Abstract
Objective: To investigate the effects of recombinant mussel adhesive protein (rMAP) on the healing of superficial second-degree acute skin injuries in female SD rats.
Methods: A superficial second-degree acute skin injury model in rats was established using a laser pen with an energy setting of 7 J/cm². Twenty SD rats were used, and the back of each rat was shaved and divided into four regions corresponding to the normal control group, model control group, rMAP group, and collagen group. Except for the normal control region, the other regions were exposed to 7 J/cm² laser irradiation to create a superficial second-degree acute skin injury model. Twenty-four hours after modeling, the corresponding experimental drugs were applied to the rMAP and collagen groups, while the normal control and model control groups received an equal volume of physiological saline (5 μL per application, twice daily) for 7 consecutive days. Histopathological changes in skin tissues were observed at D1, D3, D7, and D14 using HE staining. Fifteen additional SD rats were divided into four regions per rat corresponding to the four experimental groups, and each group received continuous drug administration for 7 days. Enzyme-linked immunosorbent assay (ELISA) was used to detect the concentrations of cytokines IL-2, IL-6, TNF-α, and IFN-γ in the skin wounds. Western blot (WB) was used to detect the expression levels of PI3K, AKT, GSK3β, and their phosphorylated forms in the wound skin. RT-PCR was used to detect the expression levels of target genes EGF, FGF-2, and VEGF.
Results: According to the "three-degree four-part" classification of skin injuries, a superficial second-degree acute skin injury model in rats could be established by irradiating the back skin with a laser at 7 J/cm². HE staining results showed that the epidermis in the model control group was hyperkeratotic and structurally disintegrated, with necrosis of dermal collagen fibers and infiltration of inflammatory cells. At different time points (D1, D3, D7, and D14), as time progressed, the repair of the epidermis and dermis and the resolution of inflammatory cell infiltration gradually improved in the rMAP and collagen groups. ELISA results showed that the concentrations of inflammatory cytokines IL-2, IL-6, TNF-α, and IFN-γ in the model control group were significantly higher than those in the normal control group (all P < 0.05). The concentrations of these inflammatory cytokines were lower in the rMAP and collagen groups compared to the model control group (P < 0.05), with no significant difference between the rMAP and collagen groups (all P > 0.05). WB results showed that the expression levels of PI3K, p-AKT, and p-mTOR proteins were significantly reduced in the model group (P < 0.05). Recombinant mussel adhesive protein significantly increased the expression levels of PI3K, p-AKT, and p-mTOR proteins, while collagen significantly increased the expression levels of p-AKT and p-mTOR (P < 0.05) and had a certain effect on increasing PI3K, although this increase was not statistically significant (P > 0.05). Recombinant mussel adhesive protein significantly increased the ratios of p-AKT/AKT and p-mTOR/mTOR. RT-PCR results showed that the expression levels of EGF, FGF-2, and VEGF genes in the skin wounds of model rats were significantly decreased, and recombinant mussel adhesive protein significantly increased the mRNA levels of EGF, FGF-2, and VEGF (P < 0.05). Collagen significantly increased the level of EGF (P < 0.05).
Conclusion: In summary, rMAP can significantly inhibit the concentrations of inflammatory cytokines IFN-γ, IL-2, IL-6, and TNF-α, thereby suppressing the inflammatory response in rat skin wounds. It can also significantly promote the expression of EGF, FGF-2, and VEGF genes, promoting cell proliferation and differentiation and the formation of granulation tissue, thus accelerating skin repair. This repair is achieved through the activation of the PI3K/AKT/mTOR signaling pathway by the phosphorylation of AKT and mTOR.
