Article Figures & Data
Figures
- Figure 1.
Astrocytes release exosomes after stimulation with brain extracts. A, Exosomes derived from astrocytes were increased after brain extract stimulation (scale bar = 100 µm). B, The expression of the exosomal markers CD9 and CD63 in the medium of primary cultured astrocytes was detected by Western blotting after 24 h of brain extract treatment (n = 3, t test). C, A representative TEM image of exosomes in the culture medium of stimulated astrocytes after 24 h of brain extract treatment (scale bar = 100 nm). D, The expression of the exosomal markers CD9 and CD63 in astrocyte-derived exosomes was detected by Western blotting. Con, control; Ext, brain extracts. Compared with the Con group, *p < 0.05 and **p < 0.01.
- Figure 2.
TBI-injured astrocyte-derived exosomes are taken up by primary microglia and promote M2 microglial polarization. A, PKH26 dye was used to assess whether exosomes were taken up by microglia (scale bar = 20 µm). B, Fluorescence confocal microscopy images showed increased expression of Iba-1 (microglia markers) and Arg-1 (M2 marker) after treatment with astrocyte-derived exosomes (scale bar = 100 µm). C, Arg-1, IL-4, and IL-10 (M2 markers) were detected by Western blot analysis (n = 3, one-way ANOVA). D–F, The mRNA expression of Arg-1, IL-4, and IL-10 (M2 markers) was detected by qRT-PCR (n = 3, one-way ANOVA). Exo, exosomes incubated with microglia for 4 h. Ext + Exo, primary microglia stimulated with brain extracts for 24 h and incubated with exosomes for 4 h. Compared with the Con group, *p < 0.05 and **p < 0.01. Compared with Con + Exo, #p < 0.05 and ##p < 0.01.
- Figure 3.
The expression of miR-873a-5p in necrotic brain tissue and edematous brain tissue in TBI was measured by qRT-PCR. Brain tissue samples from necrotic and edematous areas were collected 3 d after TBI. Compared with the edema group, **p < 0.01 and ***p < 0.001, n = 3, t test.
- Figure 4.
Effect of miR-148a-3p on LPS-induced primary microglia. A, The transfection efficiency of the miR-148a-3p mimic or miR-148a-3p inhibitor was measured by qRT-PCR (n = 3, one-way ANOVA). B–E, Western blot analysis of the protein expression of the proinflammatory factors TNF-α, IL-1β, iNOS, and IL-6 (n = 3, one-way ANOVA). F, qRT-PCR analysis of the mRNA expression of the proinflammatory factors TNF-α, IL-1β, iNOS, and IL-6 (n = 3, one-way ANOVA). LPS (1 µg/ml, 24 h) was used to induce microglial injury. Compared with the NC group, *p < 0.05, **p < 0.01, and ***p < 0.001. Compared with LPS, #p < 0.05 and ##p < 0.01.
- Figure 5.
Brain defect area, brain edema, and neurological deficits in rats after miR-148a-3p–mediated attenuation of TBI. A, MiR-148a-3p expression in the cortex was detected by qRT-PCR 1, 3, and 7 d after TBI (n = 6, one-way ANOVA). B, Neurological function of rats 1, 3, and 7 d after TBI was assessed by the mNSS (n = 6, one-way ANOVA). C, The brain water content was measured by the wet and dry method (n = 6, one-way ANOVA). D, The area of the brain defect was observed by HE staining (scale bar = 500 µm). E: Levels of phosphorylated ERK and NF-ΚB P65 were measured by Western blotting 7 d after TBI (n = 6, one-way ANOVA). Compared with the sham group, **p < 0.01 and ***p < 0.001. Compared with TBI, #p < 0.05, ##p < 0.01, and ###p < 0.001.
- Figure 6.
MiR-148a-3p suppresses the inflammatory response by promoting M2 polarization in microglia after TBI. A–C, The expression of the M1 signature genes iNOS and CD32 and the M2 signature gene CD206 was detected by qRT-PCR in the rat cortex (n = 6, one-way ANOVA). D, Immunofluorescence analysis of the expression of the M1 microglial marker iNOS in the rat cortex (scale bar = 100 µm). E, Immunofluorescence analysis of the expression of the M2 microglial marker Arg1 in the rat cortex (scale bar = 100 µm). Compared with the sham group, ***p < 0.001. Compared with TBI, #p < 0.05 and ###p < 0.001.
Tables
Gene sequence Primer name Sequence (5′ → 3′) iNOS Forward CAGGGTGTTGCCCAAACTG Reverse GGCTGCGTTCTTCTTTGCT TNF-α Forward TCTTCTCATTCCTGCTTGTGG Reverse GGTCTGGGCCATAGAACTGA Arg-1 Forward AGACAGCAGAGGAGGTGAAGTAC Reverse GGTAGTCAGTCCCTGGCTTATGGT IL-10 Forward ACCTGGTAGAAGTGATGCCC Reverse ACACCTTGGTCTTGGAGCTT IL-4 Forward GTAGGGCTTCCAAGGTGCTTC Reverse CATGATGCTCTTTAGGCTTTCCAG RTN4 Forward AGTACTTACGAAAGAAGCAGG Reverse GTATCACAGGCTCAGATGCAG GAPDH Forward ACAACTTTGGTATCGTGGAAGG Reverse GCCATCACGCCACAGTTTC IL-1β Forward TGCAGAGTTCCCCAACTGGTACA Reverse GTGCTGCCTAATGTCCCCTT-G miR-148a-3p Forward GAGACACTCCGACTCTGAGT Reverse GTTCTGTAGTGCACTGAC U6 Forward CTCGCTTCGGCAGCACA Reverse AACGCTTCACGAATTTGCGT IL-6 Forward CCGGAGAGAGACTTCACAG Reverse CATTTCCACGATTTCCCAGA β-actin Forward TGGAATCCTGTGGCATCCATGAAAC Reverse TAAAACGCAGCTCAGTAACAGTCCG CD206 Forward TCTTTGCCTTTCCCAGTCTCC Reverse TGACACCCAGCGGAATTTC CD32 Forward CCAGAAAGGCCAGGATCTAGTG Reverse GGGAACCAATCTCGTAGTGTCTGT
In this issue
