# Mechanisms underlying the role of TNF-α and IL-1β preconditioned exosomes derived from human umbilical cord mesenchymal stem cells in wound healing

## TL;DR

This study shows that exosomes from stem cells preconditioned with TNF-α and IL-1β improve wound healing by boosting collagen and blood vessel formation.

## Contribution

The study identifies miR-215-5p as a key miRNA in preconditioned exosomes that enhances wound healing via the WNK1/p-Smad3/VEGF-A pathway.

## Key findings

- TNF-α+IL-1β-Exos accelerated wound healing more than conventional exosomes.
- miR-215-5p promotes HUVEC tubulogenesis and proliferation in vitro and wound healing in vivo.
- miR-215-5p activates the WNK1/p-Smad3/VEGF-A signaling axis to enhance angiogenesis.

## Abstract

In this study, human umbilical cord mesenchymal stem cells (hUC-MSCs) were stimulated with tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) to obtain preconditioned exosomes (Exos). Comparative miRNA profiling was performed between cytokine-primed Exos and conventionally cultured counterparts to identify differentially expressed miRNAs. Functional validation of candidate miRNAs will elucidate their mechanistic roles in promoting cutaneous repair, thereby advancing the clinical translation of Exos-based regenerative therapies.

We successfully extracted and characterized hUC-MSCs derived Exos (hUCMSCs-Exos) and classified them into Exos obtained under normal culture conditions (Con-Exos) and those stimulated by TNF-α+IL-1β obtained Exos (TNF-α+IL-1β-Exos). The wound healing rate was observed and counted by establishing a mouse whole skin defect wound model in vivo. In vitro, human umbilical vein endothelial cells (HUVECs) were stimulated with two groups of hUCMSCs-Exos to observe the tube formation of HUVECs and the results of 5-ethynyl-2’-deoxyuridine (EdU) assay.Then, the differential miRNAs in the two groups of hUCMSCs-Exos were detected and validated to identify the candidate effector miRNAs, which were then analyzed by databases and searched and screened for common target genes.

TNF-α+IL-1β-Exos accelerated wound healing more than Con-Exos, mainly by increasing collagen deposition and expression of the angiogenic marker CD31. Sequencing and bioinformatics analyses revealed that the key miRNA for both co-actions was miR-215-5p. Cellular experiments showed that miR-215-5p mimics promoted HUVECs tubulogenesis and proliferation, whereas the inhibitor effect was not significant. In animal experiments, miR-215-5p mimics also significantly accelerated wound healing in mice.

TNF-α+IL-1β-Exos demonstrated superior wound healing efficacy compared to Con-Exos. This enhanced therapeutic effect may be attributed to the elevated expression of miR-215-5p in TNF-α+IL-1β-Exos. Mechanistically, miR-215-5p activates the WNK1/p-Smad3/VEGF-A signaling axis, promoting angiogenesis and accelerating cutaneous wound repair.