Search Results (1,924)

Epilepsy is a chronic neurological disorder in which current antiseizure medications are limited by incomplete seizure control and variable therapeutic responses, highlighting the need to identify new bioactive candidates. Passiflora incarnata (P. incarnata) has demonstrated neuropharmacological and anticonvulsant properties. However, the individual phytochemicals and molecular mechanisms underlying these effects remain incompletely characterized. In this study, we integrated network pharmacology with cellular validation to identify epilepsy-relevant phytochemicals of P. incarnata and evaluate their neuroprotective effects. Eleven phytochemicals underwent target prediction and were compared with epilepsy-associated genes, yielding 147 common targets. Protein–protein interaction analysis identified 134 interacting proteins and 590 interactions, with GABA_A receptor subunits prominently represented among the top hub genes. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses further identified GABA-related functions and neuroactive ligand–receptor signaling. Integration of compound–target functional profiling with structural analysis prioritized the luteolin-derived flavonoid lineage, leading to the selection of orientin, a C-glycosylflavone constituent of P. incarnata, and luteolin, its structurally related core flavone scaffold, for experimental validation. In differentiated SH-SY5Y cells, pretreatment with orientin or luteolin attenuated L-glutamic acid-induced cellular injury in the presence of glycine, as evidenced by improved cell viability and reduced lactate dehydrogenase release. Both compounds also markedly reduced glutamate-induced intracellular Ca²⁺ accumulation. Notably, thapsigargin substantially reversed these Ca²⁺-lowering effects, suggesting a potential role for ER-associated Ca²⁺ regulatory mechanisms. The predicted GABAergic targets were not directly validated, and the cellular findings demonstrate neuroprotection against glutamate-induced excitotoxicity rather than direct GABA_A receptor modulation or antiseizure efficacy. Collectively, these findings demonstrate the utility of network pharmacology-guided prioritization for identifying bioactive constituents of P. incarnata and support orientin as a neuroprotective candidate for further mechanistic and in vivo investigation. Full article

GABAergic neurotransmission plays a central role in regulating the mammalian circadian clock. However, the precise contribution of GABA type A receptor (GABA_AR) subunits to timekeeping within the suprachiasmatic nucleus (SCN) remains incompletely defined. Here, we investigated the spatiotemporal expression patterns of four GABA_AR subunits (α1, α5, β3, and γ2) in the SCN of male and female C57BL/6 mice. Our aim was to determine whether these subunits exhibit circadian, regional, and sex-specific variation under entrained (LD12:12) and free-running (DD) conditions. Fluorescence immunohistochemistry revealed the expression of all four subunits in the SCN, with several displaying rhythmic profiles. The α1 subunit peaked during the day in both sexes and was preferentially expressed in the shell region. The α5 subunit also varied across time but lacked strong regional specificity. Temporal variation in β3 and γ2 expression was observed in males but not females, indicating potential sex-dependent regulation. Notably, the γ2 subunit showed a dynamic shift in regional distribution over the circadian cycle, with higher shell expression during the day and core enrichment at night. Under DD conditions, rhythmic expression of the α1 and γ2 subunits persisted, suggesting endogenous control independent of external light cues. However, overall amplitude and regional specificity were reduced in the absence of light. These findings demonstrate that GABA_AR subunit expression within the SCN is regulated by circadian phase, SCN subregion, and biological sex. This study provides new insight into the molecular architecture of the SCN and supports the hypothesis that subunit-specific GABAergic signalling contributes to the temporal organisation of the circadian clock. Further investigation into the functional significance of these subunit patterns will help clarify their roles in SCN synchrony, photic entrainment, and sex-specific circadian regulation. Full article

