Exosomes Derived From Bone Mesenchymal Stem Cells Ameliorate Early Inflammatory Responses Following Traumatic Brain Injury

Haoqi Ni, Su Yang, Felix Siaw-Debrah, Jiangnan Hu, Ke Wu, Zibin He, Jianjing Yang, Sishi Pan, Xiao Lin, Haotuo Ye, Zhu Xu, Fan Wang, Kunlin Jin, Qichuan Zhuge, Lijie Huang, Haoqi Ni, Su Yang, Felix Siaw-Debrah, Jiangnan Hu, Ke Wu, Zibin He, Jianjing Yang, Sishi Pan, Xiao Lin, Haotuo Ye, Zhu Xu, Fan Wang, Kunlin Jin, Qichuan Zhuge, Lijie Huang

Abstract

Traumatic brain injury (TBI) is a leading cause of mortality and disability worldwide. Although treatment guidelines have been developed, no best treatment option or medicine for this condition exists. Recently, mesenchymal stem cells (MSCs)-derived exosomes have shown lots of promise for the treatment of brain disorders, with some results highlighting the neuroprotective effects through neurogenesis and angiogenesis after TBI. However, studies focusing on the role of exosomes in the early stages of neuroinflammation post-TBI are not sufficient. In this study, we investigated the role of bone mesenchymal stem cells (BMSCs)-exosomes in attenuating neuroinflammation at an early stage post-TBI and explored the potential regulatory neuroprotective mechanism. We administered 30 μg protein of BMSCs-exosomes or an equal volume of phosphate-buffered saline (PBS) via the retro-orbital route into C57BL/6 male mice 15 min after controlled cortical impact (CCI)-induced TBI. The results showed that the administration of BMSCs-exosomes reduced the lesion size and improved the neurobehavioral performance assessed by modified Neurological Severity Score (mNSS) and rotarod test. In addition, BMSCs-exosomes inhibited the expression of proapoptosis protein Bcl-2-associated X protein (BAX) and proinflammation cytokines, tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β, while enhancing the expression of the anti-apoptosis protein B-cell lymphoma 2 (BCL-2). Furthermore, BMSCs-exosomes modulated microglia/macrophage polarization by downregulating the expression of inducible nitric oxide synthase (INOS) and upregulating the expression of clusters of differentiation 206 (CD206) and arginase-1 (Arg1). In summary, our result shows that BMSCs-exosomes serve a neuroprotective function by inhibiting early neuroinflammation in TBI mice through modulating the polarization of microglia/macrophages. Further research into this may serve as a potential therapeutic strategy for the future treatment of TBI.

Keywords: bone mesenchymal stem cells; exosomes; inflammation; microglia/macrophage; neuroprotection; traumatic brain injury.

Figures

FIGURE 1
FIGURE 1
Identification of BMSCs-exosomes. (A) Transmission electron microscopy image of BMSCs-exosomes. Scale bar: 100 nm. (B) Exosomal markers (CD63, TSG101) and cytochrome c were analyzed by western blot. (C) Nanoparticles detection was performed to analyze particle size of exosomes. The percentage population of BMSC-exosomes by counts was shown.
FIGURE 2
FIGURE 2
BMSCs-exosomes improve function recovery post-TBI. Twelve to fourteen-week-old male C57BL/6 mice were subjected to TBI by CCI and treated by BMSCs-exosomes through retro-orbital injection. The neurobehavior was evaluated from day 1 to day 14 post-TBI by mNSS and Rotarod Test (A,B). ∗P < 0.05 versus the TBI + PBS group, ##P < 0.01 versus the SHAM + PBS group; one-way ANOVA. +P < 0.05; one-way repeated measures ANOVA with Dunnett T3 post hoc test for (A,B) (n = 7).
FIGURE 3
FIGURE 3
BMSCs-exosomes reduce cortical lesion post-TBI. (A) H&E staining of brain tissue from the TBI + PBS group at 14 days post-TBI showed serious tissue lesion, whereas reduced tissue lesion was observed in the TBI + EXO group. (B) The mean (±SEM) of cortical lesion area (mm2) after controlled cortical impact injury. ∗∗P < 0.01 versus the TBI + PBS group (n = 4).
FIGURE 4
FIGURE 4
BMSCs-exosomes affect cell death and inflammation post-TBI. (A) The apoptosis associated proteins Bax and Bcl-2 were analyzed by western blot at 3 days after TBI. β-actin was used as an internal control (n = 4). (B) The mRNA expressions of pro-inflammatory cytokines (IL-1β and TNF-α) were analyzed by real-time PCR and normalized with GAPDH mRNA levels at 1 day after TBI (n = 6). ∗P < 0.05, ∗∗P < 0.01 versus the TBI + PBS group, #P < 0.05, ##P < 0.01 versus the SHAM + PBS group (C).
FIGURE 5
FIGURE 5
BMSCs-exosomes inhibit the M1 polarization of microglia/macrophage at 3 days post-TBI. (A) Compared with TBI + PBS group, INOS+/iba1+ cells had significantly decreased in the TBI + EXO group at 3 days after TBI. Arrows indicate iNOS+/Iba1+ cells. Scale bar = 50 μm (n = 4). (B) The mRNA expressions of INOS (n = 6). ∗P < 0.05 versus the TBI + PBS group, ##P < 0.01 versus the SHAM + PBS group.
FIGURE 6
FIGURE 6
BMSCs-exosomes promote the M2 polarization of microglia/macrophage at 3 days post-TBI. (A) Compared with TBI + PBS group, CD206+/iba1+ cells had significantly increased in the TBI + EXO group at 3 days after TBI. Arrows indicate CD206+/Iba1+ cells. Scale bar = 50 μm (n = 4). (B) The mRNA expressions of arg-1 (n = 6). ∗P < 0.05 versus the TBI+PBS group, #P < 0.05 versus the SHAM + PBS group.

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