{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T15:31:02Z","timestamp":1787758262121,"version":"build-2784847793"},"reference-count":52,"publisher":"MDPI AG","issue":"17","license":[{"start":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T00:00:00Z","timestamp":1787702400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003627","name":"Rural Development Administration","doi-asserted-by":"publisher","award":["RS-2023-00231446"],"award-info":[{"award-number":["RS-2023-00231446"]}],"id":[{"id":"10.13039\/501100003627","id-type":"DOI","asserted-by":"publisher"}]},{"award":["RS-2023-00231446"],"award-info":[{"award-number":["RS-2023-00231446"]}],"id":[{"id":"https:\/\/ror.org\/03xs9yg50","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>Chronic stress activates the hypothalamic\u2013pituitary\u2013adrenal (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\u2013inflammatory 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.<\/jats:p>","DOI":"10.3390\/ani16172679","type":"journal-article","created":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T15:04:32Z","timestamp":1787756672000},"page":"2679","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Physiological and Proteomic Responses to Chronic Stress and the Effects of Chrysanthemum zawadskii var. latilobum (Maxim.) Kitam. Extract in Beagle Dogs"],"prefix":"10.3390","volume":"16","author":[{"given":"Ye Jin","family":"Yang","sequence":"first","affiliation":[{"name":"College of Veterinary Medicine, Gyeongsang National University, Jinju 52828, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Min Jung","family":"Kim","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Gyeongsang National University, Jinju 52828, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hee Ho","family":"Kim","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Gyeongsang National University, Jinju 52828, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ji Woong","family":"Heo","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Gyeongsang National University, Jinju 52828, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ju Lan","family":"Chun","sequence":"additional","affiliation":[{"name":"Animal Welfare Division, National Institute of Animal Science, 1500 Kongjwipatjwi-ro, Iseomyeon, Wanju-gun 55365, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eui-Cheol","family":"Shin","sequence":"additional","affiliation":[{"name":"Food Science and Technology, Gyeongsang National University, Jinju 52828, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7469-3215","authenticated-orcid":false,"given":"Kyeong Soo","family":"Kim","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Engineering, Gyeongsang National University, 501, Jinju-daero, Jinju 52725, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4397-9664","authenticated-orcid":false,"given":"Hyun-Wook","family":"Kim","sequence":"additional","affiliation":[{"name":"Division of Animal Bioscience & Integrated Biotechnology, Gyeongsang National University, Jinju 52828, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eun Ju","family":"Jeong","sequence":"additional","affiliation":[{"name":"Agri-Food Bio Convergence Institute, Gyeongsang National University, Jinju 52725, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2645-4508","authenticated-orcid":false,"given":"Dongbin","family":"Lee","sequence":"additional","affiliation":[{"name":"Department of Veterinary Surgery, Gyeongsang National University, Jinju 52725, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0199-8090","authenticated-orcid":false,"given":"Kwang Il","family":"Park","sequence":"additional","affiliation":[{"name":"College of Veterinary Medicine, Gyeongsang National University, Jinju 52828, Republic of Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"502","DOI":"10.1038\/s41380-021-01224-9","article-title":"The neuroendocrinology of stress: The stress-related continuum of chronic disease development","volume":"27","author":"Agorastos","year":"2022","journal-title":"Mol. Psychiatry"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"367","DOI":"10.1007\/s13273-020-00094-6","article-title":"Comparative analysis of acute and chronic stress-induced neurobehavioral alteration and liver injury in mice","volume":"16","author":"Oh","year":"2020","journal-title":"Mol. Cell. Toxicol."},{"key":"ref_3","unstructured":"Chu, B., Marwaha, K., Sanvictores, T., Awosika, A.O., and Ayers, D. (2026). Physiology, Stress Reaction. StatPearls, StatPearls Publishing."