Introduction

Traumatic brain injury (TBI), the dominant type of head injury, subjects more than 10 000 000 people to death or hospitalization every year globally. TBI is mainly caused by falls, traffic incidents, and beats [1]. Notably, TBI has emerged as a dominant cause of morbidity and mortality. The significant expenses on clinical caring of TBI patients and related socioeconomic issues will severely stress the healthcare system and society [2]. Much research in the past ten years suggests that mesenchymal stem cells (MSCs) are potential and efficient for brain injury treatment, in both testing models of TBI and potentially in clinic [3–6]. Despite such findings, the underlying roles of MSCs in post-TBI brain tissue repair and function recovery may not relate to the cell replacing impacts, but to the secretion-based paracrine impacts [7–9].

Exosomes are lipid bilayer membrane vesicles in diameter of ≤ 150 nm and are produced by diverse cells, such as MSCs and cancer cells [10,11]. Exosomes with abundant endosome-originating parts (e.g. microRNAs, mRNAs, proteins) are often investigated by marker proteins, transmission electron microscopy (TEM), and nanoparticle size examination [12]. Compared with the parent cells, MSC-derived exosomes (MSCs-Exo) have less immunogenicity due to limited or no proliferating ability and can be safely kept and moved without function loss [13]. The effectiveness of MSCs-Exo was verified in animals with TBI [5,14]. The function mechanism of MSCs-Exo is yet unknown but can be attributed to the post-injury inhibition of inflammatory reactions. Microglia, a significant factor in aggravating and modulating neuroinflammation, can impose a double-edge impact on TBI neurogenesis [15]. In addition to inflammatory molecules [interleukin (IL)-1β/6, tumor necrosis factor (TNF)-α] that are detrimental to neurogenesis, microglia can also secrete anti-inflammatory factors (e.g. transforming growth factor β, IL-4/10) that are beneficial to neurogenesis [16,17]. Reportedly, the use of MSCs-Exo led to neuroinflammation reduction and remarkable functional restoration in a rat TBI model [18]. Exosomes produced from MSCs may be able to limit neuroinflammation and encourage brain repair by controlling the morphologies of nearby microglia, according to some researchers [19]. Because of their safety and minimal immunogenicity, while yet displaying the same immunomodulatory and regenerative abilities as their mother cells, MSC-derived exosomes have a great deal of promise for cell-free therapy [20]. Exosomes have been utilized therapeutically to decrease neuroinflammation following TBI. It has been demonstrated that MSC-derived extracellular vesicles suppress microglia activation [21]. Thus, exosomes can relieve TBI by changing microglial roles in vivo, which is not yet clarified, however.

Nucleotide-binding oligomerization domain-like receptors (NLRs), one subgroup of pattern recognition receptors, are critically involved in inherent immune/ inflammatory reactions by producing inflammasomes [22] that are distributed discrepantly in the brain. The NLR family pyrin-domain-containing 3 (NLRP3) is mainly located in microglia [23]. NLRP3 inflammasome, a cytosolic signaling compound, is composed of NLRP3, the apoptosis-related speck-shaped protein with a caspase recruiting zone, and pro-caspase-1 [24]. NLRP3 inflammasome was up-expressed in animal models and TBI patients [23,25], and its isolation or knockdown weakened inflammatory reactions and enhance neurological roles in TBI rodents [26,27]. Studies on the control of NLRP3 by exosomes and inflammatory processes have exploded recently [28–30]. For instance, Yan et al. [31].discovered that exosomes produced by umbilical cord MSCs (UMSC-Exo) reduced the amount of cleaved caspase-1 produced, which in turn reduced the release of IL-1 and IL-18 and pyroptosis. circHIPK3 produced by UMSC-Exo down-regulated miR-421, leading in enhanced expression of fork head box class O 3a, which might suppress NLRP3 activation. Exosomes can influence immunity in disease states by keeping an eye on gp130/STAT3 signaling, the TLR4/NF-B/NLRP3 inflammasome, and programmed cell death-1 [32]. Herein, we discussed the relationship between the exosomes and NLRP3. Therefore, the NLRP3 inflammasome is a new potential target for exosomal treatment of TBI.

