Exosomes derived from bMSCs inhibit neuroinammatory after traumatic brain injury by inuencing the IL-10/STAT3 signaling pathway

Exosomes are important membranous vesicles in several physiological and pathological processes. It is postulated that the microRNAs contained in the exosomes play the major roles in these processes. Exosomal microRNAs from bone marrow mesenchymal stem cells (bMSCs) have emerged as promising therapeutic agents against inammation. However, its specic mechanism has not been claried. The present study was designed to elucidate the underlying mechanism of bone marrow mesenchymal stem cells derived exosomes on neuroinammation in vitro and in vivo. Methods: In vitro co-culture experiments were done to demonstrate the effects of bMSCs and bMSCs-derived exosomes on the polarization state of BV2 microglia cells. In vivo experiments were subsequently done to validate the role of exosomes derived from bMSCs in neuro-inammation after brain injury. MicroRNA sequencing was then done to reveal the potential expanded diversity between bMSCs and exosomes derived from bMSCs. The microRNAs were then screened and quantied by quantitative polymerase chain reaction (qPCR) to identify the most signicant miRNAs. Moreover, lentiviral transfection was performed to establish animal models with overexpressed and downregulated microRNA expression levels. The animal models were subsequently used to determine the effects of specic microRNAs on neuroinammation after TBI and their possible mechanisms of action. Results: Both bMSCs and exosomes derived from bMSCs promoted the polarization of activated BV2 microglia cells to the anti-inammatory phenotype. They inhibited the expression of pro-inammatory cytokines but promoted the expression of anti-inammatory cytokines. Exosomes derived from bMSCs played a stronger role in regulating the expression of inammatory factors. In vivo animal-based experiments further revealed that the exosomes reduced neuronal apoptosis in the cortical area. They also promoted inhibition of neuroinammation and transformation of microglia to anti-inammatory phenotype. In addition, MicroRNA sequencing and subsequent analysis revealed that microRNA-181b was actively involved in the process. Subsequent lentiviral transfection studies revealed that overexpression of miR181b effectively reduced neuronal apoptosis and neuroinammatory response after traumatic brain injury. Overexpression of microRNA181b also promoted the transformation of microglia to the anti-inammatory phenotype. These effects were achieved through activation of the IL-10/STAT3 pathway. Conclusion: Exosome derived from bMSCs promotes the polarization of microglia to anti-inammatory phenotype and inhibit neuroinammation both in vitro and in vivo. They also reduce neuronal apoptosis and protect damaged brain tissue. The microRNA-181b plays a vital role in this process by activating the IL-10/STAT3 pathway. Our study indicates that IL-10/STAT3 pathway may be involved in neuroinammatory progression and that up-regulation of the microRNA-181b is a potentially new therapeutic approach for neuroinammation. bMSCs: bone marrow mesenchymal stem cells; STAT3: signal transducer and activator of transcription 3; CNS: central nervous system; BBB: blood-brain barrier; TBI: traumatic brain injury; DMEM: dulbecco's modied eagle medium; FBS: fetal bovine serum; LPS: Lipopolysaccharide; TEM: transmission electron microscope; NTA: nanoparticle tracking analysis; TSG101: tumor susceptibility gene 101; HSP70: heatshockprotein70; MG: microglia; TUNEL: Terminal deoxynucleotidyl transferase-mediated nick end labeling; TNFα : Tumor Necrosis Factor α; TGFβ: transforming growth factor β;


Introduction
Microglia are the resident macrophages in the nervous system. They are responsible for regulating the microenvironment of the nervous system, phagocytosis of extracellular debris, and initiation of in ammation. As such, they are considered to be basic immune cells in the central nervous system (CNS) which regulates immune responses and brain function [6]. Microglia are activated in two ways after traumatic brain injury: either "classically activated" M1 phenotype or "alternatively activated" M2 phenotype. The balance between classically and alternatively activated microglial phenotypes in uence disease progression in the CNS [22]. M1 phenotype exhibits pro-in ammatory responses and expresses pro-in ammatory cytokines. In contrast, the M2 type exhibits anti-in ammatory effects and produces anti-in ammatory factors. However, there is no absolute boundary between them. There are cells that exhibit the markers of M1 and M2 phenotype at the same time as well as those that express speci c M1 and M2 markers. Nonetheless, researchers believe that demonstrating the complex phenotype of microglia in central nervous system based on M1/M2 classi cation is quite controversial because the two phenotypes exist upon stimulation and may thus reverse to the other phenotype. It is a reversible process of modi cation. Transformation of its phenotype and function may be a process with temporal and spatial heterogeneity [8,11,30].
