# TNF-α-preconditioning enhances analgesic efficacy of mesenchymal stem cell-derived extracellular vesicle in neuropathic pain via miR-101b-3p targeting Nav1.6

## TL;DR

Preconditioning mesenchymal stem cells with TNF-α improves their extracellular vesicles' ability to relieve neuropathic pain by targeting a specific microRNA that reduces nerve cell overactivity.

## Contribution

The study reveals that TNF-α preconditioning enhances analgesic efficacy of MSC-EVs via miR-101b-3p targeting Nav1.6 in neuropathic pain.

## Key findings

- TNF-α-preconditioned MSC-EVs (T-EVs) significantly improve mechanical and thermal pain thresholds in a mouse model of neuropathic pain.
- miR-101b-3p in T-EVs directly targets Nav1.6, reducing dorsal root ganglion neuron hyperexcitability.
- Nanovesicles overexpressing miR-101b-3p replicate the analgesic effects of T-EVs, suggesting therapeutic potential.

## Abstract

Mesenchymal stem cell-derived extracellular vesicle (MSC-EV) has shown promise for pain relief, but its efficacy is limited. Preconditioning MSC with tumor necrosis factor-α (TNF-α) may enhance their therapeutic potential; however, the impact on analgesia and underlying mechanisms remains unclear. Here, we investigated the analgesic effects of EV from TNF-α-preconditioned MSC (T-EV) in a chronic constriction injury (CCI) mouse model and examined the molecular mechanisms involved. Following intrathecal injection, T-EV produced greater improvements in mechanical and thermal pain thresholds than control MSC-EV (C-EV), achieving enhanced pain relief for two weeks. Whole-cell patch-clamp recordings revealed that T-EV markedly decreased both the firing rate and action potential amplitude of dorsal root ganglion (DRG) neurons. RNA sequencing revealed that T-EV was enriched in miR-101b-3p. Silencing miR-101b-3p in T-EV abolished their enhanced analgesic effects and reversed DRG hyperexcitability. Moreover, miR-101b-3p was shown by luciferase assays to bind directly to the 3′UTR of Nav1.6, suppressing its expression. Engineered MSC-derived nanovesicle overexpressing miR-101b-3p replicated the increased pain relief observed with T-EV. These findings demonstrate that TNF-α preconditioning improves the analgesic potency of MSC-EV by delivering miR-101b-3p, which downregulates Nav1.6 and decreases DRG hyperexcitability. This study supports the therapeutic potential of miR-101b-3p-enriched vesicle as a novel strategy for treating neuropathic pain.

Image 1

•T-EV exerts significantly stronger analgesic effects in the mouse model of neuropathic pain compared to the control EV.•The enhanced analgesic efficacy of T-EV is mediated by increased concentration of miR-101b-3p within the vesicles.•MiR-101b-3p directly targets and downregulates Nav1.6 expression, contributing to the potent analgesic response.•Nanovesicle-encapsulated miR-101b-3p produces a similar analgesic effect to T-EV, highlighting its therapeutic potential.

T-EV exerts significantly stronger analgesic effects in the mouse model of neuropathic pain compared to the control EV.

The enhanced analgesic efficacy of T-EV is mediated by increased concentration of miR-101b-3p within the vesicles.

MiR-101b-3p directly targets and downregulates Nav1.6 expression, contributing to the potent analgesic response.

Nanovesicle-encapsulated miR-101b-3p produces a similar analgesic effect to T-EV, highlighting its therapeutic potential.