Adipose-derived stem cells (ADSCs) offer a microenvironment for cell survival by generating neurotrophic, neuroprotective and anti-inflammatory factors, and reduce the formation of stress-related proteins, reactive oxygen, and pro-inflammatory factors [33–35]. Hence, it is suggested that ADSCs-derived exosomes (ADSCs-Exo) may also reduce neuroinflammation and facilitate functional recovery from TBI. Herein, the composition of ADSCs-Exo and the impact on microglial activation were explored in an in-vitro TBI model. Also, the potential mechanism was discussed with an in vivo mouse TBI model.

Materials and methods Ethics and approvals

A total of 62 rats were used in the study. This is due to the fact that post-trauma motor and cognitive function did not significantly differ between female and male SD. In contrast, the immunological response was more stable in female SD. This experiment involved using female SD rats [36–38]. All animal experiments were approved by the Animal Care and Use Committee of East China Normal University.

ADSC separation and characterization

ADSCs were obtained as reported [39]. Female Sprague–Dawley (SD) rats (6–8 weeks old, 180-220 g) were used for our experiment. The inguinal adipose tissue was cleaned with a phosphate buffer solution (PBS). After 5 min of centrifugation at 1200 rpm, the supernatant was isolated from the cell suspension. The pellets were cautiously suspended with PBS (10 mL) and centrifuged again. The pellets without the supernatant were dissolved again in an erythrocyte lysis solution (10 mL) and cultured for 10 min at ambient temperature. After red blood cells were lysed, the cell solution was passed via a 100-lm nylon filter to clean off cell debris. The cells were gathered and sown on culture plates with Dulbecco’s modified Eagle medium (DMEM), 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin. After 24 h, non-adhering cells were discarded, a new full medium was added and changed every third day then. Multipotency of separated cells was clarified by adipogenic differentiation, which was conducted with the cells cultured for 2 weeks in DMEM supplemented with 10% FBS, 0.5 mM isobutylmethylxanthine, 1 μM dexamethasone, 10 μM insulin and 200 μM indomethacin media and dyed by Oil Red O. DMEM medium (SH30081.01) and FBS (SH30088.02) were from Hyclone. Penicillin (5161), streptomycin (85886), isobutylmethylxanthine (I5879), dexamethasone (D1756), insulin (I1507) and indomethacin (I7378) were from Sigma.

ADSC-Exo isolation and characterization

Cells were planted with serum at 37 °C and 5% CO2 until reaching a subconfluent status (80–90%). Then the cells were slowly cleaned with PBS three times and cultivated for 48 h in serum-free DMEM. The cell-conditioned solution was collected and centrifuged successively at 300 × g, 2000 × g (both 10 min), and 10,000 × g (30 min) to discard the cell residues and debris. The exosomes were pelleted under an SW32 Ti shaking bucket rotor from the supernatant and ultracentrifuged at 100,000 × g for 70 min. To eliminate protein contamination, the pellets were suspended again in PBS, pooled, and ultracentrifuged again under the same condition. The final exosome pellets were suspended again in PBS until used.

Diameter distribution of ADSCs-Exo was analyzed under ZetaView (Particle Metrix GmbH, Meerbusch, Germany). Morphology was recorded by a Hitachi H7500 TEM device (Tokyo, Japan). Surface markers CD63 (ab193349, Abcam) and CD81 (ab109201, Abcam) encapsulated into exosomes were examined by Western blot.

Stimulation with lipopolysaccharide and co-culture with ADSCs-Exo for primary microglia

Primary microglia were separated and purified from rat cortices as described before [40]. The microglia were acquired from the astrocytic single layer by beating the flasks 10 to 20 times and planted to poly-d-lysine-coated culture plates until used. In 150-cm3 culture flasks, 5 107 cells were seeded with DMEM containing 10% heat-inactivated FBS, 2 mM l-glutamine, 1 mM sodium pyruvate, 100 mM non-essential amino acids, 50 U/ml penicillin, and 50 g/ml streptomycin. Microglial cells were exposed to 100 ng/ml lipopolysaccharide (LPS; SigmaAldrich) and cultured with 5 or 10 μg/ml ADSCs-Exo for 24 h, so as to evaluate the effect of ADSCs-Exo on LPS-stimulated primary microglial cells. Cells processed without either LPS or exosomes were set as controls. LPS (L3012), l-glutamine (G2150), sodium pyruvate (P2256), and non-essential amino acids (TMS-001) were from Sigma.

ELISA of pro-inflammatory cytokines

Different treatments were used during the 24-hour cultivation of primary microglia cells. The culture supernatant was then taken out and spun at 3000 rpm for 20 min. Finally, using the appropriate ELISA kit per the manufacturer’s instructions, the cellular abundances of TNF-α (ab236712, Abcam), IL-1β (ab255730, Abcam), and IL-6 (ab234570, Abcam) were found in the medium.

