- Discussion
After the skin surface is damaged, its healing generally requires four phases: coagulation, inflammation, proliferation, and remodeling. Histopathological examination of the injured tissue showed that in the model group, the epidermis and dermis structures disappeared and inflammatory cell infiltration occurred after day 1 (D1). In the rMAP group, partial areas exhibited new epidermal formation and inflammatory cell infiltration after D1. Over time, the epidermis and dermis gradually repaired, and after one week of treatment, both the degree and time of recovery were better than those in the model group.
During the wound healing process, activated macrophages are the main participants in the inflammatory response and key regulators of wound repair, influencing the function and state of other cells in the wound [4]. Activated macrophages in the wound mainly exist in two polarization states: M1 (pro-inflammatory) and M2 (anti-inflammatory). During the inflammatory phase, M1-type macrophages primarily promote the inflammatory response in the wound by secreting large amounts of pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, and IL-12, which regulate the inflammatory functions of immune cells like lymphocytes and dendritic cells. During the proliferative and remodeling phases, M2-type macrophages predominate, with high expression of anti-inflammatory cytokine IL-10 [5-6]. IFN-γ can activate neutrophils and natural killer cells, promote Th1 cell development, and inhibit Th2 cell activation and proliferation, inducing an inflammatory response in the body, slowing down skin wound healing, and thus affecting wound healing [7]. TNF-α is primarily synthesized by activated macrophages and T lymphocytes and is an important mediator in immune and inflammatory responses, promoting phagocytosis by neutrophils and enhancing the cascade reaction of cytokines [8]. IL-2 binds to corresponding receptors on the surface of monocytes, T cells, and B cells, accelerating T cell proliferation and activation and enhancing natural killer cell activity [9]. Under the stimulation of IL-2, natural killer cells enhance the bactericidal activity of IFN-γ. ELISA detection of inflammatory cytokines IFN-γ, IL-2, IL-6, and TNF-α in skin wounds showed that the concentrations of IFN-γ, IL-2, IL-6, and TNF-α in the model control group were significantly higher than those in the normal control group (P < 0.01). After 7 days of treatment, the concentrations of these cytokines in the rMAP group and collagen protein group were significantly lower than those in the model control group (P < 0.05), but there was no significant difference between the rMAP group and the collagen protein group (P > 0.05). These results indicate that rMAP can significantly reduce the concentration of inflammatory cytokines and has an anti-inflammatory effect on superficial second-degree acute skin injuries. Some studies have shown that collagen protein has certain antioxidant, anti-inflammatory, antipruritic, and analgesic effects during the healing and scar hyperplasia phases of burns and scalds [10-11]. The rMAP structure contains more dopamine attached to the skin surface compared to collagen protein, and arginine and lysine produce stronger adhesion through electrostatic interactions and form local stability through hydrophobic group interactions.
During the wound repair process, the wound tissue needs to accelerate the synthesis of a large amount of proteins related to healing to promote wound healing, and the activation of protein translation is considered a necessary event in the wound healing process. Abnormalities in any part of the signaling pathways that regulate protein translation, angiogenesis, and cell proliferation and differentiation can lead to chronic non-healing or deepening of the wound [12-14]. The PI3K/AKT/mTOR signaling pathway plays important physiological and pathological roles in regulating nucleic acid transcription, protein translation, cell growth and survival, proliferation, apoptosis, and angiogenesis [15-18].