In the wave of stem cell research at the beginning of the 21st century, mesenchymal stem cells from various tissues were used to treat nerve damage after brain injury or cerebral ischemia. Bone marrow mesenchymal stem cells (MSCs) are capable of repairing and replacing damaged tissues by differentiating into effector cells such as neurons and glial cells. They can also secrete some cytokines to inhibit neuroin ammation and promote nerve repair. Cognizant to this, used of MSCs is considered to be a promising method for treating diseases affecting the central nervous system [5,20,43]. In recent years, researchers have found out that the role of repair is through the paracrine effect and the exosomes secreted by MSCs rather than cell replacement [2,3,12,25,31,33]. Exosomes are membranous vesicles secreted by different cells and are capable of crossing the blood-brain barrier (BBB) [45]. They play a major role in cell-cell interactions. Exosome vesicles contain many components such as proteins, lipids, and nucleic acids. In recent years, microRNAs contained in exosome vesicles have been found to play important roles in many physiological and pathological processes [2,3,25,27,32,37]. Exosomes have been previously reported to display neuroprotective effects in rats after traumatic brain injury (TBI) [9]. Some scholars suggest that exosomes derived from mesenchymal stem cells can inhibit neuroin ammation and promote neural repair by regulating the microglial phenotype [13,38]. However, the molecular mechanisms involved have not been reported. Moreover, there is still no conclusion of the mechanism involved during exosome regulation of the microglial phenotype after TBI. As such, this study hypothesized that bMSCs derived microRNAs in the exosomes exhibits neuroprotective effects. In vivo and in vitro experiments, and microRNAs sequencing were thus conducted to explore the microRNAs involved in neuroprotection. Lentivirus was subsequently used to regulate the expression levels of related microRNAs to test the hypothesis and verify the possible downstream mechanisms.

Materials And Methods
Culturing and identi cation of BV2 and bMSCs cells BV2 cells were cultured in dulbecco's modi ed eagle medium (DMEM) high glucose culture medium supplemented with 10% Fetal Bovine Serum (FBS). Lipopolysaccharide (LPS, 1000 ng/ml) was used to activate BV2 cells for 48 hours. On the other hand, BMSCs were cultured in RPMI medium 1640 supplemented with 10% FBS. BV2 and bMSCs cells were subsequently identi ed through immunostaining using anti-Iba1 and anti-CD44 antibodies, respectively.
Extraction and identi cation of exosomes Exosomes were extracted from the cell culture supernatant of bMSCs. The bMSCs were rst washed twice with PBS. The culture medium was then replaced with exosome depleted FBS and culturing continued for 48 hours. The resultant supernatant was collected and rst centrifuged for 10 minutes at 300 g. It was then transferred to an ultracentrifuge and centrifuged for 30 minutes at 10000 g. Further centrifugation of the supernatant was done at 100000 g for 70 minutes to form an exosome pellet. The supernatant was discarded and the pellet resuspended in 500 ul PBS for use in subsequent experiments. The exosomes were then identi ed using a transmission electron microscope (TEM). Nanoparticle tracking analysis (NTA) was employed to measure the diameter and particle number of the exosomes.
In vitro co-culture experimental design Activated BV2 cells and bMSCs cells were co-cultured using a transwell system with a 0.4μm pore size. The MG group, the MG+bMSCs group, and the MG + Exo experimental groups were set up. The upper inserts were seeded with DMEM (500 ul/well), bMSCs (0.5×10 5 cells/well) or exosomes derived from bMSCs (6.3× 10 9 particles/well) while the lower chambers were seeded with activated BV2 (2×10 5 cells/well). Cell-climbing slices were set at the lower chambers in advance. The transwell plates were then incubated for 48 hours in an incubator set at 37°C and 5% CO 2 . Total RNA of BV2 cells was then extracted and the cell-climbing slices harvested for further analysis. The BV2 cells cocultured with DMEM in the MG group served as the negative control.
