From heat stress to recovery: proteomic insights into endangered Brachymystax tsinlingensis survival strategies and the ameliorative effects of anti-stress additives

Abstract

The endangered cold-water fish Brachymystax tsinlingensis (B. tsinlingensis) serves as a critical sentinel species for aquatic ecosystem responses to climate change. This study investigates heat stress impacts on behavior, physiology, and molecular homeostasis in B. tsinlingensis, and evaluates neuroprotective effects of anti-stress additives (vitamin C, gamma-aminobutyric acid, trehalose). Behavioral analysis showed a significant increase in center-zone entries under heat stress. Physiological assays showed a reduction in superoxide dismutase (SOD) and catalase (CAT) activities, alongside an upregulation of heat shock protein 70 (Hsp70), glucose-regulated protein 78 (GRP78), and caspase expression under heat stress, with a return to baseline levels following a 12 h recovery. For assays in the brain, histopathological examination identified vacuolation in cerebral tissue after heat stress. Quantitative proteomics analysis identified 831 differentially expressed proteins (DEPs) out of 8,955 proteins during the temperature change process. Pathway analysis revealed that the 'DNA replication' and 'Citrate cycle' pathways were inhibited by heat stress but reactivated during recovery, whereas the 'ECM-receptor interaction' and 'Cell adhesion molecules' pathways exhibited opposite trends. Intervention with additives showed that trehalose enhanced SOD, glutathione peroxidase (GPX), and CAT activities, as well as gene expression related to cell adhesion and barrier function. Gamma-aminobutyric acid maximally suppressed stress-related genes (Hsp70, long-chain-fatty-acid-CoA ligase, and arachidonate lipoxygenase), while vitamin C exhibited general but less targeted effects. As the first proteomic study on B. tsinlingensis, this work reveals that blood–brain barrier reconstruction and energy reallocation are key neural survival strategies under temperature change stress. Furthermore, trehalose demonstrates high potential as an anti-heat stress additive by enhancing both antioxidant defenses and blood–brain barrier integrity. These findings advance our understanding of heat adaptation mechanisms in cold-water fish species and provide scientific foundations for conserving B. tsinlingensis.

Keywords

Brachymystax tsinlingensis / Heat stress / Proteomic / Trehalose / Blood–brain barrier

References

[1] Aggarwal A, Singh I, Sandhir R. Protective effect of S-nitrosoglutathione administration against hyperglycemia induced disruption of blood brain barrier is mediated by modulation of tight junction proteins and cell adhesion molecules. Neurochem Int, 2018, 118: 205-216

[2] Campbell ID, Humphries MJ. Integrin structure, activation, and interactions. Cold Spring Harb Perspect Biol, 2011, 3: a004994

[3] Çevik ÖS, Yıldırım DD, Uzun C, Horata E. Contribution of distinctive outcome measures to the assessment of anxiety in the open field: a meta-analysis of factors mediating open-field test variability in rodent models of anxiety. Behav Brain Res, 2025, 490: 115612

[4] Dinarello CA. Proinflammatory cytokines. Chest, 2000, 118: 503-508

[5] Fan L, Liao G, Wang Z, Liu H, Cheng K, Hu J, Yang Y, Zhou Z. Insight into three water additives: revealing the protective effects on survival and stress response under cold stress for Pacific white shrimp Litopenaeus vannamei. Fish Shellfish Immunol, 2023, 139: 108845

[6] Fang M, Lei Z, Ruilin M, Jing W, Leqiang D. High temperature stress induced oxidative stress, gut inflammation and disordered metabolome and microbiome in Tsinling lenok trout. Ecotoxicol Environ Saf, 2023, 266: 115607

[7] Forster PM, Smith CJ, Walsh T, Lamb WF, Lamboll R, Hauser M, Ribes A, Rosen D, Gillett N, Palmer MD, Rogelj J, von Schuckmann K, Seneviratne SI, Trewin B, Zhang X, Allen M, Andrew R, Birt A, Borger A, Boyer T, Broersma JA, Cheng L, Dentener F, Friedlingstein P, Gutierrez JM, Guetschow J, Hall B, Ishii M, Jenkins S, Lan X, Lee J-Y, Morice C, Kadow C, Kennedy J, Killick R, Minx JC, Naik V, Peters GP, Pirani A, Pongratz J, Schleussner C-F, Szopa S, Thorne P, Rohde R, Rojas Corradi M, Schumacher D, Vose R, Zickfeld K, Masson-Delmotte V, Zhai P. Indicators of Global Climate Change 2022: annual update of large-scaleindicators of the state of the climate system and human influence. Earth Syst Sci Data, 2023, 15: 2295-2327