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"137","DOI":"10.1196\/annals.1296.016","article-title":"Fight or flight, forbearance and fortitude: The spectrum of actions of the catecholamines and their cousins","volume":"1018","author":"Arun","year":"2004","journal-title":"Ann. N. Y. Acad. Sci."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"164","DOI":"10.1016\/j.psyneuen.2019.02.021","article-title":"Stress and inflammation\u2014The need to address the gap in the transition between acute and chronic stress effects","volume":"105","author":"Rohleder","year":"2019","journal-title":"Psychoneuroendocrinology"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"525","DOI":"10.1007\/s13273-026-00617-7","article-title":"Synergistic toxicity of endocrine disruptors and environmental toxicants under climate change","volume":"22","author":"Seo","year":"2026","journal-title":"Mol. Cell. Toxicol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"243","DOI":"10.1016\/S0031-9384(98)00290-X","article-title":"Chronic stress in dogs subjected to social and spatial restriction. II. Hormonal and immunological responses","volume":"66","author":"Beerda","year":"1999","journal-title":"Physiol. Behav."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"603","DOI":"10.1002\/j.2040-4603.2016.tb00694.x","article-title":"Regulation of the Hypothalamic-Pituitary-Adrenocortical Stress Response","volume":"6","author":"Herman","year":"2016","journal-title":"Compr. Physiol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"567","DOI":"10.1007\/s13273-026-00639-1","article-title":"Toxicological implications of climate change for reproductive function in livestock","volume":"22","author":"Park","year":"2026","journal-title":"Mol. Cell. Toxicol."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Beaupere, C., Liboz, A., Feve, B., Blondeau, B., and Guillemain, G. (2021). Molecular Mechanisms of Glucocorticoid-Induced Insulin Resistance. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22020623"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"6868","DOI":"10.1038\/sj.onc.1209935","article-title":"Cross-talk between nuclear receptors and nuclear factor kappaB","volume":"25","author":"Haegeman","year":"2006","journal-title":"Oncogene"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"525","DOI":"10.1038\/s41574-019-0228-0","article-title":"The human stress response","volume":"15","author":"Russell","year":"2019","journal-title":"Nat. Rev. Endocrinol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2470547017692328","DOI":"10.1177\/2470547017692328","article-title":"Neurobiological and Systemic Effects of Chronic Stress","volume":"1","author":"McEwen","year":"2017","journal-title":"Chronic Stress"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1007\/s42000-018-0023-7","article-title":"Chronic stress and body composition disorders: Implications for health and disease","volume":"17","author":"Stefanaki","year":"2018","journal-title":"Hormones"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"363","DOI":"10.1007\/s13273-025-00585-4","article-title":"Impact of lifestyle habits and periodontal disease on metabolic syndrome in South Korean adults","volume":"22","author":"Jung","year":"2026","journal-title":"Mol. Cell. Toxicol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"H1476","DOI":"10.1152\/ajpheart.00859.2014","article-title":"Chronic stress impacts the cardiovascular system: Animal models and clinical outcomes","volume":"308","author":"Golbidi","year":"2015","journal-title":"Am. J. Physiol. Heart Circ. Physiol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"435","DOI":"10.1016\/j.cell.2025.11.031","article-title":"Hepatic adaptation to chronic metabolic stress primes tumorigenesis","volume":"189","author":"Tzouanas","year":"2026","journal-title":"Cell"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"e13857","DOI":"10.1111\/1440-1681.13857","article-title":"Overexpression of HSP27 accelerates stress-induced gastric ulcer healing via the CXCL12\/CXCR4 axis","volume":"51","author":"Lu","year":"2024","journal-title":"Clin. Exp. Pharmacol. Physiol."},{"key":"ref_19","first-page":"46","article-title":"Physiological biomarkers of chronic stress: A systematic review","volume":"15","author":"Noushad","year":"2021","journal-title":"Int. J. Health Sci."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"789","DOI":"10.1007\/s13273-022-00309-y","article-title":"Identification of miRNA expression associated with Alzheimer\u2019s disease and neurodegeneration in rat models with obstructive sleep apnea","volume":"19","author":"Kim","year":"2023","journal-title":"Mol. Cell. Toxicol."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Kang, Y.