Cell phenotype analysis BV2 cells in the lower chambers were collected after 48 hours of co-culturing for ow cytometry analysis. To verify the percentage of M1 and M2 macrophages, the cells were incubated with the following mixture of directly conjugated antibodies: uorescein isothiocyanate (FITC)-conjugated anti-IBA1 antibodies, allophycocyanin(APC)-conjugated anti-CD86 antibodies, and phycoerythrin (PE)-conjugated anti-CD206 antibodies. They were then xed with a xation buffer for 30 minutes at room temperature followed by determination of the proportions of Iba1 + CD86 + CD206and Iba1 + CD86 -CD206 + BV2 microglia cells. BV2 cells were evaluated in a ow cytometer (BD FACSCanto TM II Flow cytometer; BD Biosciences) and FlowJo v10.4 software (FlowJo LLC)

MicroRNAs sequencing
MicroRNA sequencing was performed after the exosomes were extracted to compare the different microRNA expression levels between bMSCs and exosomes derived from bMSCs. This analysis was performed by OBiO Technology (Shanghai) Corp., Ltd.
The traumatic brain injury model A TBI model was made as reported by Wen et al., 2019 [35]. A scalp incision was rst made to expose the skull. A cranial window (3 mm diameter) was then drilled on the right parietal area of the skull, 2 mm away from the midline. A lateral uid percussion injury (LFPI) device was nally used to induce TBI. Ketamine (80-100 mg/kg) was used for anesthesia.

Lentiviral transfection
The lentiviruses (1 ul) were injected into each mouse at the exposed cortex through the cranial window using a stereotaxic instrument and a microinjection pump. Lentiviral transfer vectors were used to upregulate or down-regulate the expression level of microRNA-181b based on the microRNA sequencing. The expression level of microRNA-181b was tested by real-time PCR 7 days after the transfection.
In vivo animal experimental design C57BL/6 male mice aged between 6 and 8 weeks were used in this study. The in vivo experiment was divided into two parts.
In the rst part, healthy male C57/BL6 mice were divided into 4 groups: the sham group (sham operation was performed), brain trauma group (TBI model was made, TBI group), normal saline group (TBI model was made and saline was injected through the tail vein, TBI + Saline group), and exosomes group (TBI model was made and exosomes were injected through the tail vein, TBI + Exo group).
In the second part, mice modi ed by lentiviruses were divided into three groups: the TBI group (TBI model was made in normal mice), TBI-down group (TBI model was made in microRNA down-regulated mice), and TBI-up group (TBI model was made in microRNA up-regulated mice). The brain tissues were harvested and excised on ice on days 1, 3, and 7 post initiation of brain trauma. Total RNA and proteins of the brain tissues were then extracted for follow-up rt-PCR and Western Blot tests. Brain tissues harvested on day 7 post initiation of TBI were prepared into sections for TUNEL staining and immuno uorescence analysis.

Immuno uorescence analysis
Immuno uorescence analysis was performed based on the classical protocol. Anti-iba1 and anti-CD44 were used to identify BV2 and bMSCs cells, respectively. The cell climbing slices in the in vitro experiments were double stained with anti-Arg1 and anti-iNOS followed by DAPI staining. Brain tissues collected in the rst part of the animal experiments were prepared into sections and stained with anti-Arg1 and DAPI. Evaluation of the cell-climbing slices and brain sections was done using a confocal microscope. Data was analyzed by counting the number of Iba1/DAPI, CD44/DAPI, Arg1/DAPI, and iNOS/DAPI double-positive cells with 2 high power elds in each cell climbing slice or brain section.

RNA extraction and real-time PCR
The expression levels of in ammatory factors such as IL-1β, IL-6, TNF-α, IL-10 and TGF-β was quanti ed by real-time PCR to determine whether exosomes and the micro181b could inhibit neuroin ammation.
BV2 cells were collected 48 hours after the coculture experiments. The expression factors were quanti ed to verify the effect of bMSCs and exosomes derived from bMSCs. The factors were quanti ed at 1,3, and 7 days post TBI in the in vivo experiment. This was done to observe the dynamic changes of related factors in both parts of the animal experiments. Further to this, the expression levels of the candidate microRNAs selected through microRNA sequencing and the preliminary screening results were quanti ed.
