Chili pepper-derived exosome-like nanovesicles promote macrophage M2 polarization by activating PPAR-γ to regulate mitophagy

To investigate whether Capsicum-derived exosome-like nanovesicles (CDELN) regulate mitochondrial autophagy and, consequently, macrophage polarization by activating the peroxisome proliferator-activated receptor γ(PPAR-γ) signaling pathway, along with exploring the underlying molecular mechanisms.

CDELNs were isolated and purified by ultracentrifugation combined with sucrose density gradient centrifugation. Particle size and concentration were determined by nanoparticle tracking analysis (NTA), and morphology was observed using transmission electron microscopy (TEM). Protein composition and molecular weight distribution in the nanovesicles were analyzed by SDS-PAGE with Coomassie brilliant blue staining and Western blot. Subsequently, PKH67 exosome labeling and tracing assay, zeta potential detection, and untargeted plant metabolomics profiling were performed. Mouse RAW 264.7 monocyte/macrophage leukemia cells were cultured in vitro and randomly divided into 5 groups:blank control group (NC group), LPS (1000 ng/mL) group, CDELN (10⁹ particles/mL) group, LPS+CDELN (1000 ng/mL+10⁹ particles/mL) group, and LPS+CDELN+Oleuropein (1000 ng/mL+10⁹ particles/mL+10 μmol/L) group. After 24 h of intervention, the mRNA expression levels of M1/M2 macrophage polarization-related genes and autophagy-related genes were detected by quantitative real-time polymerase chain reaction (qPCR). Mitochondrial membrane potential was measured by immunofluorescence assay. The protein expression levels of autophagy markers, M1/M2 macrophage polarization markers, and PPAR-γ pathway-related proteins were detected by Western blot. After treatment with the PPAR-γ inhibitor oleuropein, the expression of the above proteins was detected again.

CDELNs were exosome-like nanovesicles with uniform size, bilayer membrane structure, and rich in proteins and nucleic acids, which could be internalized by macrophages. CDELNs exhibited excellent stability, high sample homogeneity, and good detection reproducibility. Untargeted plant metabolomics profiling identified a total of 1477 metabolites in CDELNs, including 948 metabolites in positive ion mode and 529 metabolites in negative ion mode. qPCR results showed that, compared with the NC group, 1000 ng/mL LPS significantly upregulated the mRNA expression of M1 macrophage polarization-related genes (iNOS, TNF-α, IL-6) and autophagy-related genes (LC3 Ⅱ/Ⅰ, BECLIN) (P<0.05), while downregulating the mRNA expression of M2 macrophage polarization-related genes (ARG1, CD206) (P<0.05). Treatment with 10⁹ particles/mL CDELNs significantly reversed these gene expression changes compared with the LPS group (P<0.05). Immunofluorescence for mitochondrial membrane potential further revealed that LPS treatment decreased mitochondrial membrane potential and enhanced autophagy compared with the NC group (P<0.05), whereas CDELN intervention increased mitochondrial membrane potential and reduced autophagy in LPS-stimulated cells (P<0.05). Western blot results demonstrated that, compared with the NC group, the LPS group showed significantly elevated protein expression of autophagy markers (LC3II/I, BECLIN, PINK1, PARKIN) and M1 macrophage markers (iNOS, TNF-α) (P<0.05), as well as notably decreased protein expression of M2 macrophage markers (ARG1, CD206) and PPAR-γ pathway-related proteins (PPAR-γ, p-SMAD/SMAD) (P<0.05). In the LPS+CDELN group, the expression of autophagy and M1 polarization markers was significantly downregulated, while the expression of M2 polarization and PPAR-γ pathway-related proteins was significantly upregulated, compared with the LPS group (P<0.05). All the above changes were reversed after the addition of the PPAR-γ inhibitor.

CDELN regulates mitochondrial autophagy by activating the PPAR-γ signaling pathway, thereby modulating macrophage polarization.