretion of the anti-in ammatory factor IL-10 while miR-330-5p overexpression signi cantly rescued the secretion (Fig. 2B). Furthermore, Griess assay was used to examine the effects of miR-330-5p overexpression on nitrite concentrations. LPS stimulation signi cantly increased nitrite concentrations while miR-330-5p overexpression signi cantly decreased these levels (Fig. 2C). Next, ow cytometry and immuno uorescence were used to determine changes in M1-and M2polarization markers in BV-cells. Results showed that the expression of the M1 polarization marker CD68 was signi cantly increased while the expression of the M2 polarization marker CD206 was signi cantly decreased in BV-2 cells after LPS stimulation. In contrast, transfection with miR-330-5p mimics signi cantly downregulated CD68 expression and signi cantly upregulated CD206 expression (Fig. 2D-G). Real-time PCR results showed that the expression of the M1 polarization markers-CD11b, CD86, CD16, and MHCII-were signi cantly increased, while the expression levels of the M2 polarization markers, IL-10 and ARGINASE1, were signi cantly decreased after BV-2 cells were stimulated with LPS. miR-330-5p overexpression signi cantly inhibited M1 polarization marker expression and promoted M2 polarization marker expression (Fig. 2H, I). Collectively, miR-330-5p inhibited M1 polarization and promoted M2 polarization. Appendix File 1 shows the effects of miR-330-5p inhibitors on BV-2 polarization.
miR-330-5p targets Ehmt2 to promote CXCL14 transcription through H3K9me2 in the regulation of microglial polarization Next, we explored the downstream mechanism of miR-330-5p regulation. Three databases (Targetscan, microRNA.ORG, and miRDB) were used for target gene prediction of miR-330-5p and overlapping data from these three databases were obtained (Fig. 3A, Appendix File 2). From these data, we selected Ehmt2 -which is associated with microglia development as a candidate target gene [20]. Real-time PCR was used to measure changes in Ehmt2 expression after miR-330-5p was upregulated. Results showed that miR-330-5p signi cantly inhibited Ehmt2 expression (Fig. 3B). Western blotting was used to validate the effects of miR-330-5p upregulation on Ehmt2 protein levels. Results showed that Ehmt2 expression was decreased when miR-330-5p was upregulated (Fig. 3C, D). A luciferase reporter plasmid containing either wild-type or mutant Ehmt2 was constructed (Fig. 3E) according to its predicted binding site and was cotransfected with either miR-330-5p mimics or miR-330-5p inhibitors into 293T cells. The luciferase reporter assay was used to determine the relationship between miR-330-5p and Ehmt2 (Fig. 3F).
miR-330-5p affects microglial polarization to ameliorate traumatic brain injury in rats In order to determine whether SHED-EXs miR-330-5p has any in vivo effects, we established a rat model of TBI and used SHED-EXs and miR-330-5p for local-injection treatments at the wound site (Fig. 5A).
Motor function tests were conducted in TBI rats at 48 h, one week, two weeks, and three weeks after injury. The BBB scoring showed signi cant motor dysfunction in all groups at 48 h after TBI, except for the sham group. Motor functions in the SHED-EXs group and miR-330-5p-mimics group were signi cantly improved after one week (Fig. 5B). Sectioning of brain tissues and hematoxylin and eosin (H&E) staining were carried out in the different groups of rats at two weeks after TBI. Results showed signi cant tissue defects in brain tissues from the TBI and DMEM groups, whereas signi cant recovery in brain tissue injuries were seen in the SHED-EXs group and miR-330-5p mimics group (Fig. 5C). In ammation status was examined in the damaged brain tissues from different groups after 48 h of treatment. Results showed that the expression of the pro-in ammatory factors IL-6 and TNF-α were signi cantly lower in the SHED-EXs group and miR-330-5p mimics group compared with those in the TBI group and DMEM group, whereas the expression of the anti-in ammatory factor IL-10 was signi cantly increased (Fig. 5D, E).
Immuno uorescent staining was performed on brain tissue sections from the different groups. Results showed that the expression of the M1 polarization marker CD68 was signi cantly decreased in the SHED-EXs and miR-330-5p mimics groups, while the expression of the M2 polarization factor CD206 was signi cantly increased compared with those in the TBI group and DMEM group (Fig. 6).
Taken together, our present study found that miR-330-5p from SHED-EXs ameliorated functional impairment caused by TBI in rats. From a mechanistic perspective, TBI decreased miR-330-5p expression in microglia, and SHED-EXs delivery of miR-330-5p promoted M2 polarization and inhibited M1 polarization, thus reducing neuro-in ammation and promoting tissue repair through the downstream Ehmt2-H3K9me2-CXCL14 signaling axis (Fig. 7).