Total RNA from BV2 cells and brain tissues of mice from all groups was extracted using an RNA puri cation Kit in accordance with the manufacturer's instructions. The concentration of RNA samples was with a Nanodrop® spectrophotometer (Thermo Scienti c) and the samples stored at -80°C for preservation. First-strand cDNA synthesis was done through normal reverse transcription for mRNAs analysis. In the same line, a poly(A) tail was added to the microRNAs followed by reverse transcription for microRNA analysis. All primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The primer sequences were listed in Table 1 and 2. U6 expression was used as the internal reference gene to determine microRNAs expression while GAPDH expression served as the internal reference gene for expression of other genes. Gene expression levels were measured and calculated using the 2 -Ct method.
Detection of apoptosis TUNEL assays were done in the two in-vivo experiments to determine whether bMSCs-Exo and microRNA-181b would reduce apoptosis in the damaged cerebral cortex. TUNEL staining was performed using a TUNEL kit in accordance with the manufacturer's instructions. The number of TUNEL positive cells in 2 regions of each brain section were counted to evaluate the effect of exosomes or microRNA-181b through statistical analyses. This process was performed 7 days post TBI.

Western blot analysis
The expression levels of iNOS and Arg1 were detected to determine the phenotype of M1 and M2, respectively in the in vivo experiments. In the same line, the expression level of STAT3 as the potential responsible pathway was measured to verify its effect in the second part of animal experiments. Protein extracts were rst obtained from the brain tissue samples 7 days post TBI and their concentration determined using a BCA kit. The primary antibody anti-Arg1 was used at 1∶1000 dilution, anti-iNOS at 1∶500 dilution, anti-STAT3 at 1:200 dilution, anti-α-tubulin at 1:500, and anti-β-actin at 1∶500 dilution for western blotting (WB). The gray values were then detected using the Image J software and the value relative to the expression of the internal reference calculated. Statistical analyses based on the relative gray values were then done.

Data analysis
The Statistical Product and Service Solutions (Version 17.0, Chicago, IL, USA) was used for data analyses. Data was reported as mean values ± standard error. Variations between groups were calculated using one-way analysis of variance (ANOVA). In addition, the LSD-t test was employed for group comparisons. P values less than 0.05 (P < 0.05) indicated that there were signi cant differences between groups.
Results bMSCs and bMSCs derived exosomes induced transformation of microglial polarization towards the antiin ammatory phenotype Exosomes were isolated from bMSCs cells by ultracentrifugation. The exosomes were then identi ed using TEM, nanoparticle tracking analysis (NTA) and western blot analysis. A typical cup-shaped membrane vesicle morphology was observed ( Figure 1). The size distribution pro les from the nanoparticle tracking analysis revealed that most of the vesicles had a diameter of ~130 nm. The original concentration of the exosomes was 6.3×10 10 particles/ml. Western blotting analysis further revealed that exosome markers TSG101 and HSP70 were expressed in the exosomes (Figure 1). BV2 microglia cells were activated using LPS (1000ng/ml) and then incubated at 37℃ for 48 hours prior to coculture with bMSCs and exosomes. They were then cocultured with bMSCs and exosomes for 48 hours to facilitate the polarization of activated BV2 cells toward the anti-in ammation type ( Figure 2A). This was done to investigate the effects of bMSCs and bMSCs derived exosomes on the polarization phenotype of activated BV2 cells in a co-culture system. The expression of anti-in ammation marker Arg1 increased signi cantly in the MG+bMSCs and MG+Exo groups, while the expression of proin ammation marker iNOS decreased compared to that of the MG group. Flow cytometry analysis was then performed to determine the proportion of pro-in ammatory and anti-in ammatory phenotype cells. The BV2 cells were labelled using FITC-conjugated anti-IBA1 antibodies, APC-conjugated anti-CD86 antibodies, and PE-conjugated anti-CD206 antibodies. The Q1 area indicated Iba-1/CD86 double-positive BV2 cells while the Q3 area indicated IBA-1/CD206 double-positive BV2 cells ( Figure 2B). In the same line, activated BV2 cells were transformed to the anti-in ammatory phenotype after 48 hours of co-culturing with bMSCs and exosomes ( Figure 2C). Moreover, the exosomes were associated with a stronger antiin ammatory effect.  Exosomes decreased neuronal apoptosis after induction of TBI in mice.