Background/Objectives: Universal surgical risk calculators are not validated for use and poorly predict postoperative morbidity and mortality for women undergoing gynaecological oncology surgery. This adversely affects communication of risk resulting in poorly informed decisions and missed opportunities for medical optimization to mitigate risk preoperatively. We present the development and external validation of our novel GO SOAR surgical risk calculator for use to preoperatively predict postoperative thirty-day surgical morbidity and mortality in relation to gynaecological oncology surgeries. Methods: New logistic regression models were developed using the GO SOAR1 training cohort (n = 1811) and externally validated in an independent prospective cohort (n = 416) for two outcomes: thirty-day postoperative mortality (alive versus dead) and thirty-day postoperative morbidity (any complication (Clavien–Dindo I–V) versus none). Performance of the GO SOAR models was compared against established all-purpose surgical risk calculators (SORT/POSSUM/P-POSSUM/NSQIP). Model discrimination was assessed with sensitivity calculated at a clinically significant prespecified specificity threshold of 90%. Results: For mortality, AUROC was 0.752 (95% CI 0.570–0.935) for GO SOAR full and 0.795 (95% CI 0.660–0.930) for GO SOAR condensed; corresponding sensitivities at 90% specificity were 57.1% and 42.9%. For morbidity, AUROC was 0.698 (95% CI 0.641–0.755) and 0.703 (95% CI 0.646–0.760) for the full and condensed models, respectively, with sensitivities of 30.4% and 32.1% at 90% specificity. Conclusions: The GO SOAR model using a data-driven, gynaecological-oncology-specific approach at 90% specificity, achieved the highest observed sensitivity among the evaluated calculators. Accurate surgical risk predictions are crucial for major oncological surgery, where complications can diminish quality of life and affect long-term cancer survival. A model such as the GO SOAR surgical risk calculator that uses readily available preoperative data, regardless of income setting, is essential in reducing global disparities in surgical outcomes. Full article

γ-Aminobutyric acid (GABA) is a signaling metabolite involved in plant stress responses; its role in cadmium (Cd) stress adaptation in oilseed crops remains poorly understood. Here, we investigated the effects of exogenous GABA on Cd stress responses in two Brassica species, oilseed rape (Brassica napus, Bn) and Indian mustard (Brassica juncea, Bj). GABA alleviated Cd toxicity, as indicated by improved chlorophyll retention and reduced Cd accumulation by 22–29% across the two species. Moreover, GABA altered Cd subcellular partitioning and was accompanied by elevations in ascorbate (ASA) and glutathione (GSH) contents, respectively, together with reduced superoxide anion radical (O₂⁻) and hydrogen peroxide (H₂O₂) accumulation. GABA also altered fatty acid compositions, suggesting changes in membrane lipid metabolism under Cd stress. Transcriptome analysis identified 2502 and 352 upregulated genes under GABA + Cd treatment (relative to the control) in Bn and Bj, respectively, of which 2245 and 219 were uniquely detected in the GABA + Cd treatment groups. Moreover, GABA supplementation was associated with transcriptional changes in genes related to redox metabolism and ABC transporter-associated processes. Collectively, these findings suggest that GABA supplementation was associated with improved Cd tolerance in Brassica species, accompanied by changes in Cd subcellular partitioning, non-enzymatic antioxidant metabolism, fatty acid profiles, and stress-responsive transcriptome reprogramming. Full article

Gamma-aminobutyric acid (GABA), a non-protein amino acid widely present in plant-based foods, has gained considerable attention for its diverse physiological effects and underlying mechanisms. As a functional dietary component, GABA has been reported to exhibit antihypertensive activity in preclinical and preliminary human studies, with proposed mechanisms including angiotensin-converting enzyme (ACE) inhibition and modulation of sympathetic outflow. In nematode models, GABA has been observed to alleviate aging-related oxidative stress by enhancing antioxidant enzymes (e.g., SOD, CAT) and reducing lipofuscin accumulation. It may also alleviate certain menopausal symptoms based on limited human trial data, potentially through regulation of autonomic nervous system balance and reduction in vasomotor disturbances. Its anxiolytic and sleep-promoting effects are linked to GABAergic neurotransmission via GABAA/B receptors, which suppresses hypothalamic–pituitary–adrenal (HPA) axis hyperactivity. GABA also shows anticancer potential in cell-based assays by disrupting tumor cell cycles and metastasis pathways, while its antidiabetic role in preclinical models involves pancreatic β-cell protection and glucose homeostasis regulation. Despite the wide range of proposed physiological benefits, safety assessments indicate that GABA is generally well-tolerated at typical dietary supplement doses (≤300 mg/day), with higher intakes (up to 3000 mg/day) permitted in some regulatory frameworks, supporting its use in functional foods. Future research should focus on optimizing GABA-enriched food production and elucidating dose-related mechanisms for personalized nutrition. Full article

