NRF2介导的巨噬细胞M2型转化在低剂量ALA-PDT促进成纤维细胞增殖与迁移中的作用研究OA

Objective 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.Methods 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 co-cultured 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.Results 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<0.000 1),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<0.000 1)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).Conclusion 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.