[8] Guo J, Cai H, Zheng J, Liu X, Liu Y, Ma J, Que Z, Gong W, Gao Y, Tao W, Xue Y. Long non-coding RNA NEAT1 regulates permeability of the blood-tumor barrier via miR-181d-5p-mediated expression changes in ZO-1, occludin, and claudin-5. Biochim Biophys Acta Mol Basis Dis, 2017, 1863: 2240-2254

[9] Guo CY, Ding M, Zhang S, Wang Y, Ji YP, Xu SL, Wang YJ, Wang DL. Dietary effects of vitamin C on antioxidant capacity, intestinal microbiota and the resistance of pathogenic bacteria in cultured Silver pomfret (Pampus argenteus). PLoS ONE, 2024, 19: e0300643

[10] Guo Q, Liu Q, Zhou S, Lin Y, Lv A, Zhang L, Li L, Huang F. Apelin regulates mitochondrial dynamics by inhibiting Mst1-JNK-Drp1 signaling pathway to reduce neuronal apoptosis after spinal cord injury. Neurochem Int, 2024, 180: 105885

[11] Guo X, Chu Z, Song R, Wang Z, Li R, Xiong D, Zhang M, Jiang H, Shao J. Toxicity of common biocides used in aquaculture to embryos and larvae of Brachymystax tsinlingensis Li. J Fish Biol, 2024, 104: 463-472

[12] Guo M, Meng L, Li F, Liu Y, Zhang Z, Zhang Q, Kong W. Effects of dietary Bacillus pumilus on the growth, intestinal health, lipid metabolism, and mTOR signaling pathway of juvenile coho salmon (Oncorhynchus kisutch). Water Biology and Security, 2025, 4: 100313

[13] He Q, Yang Q, Wu L, He Y, Zeng N, Wang Z. Neurotoxic effects of per- and polyfluoroalkyl substances (PFAS) mixture exposure in mice: accumulations in brain and associated changes of behaviors, metabolome, and transcriptome. J Hazard Mater, 2025, 489: 137699

[14] Hurt E, Cheng J, Baβler J, Iwasa J, Beckmann R. Snapshot: Eukaryotic ribosome biogenesis I. Cell, 2023, 186: 2282-2282.e1

[15] IPCC (2022) Mitigation of climate change. In: Working group ii contribution to the sixth assessment report of the intergovernmental panel on climate change. Cambridge University Press

[16] Islam MJ, Puebla O, Kunzmann A. Nutritional mitigation of heatwave stress in European seabass, Dicentrarchus labrax: metabolic, cellular, and molecular responses. Aquac Rep, 2024, 36: 102168

[17] Islam SNul, Kouser S, Hassan P, Asgher M, Shah AA, Khan NA. Gamma-aminobutyric acid interactions with phytohormones and its role in modulating abiotic and biotic stress in plants. Stress Biol, 2024, 4: 36

[18] Jomova K, Alomar SY, Alwasel SH, Nepovimova E, Kuca K, Valko M. Several lines of antioxidant defense against oxidative stress: antioxidant enzymes, nanomaterials with multiple enzyme-mimicking activities, and low-molecular-weight antioxidants. Arch Toxicol, 2024, 98: 1323-1367

[19] Kadry H, Noorani B, Cucullo L. A blood-brain barrier overview on structure, function, impairment, and biomarkers of integrity. Fluids Barriers CNS, 2020, 17: 69

[20] Kaur A, Ali S, Brraich OS, Siva C, Pandey PK. State of thermal tolerance in an endangered Himalayan fish Tor putitora revealed by expression modulation in environmental stress related genes. Sci Rep, 2025, 15: 5025

[21] Klasson TD, LaGory EL, Zhao H, Huynh SK, Papandreou I, Moon EJ, Giaccia AJ. ACSL3 regulates lipid droplet biogenesis and ferroptosis sensitivity in clear cell renal cell carcinoma. Cancer Metab, 2022, 10: 14

[22] Lee S, Moniruzzaman M, Farris N, Min T, Bai SC. Interactive effect of dietary gamma-aminobutyric acid (GABA) and water temperature on growth performance, blood plasma indices, heat shock proteins and GABAergic gene expression in juvenile olive flounder Paralichthys olivaceus. Metabolites, 2023, 13: 619

[23] Lemmens B, Lindqvist A. DNA replication and mitotic entry: a brake model for cell cycle progression. J Cell Biol, 2019, 218: 3892-3902

