Proanthocyanidin capsules remodel the ROS microenvironment via regulating MAPK signaling for accelerating diabetic wound healing

Abstract

Defective diabetic wound healing is a major clinical challenge, where hyperglycemia at the wound site induces excessive reactive oxygen species (ROS) which activate the MAPK pathway (particularly p38 MAPK), resulting in sustained release of inflammatory factors and cellular damage/apoptosis. Polyphenols are efficient ROS scavengers which reduce the level of inflammation at the wound site and promote wound healing, but the low bioavailability limits their biomedical application. This study developed a simple and highly efficient method for preparing proanthocyanidin (PC) capsules through hydrogen bonding and hydrophobic interactions among PC molecules. PC capsules can continuously scavenge free radicals and release proanthocyanidins, significantly enhancing their bioavailability. A single dose of PC capsules accelerates wound healing in diabetic mice by regulating the p38 MAPK signaling cascade, reducing inflammatory mediator concentration, inhibiting cell apoptosis, and remodeling the wound microenvironment. This research makes an important contribution to the field of enhancing polyphenol bioavailability for wound healing and reveals the potential of modulating the MAPK pathway for treating other inflammation and oxidative stress-related diseases.

Keywords: Capsule; Diabetic wound; Inflammation; Proanthocyanidins; p38 MAPK pathway.

Graphical abstract

Scheme 1

Preparation and application of PC capsules for promoting wound healing in diabetic mice. (A) Schematic representation of the synthesis of PC capsules. (B) Application on diabetic mice: PC capsules are applied to the wound to enhance healing. (C) Mechanism of action: PC capsules reduce ROS, inhibit inflammation via the p38 MAPK pathway, prevent apoptosis, and then promote tissue repair.

Fig. 1

Synthesis and Characterization of PC Capsules. (A) Low-magnification SEM, (B) High-magnification SEM and (C) TEM images of PC capsules. (D) CLSM images of PC capsules. (E) EDS spectrum showing the element distribution of C, O and Ca. (F) FTIR spectra of PC and capsules. (G) UV–visible absorption spectra of PC and capsules. (H) XPS spectra and (I) elemental composition. (J) Release profiles under different pH conditions. (K–M) Antioxidant performance: (K) Free radical scavenging ability, (L) H₂O₂ scavenging ability and (M) DPPH scavenging ability of VC, PC and PC capsules on 0 h and 1 month.

Fig. 2

Cell viability and ROS scavenging capacity of PC capsules. (A–C) Cell viability of RAW 264.7 cells, DC2.4 cells and NIH3T3 cells after incubating with capsules for 24 h and 72 h. (D) Cellular uptake of capsules in RAW 264.7, DC2.4 and NIH3T3 cells. The red color represents the cytoskeleton, the blue color represents the nucleus, and the green color represents the capsule. (E–G) Cell viability induced by H₂O₂ and capsules in RAW 264.7, DC2.4 and NIH3T3 cells. (H–I) CLSM images of the ROS scavenging capacity in RAW 264.7 and DC2.4 cells using DCFH-DA probe, with the quantitative results of DCF fluorescence intensity in (J–K). (P values: ns P > 0.05, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, all the values are expressed as mean ± SD, n = 3). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Effects of PC capsules on cell apoptosis and inflammatory factors. (A) Flow cytometry analysis of NIH3T3 cells treated with H₂O₂ and different concentrations of PC capsules. (B) TUNEL staining images indicating apoptosis levels in NIH3T3 cells under various treatments. (C) Quantitative data of apoptotic cells by flow cytometry. (D) Quantitative data of apoptotic cells by TUNEL assay. (E–F) The levels of pro-inflammatory factor IL-6 and TNF-α in RAW 264.7 cells treated with LPS and PC capsules. (G–H) The levels of MnSOD and anti-inflammatory factor IL-10 in RAW 264.7 cells treated with LPS and PC capsules. (P values: ns P > 0.05, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, all the values are expressed as mean ± SD, n = 3).

Fig. 4

Therapeutic effects of PC capsules on wound healing. (A) Schematic diagram of the wound healing process and treatment groups. (B) Images of skin wounds treated with PBS (control), PC solution and PC capsules in an acute wound model. (C) Images of skin wounds treated with PBS (control), PC solution, and PC capsules in a diabetic wound model. (D) Percentage of closed diabetic wound area at different time points. (E) H&E staining of wound sections on day 14, showing tissue regeneration. (F) Masson's trichrome staining and immunohistochemical staining of IL-6, TNF-α, and MnSOD in wound tissues. (G) Quantitative analysis of area coverage by Masson's trichrome staining. (H–J) Quantitative analysis of area coverage percentages of IL-6, TNF-α, and MnSOD in wound tissues. (P values: ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, all values are expressed as mean ± SD, n = 3).

Fig. 5

The inhibition of the MAPK signaling pathway for wound healing by PC capsules. (A) Volcano plot displaying differentially expressed genes (DEGs) between the capsules and blank control groups in diabetic mouse models. (B) Heatmap of DEGs in diabetic mouse models. (C) GSEA analysis of the MAPK gene set in diabetic mouse models. (D) Western blotting results of the phosphorylation of proteins involved in the MAPK signaling pathway (JNK, p38). (E) Quantitative result of JNK and p38 in NIH3T3 cells. (F) Immunohistochemical staining analysis of p-p38 expression in skin tissues. (G) Quantitative result of p-p38 in wound skin. (H) Expression levels of total-caspase3, cleaved-caspase3, and Bcl-2. (I) Quantification result of (H). (J) Expression levels of iNOS and COX2 assessed by Western blotting. (K) Quantification result of (J). (P values: ns P > 0.05, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, all values are expressed as mean ± SD, n = 3).

Fig. 6

The schematic illustrates the signaling pathways involved in wound repair. (A) Schematic diagram of wound healing. (B) Schematic diagram of PC capsule regulatory signaling pathways.

Fig. 7

Biocompatibility of PC capsules. Serum liver and kidney function indicators: (A) Aspartate aminotransferase (AST), (B) Alanine aminotransferase (ALT), (C) Blood urea nitrogen (BUN) and (D) Creatinine (CRE). (E) H&E sections of major organs (heart, liver, spleen, lung, and kidney) at 15 days post-injection, Scale bars: 100 μm.