*3.8. Histological Observation*

In histological examination, on days 3, 7, 14, and 21 after scalding, 4 test animals in each group were euthanized, respectively, and a full layer of skin tissue of about 1.5 cm<sup>2</sup> in area around each original scald site was immediately incised as a sample. A small patch of skin sample incised from the center of the scald was fixed in 4% paraformaldehyde (PFA) for histology and collagen deposition examination (Masson staining method), while the remaining skin tissue samples were used for subsequent monitoring of biochemical parameters. The above tissue samples, which had been fixed with 4% PFA for 24 h, were embedded in para ffin and then cut with para ffin microtome (CUT 4050, Micro Tec, Inc., Walldorf, Germany) in a direction longitudinal to the tissue, followed by staining with HE and modified Masson trichrome staining kit (Beijing Leagene Biotechnology Co., Ltd., Beijing, China), respectively. Thereafter, the regeneration of epidermis and dermis, and the level and distribution of collagen fibers in each skin sample were examined via an optical microscope (DMI3000B, Leica).

#### *3.9. Determination of TP Content and Proinflammatory Cytokine Levels*

The desired amount of each remaining skin tissue sample was accurately weighed, mixed with normal saline at a ratio of weight (g): volume (mL) = 1:9, and mechanically homogenized in an ice water bath. The resulting homogenate was cryogenically centrifuged to isolate supernatant, and content of TP and levels of proinflammatory cytokines (TNFα and IL-6) in tissue in the supernatant were determined as per kit operating procedure.

#### *3.10. Determination of HYP*

Take an appropriate amount of the remaining fresh skin tissue samples from each of the above groups, accurately weighed them, and then measured the content of HYP according to the kit method.
