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    The delivery of miR-21a-5p by extracellular vesicles induces microglial polarization via the STAT3 pathway following hypoxia-ischemia in neonatal mice
  • Figure 1|Identification of EVs and MSCs.

    Analysis of the TEM images revealed that the MSCs-EVs were around 100 nm in diameter with a rounded morphology (Figure 1A). Using qNano, the size of the MSCs-EVs was seen to range from 60 to 120 nm; they were thus identified as small vesicles (Figure 1B). The Western blot analyses revealed that the MSCs-EVs contained markers of EVs, specifically CD9 and TSG101; they did not contain calnexin, GM130, cytochrome C1, or lamin A/C (Figure 1C). Using PKH67-labeled EVs, it was found that they were internalized by BV-2 cells and could be seen in the cytoplasm (Figure 1D). 


    The MSCs were found to be spindle-shaped (Figure 1E) and capable of differentiating into osteoblasts and adipocytes (Figure 1F). FACS analyses showed that the MSCs were positive for CD44, CD29, and Sca-1, and negative for CD11b, CD45, CD31, CD117, Ly6G, CD19, CD14, and CD4 (Figure 1G). These results show that the extracted cells possessed the typical characteristics of MSCs and that they were able to differentiate.


    Figure 6|MSCs-EVs decrease microglial activation in the cortex ipsilateral to the HI. 

    Immunohistochemical staining showed that the Iba-1+ cells in the sham group had a ramified shape. In the HI group, the microglia were activated and had a rounded, amoeboid-like appearance. The administration of MSCs-EVs significantly suppressed the microglial activation. The number of endpoints per Iba-1+ cell was found to be significantly lower in the HI group compared with the sham group (F(2,9) = 14.672, P < 0.01; post hoc P < 0.01); it was also found that the length of the Iba-1+ cell processes was shorter in the HI group than in the sham group (F(2,9) = 20.744, P < 0.001; post hoc P < 0.001). This difference was no longer apparent following treatment with MSCs-EVs (Figure 6).


    Figure 7|MSCs-EVs promote M2 microglial polarization and suppress HI-induced neuroinflammation in the cortex ipsilateral to the HI. 

    Treatment with MSCs-EVs was found to attenuate the mRNA levels of pro-inflammatory cytokines 72 hours after HI, including IL-1β (F(2,15) = 143.562, P < 0.001; post hoc P < 0.001) and TNF-α (F(2,15) = 119.605, P < 0.001; post hoc P < 0.001; Figure 7A); the treatment also increased the mRNA levels of anti-inflammatory cytokines, including CD206 (F(2,15) = 32.918, P < 0.001; post hoc P < 0.001) and TGF-β (F(2,15) = 122.783, P < 0.001; post hoc P < 0.001; Figure 7A). Analysis of the protein levels revealed that the treatment significantly decreased the proteinl evels of IL-1β (F(2,9) = 9.967, P < 0.01; post hoc P < 0.05) and increased the protein levels of arginase-1 (F(2,9) = 7.362, P < 0.05; post hoc P < 0.05; Figure 7B). 


    Microglial polarization was assessed using immunofluorescence staining 3 days after the HI. It was found that the number of M1 phenotypes (Iba1+CD16+ cells) in the right cortex was significantly lower in the HI + EVs group compared with the HI group (F(2,9) = 54.827, P < 0.001; post hoc P < 0.05; Figure 7C), whereas the number of M2 phenotypes (Iba1+CD206+ cells) was significantly higher (F(2,9) = 21.274, P < 0.001; post hoc P < 0.05; Figure 7C).


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  • 发布日期: 2022-03-19  浏览: 114
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