Protective mechanisms of exosomes derived from human umbilical cord mesenchymal stem cells in bronchopulmonary dysplasia

Abstract

Bronchopulmonary dysplasia (BPD) is characterized by reduced alveolar formation and disordered matrix remodeling. Currently, there are no effective therapeutic approaches for it. This study aims to explore the protective effect of exosomes derived from human umbilical cord mesenchymal stem cells on BPD by regulating the immune response and inflammatory pathways of macrophages. PKH26-labeled human umbilical cord mesenchymal stem cell line exosomes (hUCMSC-Exos) were co-cultured with RAW264.7 cells, which were assigned to the following groups: normoxia, normoxia + NLRP3 activator (Nigericin), normoxia + hUCMSC-Exos + Nigericin, hyperoxia, hyperoxia + hUCMSC-Exos, and hyperoxia + hUCMSC-Exos + Nigericin. Cell viability and cytokine expression in cell supernatant were measured for each group. PKH26 exosome staining confirmed successful uptake of hUCMSC-Exos by RAW264.7 cells. hUCMSC-Exos demonstrated protective effects against reductions in cell viability induced by both Nigericin and hyperoxia. Cells in the Hyperoxia group showed significantly increased expression of inflammatory cytokines IL-33, IL-6, IL-1beta, TNF-alpha, and IL-18 compared to those in the Normoxia group, along with elevated mRNA and protein levels of NLRP3, ASC, caspase-1, IL-18, IL-1beta, and ATF4. The Hyperoxia + hUCMSC-Exos group exhibited reduced expression of IL-33, IL-6, IL-1beta, TNF-alpha, IL-18 and IL-33, IL-6, IL-1beta, TNF-alpha, and IL-18 compared to the Hyperoxia group. In contrast, the Hyperoxia + hUCMSC-Exos + Nigericin group showed elevated levels of IL-33, IL-6, IL-1beta, TNF-alpha, and IL-18, as well as increased expression of NLRP3, ASC, caspase-1, IL-18, IL-1beta, and ATF4 compared to the Hyperoxia + hUCMSC-Exos group. hUCMSC-Exos mitigate hyperoxia-induced damage to lung macrophages by reducing endoplasmic reticulum stress, inhibiting NLRP3 inflammasome expression, and regulating inflammatory cytokine release, that may be potentially useful in BPD. Key words Bronchopulmonary dysplasia " Exosomes " Human umbilical cord mesenchymal stem cells " Macrophages " NLRP3.

Fig. 1

Identification of hUCMSC-Exos. (A) Transmission electron microscopy of exosomes (scale bar = 100 nm); (B) Detection of typical particle size of hUCMSC-Exos in submitted samples; (C) Immunoblotting detection of exosome markers.

Fig. 2

Exosomes uptake by RAW264.7 macrophages.

Fig. 3

CCK-8 Detection of cell proliferation activity. Note: ** P<0.001, * P<0.05.

Fig. 4

mRNA expression levels of NLRP3, ASC, Caspase-1 and ATF4. (A, B, C and D) mRNA expression levels of NLRP3, ASC, caspase-1 and ATF4 respectively, in monocyte macrophages. Note: ** P<0.001, * P<0.05.

Fig. 5

Expression of NLRP3, ASC, Caspase-1 and ATF4 in Monocyte Macrophages. Protein expression levels of NLRP3, caspase-1, ASC and ATF4 in monocyte macrophages, respectively. (A) Western blot images. (B) Quantitative bar graphs for each protein (NLRP3, ASC, Caspase-1, ATF4). Note: ** P<0.001, * P<0.05.

Fig. 6

Levels of cell inflammatory factors. (A, B, C, D, and E) Levels of IL-33, IL-6, IL-1β, TNF-α, and IL-18 in monocytes macrophages, respectively. ** P<0.001, * P<0.05.