*4.5. Animal Model*

The *P. americana* were raised in our lab in an appropriate environment (T: 25–30 ◦C; humidity: 70–80%). Synthetic Tβ4 was purchased from Shanghai Zhengyan Chemical Technology Co., Ltd. (Shanghai, China). A total of 75 free of viruses, bacteria and parasites KUNMING mice (7 weeks old, male) were purchased from Da Shuo (Cheng Du, China). The mice were raised in a standard laboratory pellet diet and water ad libitum about a week to adapt the environment under controlled temperature (22–25 ◦C) and humidity (about 60%) with a 12 h light/ dark cycle. All animals testing used in this study were performed in accordance with guidelines of the Animal Care Committee of Sichuan University (Chengdu, China).(20190412001, 12, April 2019).

Before the mouse model was established, all mice were anesthetized by intraperitoneal injection of 10% chloral hydrate. Under sterile conditions, the dorsal hair of the mice was shaved and completely removed with hair removal cream (Veet, Tokyo, Japan), then the skin was sterilized with 75% EtOH and a circular full-thickness excisional wound on dorsal of each mouse was made, 1.2 cm in diameter, which was created by a surgical scissor. The mice were divided into five groups of fifteen animals, each including: control group with wound and PBS treatment, positive group with wound and Tβ4 (5 μg) treatment, test group I with wound and Pa-THY1 (5 μg) treatment, test group II with wound and Pa-THY2 (5 μg) treatment, test group III with wound and Pa-THY3 (5 μg) treatment. After 24 h of surgery, they were applied to the wound by pipetting the liquid (50 μL) directly into the wound area until 11 days later. The wounds were left open with no dressing.
