Dental stem cell-derived extracellular vesicles transfer miR-330-5p to treat traumatic brain injury by regulating microglia polarization

Abstract

Traumatic brain injury (TBI) contributes to the key causative elements of neurological deficits. However, no effective therapeutics have been developed yet. In our previous work, extracellular vesicles (EVs) secreted by stem cells from human exfoliated deciduous teeth (SHED) offered new insights as potential strategies for functional recovery of TBI. The current study aims to elucidate the mechanism of action, providing novel therapeutic targets for future clinical interventions. With the miRNA array performed and Real-time PCR validated, we revealed the crucial function of miR-330-5p transferred by SHED-derived EVs (SHED-EVs) in regulating microglia, the critical immune modulator in central nervous system. MiR-330-5p targeted Ehmt2 and mediated the transcription of CXCL14 to promote M2 microglia polarization and inhibit M1 polarization. Identified in our in vivo data, SHED-EVs and their effector miR-330-5p alleviated the secretion of inflammatory cytokines and resumed the motor functional recovery of TBI rats. In summary, by transferring miR-330-5p, SHED-EVs favored anti-inflammatory microglia polarization through Ehmt2 mediated CXCL14 transcription in treating traumatic brain injury.

Fig. 1

SHED-EVs transferred miR-330-5p to activated microglia. a Transmission electron micrographs of SHED-EVs. Scale bar: 50 nm. b Particle sizes measured by nanoparticle tracking analysis. c Western blotting characterization of EV markers. d Heatmap of differentially expressed miRNAs in activated microglia co-cultured with SHED-EVs. e Volcano plot filtering of differentially expressed miRNAs. f Real-time PCR validation of candidate miRNAs. g The level of miR-330-5p in activated microglia. h GW4869 inhibited the delivery of miR-330-5p from SHED-EVs to microglia. SHED-EVs without miR-330-5p may not alter the level of miR-330-5p in microglia

Fig. 2

miR-330-5p inhibited pro-inflammatory polarization and promoted anti-inflammatory polarization of microglia. a–c The level of IL-6, TNF-α and IL-10 secreted by microglia. d Real-time PCR assay of M1 polarization marker CD86 mRNAs in microglia. e Real-time PCR assay of M1 polarization marker MHCII mRNAs in microglia. f Real-time PCR assay of M2 polarization marker IL-10 mRNAs in microglia. g, h Immunofluorescent staining for CD86 and CD206. Scale bar: 25 μm

Fig. 3

Ehmt2 was the downstream target of miR-330-5p. a Target prediction of miR-330-5p. b The mRNA level of Ehmt2 regulated by miR-330-5p. c The protein level of Ehmt2. d Western Blotting quantification. e Luciferase reporter designing. f, g Transfection of miR-330-5p mimics or inhibitors with luciferase reporter

Fig. 4

Ehmt2 reversed the effects of miR-330-5p on microglial polarization. a Ehmt2 overexpressed microglia construction. b–d The level of IL-6, TNF-α and IL-10 secreted by microglia. e NO levels quantified via Griess assay. f Real-time PCR assay of M1 polarization marker CD86 mRNAs in microglia. g Real-time PCR assay of M1 polarization marker MHCII mRNAs in microglia. h Real-time PCR assay of M2 polarization marker IL-10 mRNAs in microglia. i, j Immunofluorescent staining for CD86 and CD206. Scale bar: 100 μm

Fig. 5

miR-330-5p mediated the transcription of CXCL14 to regulate microglia polarization. a Level of H3K9me2 in microglia regulated by SHED-EVs and miR-330-5p/Ehmt2. b Quantitative analysis. c Transcription level of CXCL14 in miR-330-5p overexpressed microglia. d The enrichment of H3K9me2 regulated by miR-330-5p. e Protein level of CXCL14 in microglia regulated by SHED-EVs and miR-330-5p. f Quantitative analysis

Fig. 6

SHED-EVs/miR-330-5p ameliorated the functional damage of rat TBI. a Model construction and treatments. b Motor function analysis. c HE stained brain tissue slides. Scale bar: 200 μm. d–f The mRNA level of IL-6, TNF-α and IL-10 in brain tissues

Fig. 7

SHED-EVs/miR-330-5p shifted microglia polarization in brain tissues of TBI rats. a, c Immunofluorescent staining of brain tissues for CD86 and CD206. Scale bar: 200 μm. b, d Quantitative analysis

Fig. 8

Mechanistic diagram of SHED-EVs/miR-330-5p in treating TBI. SHED-EVs/miR-330-5p shifted microglia polarization by targeting Ehmt2-H3K9me2 mediated CXCL14 transcription, leading to mitigated neuro-inflammation and enhanced repair of brain injury