关键词: 外泌体, M2型巨噬细胞, 成纤维细胞, 光老化, 活性氧, 胶原Ⅰ型, 胶原Ⅲ型, 基质金属蛋白酶1

Abstract: 【Abstract】 Objective To investigate the protective effect of M2 macrophage-derived exosomes （M2-Exo） against ultraviolet A （UVA）-induced photoaging in human skin fibroblasts （HSFs）, and to explore the underlying mechanisms. Methods The macrophage cell line RAW 264.7 was co-cultured with interleukin-4 （IL-4, 20 ng/ml） and epigallocatechin gallate （EGCG, 20 μmol/L） for 24 hours to induce the polarization towards the M2 phenotype, which was verified by flow cytometry. Exosomes were isolated from the culture supernatant of M2 macrophages by differential centrifugation; their morphology was observed by transmission electron microscopy, and the expression of exosomal markers was determined by Western blot analysis. M2-Exo were labeled with the red fluorescent dye PKH26 and co-incubated with HSFs for 24 hours to track exosome uptake by HSFs using fluorescence microscopy. HSFs were irradiated with 8 J/cm2 UVA once daily for 5 consecutive days to establish a photoaging model, followed by the treatment with 30 and 60 ng/ml M2-Exo for 24 hours, designated as the UVA + low-dose exosome group and the UVA + high-dose exosome group, respectively; a normal control group and a UVA model group were also established. Intracellular reactive oxygen species （ROS） levels were detected using the DCFH-DA fluorescent probe. The expression of photoaging-related proteins was determined by Western blot analysis. One-way analysis of variance was used for multiple group comparisons, and the least significant difference-t test was used for pairwise comparisons. Results Flow cytometry showed that CD11b?/F4/80? cells （representing mature macrophages） accounted for 85.36% of IL-4/EGCG-induced macrophages, and CD206?/F4/80? cells accounted for 91.27%. Transmission electron microscopy revealed that the isolated exosomes exhibited typical saucer-shaped vesicular structures consistent with exosomes. Western blot analysis confirmed the expression of the exosomal markers CD63, tumor susceptibility gene 101, and apoptosis-linked gene-2-interacting protein X in M2-Exo. Fluorescence microscopy demonstrated effective uptake of PKH26-labeled exosomes （red fluorescence） by HSFs. The DCFH-DA assay showed that the ROS fluorescence intensity was significantly higher in the UVA model group （［38.76 ± 2.60］ AU） than in the normal control group, but significantly lower in the UVA + high-dose exosome group （［31.36 ± 5.36］ AU） than in the UVA model group （both P < 0.05）. As revealed by Western blot analysis, compared with the normal control group, the UVA model group showed significantly decreased relative expression levels of typeⅠ and typeⅢ collagen, but a significantly increased relative expression level of matrix metalloproteinase 1 （MMP-1） （all P < 0.05）; compared with the UVA model group, the UVA + high?dose exosome group exhibited significantly increased relative expression levels of typeⅠ and typeⅢ collagen, but a significantly decreased relative expression level of MMP?1 （all P < 0.05）. Conclusion M2-Exo could inhibit UVA-induced oxidative stress, potentially by downregulating MMP-1 expression and promoting the synthesis of typeⅠ and typeⅢ collagen, thereby ameliorating the photoaging phenotype of HSFs.

Key words: Exosomes, M2 macrophages, Fibroblasts, Photoaging, Reactive oxygen species, Collagen type I, Collagen type Ⅲ, Matrix metalloproteinase 1