Lipopolysaccharide pretreated bone marrow mesenchymal stem cells derived exosomes promote M2 macrophage polarization through CCN3/NOTCH1 pathway

AbstractBackground and Objectives:Exosomes secreted by bone marrow mesenchymal stem cells (BMSCs) pretreated with lipopolysaccharide (LPS) (L-Exo) exert a stronger anti-inflammatory effect than exosomes derived from BMSCs (Exo); exosomes are likely to exert biological effects through carrier proteins. This study aimed to investigate whether L-Exo reduces the inflammatory response after sepsis by overexpressing a specific protein.Methods:The effects of L-Exo and Exo in the treatment of sepsis models in vitro (LPS stimulating Raw264.7) were compared, and their differential proteins were analyzed by mass spectrometry. The mechanism of anti-inflammatory effect of proteins carried by exosomes was evaluated by Western blot, qRT-PCR, ELISA, cell transfection, and TUNEL.Results:ELISA showed that the concentration of TNF-a in the supernatant of septic model treated with L-Exo (131.60 mg/mL) was lower than that in the Exo group (170.85 mg/mL). WB and qRT-PCR showed that the expression of TNF-a and iNOS protein was lowest in the L-Exo group, but no obvious apoptotic cells were detected in TUNEL staining. A total of 154 proteins with significant differences were obtained; CCN3 is one of the upregulated differential proteins. In this study, we verified L-Exo’s anti-inflammatory effect by downregulating NOTCH1 signal to promote M2 polarization via cell transfection and qRT-PCR.Conclusion:L-Exo exerts an anti-inflammatory effect by promoting macrophages polarization to M2 through CCN3/NOTCH1 pathway but is not related to macrophage apoptosis pathway.