Construction of TBI rat model and application of exosomes

Under anesthesia via intraperitoneal administration of 40 mg/kg pentobarbital sodium (1%), the skull of each animal was exposed via sagittal cutting and fascia reflection at the scalp midline. Then a 4.0-mm-diameter craniotomy on the right hemisphere was made between the lambda and bregma and 3.0 mm from the sagittal suture. The skull flap was also discarded. A 2.5-mm-diameter pillar was positioned at the hit site to release a 40-g weight dropping from 10 cm high along a stainless-steel string, and then was stricken. After the injury, the scalp cut was sewn. The sham rats were received craniotomy only, except for contusion.

Rats were randomly classified into 3 groups: sham, TBI, or TBI+ADSCs-Exo (n = 7/group). ADSCs-Exo (200 µg precipitated in 200 µL of PBS) was intravenously applied over 5 min via the tail vein at 24 h after the preparation of TBI. Equal volumes of PBS (200 µL) were administered in the TBI and sham groups.

Evaluation of neurological outcome

The modified neurological severity score (mNSS) integrates sensory (vision, tactile, and proprioceptive), reflex, and motor (muscle status, abnormal motion) assays and is extensively adopted before. The mNSSs of each rat were detected at 0, 1, 4, 7, 14, 21, 28 and 35 days after TBI [41]. Neurological function was scored from 0 (normal) to 18 (maximum deficit). Each abnormal conduct or lack of a tested reflex was assigned one point. Hence, a larger score stands for a severer injury.

Foot faults were tested [42] at the same time points as mNSS. The rats can walk on a grid. Foot fault was defined as the fall or slip of a paw between the wires at each weight-bearing step. Totally 50 steps for the right forelimb were observed.

Immunofluorescence

Cells were fixated in 4% paraformaldehyde, and cleaned two times in 1× PBS. The primary antibody was anti-Iba1 (ab178846, 1 : 100, Abcam). After nuclei were dyed with DAPI (ab104139, 1 : 1000, Abcam), the cells were examined by fluorescence microscopy. All images were processed on ImageJ (software version 1.52; National Institutes of Health, Bethesda, MD, USA) [43].

Western blot

The total protein of the cortical tissue close to or in the vicinity of the damage site was taken for assay. Brain tissues were treated with a RIPA protein lysis solution and total proteins were harvested via centrifugation. Western blot was carried out as per standard steps. Primary antibodies were NLRP3 (ab263899, 1 : 1000, Abcam), Caspase-1(ab286125, 1 : 1000, Abcam), and β-actin (ab8227, 1 : 5000, Abcam). Membranes were observed by an enhanced chemiluminescence Western blot device (Thermo Fisher Scientific). Gray values were tested on Image J (Rawak Software, Germany).

Statistical analysis

GraphPad Prism Software 8.0 (GraphPad Software, USA) was used to conduct the statistical analysis. All data were analyzed using Student’s t-test for two groups, one-way analysis of variance (ANOVA), or two-way ANOVA with Bonferroni’s multiple comparison test for more than two groups. The data are all reported as the mean ± SD. When P < 0.05, differences between means were regarded as statistically significant.

Results Characterization of ADSCs-Exo

The ADSCs are plastic-adhering and look like fibroblasts (Fig. 1a). The cells were processed by adipogenic solutions and confirmed by Oil Red O dyeing of lipid drops (Fig. 1b). The ADSCs-Exo were comprehensively characterized by TEM and Western blot. ADSCs-Exo exhibits a round shape under TEM (Fig. 1c), and CD63 and CD81 are positive in ADSCs-Exo as per Western blot (Fig. 1d), which both confirms the existence of exosomes. The above results reveal the triumphant isolation and recognition of ADSCs-Exo.

Fig. 1:

Characterization of ADSCs and ADSCs-Exo. (a) ADSCs as monolayer fibroblast-like cells. (b) Lipid vacuoles stained with Oil red O after adipocytic division. (c) TEM of ADSCs-Exo. (d) Western blotting was used to identify exosome-specific markers (CD63 and CD81). Results are representative of n = 3 independent experiments.

