Hypoxic Bone Mesenchymal Stem Cell-Derived Exosomes Direct Schwann Cells Proliferation, Migration, and Paracrine to Accelerate Facial Nerve Regeneration via circRNA_Nkd2/miR-214-3p/MED19 Axis

Abstract

Background

Facial nerves have the potential for regeneration following injury, but this process is often challenging and slow. Schwann cells (SCs) are pivotal in this process. Bone mesenchymal stem cells (BMSC)-derived exosomes promote tissue repair through paracrine action, with hypoxic preconditioning enhancing their effects. The main purpose of this study was to determine whether hypoxia-preconditioned BMSC-derived exosomes (Hypo-Exos) exhibit a greater therapeutic effect on facial nerve repair/regeneration and reveal the mechanism.

Methods

CCK-8, EdU, Transwell, and ELISA assays were used to evaluate the functions of Hypo-Exos in SCs. Histological analysis and Vibrissae Movements (VMs) recovery were used to evaluate the therapeutic effects of Hypo-Exos in rat model. circRNA array was used to identify the significantly differentially expressed exosomal circRNAs between normoxia-preconditioned BMSC-derived exosomes (Nor-Exos) and Hypo-Exos. miRDB, TargetScan, double luciferase assay, qRT-PCR and WB were used to predict and identify potential exosomal cirRNA_Nkd2-complementary miRNAs and its target gene. The function of exosomal circRNA_Nkd2 in facial nerve repair/regeneration was evaluated by cell and animal experiments.

Results

This study confirmed that Hypo-Exos more effectively promote SCs proliferation, migration, and paracrine function, accelerating facial nerve repair following facial nerve injury (FNI) compared with Nor-Exos. Furthermore, circRNA analysis identified significant enrichment of circRNA_Nkd2 in Hypo-Exos compared with Nor-Exos. Exosomal circRNA_Nkd2 positively regulates mediator complex subunit 19 (MED19) expression by sponging rno-miR-214-3p.

Conclusion

Our results demonstrated a mechanism by which Hypo-Exos enhanced SCs proliferation, migration, and paracrine function and facial nerve repair and regeneration following FNI through the circRNA_Nkd2/miR-214-3p/Med19 axis. Hypoxic preconditioning is an effective and promising method for optimizing the therapeutic action of BMSC-derived exosomes in FNI.

Graphical Abstract

Keywords:

• hypoxic
• BMSCs
• exosomes
• Schwann cells
• facial nerve injury

Abbreviations

FNI, Facial nerve injury; SCs, Schwann cells; BMSCs, Bone mesenchymal stem cells; miRNAs, microRNAs; circRNAs, circular RNAs; MED19, Mediator complex subunit 19; pSCs, Primary Schwann cells; SD, Sprague Dawley; CCK8, Cell counting kit-8; EdU, Ethynyldeoxyuridine; OD, Optical density; IHC, Immunohistochemistry; VMs, Vibrissae Movements; qRT-PCR, Quantitative real-time polymerase chain reaction; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; FISH, Fluorescence in situ hybridization; WT, wild type; MUT, Mutant type; WB, Western Blotting; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Disclosure

The authors report no conflicts of interest in this work.

Additional information

Funding

This study was supported by the National Natural Science Foundation of China (No. 82171360 and No. 81974186), Science and Technology Commission of Shanghai Municipality Project (No. 21Y21900500).