The Office of Scientific Research Management of Wuhan University of Science and Technology approved the experimental protocol, with approval ID: WUST-IACUC-201814. and airway hyperresponsiveness (AHR), we were able to validate the successful establishment of the model. Furthermore, by detecting the attenuating effects of melatonin (MT) and the levels of oxidative stress in Ferrostatin-1 (Fer-1) the atopic march mice, we explored the potential molecular mechanisms involved in the development of atopic march. Results: By successfully establishing an experimental atopic march mouse model, we were able to demonstrate that overproduction of oxidative stress in the lung significantly up-regulated the activation of nuclear factor-B (NF-B) signaling pathways causing thymic stromal lymphopoietin (TSLP) release, which further promotes the development of atopic march. Conclusions: To mitigate the development of the atopic march, antioxidants such as Ferrostatin-1 (Fer-1) MT may be imperative to inhibit NF-B activation in the lung, especially after the onset of AD. access to water and food. All animal experiments took place at the Animal Experimental Center of Wuhan University of Science and Technology following relevant guidelines and regulations. The Office of Scientific Research Management of Wuhan University of Science and Technology approved the experimental protocol, with approval ID: WUST-IACUC-201814. Mice were killed by cervical dislocation under pentobarbital sodium anesthesia. Main reagents and kits Acetone, dibutyl phthalate (DBP, 99%), fluorescein isothiocyante (FITC), MT and OVA were obtained from SigmaCAldrich (St. Louis, MO, U.S.A.). All other chemicals were of analytical grade. Mouse enzyme-linked immunosorbent assay (ELISA) kits for total IgE were obtained from Biolegend (San Diego, CA, U.S.A.), and OVA-IgE and OVA-IgG1 were Ferrostatin-1 (Fer-1) purchased from BlueGene (Shanghai, China). ELISA kits for IL-1, TNF-, IL-4, IL-5, IL-13, interferon (IFN)-, IL-33 and TSLP were all obtained from eBioscience (San Diego, CA, U.S.A.). The glutathione (GSH) and malonaldehyde (MDA) test kits were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). The protein test kit was provided by Sangon Biotech (Beijing, China). Experimental design and procedure A 0.5% FITC-mediated contact hypersensitivity (CHS) is a Th2-dominant immune system and elevated levels of IL-4 expression in the inflamed skin and increased IgE levels in the serum. Because AD shares many features with CHS to FITC, this model of Th2-type CHS was used in the present study to induce AD-like skin lesions and then aerosol challenge of OVA to establish the mice model of atopic march. There are six groups in this experiment with six mice per group. (1) Control group (Control) mice were treated with 120 l of vehicle (1:1 DBP/acetone) on their shaven backs on days 5 and 6. On day 11, their shaven backs and left ears were subjected to 40 l of vehicle. In addition, the mice were exposed to an aerosol challenge of Ferrostatin-1 (Fer-1) saline (30 min/day) using an ultrasonic nebulizer (Yuyue 402A type I, China) on days 19 through 25. (2) MT control group (MT) mice received the same treatment as the control group, but in addition the mice were treated with 5 mg/(kg.day) MT by intratracheal instillation, from days 19 to 25. (3) A 0.5% FITC-induced AD group (0.5% FITC) mice were sensitized with 120 l of 0.5% FITC on days 5 and 6 on their shaven backs, and on day 11 their shaven backs and left ear were subjected to 40 l of 0.5% FITC . From days 19 to 25, the mice were exposed to saline (30 min/day) using an ultrasonic nebulizer. (4) OVA exposure group (OVA) mice were exposed to an aerosol challenge of 1% OVA (30 min/day) using an ultrasonic nebulizer on days 19 through 25. (5) A 0.5% FITC and OVA co-treatment group (0.5% FITC+OVA) mice were Ferrostatin-1 (Fer-1) treated with 120 l of 0.5% FITC on days 5 and 6 on their shaven backs, and on day 11, their shaven backs and left ear were subjected to 40 l of 0.5% FITC. The mice were then challenged with 1% OVA using an ultrasonic nebulizer from days 19 to 25. (6) A 0.5% FITC and MT exposure combined with OVA challenged group (0.5% FITC+OVA+MT). These mice were treated with 120 l of 0.5% FITC on days 5 and 6 on their shaven backs, and on day 11 their shaven backs and left ear were subjected to 40 l of 0.5% FITC. From days 19 to 25, these mice were challenged with 1% Rabbit Polyclonal to Trk A (phospho-Tyr680+Tyr681) OVA using an ultrasonic nebulizer. In addition, 5 mg/(kg.day) MT was administered by intratracheal instillation from days 19 to 25. The detailed protocol is shown in Figure 1. Open in a separate window Figure 1 Exposure and sensitization protocol Quantitative analyses of total serum IgE, OVA-IgE, and OVA-IgG1 Heart blood was collected after the mice were anesthetized with pentobarbital on day 18. Immediately after collection, the blood serum.