Ac2-26 Hydrogel Modulates IL-1β-Driven Inflammation via Mast Cell-Associated and Immune Regulatory Pathways in Diabetic Wounds

Abstract

Chronic, non-resolving inflammation is a major contributor to impaired wound healing in diabetes. Annexin A1 (AnxA1), a pro-resolving mediator, and its mimetic peptide Ac_2-26 have demonstrated therapeutic potential in modulating inflammatory responses. In this study, we evaluated the effects of topical Ac_2-26 hydrogel in a streptozotocin-induced diabetic wound model. Treatment significantly accelerated wound closure, improved tissue architecture, and reduced leukocyte infiltration. Immunohistochemical analysis revealed diminished mast cell accumulation and IL-1β expression in treated wounds. Complementary transcriptomic profiling supported the downregulation of pro-inflammatory genes, including Il1b and mast cell-related mediators, confirming the peptide's regulatory effect on the wound immune landscape. Mounting evidence suggests that dysregulated mast cell activity plays a role in the heightened inflammatory tone and delayed tissue repair observed in diabetic wounds. In our model, Ac_2-26 hydrogel treatment attenuated IL-1β expression, suggesting an indirect downregulation of NLRP3 inflammasome activation, potentially mediated through mast cell modulation, though effects on other cell types within the wound microenvironment cannot be excluded. While definitive causality cannot be assigned, the integration of histological and transcriptomic data highlights mast cells as contributors to the IL-1β-driven inflammatory burden in diabetic wounds. These findings underscore the immunomodulatory capacity of Ac_2-26 and its potential to restore resolution pathways in chronic wound settings, positioning it as a promising candidate for future therapeutic development.

Keywords: IL-1β; annexin A1; diabetes; mast cell; wound healing.

Figure 1

Histological analysis of the wound healing process (representative images of days 3 and 14). (A,C,E,G) Inflammatory influx in the ND-V and D-V groups. (B,D) Reduction of inflammatory infiltrate in the ND-A and D-A groups (day 3) and (F,H) improvement in tissue regeneration after 14 days of hydrogel treatment. Arrows indicate inflammatory influx in the ND-V and D-V groups. Inset shows a higher magnification of the indicated region, highlighting the inflammatory cells. Hematoxylin and eosin (HE) staining. Bars: 20 µm. Ep, epidermis; D, dermis. Non-diabetic vehicle hydrogel (ND-V); Non-diabetic Ac_2–26 hydrogel (ND-A); Type 1 diabetes vehicle hydrogel (D-V) and Type 1 diabetes Ac_2–26 hydrogel (D-A), (n = 5 animals/group).

Figure 2

Quantification of mast cells in the healing process (representative images of days 3 and 14). Intact (arrows) (A–D) and degranulated mast cells (inset) (E–H). Quantification of intact (I), degranulated (J) mast cells. Staining: Toluidine blue. Bars: 50 μm. Non-diabetic vehicle hydrogel (ND-V); Non-diabetic Ac_2–26 hydrogel (ND-A); Type 1 diabetes vehicle hydrogel (D-V) and Type 1 diabetes Ac_2–26 hydrogel (D-A), (n = 5 animals/group). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001 versus treated animals and/or non-diabetic vehicle hydrogel.

Figure 3

Quantification of mast cells during the healing process. Safranin staining (representative images from days 3 and 14) showing mast cells (A–H). Black arrows indicate mast cells (E–H), with an enlarged view of a mast cell in the inset (G). An increase in mast cell numbers is observed in (E,G), while a reduction after treatment is shown in (F,H). Quantification of safranin-positive mast cells is presented in (I). Berberine sulfate staining reveals mast cell granules with intense fluorescence in the ND-V and D-V groups (white arrows), and weaker fluorescence in the ND-A and D-A groups (J). Stainings: safranin and berberine sulfate. Scale bars: 20 μm (A–I) and 50 μm (J). Non-diabetic vehicle hydrogel (ND-V); Non-diabetic Ac_2–26 hydrogel (ND-A); Type 1 diabetes vehicle hydrogel (D-V) and Type 1 diabetes Ac_2–26 hydrogel (D-A), (n = 5 animals/group). * p ≤ 0.05 and **** p ≤ 0.0001 versus treated animals and/or non-diabetic vehicle hydrogel.

Figure 4

Expression of NLRP3 in the dermis and dosage of IL-1β mediator levels in the healing process. Immunohistochemistry (representative images of days 3 and 14) of NLRP3 positive cells (A–H). The inset shows the immunostained cell in detail. Bars: 20 µm. Levels of IL-1β (I). The data show means ± S.D of pg per mL of mediator (n = 5 animals/group), obtained using a multiplex assay for IL-1β quantification. Non-diabetic vehicle hydrogel (ND-V); Non-diabetic Ac_2–26 hydrogel (ND-A); Type 1 diabetes vehicle hydrogel (D-V) and Type 1 diabetes Ac_2–26 hydrogel (D-A). * p ≤ 0.05, *** p ≤ 0.001 and **** p ≤ 0.0001 versus treated animals and/or non-diabetic vehicle hydrogel.

Figure 5

The mRNA expression levels of Il1-b (A), Casp1 (B), Nlrp3 (C), Tpsab1, and Tpsb2 (D) in spontaneous diabetic db/db animal skin lesions were analyzed using in silico analysis based on the publicly available transcriptome data from the GSE182906 study. * p ≤ 0.05 versus healthy wild-type C57BL/6J animals. Statistical analysis was performed using one-way ANOVA.