Exosome internalization reduces LPS-initiated generation of pro-inflammatory molecules in microglial cells

The direct impact of ADSCs-Exo on pro-inflammatory molecule alteration was further verified in the primary microglia (Fig. 2a–i). The LPS-induced morphology of microglial cells is altered by ADSCs-Exo. LPS-treated (100 ng/ml) microglial cells showed long thin processes extending from the cell body, in contrast to the control group. The number of long thin processes extending from the cell body in cells cultured with ADSCs-Exo (10 µg/ml) followed by LPS treatment was decreased, and these cells had the similar appearance as the control group.

Fig. 2:

Functional influence of ADSCs-Exo on LPS-induced microglia in vitro. (a–i) Primary microglia were co-cultured 24 h in three groups: PBS control, LPS (100 ng/ml LPS), and ADSCs-Exo (100 ng/ml LPS + 10 µg/ml ADSCs-Exo). Primary microglia detected through specific markers Iba1 (green) by immunostaining analysis. Levels of (j) TNF-α, (k) IL-6 and (l) IL-1β in the cell supernatant of each group. The pro-inflammatory molecules were detected by ELISA. The data are expressed as the mean ± SD (n = 7/group). *P < 0.001 vs. the control group; #P < 0.05, ##P < 0.001 vs. the LPS group.

In the microglia, 100 ng/ml LPS stimulation initiated the secretion of IL-1β/6 and TNFα. Compared with LPS-processed cells (Fig. 2j), ADSCs-Exo restricted the LPS-caused TNFα production by 5 µg/ml (1445 pg/ml) and 10 µg/ml (913 pg/ml) (both P = 0.001). Additionally, ADSCs-Exo decreased the LPS-induced IL-6 secretion by 5 µg/ml (290 pg/ml; P = 0.035) and 10 µg/ml (178 pg/ml; P = 0.001) (Fig. 2k). Finally, ADSCs-Exo reduced the LPS-caused IL-1β formation by 5 µg/ml (503 pg/ml) and 10 µg/ml (485 pg/ml) (both P = 0.001) (Fig. 2l). Such results indicate that ADSCs-Exo inhibit pro-inflammatory responses in primary microglia by LPS.

ADSCs-Exo application significantly enhances post-TBI sensorimotor functional restoration in rats

The mNSSs on Day 1 post-TBI were near 12 in all rats, implying no obvious difference in the neuro-deficits among the TBI rats at baseline. When compared to day 1 post-injury, the mNSS score in the ADSCs-Exo group animals decreased significantly over time, starting at Day 7 and continuing through Day 35 (P < 0.05), indicating a considerable improvement in mNSS following TBI (Fig. 3a). Before the assignment of treatments (PBS or ADSCs-Exo), there was no discernible difference in the footfault tests between the TBI groups on day 1 post-injury. In the ADSCs-Exo group animals, a substantial decrease in the number of foot faults was seen over time, commencing on day 14 and continuing until day 35 compared to day 1 post-injury (P < 0.05), indicating the presence of a considerable recovery in foot faults following TBI (Fig. 3b). Thus, ADSCs-Exo administration contributed to sensorimotor functional recovery in the TBI rats.

Fig. 3:

Administration with ADSCs-Exo significantly improved sensorimotor functional restoration as detected by modified neurological severity score (a), right forelimb foot fault (b) in rats after TBI. *P < 0.05 vs. PBS group. Data were expressed as mean ± SD (n = 7/group).

ADSCs-Exo inhibit NLRP3-mediated inflammasome activity after TBI

Post-TBI stimulation of NLRP3 inflammasome and the inhibitive effect of ADSCs-Exo on NLRP3 were examined by detecting the expressions of the parts of NLRP3 inflammasome and cleaved activated caspase-1 using Western blot (Fig. 4a). NLRP3, caspase-1 were decreased in ADSCs-Exo rats after injury compared to the TBI group, as shown in Fig. 4b and c (P < 0.05). This implies that NLRP3 activation and its downstream activities are blocked by ADSCs-Exo downregulation.

Fig. 4:

Effect of ADSCs-Exo on NLRP3 inflammasome expression post-TBI. (a) Western blot of NLRP3 and caspase-1 at 24 h post-TBI, and relative expressions of (b) NLRP3 and (c) caspase-1 in pericontusional cerebral cortex. Data were expressed as mean ± SD (n = 7/group). *P < 0.001 vs. sham group; #P < 0.05 vs. TBI group.

