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. 2020 Dec 4:8:585132.
doi: 10.3389/fcell.2020.585132. eCollection 2020.

Effects of ALA-PDT on the Healing of Mouse Skin Wounds Infected With Pseudomonas aeruginosa and Its Related Mechanisms

Tao Yang  1 , Yang Tan  1 , Wentao Zhang  1 , Weijiang Yang  1 , Jiefu Luo  1 , Ling Chen  1 , Hong Liu  1 , Guihong Yang  1 , Xia Lei  1
Affiliations

Effects of ALA-PDT on the Healing of Mouse Skin Wounds Infected With Pseudomonas aeruginosa and Its Related Mechanisms

Tao Yang et al. Front Cell Dev Biol. .

Abstract

Photodynamic therapy (PDT) is a promising new method to eliminate microbial infection and promote wound healing. Its effectiveness has been confirmed by some studies; however, the mechanisms of PDT in wound healing remain obscure. We used mouse skin wounds infected with Pseudomonas aeruginosa as a research object to explore the therapeutic effects and mechanisms of 5-aminolevulinic acid photodynamic therapy (ALA-PDT). ALA-PDT treatment significantly reduced the load of P. aeruginosa in the wound and surrounding tissues and promoted the healing of skin wounds in mice. Hematoxylin-eosin (HE) and Sirius red staining showed that ALA-PDT promoted granulation tissue formation, angiogenesis, and collagen regeneration and remodeling. After ALA-PDT treatment, the expression of inflammatory factors (TNF-α and IL-1β) first increased and then decreased, while the secretion of growth factors (TGF-β-1 and VEGF) increased gradually after treatment. Furthermore, ALA-PDT affected the polarization state of macrophages, activating and promoting macrophages from an M1 to an M2 phenotype. In conclusion, ALA-PDT can not only kill bacteria but also promote wound healing by regulating inflammatory factors, collagen remodeling and macrophages. This study further clarifies the mechanism of PDT in the healing of infectious skin wounds and provides further experimental evidence for its clinical treatment of skin wounds infected by P. aeruginosa.

Keywords: Pseudomonas aeruginosa; inflammatory factor; macrophagocyte; photodynamic therapy; wound healing.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Bacterial counting on wounds. The amounts of P. aeruginosa were counted on wounds before treatment (D0) and on the 1st (D1), 3rd (D3), 7th (D7), 14th (D14) day after different treatments. ∗P < 0.05 versus the other groups.
FIGURE 2
FIGURE 2
Effects of ALA-PDT for the healing rate on wounds. (A) The wound pictures of healing process. Scale bar indicate 1 cm. (B) The healing rate was counted before treatment (D0) and on the 1st (D1), 3rd (D3), 7th (D7), 14th (D14) day after different treatments. ∗P < 0.05 versus the other groups.
FIGURE 3
FIGURE 3
HE staining assay. The HE staining results of each group before treatment (D0) and on the 1st (D1), 3rd (D3), 7th (D7), 14th (D14) day after different treatments. The blue arrows indicate the structure of the new epithelium. The yellow arrows indicate relatively thickened and increased collagen fibers. The red arrows indicate new capillaries. Scale bar indicate 100 μm.
FIGURE 4
FIGURE 4
Sirius red polarized light method assay. The picric acid Sirius red staining results of each group before treatment (D0) and on the 1st (D1), 3rd (D3), 7th (D7), 14th (D14) day after different treatments. Scale bar indicate 50 μm.
FIGURE 5
FIGURE 5
Effects of ALA-PDT on TNF-α, IL-1β, TGFβ-1, and VEGF. (A) Western blot assay of TNF-α, IL-1β, TGFβ-1, and VEGF proteins on the 3rd (D3), 7th (D7), 14th (D14) day after different treatments in wounds. (B) Western blot analysis of IL-1β. ∗P < 0.05 versus the other groups. (C) Western blot analysis of TNF-α. ∗P < 0.05 versus the other groups. (D) Western blot analysis of TGFβ-1. ∗P < 0.05 versus the other groups. (E) Western blot analysis of VEGF. ∗P < 0.05 versus the other groups.
FIGURE 6
FIGURE 6
Effects of ALA-PDT on M1 and M2 macrophages in wounds. (A) Immunofluorescence staining of M1 and M2 macrophages before treatment (D0) and on the 1st (D1), 3rd (D3), 7th (D7), 14th (D14) day after treatment in wounds. The primary antibodies anti-iNOS (rabbit polyclonal antibody, 1:100) and anti-CD163 (goat polyclonal antibody, 1:100) from Servicebio (China) and the fluorescent second antibodies goat anti-rabbit (1:300) and rabbit anti-goat (1:300) from Servicebio (China) and DAPI from Abcam (United States) were used. The marker of M1 and M2 macrophages are iNOS and CD163, respectively. Cells stained red were considered M1 macrophages, and cells stained green were considered M2 macrophages. (B,C) Immunofluorescence staining analysis of M1 and M2 macrophages. ∗P < 0.05 versus the control groups. Scale bar indicate 20 μm.
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