Cancer progression and therapeutic resistance are shaped by reciprocal dynamic interactions between malignant cells and the metabolically altered tumor microenvironment (TME). Tumor-associated hypoxia, glucose and amino acid competition, extracellular acidity, lactate accumulation, adenosine, prostaglandin E2 (PGE2) and other metabolic signals can remodel immune cell function and determine whether inflammation is tumoricidal or tumor-supportive. Although B lymphocytes have traditionally been viewed as antibody-producing cells, tumor-infiltrating B (TIL-B) cells comprise functionally heterogeneous populations that can act as antigen-presenting cells, cytokine and chemokine producers, antibody-secreting cells, cytotoxic effectors, regulatory B cells (Bregs) and organizers of tertiary lymphoid structures (TLSs). Their functional state is strongly influenced by the metabolic and spatial context in which they reside. This review focuses on the intersection of B cell biology and tumor immunometabolism, emphasizing how hypoxia, lactate, nutrient limitation, adenosine, PGE2, kynurenine and B cell-derived γ-aminobutyric acid (GABA) may shape B cell states and their interactions with myeloid and lymphoid cells. We discuss how metabolically conditioned Bregs and immunoglobulin (Ig)A-skewed humoral responses can contribute to immune suppression, whereas metabolically competent antigen-presenting, IgG-biased and TLS-associated B cell responses may support effective anti-tumor immunity. Importantly, the effects are tumor type- and context-dependent: B cell/TLS signatures are associated with favorable outcomes in several breast, lung and other solid tumors, whereas B cell-centered immune landscapes can be suppressed or neutral in pancreatic cancer and IgA-dominated responses may be unfavorable in selected malignancies. We further examine how these states may influence sensitivity or resistance to immune checkpoint blockade, chemotherapy, radiotherapy and cellular therapies. Finally, we highlight B cell metabolic pathways as potential therapeutic entry points and identify priorities for spatial metabolomics and prospective interventional studies. Full article

Zeugodacus tau (Walker) is a polyphagous invasive tephritid capable of cross-family host shifts from ancestral Cucurbitaceae crops to phylogenetically divergent commercial fruits, causing severe horticultural economic losses. As an obligate fruit-boring pest, Z. tau largely relies on larval metabolic plasticity to achieve successful colonization of novel hosts during host shifts. However, few matched fruit–larva metabolomic studies integrate host chemistry to larval physiology, hindering sustainable pest control development. We performed untargeted LC–MS metabolomics on matched fruit and third-instar larval samples from eight hosts after multi-generational acclimation. Principal component analysis (PCA) split fruit and larval metabolomes into cucurbit and non-cucurbit clusters, indicating that larval metabolic profiles align with host fruit chemistry during host shifts. Cucurbit fruits contained uniformly high γ-aminobutyric acid (GABA), and their larvae exhibited enriched glutamate (Glu)-centred amino acid pathways. Non-cucurbit fruits showed heterogeneous chemical composition: larvae-fed banana, mango and pitaya all possessed high UDP-glucose (UDPG), whose levels correlated with fruit glucose-6-phosphate (G6P) enriched in banana and mango. Overall, larvae colonizing distinct non-cucurbit hosts deployed a suite of divergent carbohydrate metabolic modules to respond to variable fruit chemical microenvironments. Banana and pitaya larvae showed enhanced starch–sucrose turnover, whereas mango larvae featured prominent ascorbate and aldarate metabolism; both pathways are closely connected to carbohydrate homeostasis. Orange and guava larvae activated distinct gluconeogenic branches. Gluconeogenesis itself constitutes an important component of carbohydrate metabolism. This dual-metabolome profiling uncovered host-specific biomarkers and two divergent metabolic strategies underlying the host shift in Z. tau, characterizing host-linked metabolic variation after long-term laboratory acclimation, which delivers correlative biochemical clues for subsequent pest management research. Full article

GABA (γ-aminobutyric acid) is the most abundant inhibitory neurotransmitter in the central nervous system of insects. GABAergic signal transduction is involved in the control and modulation of various vital functions such as salivation, locomotion, vision and olfaction, circadian timekeeping and sleep, arousal as well as learning and memory. GABA exerts its effects by binding to specific receptors, which are divided into GABA_A and GABA_B receptors. GABA_A receptors are ligand-gated Cl⁻ channels and molecular targets for a variety of small-molecule insecticides (polychlorocycloalkanes such as dieldrin, phenylpyrazoles such as fipronil, isoxazolines, and meta-diamides) that are widely used in agriculture. GABA_B receptors are seven-transmembrane G-protein-coupled receptors. GABA_B receptors function as obligate heteromers consisting of the two subunits, GABA_B-R1 and GABA_B-R2, whereby GABA_B-R1 binds the ligand and GABA_B-R2 is coupled to the G protein. Usually, adenylyl cyclase activity is inhibited via the G_αi subunits of heterotrimeric G proteins; this leads to a reduction in the intracellular cAMP level. This review summarizes the current knowledge on the molecular and pharmacological properties of insect GABA receptors. Finally, two established model organisms for studying the effects of GABA in insects are presented as examples. The American cockroach (Periplaneta americana) has a long tradition as an object to study GABAergic circuits in the olfactory pathway and the role of GABA in the control of salivary secretion. The fruit fly (Drosophila melanogaster) has proven to be an unsurpassed model system to study the effects of GABA and specific GABA receptors on a variety of physiological functions and behavioral processes. In particular, D. melanogaster is used as a disease model for several human diseases in which GABAergic signaling is involved. Full article

