Role of NRF2-mediated M2 macrophage polarization in promoting fibroblast proliferation and migration by low-dose ALA-PDT

To investigate whether low-dose 5-aminolevulinic acid photodynamic therapy (ALA-PDT) promotes fibroblast proliferation and migration by activating nuclear factor erythroid 2-related factor 2 (NRF2)-mediated M2 macrophage polarization and thereby accelerates tissue repair in order to clarify the role of NRF2 in fibroblast proliferation and migration.

Human monocytic leukemia cell line Tamn-Horsfall Protein 1 (THP-1) was randomly divided into 4 groups: ① Control group: THP-1 cells were differentiated into M0 macrophages by using 100 ng/mL phorbol 12-myristate 13-acetate (PMA) for 48 h, and then cultured normally without further treatment; ② ALA group: M0 macrophages were incubated with 0.1 mmol/L ALA under light-shielded conditions for 2.5 h; ③ Red light group: M0 macrophages were cultured normally and then exposed to 50 mW/cm² red light irradiation for 30 s; ④ ALA-PDT group: M0 macrophages were incubated with 0.1 mmol/L ALA under light-shielded conditions for 2.5 h, followed by exposure to 50 mW/cm² red light irradiation for 30 s. To verify the pivotal role of NRF2, an NRF2-IN-1 intervention group was established, in which 10 μmol/L NRF2-specific inhibitor NRF2-IN-1 was added in the culture medium for 10 h prior to ALA-PDT treatment. The experimental indicators included: Immunofluorescence (IF) staining to observe the protein localization and expression intensity of the M2 marker CD206; Western blotting to quantify the expression levels of macrophage markers (ARG-1, iNOS) and NRF2 and HO-1; and ELISA to detect the secretion concentrations of anti-inflammatory and pro-repair factors (IL-4, IL-10, IL-13, TGF-β, and VEGF) in the supernatant. Subsequently, conditioned media from the macrophages of each group were indirectly cocultured with fibroblasts, and CCK-8 assay was employed to detect fibroblast proliferation at 24 h, and cell scratch assay was performed to quantitatively analyze fibroblast migration rates at 0 and 24 h.

The experimental results showed that compared with the control, ALA, and red light groups, the ALA-PDT group exhibited significantly upregulated NRF2 protein level in macrophages (total NRF2 relative expression approximately 3.6-fold higher than that of the control group, P<0.001), manifested by enhanced CD206 fluorescence intensity (relative fluorescence intensity approximately 2.5; P<0.001), elevated ARG-1 expression level (relative expression approximately 4.0; P<0.0001), and significantly increased secretion of IL-4 (approximately 260 pg/mL; P<0.05), IL-10 (approximately 45 pg/mL; P<0.01), IL-13 (approximately 75 pg/mL; P<0.001), TGF-β(approximately 30 pg/mL; P<0.01), and VEGF (approximately 70 pg/mL; P<0.0001) in the supernatant. After co-culture with conditioned medium from the ALA-PDT group, fibroblast proliferation activity (cell viability approximately 1.7; P<0.05) and migration rate (migration rate approximately 0.21; P<0.05) were both significantly promoted. However, after intervention with the NRF2 inhibitor NRF2-IN-1, these effects were significantly reversed: NRF2 (total NRF2 relative expression decreased to approximately 2.5; P<0.01) and HO-1 (relative expression decreased to approximately 2.2; P<0.01) expression in the ALA-PDT group was inhibited, CD206 expression was attenuated (relative fluorescence intensity decreased to approximately 1.8; P<0.05), and the secretion levels of IL-4 (decreased to approximately 17 pg/mL; P<0.05), IL-10 (decreased to approximately 35 pg/mL; P<0.05), IL-13 (decreased to approximately 24 pg/mL; P<0.05), TGF-β(decreased to approximately 22 pg/mL; P<0.05), and VEGF (decreased to approximately 50 pg/mL; P<0.01) were reduced. Correspondingly, the conditioned medium from the macrophages of the inhibitor-treated ALA-PDT group showed significantly diminished capacity to promote fibroblast proliferation (cell viability decreased to approximately 1.0; P<0.05) and migration (migration rate decreased to approximately 0.3; P<0.05).

Low-dose ALA-PDT activates the NRF2/HO-1 signaling pathway in macrophages, inducing their polarization toward the M2 anti-inflammatory phenotype, and increase the secretion of anti-inflammatory and pro-repair factors, thereby significantly promoting fibroblast proliferation and migration through a paracrine mechanism, which provides crucial theoretical basis and potential molecular targets for the clinical application of low-dose PDT strategies in the treatment of refractory wounds.