Annexin A1-Mediated Therapeutic Effect of Extracellular Vesicles Derived from Thrombin- Preconditioned Mesenchymal Stromal Cells in Experimental Necrotizing Enterocolitis

Abstract Background Necrotizing enterocolitis (NEC) is a devastating inflammatory disease in premature infants lacking specific treatment. Extracellular vesicles from thrombin-preconditioned mesenchymal stromal cells (thMSC-EVs) show enhanced therapeutic efficacy over naïve MSC-EVs. This study aimed to identify the key enriched therapeutic factor and its mechanism in experimental NEC. Methods Comparative proteomic profiling (LC-MS/MS) of thMSC-EVs was performed to identify candidate proteins. NEC was induced in neonatal mice (P4) using formula feeding, LPS, and stress. thMSC-EVs were administered intraperitoneally. Bulk RNA-sequencing and multi-omics ligand-receptor analyses of the mouse ileum integrated proteomic and transcriptomic data. The candidate, Annexin A1, was validated using in vitro LPS-induced inflammation and multiple-hit (HIT) models, assessing viability, barrier integrity, and NF-κB and Wnt/β-catenin signaling following treatment with thMSC-EVs, the Annexin A1 mimetic peptide (Ac2-26), and ANXA1-knockdown MSC-derived EVs (siANXA1-MSC-EVs). Results Proteomic analysis showed thMSC-EVs were significantly enriched with anti-inflammatory proteins, including a 3.8-fold increase in ANXA1. In the NEC model, thMSC-EVs attenuated intestinal necrosis and apoptosis, suppressed the mucosal antimicrobial response, and promoted tissue repair. Multi-omics ligand-receptor analysis identified the Annexin A1–formyl peptide receptor 1 interaction as the most prominent therapeutic axis. In vitro, Ac2-26 replicated the effects of thMSC-EVs, restoring cell viability and ZO-1 expression and suppressing NF-κB while restoring Wnt/β-catenin signaling. Conversely, siANXA1-MSC-EVs showed a significantly, though partially, diminished therapeutic effect. Conclusion Thrombin preconditioning amplifies the ANXA1 content within MSC-EVs. This enriched ANXA1 exerts a dual therapeutic effect in NEC by suppressing TLR4/NF-kB-mediated inflammation and promoting Wnt/β-catenin-mediated epithelial regeneration via the FPR signaling. Our findings establish ANXA1 as a major, though not exclusive, effector of thMSC-EVs and as a novel therapeutic target for NEC, supporting thMSC-EVs as a potent cell-free therapeutic agent.