Abstract: Spinal cord injury is a disabling condition with limited treatment options. Multiple studies have provided evidence suggesting that small extracellular vesicles (SEVs) secreted by bone marrow mesenchymal stem cells play a role in eliciting the beneficial effects conferred by MSC transplantation following spinal cord injury. Strikingly, hypoxic preconditioned bone marrow mesenchymal stem cell-derived small extracellular vesicles (HSEVs) appears to increase their therapeutic potency. We thus explored the role of hypoxic preconditioned bone marrow mesenchymal stem cell-derived small extracellular vesicles in macrophage immune regulation after spinal cord injury in rats and its significance in spinal cord repair. Small extracellular vesicles or hypoxic preconditioned bone marrow mesenchymal stem cell-derived small extracellular vesicles were isolated from bone marrow mesenchymal stem cell supernatants by density gradient ultracentrifugation. Hypoxic preconditioned bone marrow mesenchymal stem cell-derived small extracellular vesicles administration to rats via tail vein injection after spinal cord injury reduced the lesion area and attenuated spinal cord inflammation. Hypoxic preconditioned bone marrow mesenchymal stem cell-derived small extracellular vesicles regulate macrophage polarization towards M2 macrophages in vivo and in vitro. miRNA sequencing and bioinformatics analyses of small extracellular vesicles and hypoxic preconditioned bone marrow mesenchymal stem cell-derived small extracellular vesicles revealed that miR-146a-5p is a potent mediator of macrophage polarization, with interleukin-1 receptor-associated kinase 1 (IRAK1) as a downstream target. Reducing miR-146a-5p expression in hypoxic preconditioned bone marrow mesenchymal stem cell-derived small extracellular vesicles partially attenuates macrophage polarization. Our data suggest that hypoxic preconditioned bone marrow mesenchymal stem cell-derived small extracellular vesicles attenuate spinal cord inflammation and injury in rats by transporting miR-146a-5p, which alters the polarization state of macrophages. This study provides new insights into the application of hypoxic preconditioned bone marrow mesenchymal stem cell-derived small extracellular vesicles as a therapeutic tool for spinal cord injury.

Key words: bone marrow mesenchymal stem cell, hypoxic precondition, interleukin-1 receptor-associated kinase 1, mesenchymal stem cells, macrophages, small extracellular vesicles, spinal cord injury