-M., and Kim, K. (2026). Diagnostic indicators and biomarkers of static blood-related disorders: A comprehensive review. Mol. Cell. Toxicol.","DOI":"10.1007\/s13273-026-00636-4"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1129","DOI":"10.1007\/s13273-025-00519-0","article-title":"Combined effects of prenatal stress and lead exposure on hippocampal protein expression and behavior in rats: Implications for psychiatric disorders","volume":"21","author":"Lee","year":"2025","journal-title":"Mol. Cell. Toxicol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"113","DOI":"10.1007\/s13273-018-0014-4","article-title":"Exosomes as diagnostic biomarkers in cancer","volume":"14","author":"Kim","year":"2018","journal-title":"Mol. Cell. Toxicol."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Kim, J.H., Park, B.I., Kim, Y.H., Yoon, J.S., Choi, N.Y., and Kim, K.J. (2025). Chrysanthemum zawadskii var. latilobum Flower Essential Oil Reduces MRSA Pathogenicity by Inhibiting Virulence Gene Expression. Molecules, 30.","DOI":"10.3390\/molecules30030553"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"128","DOI":"10.3892\/etm.2023.11827","article-title":"Ethanol extract of Chrysanthemum zawadskii inhibits the NLRP3 inflammasome by suppressing ASC oligomerization in macrophages","volume":"25","author":"Jang","year":"2023","journal-title":"Exp. Ther. Med."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"513","DOI":"10.1007\/s13273-026-00614-w","article-title":"Circadian-dependent UV-B stress responses reveal differential antioxidant and photoprotective mechanisms in Achyranthes japonica microgreens","volume":"22","author":"Kim","year":"2026","journal-title":"Mol. Cell. Toxicol."},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Nunez, S.G., Rabelo, S.P., Subotic, N., Caruso, J.W., and Knezevic, N.N. (2025). Chronic Stress and Autoimmunity: The Role of HPA Axis and Cortisol Dysregulation. Int. J. Mol. Sci., 26.","DOI":"10.3390\/ijms26209994"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1002\/jat.727","article-title":"A good practice guide to the administration of substances and removal of blood, including routes and volumes","volume":"21","author":"Diehl","year":"2001","journal-title":"J. Appl. Toxicol."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Marza, S.M., Munteanu, C., Papuc, I., Radu, L., Diana, P., and Purdoiu, R.C. (2024). Behavioral, Physiological, and Pathological Approaches of Cortisol in Dogs. Animals, 14.","DOI":"10.3390\/ani14233536"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"75","DOI":"10.2478\/fv-2022-0009","article-title":"Chronic Stress Indicators in Canines","volume":"66","author":"Dolan","year":"2022","journal-title":"Folia Vet."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Cobb, M.L., Jimenez, A.G., and Dreschel, N.A. (2025). Beyond Cortisol! Physiological Indicators of Welfare for Dogs: Deficits, Misunderstandings and Opportunities. J. Appl. Anim. Welf. Sci., 1\u201330.","DOI":"10.1080\/10888705.2025.2572616"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Alotiby, A. (2024). Immunology of Stress: A Review Article. J. Clin. Med., 13.","DOI":"10.3390\/jcm13216394"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"114743","DOI":"10.1016\/j.physbeh.2024.114743","article-title":"Plasma proteomic signature of chronic psychosocial stress in mice","volume":"289","author":"Melo","year":"2025","journal-title":"Physiol. Behav."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"coae075","DOI":"10.1093\/conphys\/coae075","article-title":"The plasma proteome reveals markers of recent and repeated stress in free-ranging seals","volume":"12","author":"Avalos","year":"2024","journal-title":"Conserv. Physiol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"118457","DOI":"10.1016\/j.biopha.2025.118457","article-title":"The role of MMPs and TIMPs in regenerative medicine: From pathological ECM remodeling to therapeutic applications","volume":"191","author":"Kazemi","year":"2025","journal-title":"Biomed. Pharmacother."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"a009712","DOI":"10.1101\/cshperspect.a009712","article-title":"The thrombospondins","volume":"3","author":"Adams","year":"2011","journal-title":"Cold Spring Harb. Perspect. Biol."