MYD88-silencing hUCMSC-derived extracellular vesicles alleviate sepsis- associated encephalopathy by modulating neurovascular and microglial inflammation

Background: Sepsis-associated encephalopathy (SAE) is a common complication of sepsis and is closely associated with persistent neuroinflammation, neurovascular injury and long-term cognitive impairment. Myeloid differentiation primary response 88 (MYD88) is a shared signaling adaptor for the Toll-like receptor family and the interleukin-1 receptor (IL-1R) family, and its expression remains persistently elevated in microglia and vascular endothelial cells in surviving mice after cecal ligation and puncture (CLP). Methods: hUCMSC-derived EVs were isolated from serum-free conditioned medium, loaded with MYD88-targeting siRNA (siMYD88) or negative-control siRNA by electroporation, and characterized by nanoparticle tracking analysis, transmission electron microscopy, and EV marker profiling. siRNA protection and MYD88 knockdown were evaluated in BV2 cells and primary microglia. Therapeutic efficacy was examined in a CLP survivor model using a rescue intervention initiated 72 h after CLP. Brain delivery, blood-brain barrier permeability, inflammatory and antioxidant signaling, hippocampal ultrastructure, and behavioral outcomes were assessed. Results: siMYD88-loaded hUCMSC-EVs protected siMYD88 from RNase degradation and mediated functional MYD88 knockdown in microglial cells. After tail-vein administration, siMYD88-EVs entered the brain and were taken up by microglia. Rescue treatment with siMYD88-EVs at 72 h after CLP reduced MYD88 overexpression in vascular endothelial cells and protected against sepsis-induced disruption of blood-brain barrier integrity. siMYD88-EVs also suppressed hippocampal microglial activation, attenuated MYD88/NF-κB/COX2 inflammatory-redox signaling, and restored NRF2/HO-1-associated antioxidant responses impaired after CLP. MYD88 silencing further enhanced TREM2-associated microglial responses, preserved hippocampal mitochondrial and synaptic ultrastructure, and improved anxiety-like behavior and cognitive deficits after CLP. Conclusions: These findings indicate that sustained MYD88 signaling is a targetable driver of post-septic neurovascular injury and neuroinflammatory damage, and support engineered EV-mediated MYD88 silencing as a potential cell-free RNA delivery strategy for SAE.

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