Glial mechanisms regulate neuronal excitability through multiple mechanisms, including the control of extracellular inhibitory signaling. One such mechanism is the Glu/GABA exchange process, in which glutamate uptake is coupled to GABA release, with GABA being synthesized from the polyamine putrescine, placing putrescine at a central position in metabolic pathways that may influence epileptiform activity. Here, we examined how pharmacological manipulation of key enzymes of putrescine metabolism affects seizure-like activity in the low-[Mg²⁺] in vitro model of frontotemporal epilepsy, complemented by in vivo recordings in the non-convulsive absence epilepsy model Wistar Albino Glaxo/Rijswijk (WAG/Rij) rats. Increasing putrescine availability by inhibiting spermidine synthase with trans-4-methylcyclohexylamine significantly reduced both the duration and appearance of seizure-like events. Strikingly, simultaneous inhibition of monoamine oxidase B (MAO-B) and diamine oxidase (DAO) with deprenyl and aminoguanidine almost completely abolished seizure-like events in vitro and markedly suppressed spike–wave discharges in WAG/Rij rats. This effect was largely reversed by blockade of GAT-2/3 transporters with SNAP-5114, suggesting the presence and significant anti-epileptic potential of a MAO-B- and DAO-independent putrescine–GABA synthesis pathway. Together, these findings indicate that the anticonvulsant effects of putrescine metabolism arise from the coordinated engagement of multiple parallel pathways rather than from a single dominant enzymatic route. Putrescine thus appears to function as a metabolic hub whose increased availability can be channeled into several anticonvulsant processes, suggesting that therapeutic strategies enhancing putrescine-dependent inhibitory pathways at the network level may offer promising avenues for the modulation of epileptiform activity. Full article

Soft fruits are highly perishable due to their delicate surface barriers, rapid cell-wall disassembly, turgor loss, oxidative imbalance, and tightly coordinated ripening and senescence processes that collectively accelerate water loss, softening, and decay after harvest. This review examines how melatonin (MLT) interacts with these deterioration processes in blueberries, raspberries, strawberries, grapes, and sweet cherries. Current evidence identifies redox regulation as the most consistent mechanism of MLT action, including modulation of reactive oxygen species, enhancement of antioxidant enzymes and the ascorbate–glutathione cycle, and maintenance of phenolic and other non-enzymatic antioxidants. These responses are linked with lower lipid peroxidation, reduced membrane leakage, delayed cell-wall degradation, and improved firmness, water retention, and decay resistance. More limited and species-specific evidence suggests that MLT may influence cuticular wax metabolism, endogenous MLT biosynthesis, H₂S signaling, GABA and polyamine metabolism, phenylpropanoid pathways, and host-defense responses. However, direct evidence for effects on cuticular permeability, cellular compartmentation, and hormonal signaling remains limited. Overall, MLT should be regarded as a context-dependent regulator of redox homeostasis, tissue integrity, ripening, and defense rather than as a universal anti-senescence treatment, because its effects vary with species, cultivar, concentration, maturity stage, and storage conditions and still require validation under commercial conditions. Full article

