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MiR-126-3p-Enriched Extracellular Vesicles from Hypoxia-Preconditioned VSC 4.1 Neurons Attenuate Ischaemia-Reperfusion-Induced Pain Hypersensitivity by Regulating the PIK3R2-Mediated Pathway

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Abstract

Recent evidence suggests that hypoxia preconditioning can alter the microRNA (miRNA) profile of extracellular vesicles (EVs) and has better neuroprotective effects when enriched miRs are delivered to recipients. However, the roles of exosomal miRNAs in regulating ischaemia-reperfusion (IR)-induced pain hypersensitivity are largely unknown. Thus, we isolated EVs from normoxia-conditioned neurons (Nor-VSC EVs) and Hypo-VSC EVs by ultracentrifugation. After the initial screening by a microarray analysis and quantitative RT-PCR (qRT-PCR), miR-126-3p, which was detected as the most altered miR in the Hypo-VSC EVs, was further confirmed by applying GW4869 to inhibit exosomal secretion. Moreover, transfection with a miR-126 mimic obviously increased miR-126-3p expression in Nor-VSC EVs, whereas a miR-126 inhibitor prevented the increase in miR-126-3p in Hypo-VSC EVs. A rat model of pain was established by performing 8-min occlusion of the aorta. Following IR, compared with the Nor-VSC EVs- or antagomir-126-injected rats, the Hypo-VSC EVs-injected rats displayed improved pain hypersensitivity demonstrated as higher PWT and PWL values. Mechanistically, PIK3R2 is a target of miR-126-3p and might be a modulator of the phosphoinositide 3-kinase (PI3K)/Akt pathway as the PIK3R2 and PI3K immunoreactivities in each group were changed in opposite directions. Compared with the controls, higher protein levels of PI3K and phosphorylated Akt but lower levels of phosphorylated nuclear factor-κ B (NF-κB), tumour necrosis factor (TNF)-α and interleukin (IL)-1β were detected in the spinal cords of the Hypo-VSC EVs-injected rats, and these effects were impaired by an injection of Hypo-VSC EVs combined with antagomir-126. Collectively, the miR-126-3p-enriched Hypo-VSC EVs attenuated IR-induced pain hypersensitivity by restoring miR-126-3p expression in the injured spinal cord and subsequently modulating PIK3R2-mediated PI3K/Akt and NF-κB signalling pathways.

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Data Availability

The datasets used in this study are available from the corresponding author upon reasonable request.

Abbreviations

antagomir-126:

miR-126 antagomir

DMEM:

Dulbecco’s modified Eagle’s medium

EVs:

Extracellular vesicles

FBS:

Foetal bovine serum

FISH:

Fluorescence in situ hybridization

GM:

Golgi auto-antigen

HBSS:

Hank’s balanced salt solution

Hypo-VSC EVs:

Hypoxia-preconditioned neurons-derived extracellular vesicles

IL:

Interleukin

IR:

Ischaemia-reperfusion

miR:

microRNA

MSC-Exos:

Mesenchymal stem cell-derived exosomes

MT:

Mutant

NTA:

Nanoparticle tracking analysis

NC:

Negative control

NF-κB:

Nuclear factor-kappa B

Nor-VSC EVs:

Normoxia-conditioned neuron-derived extracellular vesicles

OGD/R:

Oxygen-glucose deprivation and reperfusion

p-Akt:

Phosphorylation of Akt

PI3K:

Phosphatidylinositol 3-kinase

PIK3R2:

Phosphoinositide-3-kinase regulatory subunit

PI3K:

Phosphoinositide 3-kinase

p-NF-κB:

Phosphorylated NF-κB

t-Akt:

Total Akt

TEM:

Transmission electron microscopy

t-NF-κB:

Total NF-κB

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Funding

This work was supported by the National Natural Science Foundation of China (no. 81601053 and 81771342) and the Scientific Research Fund of Liaoning Provincial Education Department (no. LK201636).

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Authors and Affiliations

  1. Department of Anesthesiology, First Affiliated Hospital, China Medical University, Shenyang, 110001, Liaoning, China

    He Wang, Feng-Shou Chen, Zai-Li Zhang, Hong-Xu Zhou, Hong Ma & **ao-Qian Li

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  1. He Wang
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Contributions

H.W. and Z.-L.Z. participated in the animal care and generated the animal models. H.W. and C.-F.C. performed the cell models and prepared the samples for the RT-PCR and immunohistochemistry assays; C.-F.C., Z.H.-X. and Z.-L.Z performed the experiments and statistical analysis; X.-Q.L. isolated and identified the neuron-derived EVs; and H.M. guided the study design and provided important suggestions regarding the manuscript writing.

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Correspondence to **ao-Qian Li.

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All experiments performed in this study were approved by the Ethics Committee of China Medical University and performed in compliance with the Care and Use of Laboratory Animals under approved protocols.

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Wang, H., Chen, FS., Zhang, ZL. et al. MiR-126-3p-Enriched Extracellular Vesicles from Hypoxia-Preconditioned VSC 4.1 Neurons Attenuate Ischaemia-Reperfusion-Induced Pain Hypersensitivity by Regulating the PIK3R2-Mediated Pathway. Mol Neurobiol 58, 821–834 (2021). https://doi.org/10.1007/s12035-020-02159-y

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