Western blot (WB) analysis of protein expression levels in the PI3K/AKT/mTOR signaling pathway showed that the protein expression levels of PI3K, p-AKT, p-mTOR, and the p-AKT/AKT ratio in the model group were significantly lower than those in the normal group (P < 0.05). The expression level of PI3K decreased from 1.042 ± 0.395 to 0.283 ± 0.146, the expression level of p-AKT decreased from 0.994 ± 0.051 to 0.283 ± 0.068, and the expression level of p-mTOR decreased from 1.023 ± 0.207 to 0.144 ± 0.114. Compared with the model control group, the expression level of PI3K in the recombinant mussel adhesive protein (rMAP) wound tissue was significantly increased (P < 0.05), and the expression levels of p-AKT and p-mTOR in each drug group were significantly increased (P < 0.05). The p-AKT content in the rMAP group increased from 0.283 ± 0.068 to 0.632 ± 0.095, and the p-mTOR content increased from 0.144 ± 0.114 to 0.533 ± 0.086. There was no significant difference in the expression levels of AKT and mTOR among the experimental groups (P > 0.05). The p-AKT/AKT ratio in the rMAP group was significantly higher than that in the model group (P < 0.05), increasing from 0.367 ± 0.044 to 0.596 ± 0.093, and the p-mTOR/mTOR ratio increased from 0.961 ± 1.188 to 1.373 ± 1.140, although this difference was not statistically significant. These results indicate that the establishment of a rat model of superficial second-degree non-chronic skin injury leads to reduced expression levels of PI3K/AKT/mTOR-related proteins, signal pathway dysregulation, and delayed wound healing. rMAP promotes wound tissue healing by activating the PI3K/AKT/mTOR signaling pathway through the phosphorylation of AKT and mTOR, participating in cell proliferation and differentiation, and regulating protein expression.
The main cytokines involved in angiogenesis include epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and transforming growth factor. These cytokines interact to promote angiogenesis. EGF plays different roles at different stages of wound repair: during the coagulation phase, it chemotaxes epithelial cells to migrate to the wound site via platelet release; during the inflammatory phase, it chemotaxes macrophages and fibroblasts to gather at the wound site, promoting the inflammatory response; during the proliferative phase, it binds to receptors and promotes receptor phosphorylation, thereby promoting protein synthesis and cell proliferation [19-20]. EGF also stimulates fibronectin production and increases the number of fibroblasts at the wound site, promoting wound proliferation [21]. EGF has a strong effect on various interstitial cells, including fibroblasts, vascular endothelial cells, vascular smooth muscle cells, and chondrocytes, enhancing the synthesis of extracellular macromolecules in granulation tissue, such as DNA, RNA, proteins, and collagen, promoting granulation tissue formation and accelerating wound healing [22].
FGF-2 has angiogenic and chemotactic effects. Similar to EGF, it chemotaxes neutrophils, macrophages, and fibroblast growth factors to the wound site during the inflammatory phase, enhancing the inflammatory response in wound healing [23-25]. FGF-2 promotes the proliferation and differentiation of fibroblasts, vascular endothelial cells, and smooth muscle cells, accelerating granulation tissue growth and promoting wound healing. VEGF is one of the strongest known promoters of angiogenesis, serving as a crucial chemotactic factor for endothelial cells, primarily synthesized by keratinocytes and macrophages [26]. VEGF increases the content of plasminogen activator and collagenase in endothelial cells, promoting capillary fusion and the formation of larger blood vessels, playing a vital role in endothelial cell migration and proliferation, and promoting the formation of new blood vessels [27], providing sufficient oxygen and nutrients to the wound.
Real-time PCR was used to detect the expression levels of EGF, FGF-2, and VEGF genes. The results showed that compared to the normal group, the mRNA expression levels of EGF, FGF-2, and VEGF in the skin tissue of rats in the model group were significantly reduced (P < 0.01). The EGF mRNA expression level decreased from 1.051 ± 0.342 to 0.157 ± 0.067, the FGF-2 mRNA expression level decreased from 1.005 ± 0.123 to 0.521 ± 0.127, and the VEGF mRNA expression level decreased from 1.019 ± 0.231 to 0.226 ± 0.031. Compared to the model control group, the mRNA expression levels of EGF in the skin tissue of rats treated with rMAP and collagen protein were significantly increased (P < 0.05), rising from 0.157 ± 0.067 to 0.412 ± 0.182. The mRNA expression levels of FGF-2 and VEGF in the skin tissue of rats treated with rMAP were also significantly increased (P < 0.05), with FGF-2 mRNA expression increasing from 0.521 ± 0.127 to 0.879 ± 0.179 and VEGF mRNA expression increasing from 0.226 ± 0.031 to 0.435 ± 0.102. In summary, rMAP can promote wound healing by increasing the expression levels of VEGF, FGF-2, and EGF mRNA in granulation tissue, exerting the combined regulatory effects of these factors, promoting the proliferation of fibroblasts and new capillaries, improving blood circulation at the wound site, promoting granulation tissue growth and filling, accelerating epithelial cell growth, and facilitating wound healing.
