ALA-PDT regulates macrophage M1 polarization via ERK/MAPK-NLRP3 pathway to promote the early inflammatory response.

BACKGROUND: Photodynamic therapy (PDT) is a promising new approach to promote wound healing and its effectiveness has been demonstrated in both clinical and animal studies. Macrophages are the key cells in wound healing and inflammatory response. However, the mechanism of action of PDT on macrophages in promoting wound healing is still unclear. METHODS: In this study, RAW264.7 cells were used. We analyzed the expression levels of macrophage markers arginase 1 (Arg-1), CD206, iNOS, CD86, and inflammatory factors IL-6, TNF-α, and IL-1ß by reverse transcription-polymerase chain reaction and Western blot, Milliplex microtubule-associated protein multiplex assay was performed to analyze the expression of inflammatory factors in the supernatant. Live cell Imaging System to observe the dynamic process of macrophage phagocytosis. Western blot was performed to observe the activation of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) and NOD-like receptor protein 3 (NLRP3) inflammasome. RESULTS: 5-Aminolevulinic acid (ALA)-PDT increased the expression of M1 marker iNOS/CD86 and decreased the expression of Arg-1/CD206 in RAW264.7 cells, while, proinflammatory factors IL-6, TNF-α, and IL-1ß expression was enhanced and macrophage phagocytosis was increased. We also found that these phenomena were associated with activation of the ERK/MAPK-NLRP3 pathway. CONCLUSION: ALA-PDT promotes early inflammatory responses by regulating macrophage M1 polarization through the ERK/MAPK-NLRP3 pathway. It also promotes macrophage phagocytosis.

ALA_PDT Promotes Ferroptosis-Like Death of Mycobacterium abscessus and Antibiotic Sterilization via Oxidative Stress.

Mycobacterium abscessus is one of the common clinical non-tuberculous mycobacteria (NTM) that can cause severe skin infection. 5-Aminolevulinic acid photodynamic therapy (ALA_PDT) is an emerging effective antimicrobial treatment. To explore whether ALA_PDT can be used to treat M. abscessus infections, we conducted a series of experiments in vitro. We found that ALA_PDT can kill M. abscesses. Mechanistically, we found that ALA_PDT promoted ferroptosis-like death of M. abscesses, and the ROS scavenger N-Acetyl-L-cysteine (NAC) and ferroptosis inhibitor Ferrostatin-1 (Fer-1) can mitigate the ALA_PDT-mediated sterilization. Furthermore, ALA_PDT significantly up-regulated the transcription of heme oxygenase MAB_4773, increased the intracellular Fe2+ concentration and altered the transcription of M. abscessus iron metabolism genes. ALA_PDT disrupted the integrity of the cell membrane and enhanced the permeability of the cell membrane, as evidenced by the boosted sterilization effect of antibiotics. In summary, ALA_PDT can kill M. abscesses via promoting the ferroptosis-like death and antibiotic sterilization through oxidative stress by changing iron metabolism. The study provided new mechanistic insights into the clinical efficacy of ALA_PDT against M. abscessus.

In vitro study of the effect of ALA-PDT on Mycobacterium abscessus and its antibiotic susceptibility.

BACKGROUND: The skin infection caused by Mycobacterium abscessus (M. abscessus) is extremely difficult to treat in clinical practice. PDT (photodynamic therapy) is a promising antibacterial treatment. We evaluated the effect of photodynamic therapy using 5-aminolevulinic acid (ALA) as a photosensitizer on M. abscessus and its antibiotic resistance in this study. METHODS: M. abscessus and biofilm were treated with different concentrations of ALA and then irradiated with LED light (635 nm, 80 J/cm2), while there were ALA-only group, light-only group, and negative control group. The effects were observed by colony counting, crystal violet staining, confocal laser scanning microscope (CLSM), and scanning electron microscope (SEM). The changes of drug susceptibility of M. abscessus at sublethal doses were detected by micro-broth dilution method, and the possible mechanism was explored by fluorometer and real-time fluorescence quantitative Polymerase Chain Reaction (RT-qPCR). RESULTS: ALA-PDT showed a significant killing effect on M. abscessus at ALA concentrations greater than 50 µg/ml and the effect increased with increasing photosensitizer concentrations. ALA-PDT also showed a notable scavenging effect on M. abscessus biofilm, which was also enhanced with increasing ALA concentrations. At sublethal doses, the susceptibility of M. abscessus to antibiotics was increased, and ALA-PDT greatly increased the cell wall permeability of M. abscessus and decreased the mRNA expression of drug resistance genes whiB7 and erm (41), as well as efflux pump genes MAB_1409c and MAB_3142c at the transcriptional level. CONCLUSIONS: ALA-PDT has a significant killing effect on M. abscessus and can increase its antibiotic susceptibility.

Dermal Fibroblast Migration and Proliferation Upon Wounding or Lipopolysaccharide Exposure is Mediated by Stathmin.

The dermal fibroblast is a crucial executor involved in wound healing, and lipopolysaccharide is a key factor in initiating the migration and proliferation of the dermal fibroblasts, followed by wound healing. However, the underlying molecular mechanism is still unknown. In this study, we demonstrated that stathmin increased concomitantly with p38/MAPK pathway activation by lipopolysaccharide stimulation of the human dermal fibroblast (HDF), which induced microtubule (MT) depolymerization followed by increased HDF migration and proliferation. In contrast, the application of taxol, the small interfering RNA transfection of stathmin, or the application of the p38/MAPK inhibitor SB203580 suppressed MT depolymerization and HDF migration and proliferation. Additionally, the overexpression of a MKK6(Glu) mutant, which constitutively activated p38/MAPK, resulted in MT depolymerization and, subsequently, promoted HDF migration and proliferation. Our data reveal a crucial role of stathmin in HDF migration and proliferation. These findings will provide new targets and strategies for clinical interventions in wound healing.