Characterization of MSCs and MSCs-EVs
TEM analysis revealed that MSCs-EVs were almost around 100 nm and a rounded morphology (Figure 1A). MSCs-EVs were identified as small vesicles ranging from 60 to 120 nm with qNano (Figure 1B). MSCs-EVs expressed EVs markers such as CD9 and TSG10, with absence Calnexin, GM130, Cytochrome C1 or Lamin A/C with Western blot (Figure 1C). Additionally, PKH67-EVs were internalized in BV-2 cells and localized in the cytoplasm (Figure 1D).
MSCs exhibited a spindle-shaped morphology (Figure 1E) and were found to be capable of differentiating into osteoblasts and adipocytes (Figure 1F). MSCs were positive for CD44, CD29 and Sca-1, but negative for CD11b, CD45, CD31, CD117, Ly6G, CD19, CD14, or CD4 by FACS analysis (Figure 1G)
MSCs-EVs treatment inhibited OGD-induced microglial apoptosis
We first investigated whether MSCs-EVs exerted protective effects against OGD insult in BV-2 cells. The result showed that OGD followed by 24 h reoxygenation significantly decreased the viability of BV-2 cells at 1 h (t=6.041, df=10, p<0.01), 3 h (t=8.095, df=10, p<0.001) and 5 h (t=10.364, df=10, p<0.001) post-OGD.
MSCs-EVs (10, 20 μg/mL) increased the viability of the OGD-treated BV-2 cells partly at 3 h post-insult (Figure 2B) (10 μg/mL: t=-4.426, df=10, p<0.01), 20 μg/mL: (t=-9.253, df=10, p<0.001). However, no significant differences exist at 5 μg/mL MSCs-EVs group (t=-1.591, df=10, p>0.05) (Figure 2B). Further, in vitro experiments were performed using 10 μg/mL MSCs-EVs.
When MSCs-EVs (10 μg/mL) were pretreated with RNase A did not affect the neuroprotection of MSCs-EVs on cell viability at 3 h following OGD exposure (p>0.05). In addition, pretreated MSCs-EVs with 0.5% Triton X‐100 (detergent to lyse membrane vesicles) caused decrease in the protective properties of MSCs-EVs ([F(4,25)=90.701, p<0.001]; post hoc p<0.001). And pretreated EVs with Triton X‐100 and RNase A reversed the protective properties of MSCs-EVs on cell viability (post hoc p<0.001). Moreover, pretreated EVs with Triton X‐100 and RNase A significantly decreased cell viability compared with Triton X‐100 pre-treatment (post hoc p<0.001) (Figure 2C).
MSCs-EVs treatment promoted M2 polarization in BV-2 cells following OGD exposure
Because OGD induces microglia into a pro-inflammatory state, we examined the influence of EVs on BV-2 cells following OGD exposure. The qRT-PCR results showed that OGD exposure increased the levels of pro-inflammatory cytokines post-OGD, including TNFα (t=-4.034, df=10, p<0.01 at 1 h; t=-2.360, df=10, p<0.05 at 3 h; t=-1.902, df=10, p>0.05 at 5 h), IL-1β (t=-3.124, df=10, p<0.05 at 1 h; t=-5.696, df=10, p<0.01 at 3 h; t=-4.932, df=10, p<0.01 at 5 h), iNOS (t=-0.393, df=10, p>0.05 at 1 h; t=-10.290, df=10, p<0.001 at 3 h; t=-4.490, df=10, p<0.01 at 5 h) (Figure 3A).
OGD dramatically decreased the levels of anti-inflammatory cytokines at 1 h, 3 h, and 5 h post-insult: IL-10 (t=3.000, df=10, p<0.05 at 1 h; t=2.490, df=10, p<0.05 at 3 h; t=2.847, df=10, p<0.05 at 5 h), TGF-β (t=5.071, df=10, p<0.001 at 1 h; t=3.936, df=10, p<0.01 at 3 h; t=5.737, df=10, p<0.001 at 5 h), M2 microglia surface marker CD206 (t=5.134, df=10, p<0.001 at 1 h; t=3.882, df=10, p<0.01 at 3 h; t=3.317, df=10, p<0.01 at 5 h) (Figure 3A).
MSCs-EVs treatment attenuated the levels of pro-inflammatory cytokines at 3 h post-OGD, including TNFα ([F(2,15)=157.208, p<0.001]; post hoc p<0.001), IL-1β ([F(2,15)=19.931, p<0.001]; post hoc p<0.01), and iNOS ([F(2,15)=104.828, p<0.001]; post hoc p<0.001) at 3 h following OGD insult (Figure 3B). Moreover, EVs treatment increased the levels of anti-inflammatory cytokines at 3 h post-OGD, including IL-10 ([F(2,15)=6.850, p<0.01]; post hoc p<0.05), TGF-β ([F(2,15)=7.367, p<0.01]; post hoc p<0.05) and CD206 ([F(2,15)=17.170, p<0.001]; post hoc p<0.001) (Figure 3B). Taken together, these data indicate that MSCs-EVs treatment are a homeostatic regulator of microglia polarization and facilitate the differentiation of microglia to M2, but not M1.
miR-21a-5p in MSCs-EVs attenuated inflammatory response following OGD exposed in BV-2 cells
EVs-mediated miRNA transport has been proposed to be an essential mechanism that regulates target gene expression for cell-to-cell communication . We previously reported that miR-21a-5p was found to be highly abundant in MSCs-EVs . Next, we observed that miR-21a-5p in MSCs-EVs account for its immune-modulatory property. First, in order to observe miR-21a-5p located in in the lumen, MSCs-EVs were treated with RNase A, Triton-X-100, and RNase A + TritonX-100 . RT‐qPCR analysis showed that pretreated MSCs-EVs with RNase A or Triton-X-100 alone did not abolish detection of miR-21a-5p in MSCs-EVs. Conversely, pretreated MSCs-EVs with RNase A and TritonX-100 could abolish the miR-21a-5p in MSCs-EVs (Figure 4A). These data confirmed that miR-21a-5p is inside of MSCs-EVs, which resistance to digestion. Following OGD exposure, miR-21a-5p levels were significantly decreased at 1 h (t=2.687, df=10, p<0.05), 3 h (t=2.969, df=10, p<0.05) and 5 h (t=1.655, df=10, p>0.05) post-HI (Figure 4B). MSCs-EVs treatment remarkably augmented miR-21a-5p expression levels at 3 h post-OGD ([F(2,15) = 20.783, p<0.001]; post hoc p<0.001) (Figures 4C).