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Diaz-Jimenez, D., Kolb, J.P., and Cidlowski, J.A. (2021). Glucocorticoids as Regulators of Macrophage-Mediated Tissue Homeostasis. Front. Immunol., 12.","DOI":"10.3389\/fimmu.2021.669891"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1","DOI":"10.2174\/1570161043476483","article-title":"Glucocorticoids and vascular reactivity","volume":"2","author":"Yang","year":"2004","journal-title":"Curr. Vasc. Pharmacol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"e200527","DOI":"10.1212\/NXI.0000000000200527","article-title":"Decoding TREM2 Signaling Pathways: Linking Macrophage Glycolysis to Inflammatory Diseases in the CNS","volume":"13","author":"Che","year":"2026","journal-title":"Neurol. Neuroimmunol. Neuroinflammation"},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Paulson, R.F., Ruan, B., Hao, S., and Chen, Y. (2020). Stress Erythropoiesis is a Key Inflammatory Response. Cells, 9.","DOI":"10.3390\/cells9030634"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1016\/j.exphem.2020.07.011","article-title":"Stress erythropoiesis: Definitions and models for its study","volume":"89","author":"Paulson","year":"2020","journal-title":"Exp. Hematol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"2900","DOI":"10.1038\/s41467-023-38624-0","article-title":"Platelet-derived chemokines promote skeletal muscle regeneration by guiding neutrophil recruitment to injured muscles","volume":"14","author":"Graca","year":"2023","journal-title":"Nat. Commun."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Wu, Q., Tu, H., and Li, J. (2022). Multifaceted Roles of Chemokine C-X-C Motif Ligand 7 in Inflammatory Diseases and Cancer. Front. Pharmacol., 13.","DOI":"10.3389\/fphar.2022.914730"},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Martinez-Arroyo, O., Selma-Soriano, E., Ortega, A., Cortes, R., and Redon, J. (2021). Small Rab GTPases in Intracellular Vesicle Trafficking: The Case of Rab3A\/Raphillin-3A Complex in the Kidney. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22147679"},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Jin, H., Tang, Y., Yang, L., Peng, X., Li, B., Fan, Q., Wei, S., Yang, S., Li, X., and Wu, B. (2021). Rab GTPases: Central Coordinators of Membrane Trafficking in Cancer. Front. Cell Dev. Biol., 9.","DOI":"10.3389\/fcell.2021.648384"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"321","DOI":"10.2174\/1871530323666230803095118","article-title":"Chronic Stress as a Risk Factor for Type 2 Diabetes: Endocrine, Metabolic, and Immune Implications","volume":"24","author":"Lisco","year":"2024","journal-title":"Endocr. Metab. Immune Disord. Drug Targets"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"754","DOI":"10.1038\/jid.2013.356","article-title":"Keratin 9 is required for the structural integrity and terminal differentiation of the palmoplantar epidermis","volume":"134","author":"Fu","year":"2014","journal-title":"J. Investig. Dermatol."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"7","DOI":"10.1186\/1755-1536-5-7","article-title":"Heat shock protein 27 (HSP27): Biomarker of disease and therapeutic target","volume":"5","author":"Vidyasagar","year":"2012","journal-title":"Fibrogenesis Tissue Repair."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1007\/978-0-387-39975-1_2","article-title":"The cellular \u201cnetworking\u201d of mammalian Hsp27 and its functions in the control of protein folding, redox state and apoptosis","volume":"594","author":"Arrigo","year":"2007","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1007\/s12192-013-0452-9","article-title":"Reaction of small heat-shock proteins to different kinds of cellular stress in cultured rat hippocampal neurons","volume":"19","author":"Golenhofen","year":"2014","journal-title":"Cell Stress. Chaperones"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"375","DOI":"10.1007\/s13273-025-00586-3","article-title":"Tenuigenin exerts neuroprotective effects by activating the Nrf2\/HO-1 pathway to perform antioxidant activity and inhibit neuronal ferroptosis, preventing epilepsy in immature mice","volume":"22","author":"Zhang","year":"2026","journal-title":"Mol. Cell. Toxicol."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1177\/117693510500100102","article-title":"Proteomics: Analysis of spectral data","volume":"1","author":"Burke","year":"2005","journal-title":"Cancer Inform."}],"container-title":["Animals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2615\/16\/17\/2679\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T15:11:58Z","timestamp":1787757118000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2615\/16\/17\/2679"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,26]]},"references-count":52,"journal-issue":{"issue":"17","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["ani16172679"],"URL":"https:\/\/doi.org\/10.3390\/ani16172679","relation":{},"ISSN":["2076-2615"],"issn-type":[{"value":"2076-2615","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,26]]}}}