‌Dual-target hUCMSCs/EVs therapy for autism spectrum disorder: remodeling gut microbiota and modulating neuroimmune crosstalk in a valproic acid-induced C57BL/6 mice model.

INTRODUCTION: Autism Spectrum Disorder (ASD) is a complex neurodevelopmental condition characterized by deficits in social communication and repetitive behaviors. Accumulating evidence implicates neuroimmune dysregulation and gut microbiota dysbiosis in its pathogenesis, yet effective therapies targeting these mechanisms are lacking. This study investigated the therapeutic efficacy of human umbilical cord-derived mesenchymal stem cells (hUCMSCs) and their extracellular vesicles (EVs), both alone and in combination, in a valproic acid (VPA)-induced ASD mouse model. METHODS: VPA-exposed C57BL/6 mice were randomized into five groups: control, VPA, hUCMSCs alone, EVs alone, and hUCMSCs + EVs combination. Behavioral tests, biochemical analyses, 16 S rRNA sequencing, immunofluorescence, and transmission electron microscopy (TEM) were performed RESULTS: While both hUCMSCs and EVs alone showed some beneficial effects on certain ASD-like symptoms, each exhibited limited efficacy in achieving comprehensive remediation. In contrast, the combined hUCMSCs + EVs therapy yielded the most robust improvements across multiple domains, including social interaction and repetitive behaviors. Furthermore, the combination therapy synergistically normalized neuroinflammatory cytokine levels and oxidative stress, restored synaptic and mitochondrial ultrastructure in key brain regions, and promoted gut microbiota homeostasis by enriching beneficial bacteria such as Lactobacillus and reducing pathogens. DISCUSSION: These results highlight that although individual treatments offer partial relief, only the combined strategy fully restores neuroimmune-microbiota homeostasis, demonstrating a complementary and synergistic therapeutic effect. This study establishes a novel dual-target approach leveraging systemic hUCMSCs and CNS-targeted EVs, providing a promising translational strategy for ASD through orchestrated regulation of the neuro-immune-microbiota axis.