To confirm whether or not miR-21a-5p has a critical role in the aforementioned neuroprotection of MSCs-EVs, MSCs-EVs were pretreated with an anti‐miR‐21a-5p inhibitor (EVs-miR-21ainhibitor) or with its negative control (EVs-miR-21aINC). qRT-PCR results showed that miR-21a-5p inhibitor remarkable decreased miR-21a-5p expression ([F(2,15) =162.026, p<0.001]; post hoc p<0.001) in MSCs-EVs (Figure 4D). Morever, miR-21a-5p inhibitor remarkable reversed EVs-induced miR-21a-5p levels post-OGD ([F(3,20) =15.773, p<0.001]; post hoc p<0.01) (Figures 4E).
EVs-miR-21ainhibitor was significantly suppressed the cell viability following OGD in BV-2 cells as compare with EVs-miR-21aINC, ([F(3,20)=31.199, p<0.001]; post hoc p<0.001) (Figure 4F).
EVs-miR-21ainhibitor suppressed the anti-inflammatory effects of EVs-miR-21aNC upon: TNF-α ([F(3,20)=37.149, p<0.001]; post hoc p<0.001), IL-1β ([F(3,20)=23.528, p<0.001] ; post hoc p<0.001), and iNOS ([F(3,20)= 8.357, p<0.01] ; post hoc p<0.05) and, while IL-10 ([F(3,20)=8.387, p<0.01]; post hoc p<0.05), TGF-β ([F(3,20)=14.381, p<0.001] ; post hoc p<0.001) and CD206 ([F(3,20)=12.117, p<0.001]; post hoc p<0.05) expression was significantly increased following OGD exposure (Figure 4G).
MiR-21a-5p in MSCs-EVs targets STAT3 signaling pathway following OGD exposed in BV-2 cells
Next, to investigate the targets of miR-21a-5p that may be potentially associated with microglia polarization and inflammation, we used the available databases to search for potential miR-21a-5p targeted genes. Notably, the miR-21a-5p/STAT3 pathway has been shown to be the most critical cellular pathway related to the modulation of the immune system and inflammation [21, 22]. Subsequently, we evidenced that OGD increased STAT3 phosphorylation (p-STAT3) at 1 h (t=-4.209, df=6; p<0.01), 3 h (t=-3.198, df=6; p<0.05) and 5 h (t=-4.348, df=6; p<0.01) post-OGD (Figure 5A). MSCs-EVs treatment significantly decreased the levels p-STAT3 ([F(3,12)=8.368, p<0.001]; post hoc p<0.05) (Figure 5C). Furthermore, inhibition of miR-21a-5p in the MSCs-EVs group had exactly the reverse effect (post hoc p<0.05) (Figure 5C).
TargetScan analysis predicted binding of miR-21a-5p to 3′-UTR of STAT3 (Figure 5D). MiR-21a-5p mimics decreased luciferase activity associated with WT STAT3 3’ UTR, but not luciferase activity associated with MUT STAT3 3’ UTR in BV-2 cells (t=25.649, df=10; p<0.001) (Figure 5E).
MiR-21a-5p in MSCs-EVs promoted M2 microglia polarization following HI insult
In order to confirm that the anti-inflammatory effect associated with MSCs‐EVs administration is mediated by the EVs‐delivery of miR‐21a‐5p, mice were injected with either PBS, MSCs‐EVs, EVs-miR-21ainhibitor and EVs-miR-21aINC. Like that ours reported previously [16, 17], MSCs-EVs administration significantly decreased the water content ([F(2,9)=50.806, p<0.001] ; post hoc p<0.001) and infarct volumes ([F(2,9)=189.879, p<0.001] ; post hoc p<0.001) in HI neonatal mice (Figure S1 in supplementary material). EVs-miR-21ainhibitor abrogated this effect of MSCs-EVs (Figure S2 in supplementary material).
Consistently, micrglia polarization was as assessed by immunofluorescence staining. The results revealed that the number of M1 phenotypes (CD16+/Iba1+ cells) the right cortex was significantly reduced (Figure 6A) and the number of M2 phenotypes (CD206+/Iba1+ cells) in the ipsilateral cortex significantly increased (Figure 6B) in the HI + EVs group as compared with that in response to HI alone when determined at 3 d post-HI.
MiR-21a-5p in MSCs-EVs regulated STAT3 signaling pathway in the right cortex following HI insult
Like that reported previously , p-STAT3 staining is localized in the Iba1+ cells microglia/macrophages in the right hemisphere (Figure 7A). Consistent with in vitro data, EVs treatment significantly reduced p-STAT3 ([F(2,9)=17.488, p<0.01] ; post hoc p<0.01) in the right cortex at 3 d post-HI (Figure 7B). EVs-miR-21ainhibitor abrogated this effect of EVs ([F(3,12)=32.603, p<0.001] ; post hoc p<0.01) (Figure 7C).