[24] Li W, Chen Z, Chin I, Chen Z, Dai H. The role of ve-cadherin in blood-brain barrier integrity under central nervous system pathological conditions. Curr Neuropharmacol, 2018, 16: 1375-1384

[25] Li Q, Huang Y, Xie X, Zhai X, Chen M, Liang D, Cao M, Liang S, Lin L. Comparison of growth, and non-specific immune of crisp and ordinary tilapia (Oreochromis niloticus). Aquaculture, 2023, 562: 738827

[26] Liu Y, Li B, Chen T, Tian S, Zhang Z. The synthesis, degradation and biological function of trehalose- 6-phosphate. Stress Biol, 2025, 5: 38

[27] Ma F, Ma R, Zhao L. Effects of antimicrobial peptides on antioxidant properties, non-specific immune response and gut microbes of Tsinling lenok trout (Brachymystax lenok tsinlingensis). Biochem Genet, 2024

[28] Mata-Bermudez, A., Trejo-Chávez, R., Martínez-Vargas, M., Pérez-Arredondo, A., Martínez-Cardenas, M. de L.Á., Diaz-Ruiz, A., Rios, C., Navarro, L., 2024. Dysregulation of the dopaminergic system secondary to traumatic brain injury: implications for mood and anxiety disorders. Front. Neurosci. 18. https://doi.org/10.3389/fnins.2024.1447688

[29] Miao Q, Fang Z, Zhou J, Wang Y, Wan Z, Xu S, Guo C. Study on tolerance, tissue damage and oxidative stress of juvenile silver pomfret (Pampus argenteus) under copper sulfate, formaldehyde and freshwater stress. Aquaculture, 2025, 599: 742134

[30] Nawaz M, Hassan MU, Chattha MU, Mahmood A, Shah AN, Hashem M, Alamri S, Batool M, Rasheed A, Thabit MA, Alhaithloul HAS, Qari SH. Trehalose: a promising osmo-protectant against salinity stress—physiological and molecular mechanisms and future prospective. Mol Biol Rep, 2022, 49: 11255-11271

[31] Nikjoo M, Farhangi M, Patimar R, Adineh H, Alizadeh M. The protective effect of vitamin C on growth, digestive enzymes, immune response, and gill histology in Caspian roach (Rutilus rutilus caspicus) under diazinon stress. Aquac Rep, 2023, 32: 101698

[32] Ogun AO, Kim H, Yoon S, Lee S, Jeon H, Aulia D, Hur J, Lee S. Effects of dietary gamma-aminobutyric acid (GABA) inclusion on acute temperature stress responses in juvenile olive flounder (Paralichthys olivaceus). Animals, 2025, 15: 809

[33] Rathore SS, Hanumappa SM, Yusufzai SI, Suyani NK, Abdullah-Al-Mamun M, Nasren S, Sidiq MJ, Hanumanthappa SK, Kalyani R. Dietary administration of engineered nano-selenium and vitamin c ameliorates immune response, nutritional physiology, oxidative stress, and resistance against Aeromonas hydrophila in Nile Tilapia (Oreochromis niloticus). Biol Trace Elem Res, 2023, 201: 4079-4092

[34] Rihacek M, Kosaristanova L, Fialova T, Rypar T, Sterbova DS, Adam V, Zurek L, Cihalova K. Metabolic adaptations of Escherichia coli to extended zinc exposure: insights into tricarboxylic acid cycle and trehalose synthesis. BMC Microbiol, 2024, 24: 384

[35] Ruenkoed S, Nontasan S, Phudkliang J, Phudinsai P, Pongtanalert P, Panprommin D, Mongkolwit K, Wangkahart E. Effect of dietary gamma aminobutyric acid (GABA) modulated the growth performance, immune and antioxidant capacity, digestive enzymes, intestinal histology and gene expression of Nile tilapia (Oreochromis niloticus). Fish Shellfish Immunol, 2023, 141: 109056

[36] Rui L. Energy metabolism in the liver. Compr Physiol, 2014, 4: 177-197

[37] Sevriev B, Dimitrova S, Kehayova G, Dragomanova S. Trehalose: neuroprotective effects and mechanisms—an updated review. NeuroSci, 2024, 5: 429

[38] Stevens AM, Gobler CJ. Interactive effects of acidification, hypoxia, and thermal stress on growth, respiration, and survival of four North Atlantic bivalves. Mar Ecol-Prog Ser, 2018, 604: 143-161

[39] Sung W, Peiqi Y, Yiyu C (1998) China Red Data Book of Endangered Animals: Pisces. Science Press, Beijing

