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2. Experimental Methods
2.1 Establishment of a Superficial Second-Degree Acute Injury Wound Model in Rats: SD rats weighing 180 to 200 g were used. The dorsal hair of the rats was removed using an electric clipper, followed by the application of 8% sodium sulfide evenly over the skin. After 30 seconds, the skin was washed with warm water to remove the sodium sulfide and residual hair. Once the dorsal area was dry, the rats were anesthetized with 60 mg/kg pentobarbital sodium via intraperitoneal injection. Four 1.5 cm × 1.5 cm square regions were marked on the dorsal area of each rat, which were designated as the normal control group, model control group, rMAP group, and collagen group. Except for the normal control region, the other regions were irradiated with a laser pen set at level 7, approximately 1 mm from the surface, for 2 to 3 seconds to establish the acute skin injury model. The success of the superficial second-degree skin acute injury model was evaluated according to the "three degrees and four divisions" classification of burn injuries described in Surgery [2].
2.2 Histopathological Examination of Wound Tissue in Each Group of Rats [3]: Twenty SD rats were used to establish the superficial second-degree skin acute injury model as described in section 2.1. The rats were allocated to time points D1, D3, D7, and D14, with 5 rats at each time point. The dorsal area of each rat was divided into four regions: normal control group A, model control group B, rMAP group C, and collagen group D. The rMAP and collagen groups received the corresponding experimental drugs, while the normal control and model control groups received equal volumes of physiological saline solution (5 µL per region, twice daily, once in the morning and once in the afternoon). The continuous treatment periods for each time point were 1, 3, 7, and 14 days, respectively. At the appropriate time points, the rats were euthanized with 60 mg/kg pentobarbital sodium via intraperitoneal injection, and skin tissue samples from each region were collected and fixed in 4% paraformaldehyde solution. The tissues were stained with HE, and images were captured using a Pannoramic 250 digital slide scanner under a 200× microscope to observe histopathological changes.
2.3 Cytokine Detection: Fifteen SD rats were used to establish the superficial second-degree skin acute injury model as described in section 2.1. Twenty-four hours after the model was established, the corresponding experimental drugs were applied to each region of the dorsal area of each rat. The normal control and model control groups received equal volumes of physiological saline (5 µL per region, twice daily, once in the morning and once in the afternoon) for 7 consecutive days. Twenty-four hours after the last dose, the rats were euthanized with 60 mg/kg pentobarbital sodium via intraperitoneal injection, and the wound areas from the marked regions on the dorsal area were excised and immediately stored at -80°C for subsequent use.
2.4 Western Blot Analysis of PI3K, AKT, p-AKT, mTOR, and p-mTOR Protein Expression in Rat Skin Wounds: The wound tissues from each group of rats stored at -80°C were used to extract total protein using a protein extraction kit. The protein concentration was determined using a BCA protein quantification kit. SDS-polyacrylamide gel electrophoresis, membrane transfer, and incubation with primary antibodies (PI3K, AKT, p-AKT, mTOR, p-mTOR, β-actin) were performed sequentially. The membranes were blocked overnight at 4°C and then incubated with secondary antibodies (1:5000) at room temperature for 2 hours. The proteins were visualized using an ECL detection kit, and images were captured using a chemiluminescence imaging system to analyze the relative expression levels of the target proteins.
2.5 PCR Analysis of EGF mRNA, FGF-2 mRNA, and VEGF mRNA Expression in Rat Skin Wounds: Approximately 10-20 mg of the samples stored at -80°C were placed in EP tubes and 350 µL of lysis buffer LB was added. The samples were homogenized using a high-speed low-temperature tissue grinder, and the homogenate was transferred to a binding column and centrifuged. The supernatant was discarded, and the process was repeated to extract total RNA. cDNA was synthesized from the total RNA, and PCR amplification was performed using the first-strand cDNA as the template to obtain gene cDNA clones. The full sequences of the genes were retrieved from the NCBI database, and specific primers for each gene were designed using Primer software. All primers were synthesized and purified by Shanghai Sangon Biotech Co., Ltd. (see Table 4). The CT (threshold cycle) values of the PCR products were analyzed using Thermo Scientific PikoReal software. The relative mRNA expression levels were calculated using the 2-ΔΔCT method: ΔCT = CT target gene - CT reference gene, ΔΔCT = ΔCT experimental - ΔCT control, and the fold change in mRNA expression was represented as 2-ΔΔCT.