Discussion

MSCs-Exo can be applied in ideal cell-free treatment of TBI. Investigation into the effects and mechanisms of ADSCs-Exo on TBI reveals that ADSCs-Exo can resist inflammation in TBI. The exosomes can lower the formation of pro-inflammatory factors by microglia in vitro following LPS activation. Vein administration of ADSCs-Exo in vivo is an efficient cure for neurological function recovery by reducing NLRP3 inflammasome activation.

Testing models and clinical applications of TBI indicate that MSCs are a promising and prospective treatment for brain injuries. However, as reported recently, the main mechanisms of MSCs involved in post-TBI cerebral reconstruction and functional repair may be the paracrine impacts of secretion-related factors (e.g. MSCs-Exo) rather than cell replacing effects. Exosomes comprise of proteins, nucleic acids, miRNAs, lipids, and mRNAs [44]. MSCs-Exo can relieve neuroinflammation, facilitate neurogenesis, angiogenesis and functional restoration, and save pattern isolation and spatial learning injuries after TBI in animal models [14,45,46]. Herein, the shape, diameter layout, and surface marker proteins of ADSCs-Exo were detected by TEM and Western blot. Moreover, ADSCs-Exo were shown to reduce inflammasome activation in rats after TBI, which contributed to significant sensorimotor functional recovery.

Neurological inflammation is a major molecular and cellular response of the central nervous system (CNS) to trauma. Microglia, the inherent immune cells of the CNS, are a regulator of this response after TBI. Microglial activation initiates the generation of TNF-α and IL-1β/6 [47]. These pro-inflammatory molecules are harmful and activate the auto- and paracrine formation of glutamate from microglia. As such, such activity can directly and neurotoxically impact neurons, synapses and dendrites, depending on the released quantity. LPS-treated primary microglia were used to simulate such neurotoxic microglial behavior [48,49]. The LPS model initiated alterations in microglial activation pro-inflammatory phenotypes. Herein, ADSCs-Exo relieved LPS-caused microglial motion and the secretion of pro-inflammatory factors in vitro. Such results support that microglial inhibition facilitates the treatment effects of ADSCs-Exo on TBI.

Moreover, the secretion of inflammatory cytokines requires inflammasome actions. The NLRP3 inflammasome, consisting of NLRP3, pro-caspase-1 and the adaptor ASC [50], is a significant factor in microglial activation [51] and the best-featured part in microglia. The action of this inflammasome in post-injury neurological inflammation was confirmed in TBI models in vivo and in people with modest/severe TBI. The NLRP3 inflammasome, ASC and caspase-1 mRNA were up-expressed at 6 h and cerebral NLRP3 and caspase-1 protein levels rose at 24 h after a fluid percussion injury [23]. NLRP3, caspase-1 and IL-1β were considerably upregulated in rats at 12 h and 24 h after blast-initiated TBI and in a mouse TBI model [52]. Based on the discovery of the significant function of NLRP3 inflammasome in TBI, it is hypothesized that NLRP3 is a potentially critical target for neuroinflammation modulation and TBI recovery. Actually, NLRP3 silence considerably weakened neuroinflammatory procedures and significantly improved the injured functions in mice [26]. As such, we hypothesize that the anti-TBI function of ADSCs-Exo may also be associated with the start of NLRP3 inflammasome activation. To prove this hypothesis, we examined the inflammatory reaction in a testing model. After the improved sensorimotor functional restoration, ADSCs-Exo blocked the generation of IL-1β/6. ADSCs-Exo also restricted the expression of NLRP3 and caspase-1. The above results infer that ADSCs-Exo may protect rats from TBI by blocking NLRP3-modulated inflammasome stimulation, which then suppresses caspase-1 expression.

Conclusion

In the present study, ADSCs-Exo can improve LPS-caused inflammatory activation by decreasing pro-inflammatory molecules in microglia. Moreover, the neuroprotective role may be ascribed a part to the inhibition on the formation of NLRP3-adjusted inflammasome. This study offers new information about ADSCs-Exo-based therapy and the potential mechanism.

Acknowledgements

This study was funded by supported by a grant from the Scientific Research Project of the Second People’s Hospital of Wuhu (2020A02, 2020D24, 2019D08) and the Research Project of Teaching Hospital of Wannan Medical College (JXYY202002).

L.T. and Y.X. conceived and designed the experiments. L.T., Y.X., L.W., and J.P. performed experiments. L.W. and L.T. were involved in the analysis and interpretation of data. L.T. and Y.X. were involved in writing and review of the manuscript. L.T. and Y.W. were involved in study supervision.

Conflicts of interest

There are no conflicts of interest.

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