[40] Wang T, Yuan W, Liu Y, Zhang Y, Wang Z, Zhou X, Ning G, Zhang L, Yao L, Feng S, Kong X. The role of the JAK-STAT pathway in neural stem cells, neural progenitor cells and reactive astrocytes after spinal cord injury. Biomed Rep, 2015, 3: 141-146

[41] Wang Z, Zhou J, Li J, Lv W, Zou J, Fan L. A new insight into the intestine of Pacific white shrimp: Regulation of intestinal homeostasis and regeneration in Litopenaeus vannamei during temperature fluctuation. Comp Biochem Physiol Part D Genomics Proteomics, 2020, 35: 100687

[42] Wang Y, Wu J, Wang J, He L, Lai H, Zhang T, Wang X, Li W. Mitochondrial oxidative stress in brain microvascular endothelial cells: triggering blood-brain barrier disruption. Mitochondrion, 2023, 69: 71-82

[43] Wang Z, Guo X, Tu J, Shi X, Gan L, Zhang M, Jiang H, Zhang X, Shao J. Responses of Micropterus salmoides under ammonia stress and the effects of a potential ammonia antidote. Animals, 2023, 13: 397

[44] Wang Q, Pan L, Chen S, Zhang Y, Liu G, Wu Y, Qin X, Zhang P, Zhang W, Zhang J, Kong D. BBB proteomic analysis reveals that complex febrile seizures in infancy enhance susceptibility to epilepsy in adulthood through dysregulation of ECM-receptor interaction signaling pathway. Fluids Barriers CNS, 2025, 22: 49

[45] Wang Z, Ye H, Liu P, Lin S, Wang Y, Zhou Q, Jiang H, Shao J. Surviving the heat: the homeostatic regulation mechanism of endangered Brachymystax tsinlingensis. J Therm Biol, 2025, 127: 104023

[46] Xia J, Peng M, Huang Y, Elvidge CK. Acute warming in winter eliminates chemical alarm responses in threatened Qinling lenok Brachymystax lenok tsinlingensis. Sci Total Environ, 2021, 764: 142807

[47] Xing YC, Lv BB, Ye EQ, Fan EY, Li SY, Wang LX, Zhang CG, Zhao YH. Revalidation and redescription of Brachymystax tsinlingensis Li, 1966 (Salmoniformes: Salmonidae) from China. Zootaxa, 2015, 3962: 191-205

[48] Yan D, Long X, Zhang X, Dong X, Wang Z, Jiang H, An M, Chen J, Gan L. Identification and characterization of long non-coding RNAs in intestinal immune regulation of largemouth bass, Micropterus salmoides, under acute heat stress. Comp Biochem Physiol d: Genomics Proteomics, 2023, 48: 101132

[49] Yang Q, Zhong Y, Yang F, Li H, Tran NT, Zhang M, Wang L, He L, Zhang Z, Ge H, Wu J, Ai C, Li S, Lin Q. Transcriptome analysis of sea cucumber (Apostichopus japonicus) in southern China under heat stress. Aquac Rep, 2024, 36: 102036

[50] Yohana MA, Ray GW, Yang Q, Kou S, Tan B, Wu J, Mao M, Bo Ge Z, Feng L. Protective effects of butyric acid during heat stress on the survival, immune response, histopathology, and gene expression in the hepatopancreas of juvenile Pacific shrimp (L. vannamei). Fish Shellfish Immunol, 2024, 150: 109610

[51] Yuan, B., Bi, Y., 2019. Multiple factors shape the neural responses to brain damage: anatomy, topology, etiology and demography. Front. Hum. Neurosci. 13. https://doi.org/10.3389/conf.fnhum.2019.01.00050

[52] Zhang F, Dou J, Zhao X, Luo H, Ma L, Wang L, Wang Y (2023) Identification of key genes associated with heat stress in rats by weighted gene co-expression network analysis. Animals 13(10):1618. https://doi.org/10.3390/ani13101618

[53] Zheng Z, Li Y, Jin G, Huang T, Zou M, Duan S. The biological role of arachidonic acid 12-lipoxygenase (ALOX12) in various human diseases. Biomed Pharmacother, 2020, 129: 110354

Funding

National Natural Science Foundation of China(no. 32002392)

Natural Science Research Project of Guizhou Provincial Department of Education(QJJ-2024-37)

Guizhou Provincial Basic Research Program (Natural Science)(No. MS[2025]697)

Guizhou Provincial Science and Technology Project(QKHJC[2024]youth105)

Cultivation Project of Guizhou University(No. [2023]15)

Open Project for Key Lab of Freshwater Biodiversity Conservation, Ministry of Agriculture of China(no: LFBC1109)

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