Lu Wang, Min Wang, Yuyan Liu, Yang Bai, Yun Li, Yuan Cao, Chengjin Wang, Zhen Gao, Yiqi Wu, Zihui Deng, Hongjun Kang
Background Central nervous system (CNS) injury is a major determinant of mortality and persistent disability after heat stroke. Mesenchymal stromal/stem cell-derived extracellular vesicles (MSC-EVs) are candidate cell-free immunomodulatory therapeutics, but their effects and molecular correlates in heat stroke-associated brain injury remain incompletely defined. We investigated whether MSC-EVs attenuate hippocampal inflammation and alter microglial phenotype-associated markers after heat stroke. Methods Human umbilical cord MSC-EVs were isolated by differential ultracentrifugation and characterized by transmission electron microscopy, nanoparticle tracking analysis, and immunoblotting for EV-enriched and non-EV markers. In a mouse heat stroke model, MSC-EVs (200 μg total protein per mouse) were administered intravenously. Neurological outcomes, hippocampal pathology, inflammatory mediators, IBA1 immunoreactivity, and phenotype-associated markers were evaluated. BV2 heat-stress and MSC co-culture experiments were used to examine VIM-AS1-associated miR-34a-5p/Per2 signaling. Results MSC-EV treatment improved survival and neurological scores and reduced hippocampal injury and inflammatory readouts after heat stroke. In mouse brain and BV2 models, treatment was associated with lower expression of inflammatory-associated markers and higher expression of repair-associated markers. EV RNA sequencing identified abundant VIM-AS1. VIM-AS1 gain- and loss-of-function experiments altered miR-34a-5p and Per2 expression, while miR-34a-5p directly repressed a conserved mouse Per2 3′UTR reporter. VIM-AS1 overexpression also improved inflammatory and neurological readouts in vivo . Conclusions MSC-EVs attenuated heat stroke-induced hippocampal injury and were associated with coordinated changes in VIM-AS1, miR-34a-5p, and Per2. These findings support MSC-EVs and VIM-AS1-associated signaling as candidates for further mechanistic investigation, while not establishing VIM-AS1 as the sole or dominant active EV cargo.