Human Uterine Smooth Muscle Cells (HUSMCs)

ASC-Derived Extracellular Vesicles Suppress Macrophage-Driven Inflammatory Amplification and Contractile Activation of Uterine Smooth Muscle Cells

Preterm labor is a major cause of neonatal morbidity and mortality and is frequently driven by infection-associated inflammation that promotes premature uterine activation. In this study, we investigated the effects of adipose stem cell-derived extracellular vesicles (ASC-EVs) on macrophage-mediated inflammatory signaling in uterine smooth muscle cells (HUtSMCs). An in vitro model was established by treating HUtSMCs with conditioned media derived from LPS-stimulated RAW264.7 macrophages. Activation of signaling pathways was assessed by Western blotting and immunofluorescence, and functional responses were evaluated using calcium flux and collagen gel contraction assays.

Conditioned media from LPS-stimulated macrophages induced robust activation of MAPK (ERK1/2 and JNK) and NF-κB signaling, accompanied by IκB degradation and nuclear translocation of phosphorylated p65, whereas ASC-EVs pretreatment significantly attenuated these responses and reduced the expression of pro-inflammatory cytokines, including IL-6, IL-8, and MCP-1. Furthermore, macrophage-conditioned media enhanced intracellular calcium flux and contractile activity in HUtSMCs, both of which were suppressed by ASC-EVs. Inhibition of TLR4 signaling in macrophages reduced the inflammatory potency of conditioned media, indicating a key upstream role of macrophage TLR4 activation. Collectively, these findings demonstrate that ASC-EVs suppress macrophage-mediated inflammatory activation and downstream contractile responses, suggesting their potential as a cell-free therapeutic strategy for preventing inflammation-associated preterm labor.