Physiological and Proteomic Responses to Chronic Stress and the Effects of Chrysanthemum zawadskii var. latilobum (Maxim.) Kitam. Extract in Beagle Dogs

Chronic stress activates the hypothalamic–pituitary–adrenal (HPA) axis and induces systemic physiological changes. This study aimed to identify stress-associated serum biomarker candidates in chronically stressed beagle dogs and to evaluate the anti-stress effects of Chrysanthemum zawadskii var. latilobum (Maxim.) Kitam. extract (CZE). Beagle dogs were subjected to movement restriction-induced chronic stress for 42 days, with or without repeated oral administration of CZE. Serum cortisol and corticosterone concentrations were measured using ELISA, and hematological and serum biochemical parameters were analyzed. Serum samples collected at day 0 and day 42 of the 84-day chronic stress model were subjected to LC-MS/MS-based proteomic analysis. Chronic stress increased serum cortisol and corticosterone concentrations and induced alterations in hematological and serum biochemical parameters, whereas CZE administration tended to attenuate or stabilize these stress-associated changes. Proteomic analysis identified 380 serum proteins, of which 348 quantified proteins were subjected to differential expression analysis. The differentially expressed proteins were primarily associated with immune response, inflammatory response, extracellular matrix organization, angiogenesis, neurogenesis, and secretion. TIMP1, TREM2, RAB8A, KRT9, PPBP, THBS1, and HSPB1 were identified as candidate stress-associated serum biomarkers. These findings indicate that chronic stress induces endocrine, hematological, biochemical, and serum proteomic alterations in beagle dogs. Changes in proteins related to immune–inflammatory regulation and extracellular matrix organization suggest that prolonged stress may affect systemic immune responses and tissue remodeling. CZE may contribute to the maintenance of physiological homeostasis under chronic stress conditions and may have potential as a functional anti-stress material, while the identified serum proteins may provide a basis for the development of biomarkers for chronic stress.