The lesion area and the neuron apoptosis in each group was determined after 7 days of TBI to detect the effects of exosomes on the nervous system after TBI in vivo (Figure 4). The damaged area of the mice in the TBI group and TBI + Saline group were larger compared with those of mice in the TBI + Exo group ( Figure 4A   Exosomes inhibited in ammation of the brain tissue after TBI An in-vivo experiment was designed in which exosomes were injected through the tail vein followed by induction of the TBI model to examine whether exosomes could inhibit in ammation in the CNS after induction of TBI.The expression levels of Arg1, iNOS, and in ammatory factors were then detected by immuno uorescence, western blotting and real time qPCR ( Figure 5). The proportion of Arg1 + cells in the TBI+Exo group was signi cantly higher than that of the other groups ( Figures 5A & B). In the same line, the expression level of Arg1 in the TBI+Exo group was signi cantly higher than that of the other groups. However, the expression of iNOS in the TBI+Exo group was signi cantly lower than that of the TBI and TBI+Saline group (Figure 5C, P<0.05). Figure 5D-( , , ) shows the expression levels of the related in ammatory factors after 1 day, 3 days, and 7 days, respectively from the onset of TBI.Evidently, the expression levels of IL-10 and TGF-β in the TBI +Exo group were higher than that of the other two groups on day1, 3, and 7 post TBI. Moreover, the expression levels of IL-1β and TNF-α in the TBI+Exo group were lower than those of the TBI and TBI+Saline groups on day 3 and 7 post TBI. In the same line, injection of exosomes upregulated the expression of STAT3 but inhibited the expression of NFκB (Figure 5D-). miRNA-181b was highly expressed in both exosomes and brain tissues miRNA sequencing of bMSCs cells and exosomes derived from bMSCs cells was done to explore the effective components of exosomes. Sequencing results revealed that there were more than 500 miRNAs highly expressed and more than 300 microRNAs lowly expressed in the exosomes compared to the bMSCs cells. Parts of the differentially expressed microRNAs are shown in the heat map and volcano plot ( Figures 6A & B). Based on microRNAs reported in other relevant studies, let-7c, miR-124, miR-21a, and miR-181b were chosen to be intensively studied. The expression levels of these microRNAs in brain tissues of mice in the TBI + Exo group at day 7 post TBI were thus determined. miR-181b was found to be highly expressed in the TBI + Exo group ( Figure 6C). This strongly suggested that miR-181b could inhibit neuroin ammation and regulate the phenotype of the microglia by up-regulating the STAT3 related pathway. Up-regulation of microRNA-181b inhibited neuronal apoptosis, reduced neuroin ammation and regulated the phenotype of microglia Lentiviral vectors were used to obtain different expression levels of miR-181b to verify the role of miR-181b in the neuroin ammation process post TBI. The expression level of miR-181b was detected 7 days after transfection of the mice with the lentivirus. The expression level of miR-181b in the up-regulation and down-regulation group was 2.7 times higher and 0.4 times lower than that of the normal C57BL/6 mice, respectively ( Figure 7B). Neuron apoptosis detected in each group at day 7 post TBI revealed that the numbers of TUNEL-positive cells in the three groups were 38.2±3.76, 35.6±4.50, and 24.8±1.94, respectively (Figures 7A & C). Notably, the number of TUNEL-positive cells in the miR-181b up-regulation group was signi cantly low than the other two groups. In the same line, the microglia markers Arg1 and iNOS, and the transcriptional regulator STAT3 were detected by Western Blotting (Figure 7D). The TBI-up group had a higher expression level of Arg1 and STAT3, but a lower expression level of iNOS. In ammatory factors were also detected at day 1, 3, and 7 post TBI (Figure 7 E-I, II & III). The expression level of IL-10 and TGF-β in the TBI-up group was signi cantly higher than that of the other two groups on the 1 st , 3 rd , and 7 th day post TBI (P<0.05). However, the expression level of IL-1β in the TBI-up group was signi cantly lower than that of the other two groups on the 1 st , 3 rd and 7 th day post TBI (P<0.05). Similarly, the expression level of TNF-α in the TBI-up group was signi cantly lower than that of the other two groups on the 3 rd and 7 th day post TBI (P<0.05). Nonetheless, the expression level of IL-6 was not signi cantly different among the groups on the 1 st , 3 rd , and 7 th day post TBI. Further analysis of the TBIup group revealed that the expression levels of IL-10 and TGF-β gradually increased after TBI (Figure 7E-).