Discussion
Our in-vitro and in-vivo study demonstrated that SHED affected microglial polarization through miR-330-5p in exosomes to ameliorate neurological impairment caused by TBI in rats. In the present study, SHED was chosen to repair neurological damage. In addition to their potential clinical signi cance, our ndings suggest that we may consider the perspective of an embryonic origin of stem cells in the treatment of disease. SHED and other odontogenic stem cells, such as dental pulp stem cells (DPSCs), all originate from neural crest cells during the embryonic stage [24,25]. This may be the reason that the signals inside had better functionality. Therefore, odontogenic stem cells may be used as treatment substitutes for neural stem cells, the latter of which are ethically controversial to some extent. The application range of odontogenic stem cells was somehow expanded. From this perspective, future studies may focus on other types of odontogenic stem cells to detect their neuro-restorative functions, which may provide more possible sources of stem cells in the treatment of TBI.
In order to clarify the potential mechanism of SHED-EXs in regulating microglial polarization, we performed a miRNA array to detect differentially expressed miRNAs. Several studies have shown that stem-cell-derived EXs perform their biological functions on target cells by delivering speci c miRNAs. For example, the key to mesenchymal stem cell transplantation for myocardial infarction is in the miR-125b-5p contained within secreted EXs [26]. Bone marrow mesenchymal stem cells (BMSCs) exert their protective effects on ischemia-reperfusion via miR-199a-5p in their EXs [27]. BMSCs-derived EXs regulate age-related insulin resistance through the transfer of functional miR-29b-3p [28]. The present study showed that compared with BV-2 after LPS activation, the addition of SHED-EXs caused a signi cant upregulation of miR-330-5p expression in activated BV-2. We hypothesize that miR-330-5p was the key constituent in SHED-EXs that was effectively transferred to microglia. miR-330-5p expression was signi cantly down-regulated in BV-2 after LPS activation, suggesting that the effects of external stimuli on microglia may be mediated by miR-330-5p.
It has been reported that M2 polarization of microglia, which is bene cial for recovery, depends on the STAT6-mediated IL-4/IL-13 signaling pathway [29]. In a mouse autoimmune encephalomyelitis model, deletion of IL-4 inhibits M2 polarization and aggravates disease-related damage. In addition, the TGF-β1 pathway is active during M2 polarization [30]. Transcription factors such as NF-kB, C/EBP-β, and Notch/RBP-J also have important regulatory effects on the polarization of microglia [31][32][33]. In the present study, we demonstrated that Ehmt2 was responsible for the regulation of miR-330-5p in microglial polarization. Ehmt2 promoted M1 polarization, inhibited M2 polarization, and thus may also represent a potential target for TBI therapy.
Ehmt2 is a major histone methyltransferase that speci cally catalyzes H3K9 dimethylation [34]. H3K9me2 has a binding site for heterochromatin protein 1 (HP1), which recruits transcriptional repressors to prevent gene activation [35]. In the adult central nervous system, chemokines and their receptors participate in developmental, physiological, and pathological processes, promote cellular interactions and activate and maintain central-nervous-system homeostasis [36]. CXCL14 is one of the oldest chemokines, but its function is not well known [37]. CXCL14 regulates hippocampal integrity and maintains balance in the adult brain. Therefore, the imbalance of CXCL14 can seriously affect the homeostasis of the nervous system. Our present study found that miR-330-5p inhibited the enrichment of Ehmt2 and H3K9me2 in the CXCL14 promoter region, thereby promoting the transcription of CXCL14, suggesting that the mechanism by which CXCL14 maintains nervous-system stability may be closely related to microglial polarization.
Cell-cell interactions are the primary mechanisms during stem cell therapy. Stem cells may directly secrete cytokines to remotely regulate the functions of recipient cells or indirectly carry out their functions through EXs [38,39]. However, a continuous supply of effective factors is required for long-term treatment results [40]. In the present study, the effect of SHED-EXs/miR-330-5p was determined by a one-time injection, suggesting an e cacy "memory" mechanism of SHED-EXs/miR-330-5p transplantation.
Histone methylation-mediated epigenetic regulation may produce long-term effects as a stable regulatory factor.
Our present study demonstrated that SHED-EXs/miR-330-5p repaired motor function of rats with TBI. Hence, SHED-EXs/miR-330-5p is expected to represent a new strategy for treating TBI. Moreover, miR-330-5p and SHED-EXs could be combined to play their corresponding advantages in the system, thereby creating a new method for treating TBI. In addition, traditional drugs cannot cross the blood-brain barrier to function at the injury site. Due to the nanoscale size of SHED-EXs, future studies using intravenous injections of SHED-EXs are needed to determine their potential e cacies.
Conclusions SHED-EXs delivery of miR-330-5p ameliorates functional impairment caused by TBI by promoting M2 polarization and inhibiting M1 polarization to reduce neuro-in ammation and promote tissue repair through the Ehmt2-H3K9me2-CXCL14 axis.