Autism spectrum disorder (ASD) is a highly heterogeneous neurodevelopmental disorder characterized by core features of social communication deficits and high prevalence of gastrointestinal comorbidities. With its continuously rising global prevalence, current therapeutic modalities remain unable to target and ameliorate the core symptoms of ASD. The microbiota–gut–brain axis (MGBA), a critical pathway mediating crosstalk between the gut microbiota and the brain, has been extensively documented to be deeply involved in the pathological progression of ASD in recent years. However, prior studies have predominantly focused on the unidirectional regulation of the brain by gut microbiota, lacking an integrated account of the bidirectional regulation across immune, metabolic, and endocrine systems. Centered on the immune–metabolic–endocrine interactive network, this review systematically delineates the bidirectional regulatory mechanisms of the MGBA in ASD by integrating recent evidence from microbiota sequencing, animal models, and clinical intervention studies, with the aim of clarifying the bidirectional causal controversy between intestinal microecological disturbance and ASD behavioral abnormalities. This review proposes that in children with ASD, decreased abundance of beneficial intestinal bacteria and disrupted metabolic profiles of short-chain fatty acids synergistically impair intestinal barrier integrity, triggering peripheral chronic inflammation that further drives excessive microglial activation-mediated central neuroinflammation. Subsequently, disturbances in the homeostasis of multiple neurotransmitters including 5-hydroxytryptamine (5-HT), γ-aminobutyric acid (GABA), histamine, and dopamine occur via the vagus nerve and hypothalamic–pituitary–adrenal (HPA) axis, ultimately driving ASD behavioral abnormalities. Conversely, chronic stress and behavioral characteristics associated with ASD reshape the intestinal microecology through neuroendocrine pathways, forming a vicious cycle of “microbiota dysbiosis—immune inflammation—HPA axis hyperactivity—further intestinal microecological imbalance”. This review summarizes the therapeutic efficacy and translational bottlenecks of three types of microecological interventions: fecal microbiota transplantation (FMT), probiotics, and ketogenic diet, and analyzes the current limitations in the field, including pronounced population heterogeneity, unclear cross-talk mechanisms among multiple pathways, and the scarcity of large-sample clinical evidence. Collectively, this review preliminarily elucidates the complete multi-system interactive framework of MGBA regulation in ASD, providing theoretical support for mechanistic research and gut-targeted individualized interventions for ASD. Full article

Salt stress is frequently encountered by plants and is a major constraint on crop production. Wheat (Triticum aestivum L.) is a staple cereal crop that is important for human health and food security. However, the response mechanisms of roots from different wheat varieties to salt stress remain unclear. In this study, we analyzed the complex response mechanisms of the salt-tolerant variety Longjian 114 (LJ114) and the salt-sensitive variety Chinese Spring (CS) to salt stress using integrated transcriptomic and metabolomic analyses. The results showed that LJ114 maintained better growth and exhibited higher antioxidant enzyme activities and greater osmotic adjustment capacity under salt stress. Differentially expressed genes in LJ114 were predominantly enriched in flavonoid biosynthesis, alpha-linolenic acid metabolism, and zeatin biosynthesis under salt stress. Metabolomic profiling identified 1366 differential metabolites, with LJ114 specifically enriched in tyrosine metabolism, tryptophan metabolism, glycerophospholipid metabolism, and fatty acid metabolism under salt stress. Integrated analyses further highlighted ABC transporters and alanine, aspartate, and glutamate metabolism as hub pathways, with notably higher GAD expression and GABA accumulation under salt stress. These findings suggest new insights into the molecular mechanisms underlying wheat salt tolerance and propose potential candidate targets for enhancing crop salinity tolerance. Full article

Temperature stress is a major constraint on the productivity of microalgae used in aquaculture. γ-Aminobutyric acid (GABA) is well-established as a key player in the stress tolerance of higher plants, yet its role in microalgae remains largely unexplored. Here, we examined the effects of low (15 °C), optimal (25 °C), and high (35 °C) temperatures on the GABA shunt in Isochrysis zhanjiangensis during the initial and mid-exponential growth phases. The results demonstrated that temperature stress significantly inhibited cell growth and photosynthetic efficiency (assessed by Fv/Fm and Fv’/Fm’), with soluble protein decreasing and soluble sugar accumulating. During the initial exponential phase, both low and high temperature stress triggered marked GABA accumulation, accompanied by coordinated increases in glutamate decarboxylase (GAD) and diamine oxidase (DAO) activities. Interestingly, the transcript levels of IzGAD and IzDAO decreased under these conditions, suggesting that GABA accumulation at this stage is predominantly governed by post-translational activation rather than transcriptional upregulation. Upon entry into the mid-exponential phase, a distinct phase-dependent shift in GABA biosynthetic regulation emerged. Under low temperature stress, GAD activity and IzGAD expression were both suppressed, whereas DAO activity and IzDAO transcripts increased significantly, indicating the transition to DAO-mediated GABA production as the dominant route. Under high temperature stress, both GAD and DAO activities increased, yet their corresponding gene transcription remained repressed, revealing a persistent asynchrony between enzyme activities and gene expression across both phases. Meanwhile, the expression of catabolic genes (IzGABA-T, IzSSADH1, and IzSSADH2) was consistently downregulated, further facilitating the net accumulation of GABA. Promoter analysis revealed multiple stress- and hormone-responsive cis-elements in these genes, implying a complex regulatory network. Collectively, our findings uncover a growth-phase-dependent reconfiguration of GABA biosynthetic pathways in I. zhanjiangensis under temperature stress. These insights provide a mechanistic basis for strain-specific temperature management in aquaculture applications. Full article