Discussion
Microglia plays an important role in regulating neuronal functions such as cell survival, neurogenesis, and neuroin ammation [1,15,24,40]. In healthy brain tissues, microglia can phagocytose cell debris and damaged neurons. Activated microglia can also release various pro-in ammatory and anti-in ammatory cytokines and mediators such as interleukin-1β, interleukin-6, interleukin-10, arginase-1, cyclooxygenase-2 and inducible nitric oxide synthase among others [10,21] Exosomes derived from MSC protect various tissues from damage [2,9,23,31,44] Nonetheless, there are still many unanswered questions regarding their role in the pathophysiological process. Kim reported that exosomes isolated from MSCs could improve the cognitive impairment in TBI mice [9]. However, the molecular signals that mediated interactions between exosomes and neurons to promote neuronal survival were still unclear.
On the other hand, neuroin ammation affects the recovery of nerve function and the survival of neurons. As such, it is an important process in secondary brain injury. Herein, exosomes co-cultured with bMSCs inhibited the pro-in ammatory effect of activated microglia-like BV2 cells. They also played a crucial role in promoting macrophage polarization towards the M2 phenotype. Moreover, the BV2 cells expressed both Arg1 and iNOS at the same time. This result was consistent with that of Pettersen's, Vogel's, and Wong's reports [19,34,36]. In the same line, Italiani reported that human macrophages could be polarized to the M1 phenotype and then mature to the M2 phenotype with continuous changes in culture conditions [7]. Similarly, after exposure to classic M1 activation signals or interferon-γ, M2 macrophages can express M1 speci c cytokines and markers thereby transforming to the M1 phenotype [18,29]. Although there is no universal consensus on the transformation of microglia phenotype, phenotype changes of macrophages induced by external factors are dynamic, and show heterogeneity in space and time. Cognizant to this, the phenotypic de nition of macrophages should be interpreted based on speci c markers and function. As such, the real situation is by no means as simple as M1/M2. Zhang et al. reported that TBI rats treated with bMSCs exosomes had signi cantly stronger learning ability than those in other groups when tested using Morris water maze at 34-35 days after injury [41]. In addition, their sensory function and behavioral scores were higher than those of other groups at 14-35 days post TBI. In the same study, the exosome treatment group also had a signi cant increase in the number of neovascular endothelial cells in the dentate gyrus that effectively reduced neuroin ammation. This was the rst in vivo study of exosomes reporting their role in treatment of TBI. Herein, the damaged area in the TBI + Exo group was signi cantly less than that of the TBI and TBI + Saline groups. In addition, the number of TUNEL-positive cells in the damaged area was also signi cantly reduced after treatment with exosomes. This strongly indicated that the use of exosomes in the acute phase after TBI could reduce brain tissue damage and neuronal apoptosis. In the same line, the neuroin ammatory response in the exosome treatment group was inhibited. The expression of pro-in ammatory factors IL-1β and TNF-α was signi cantly suppressed while that of the anti-in ammatory factors IL-10 and TGF-β was signi cantly increased. The expression of M2 type microglia marker Arg1 was also signi cantly increased in the exosome treatment group.
There are numerous studies on neuroin ammatory signal pathways. On one hand, Man reported that the expression of the Notch1/NFκB pathway protein was signi cantly increased in the cerebral ischemic stroke models [4]. On the other hand, Zeng reported that hypertonic saline attenuated the expression of pro-in ammatory mediators and down-regulate the Notch signaling pathway [39]. The role of STAT3 signaling pathway during in ammation is controversial. Ryu reported that dasatinib regulated the neuroin ammatory response of LPS-induced microglia and astrocytes by inhibiting the expression of STAT3 and the pro-in ammatory factors [26]. In the same line, Zhang reported that ginkgo biloba extract protected the brain tissues after ischemic stroke by signi cantly reducing the expression of proin ammatory cytokines [42]. These effects may have been achieved by inhibiting the JAK2/STAT3 pathway. However, Staples reported that IL-10 activated the phosphorylation of transcription factor STAT3 through the autocrine feedback. As such, activation of STAT3 could up-regulate the expression of IL-10. The autocrine feedback enhanced the anti-in ammatory effects of IL-10 [28].
