Mesenchymal stem cell-conditioned medium accelerates type 2 diabetic wound healing by targeting TNF and chemokine signaling

Abstract

Introduction: Given the crucial role of paracrine signaling in the therapeutic function of adipose tissue-derived mesenchymal stem cells (ADSCs) for skin wound repair, this study aimed to evaluate the efficacy of ADSC-conditioned medium (ACM) in enhancing type 2 diabetic (T2D) wound healing.

Methods: The effect of ACM on the viability and angiogenesis of human umbilical vein endothelial cells (HUVECs) was first evaluated using the CCK-8 assay and q-PCR analysis, respectively. Next, a T2D rat model was established through the combination of a high-fat diet and streptozotocin (STZ). Following the establishment of full-thickness skin defects in T2D rats, ACM or serum-free cultured medium was daily injected around the wound edges for 7 days. Afterward, the skin wound healing rate was analyzed, and the skin tissues were assessed by histopathological examination. The mRNA levels of TNF-α, IL-1β, IL-6, COX-2, IL-12, and IFN-γ were evaluated by q-PCR analysis. Additionally, transcriptome sequencing and immunohistochemistry were performed to reveal the potential mechanisms of ACM in T2D skin wound healing.

Results: ACM significantly enhanced HUVEC proliferation and angiogenesis while upregulating the expression of EGF, bFGF, VEGF, and KDR. In T2D rats, ACM accelerated wound closure and suppressed pro-inflammatory mediators (TNF-α, IL-1β, IL-6, COX-2, IL-12, and IFN-γ). Notably, transcriptome analysis revealed ACM-mediated downregulation of TNF and chemokine signaling pathways.

Discussion: ACM promotes diabetic wound healing through dual mechanisms: (1) stimulating vascularization by inducing growth factor expression and (2) modulating the inflammatory microenvironment by inhibiting TNF/chemokine cascades. These findings position ACM as a promising cell-free therapy for impaired wound healing in diabetes.

Keywords: adipose tissue-derived mesenchymal stem cells; conditioned medium; regeneration; skin wound; type 2 diabetes.

FIGURE 1

ACM promotes vascular cell proliferation and angiogenesis. (A) ACM promotes HUVEC proliferation. (B) Representative images of HUVECs after ACM treatment (scale bar, 100 μm). (C) Relative mRNA expression of EGF, bFGF, VEGF, and KDR in HUVECs after ACM treatment.

FIGURE 2

ACM accelerates T2D skin wound healing in rats. (A) General observation of skin wounds after ACM treatment. (B) Skin wound healing rate after ACM treatment. (C) Histopathological changes in skin wounds after ACM treatment by HE and Masson staining, respectively (scale bar, 1 mm). The area within the blue dotted line represents the damaged region. The green oval shape represents the area of inflammation. (D) Immunofluorescence expression of CD31, FGF-2, and VEGF in skin wounds after ACM treatment. Scale bar = 200 μm and 50 μm, respectively. (E) Number of CD31⁺ cells and the relative MFI of FGF-2 and VEGF expression in skin wounds after ACM treatment.

FIGURE 3

ACM inhibits the mRNA level of inflammatory factors in T2D skin wounds. The relative mRNA expression of TNF-α (A), IL-1β (B), IL-6 (C), COX-2 (D), IL-12 (E), and IFN-γ (F) in skin wounds.

FIGURE 4

ACM inhibits the expression of inflammatory factors in macrophages. The immunofluorescence expression of TNF-α, IL-1β, and IL-6 in F4/80⁺ macrophages in skin wounds after ACM treatment. Scale bar = 200 μm and 50 μm, respectively.

FIGURE 5

Transcriptome sequencing analysis of T2D skin wound tissues in rats. The volcano plot for differential gene expression of normal vs. model groups (A) and ADSC vs. model (B) groups. The gray pixel represents a gene where the difference in expression is not significant, while red and green pixels represent those that are significant. (C) Venn diagrams exhibiting the number of identified genes and the overlay of these identified genes. GO annotation and pathway enrichment analysis in normal vs. model groups (D) and ADSC vs. model (E) groups.

FIGURE 6

ACM downregulates TNF and chemokine signaling in T2D skin wound tissues. (A) Representative images of TNF-α, NF-κB, p-NF-κB, MAPK, and p-MAPK expression in skin tissues. Scale bars = 200 μm. (B) Relative expressions of TNF-α, NF-κB, p-NF-κB, MAPK, and p-MAP . (C) Representative images of CXCL1, CXCL2, and CXCL8 expression in skin tissues. Scale bars = 200 μm. (D) Relative expressions of CXCL1, CXCL2, and CXCL8.