Mouse model
Male C57BL/6 mice aged 8 weeks were purchased from the Experimental Animal Center of Xi’an Jiaotong University. Mast Cell-Deficient C57BL/6 STOCK KitW-sh/W-shmice and MrgprB2 deficient (MUT) mice were purchased from the Model Animal Research Center of Nanjing University. The mice were housed in individual cages in a colony room with 20–25 ℃, a relative humidity of 40%, and were fed standard dry food twice per day.
Adult male mice weighing 18-22 g were anesthetized with an intraperitoneal injection of pelltobarbitalum natricum. Dorsal skin was shaved off a day before the experiment start, and the mice were randomly divided into different groups (6 mice per group).
For tacrolimus short-term application, experimental groups were treated with topical 0.03%, 0.1%, 0.3% tacrolimus prepared by glycerine respectively twice a day, while glycerine as solvent was given to control group. Scratching bouts were counted, skin and serum samples of each treated and control mouse were collected at the end of 1 day. For tacrolimus long-term application, experimental groups were treated with topical 0.03% twice a day. C48/80 was used for subcutaneous injection on day 7 and 11, scratching bouts were counted later and skin samples were collected respectively. The experimental protocols involving mice were approved by the Animal Ethics Committee at Xi’an Jiaotong University, Xi’an, China (Permit Number: XJTU 2019-711).
Finally, serum and skin samples were further used to detect inflammatory cytokines release and MrgprB2 mRNA expression. What’s more, H&E staining and avidin staining were performed using collected skin samples.