The relationship between IL-10 and STAT3 seems to be particularly subtle. IL-10 plays a role in the antiin ammatory response by binding to its receptor (IL-10R). The Activation of the IL-10/JAK1/STAT3 cascade pathway in turn causes activation of the phosphorylated STAT3 within seconds. Though STAT3 has no anti-in ammatory effects, it can activate many anti-in ammatory effector genes which can inhibit pro-in ammatory genes at the transcriptional level [16,17]. Herein, the IL-10 level of the exosomes treatment group was signi cantly higher than that of the other two groups on the 1st, 3rd and 7th day post TBI. Moreover, the expression of STAT3 in the exosome treatment group was signi cantly higher than that in the TBI group on the 7th day post TBI. Based on the interaction between IL-10 and STAT3, it can be concluded that IL-10 activates the STAT3 cascade pathway to inhibit neuroin ammation and to promote the transformation of microglia to an anti-in ammatory phenotype.
Recently, researchers have postulated that the therapeutic effect of exosomes depends on large amounts of miRNAs [14,27]. Cognizant to this, miRNAs sequencing was done to measure the differential expression level of miRNAs between exosomes and bMSCs. Based on microRNAs reported in other relevant studies and the sequencing results, let-7c, miR-124, miR-21a, and miR-181b were screened as those strongly associated with neuroin ammation. Subsequent qPCR results of the brain tissue revealed that the expression of miR-181b in the exosome treatment group was signi cantly higher than that of the other groups. It was thus concluded that miR-181b is involved in regulation of neuroin ammation as well as regulation of the microglial phenotype.
Further investigations of the effects of miR-181b using lentiviral vectors revealed that there were signi cantly fewer apoptotic cells in the TBI-up group compared to the other groups. The TBI-up group had signi cantly higher levels of IL-10 and TGF-β and lower levels of IL-1β and TNF-α in the injured area. Further analysis revealed that the expression levels of IL-10 and TGF-β gradually increased after TBI.
Compared with the TBI group, the expression of ARG1 and STAT3 in the TBI-up group increased signi cantly, while the expression of iNOS decreased. Taken together, these results strongly suggested that mirR-181b could activate the IL-10/STAT3 pathway, promote phenotype transformation of microglia, and inhibit neuroin ammation and neuronal apoptosis.
Nonetheless, this study was limited by several factors. The direct targets of miR181b and the mechanism of how miR-181b activate the IL-10/STAT3 pathway were not identi ed. The transfection e cacy in vivo was limited by the injection dose and the titer of lentivirus. Further studies on miR181b targets and their mechanisms of action are therefore needed to decipher its role in neuroprotection. Modi ed transfection process was needed as well. Despite these limitations, our current study disclosed that exosomes derived from bMSCs can inhibit neuroin ammation both in vitro and in vivo as well as depressing the apoptosis after TBI. And microRNA-181b palys as a potential target which may regulate the IL-10/STST3 pathway, and subsequently in uence the neuroin ammation post TBI.

Conclusion
Exosomes derived from bMSCs promotes the polarization of microglia to the anti-in ammatory phenotype and inhibit the neuroin ammatory response both in vitro and in vivo. The miR-181b may plays a vital role in this process by activating the IL-10/STAT3 pathway. The prominent role of microRNAs in neuroprotective offers new potential avenues of research into the treatment of neuroin ammation post TBI.    Figure 1 Identi cation and NTA analysis of bMSCs derived exosomes. TEM results revealed the membrane vesicle had a typical cup-shaped morphology. Scale bar =100 nm. NTA revealed that most vesicles had a diameter of ~130 nm. The original concentration of the exosomes was 6.3×1010 particles/ml. Western Blot further revealed that exosome markers TSG101 and HSP70 were expressed in the exosomes.