Background: Chrysin (5-7-dihydroxyflavone) exerts anxiolytic-like effects via the GABA_A/benzodiazepine receptor complex after acute administration, similar to diazepam. Chronic diazepam treatment produces pharmacological tolerance, likely due to structural receptor modification. Whether chronic chrysin treatment also produces pharmacological tolerance remains unknown. This study evaluated GABA_A/benzodiazepine receptor-mediated behavioral responses after a pharmacological challenge with a sedative dose of diazepam (5 mg/kg) in rats chronically treated with chrysin or diazepam. Methods: Thirty-five male Wistar rats were divided into five groups: vehicle, chrysin (1, 2.5, or 5 mg/kg), and diazepam (2 mg/kg, pharmacological control). Treatments were injected intraperitoneally for 32 days. On day 28, rats were evaluated in the elevated plus maze and locomotor activity tests. On day 32, all groups received a pharmacological challenge with 5 mg/kg diazepam and were assessed via sedative scale and the rota-rod test. Results: Chronic Chrysin (5 mg/kg) but not diazepam, maintained anxiolytic-like effects. No doses of chrysin showed pharmacological cross-tolerance to 5 mg/kg of diazepam, which produced incoordination and sedation, similar to vehicle. Conclusions: Chronic treatment with chrysin (5 mg/kg) maintained their anxiolytic-like effects without pharmacological tolerance or cross-tolerance, unlike diazepam. This result supports the potential anxiolytic-like effects of chrysin in the long-term, as an alternative for anxiety disorders that require chronic treatment. This could contribute to the translational studies that could be applied to humans in the future. Full article

γ-aminobutyric acid (GABA), an inhibitory neurotransmitter, plays an important role in regulating stress responses through modulation of the hypothalamic–pituitary–adrenal axis, neuroendocrine activity, antioxidant defense, and immune function. Therefore, this study evaluated the effects of dietary rumen-protected γ-aminobutyric acid (RP-GABA) supplementation on alleviating long-distance transport-induced stress in beef cattle. Forty-eight healthy Simmental bulls were randomly assigned to four treatment groups: CON, G-L, G-M, and G-H, receiving RP-GABA at 0, 300, 500, and 700 mg/kg of concentrate dry matter, respectively. The experiment lasted 53 days, including a 21-day pre-transport feeding period, a 2-day transportation period, and a 30-day post-transport feeding period. Samples were collected on experimental Days 21, 23, and 54. The results showed that long-distance transport impaired antioxidant status, immune function, and stress hormone secretion in beef cattle. The responses to different dietary RP-GABA supplementation levels varied among serum parameters. Long-distance transport reduced triiodothyronine and thyroxine (T4) concentrations, while the G-L group alleviated these reductions and maintained higher levels than the CON group after transport (Day 23). The G-M group enhanced glutathione peroxidase (GSH-Px) activity after transport (Day 23) (p < 0.001) and promoted the elevation of interleukin (IL)-4 levels both before and after long-distance transport (Days 21 and 23). The G-H group significantly decreased serum cortisol concentration after transport (Day 23), while reducing epinephrine levels and increasing glutathione peroxidase activity (all p < 0.001). Moreover, the G-H group further enhanced the increases in IL-4 and total antioxidant capacity before and after transport (Days 21 and 23), and increased IL-6 concentrations before and after transport (Days 21 and 54). Regarding dose-specific effects, the G-L group showed limited effects on stress-related hormones, immune function, and antioxidant capacity, whereas the G-H group enhanced some antioxidant and immune-related responses but had higher pre-transport stress hormone concentrations. In contrast, the G-M group exhibited a relatively balanced response across stress hormone levels, antioxidant status, and immune regulation, characterized by higher T4 (p = 0.003), GSH-Px (p = 0.001), and IL-4 (p = 0.006) concentrations and lower alkaline phosphatase (p = 0.005) and tumor necrosis factor-alpha concentrations. Therefore, under the conditions of this study, dietary supplementation with 500 mg/kg RP-GABA in concentrate may represent a potential level for maintaining balanced physiological responses in beef cattle. Full article