In vitro wound healing effects of postbiotics derived from the gut microbiota of long-lived blind mole rats, a model of healthy ageing

Abstract

Chronic wounds represent a global public health burden to patients and healthcare professionals worldwide. Considering the unmet need for safe and effective therapeutic approaches for wound healing, research on discovering new bioactive materials that support all stages of wound healing is gaining importance. In this study, the wound-healing activity of postbiotics obtained from Limosilactobacillus reuteri EIR/Spx-2, isolated from the gut microbiota of long-lived blind mole rats (Nannospalax xanthodon), was investigated. Our results demonstrated that postbiotics exhibited a strong inhibitory effect against important skin pathogens, eliminated their biofilm formation, and downregulated the expression of genes involved in their quorum-sensing regulatory mechanisms. Furthermore, treatment with postbiotics resulted in a significant increase (23.82% ± 2.11%) in L929 fibroblast cell proliferation. Additionally, postbiotics applied on scratched fibroblast monolayer significantly accelerated the re-epithelialization by 66.78% ± 3.74%. The treatment also increased the mRNA expression and protein levels of COL1A1 in the early healing phase. Moreover, the intracellular ROS levels of L929 cells suppressed by H₂O₂ were significantly reduced, which could be attributed to the content of flavonoids (4.8 mg/g) and phenolic compounds (7.12 mg/g) in postbiotics, as well as their DPPH scavenging activity. After treatment with postbiotics, the mRNA levels of IL-6 (5.77-fold) and TNF-α (1.76-fold) and the amount of NO (79.25% ± 3.18%) were significantly decreased in LPS-induced murine macrophages. The diverse metabolite profile of postbiotics, as characterised using chromatographic techniques, exhibited a strong correlation with their biological activity across all stages of the wound healing process, highlighting their potential as promising candidates for wound healing applications.

Keywords: biofilms; infection; inflammation Limosilactobacillus reuteri; postbiotics; proliferation; wound healing.

FIGURE 1

Inhibitory effects of postbiotics against skin pathogens (A) Inhibition zones (mm) one agar plates (B, C) Biofilm formation displayed with crystal violet assay (D) Relative gene expression of quorum sensing‐related genes and (E) the cell viability of pathogens in the different groups of treatments. Data are presented as the mean ± SD (n = 3). Statistical significance was determined using the following symbols: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, n.s., non‐significant.

FIGURE 2

(A) Confocal laser microscopy images and (B) scanning electron micrographs illustrating the effect of postbiotics on the cell viability of pathogens and their biofilm formations in the different groups of treatments.

FIGURE 3

(A) Cell viability of L929 cells upon treatment with postbiotics determined by (A) MTT assay and (B) flow cytometry analysis using Annexin V‐FITC/PI staining (C) The quadrants define necrotic (single PI‐positive) cells, late apoptotic cells (annexin V and PI double‐positive), early apoptotic cells (annexin V single‐positive), and healthy cells (nonapoptotic cells) (D) Photomicrographs of L929 cells during in vitro wound‐healing/scratch assay at the indicated time points using a 10× objective on an inverted microscope (E) Wound area and wound closure (%) of each treatment group at 24 h of postinjury. Data are presented as the mean ± SD (n = 3). Statistical significance was determined using the following symbols: *p < 0.05, **p < 0.01, ***p < 0.001, n.s., non‐significant.

FIGURE 4

Collagen type 1 (COL1A1) production in fibroblast cell line L929 when treated with postbiotics. (A) Gene expressions determined by qRT‐PCR analysis. (B) Protein fold‐change evaluated by western blotting. (C) Immunofluorescence detection investigated by fluorescence microscopy (COL1A1, green fluorescence; DAPI for cellular nuclei, blue fluorescence.

FIGURE 5

(A) Cell viability of L929 cells upon treatment with different H₂O₂ concentrations with or without postbiotics determined by MTT assay. (B) Fold chance of fluorescent intensity of intracellular ROS determined by flow cytometry. (C) Cell viability of RAW264.7 murine macrophages cells upon treatment with different LPS concentrations and postbiotics determined by MTT assay (D) NO (E) TNF‐α and IL‐6 productions in LPS‐induced RAW264.7 murine macrophages with or without probiotics. Data are presented as the mean ± SD (n = 3), Statistical significance was determined using the following symbols: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001, n.s., non‐significant.