Search Results (495)

This study evaluated the effects of crayfish shell meal (CSM) processed by drying, enzymatic hydrolysis, or fermentation on juvenile largemouth bass (Micropterus salmoides). A total of 320 fish (initial body weight, approximately 5.69 g) were distributed into 16 tanks, with 20 fish per tank and four replicates per treatment. Four isonitrogenous and isoenergetic diets were prepared: a fish meal-based control diet (FM) and three diets containing 4.5% dried CSM (DCSM), enzymatically hydrolyzed CSM (ECSM), or fermented CSM (FCSM), in which fish meal was reduced from 45.0% to 39.6%, and were fed for 8 weeks. Compared with the FM group, DCSM significantly reduced final body weight, weight gain rate, specific growth rate, and protein efficiency ratio, and increased feed conversion ratio (p < 0.05). ECSM generally showed intermediate responses, whereas FCSM did not differ significantly from FM in these growth and feed utilization indices (p > 0.05). DCSM also significantly increased serum aspartate aminotransferase and blood urea nitrogen levels and induced a pro-inflammatory intestinal response, as indicated by decreased il10 and increased il1b and tnfa expression (p < 0.05). Compared with DCSM, FCSM significantly improved these serum and inflammatory indices, increased intestinal trypsin, Na⁺/K⁺-ATPase and γ-glutamyl transferase activities (p < 0.05), and showed more favorable antioxidant and gut microbiota responses. Overall, fermentation was more effective than enzymatic hydrolysis in improving the feeding value of CSM for largemouth bass. These results suggest that FCSM is a promising alternative ingredient for aquafeeds and may contribute to the high-value utilization of crayfish processing by-products. Full article

Nitrite is a key environmental challenge in intensive shrimp aquaculture, adversely affecting physiological regulation and survival. Although tolerant Penaeus vannamei families have been established by selective breeding, the basis of family-level variation in tolerance has yet to be clarified. In this study, nitrite-tolerant and nitrite-sensitive families were compared using survival analysis, transcriptomics, targeted qPCR validation, physiological assays, and RNA interference of representative transport-related genes. Under nitrite exposure, the tolerant family exhibited significantly higher survival and a distinct gill transcriptional response, characterized by stronger induction of acid–base and ion-transport genes, including carbonic anhydrase 2 (CA2), the Na⁺/K⁺-ATPase subunits ATP1A and ATP1B, as well as several V-type H⁺-ATPase-related genes. These transcriptional changes were accompanied by elevated ATP content and Na⁺/K⁺-ATPase activity, improved hemolymph pH stability, and reduced nitrite accumulation in both gill and hemolymph. RNAi-mediated knockdown of CA2 or ATP1B attenuated the nitrite-induced transport response, decreased ATP content and NKA activity, exacerbated hemolymph acidification, promoted internal nitrite accumulation, and ultimately reduced shrimp survival under nitrite stress. Family-based validation further showed that the tolerant family displayed higher survival than the sensitive family in the dsEGFP group, whereas this advantage was markedly reduced after CA2 or ATP1B knockdown under nitrite stress. These findings highlight that strengthened branchial ion transport and acid–base regulation represent key physiological mechanisms underlying nitrite tolerance in resistant shrimp families. Full article

Although the effectiveness of plant-derived essential oils (EOs) against several insect pests is well-documented, their high volatility presents a challenge. In this study, the potential to enhance the insecticidal activity of Satureja hortensis L. EO, an accessible natural agent, through nanoemulsification was assessed against the cosmopolitan pest Spodoptera frugiperda (J. E. Smith, 1797). The nanoemulsion of the EO (NEEO) was prepared using Tween 80 as the emulsifying agent and high-intensity ultrasonication. Oral bioassays indicated that the NEEO was more toxic (LC₅₀ = 0.922%) than the pure EO (LC₅₀ = 1.186%). Sublethal exposure to LC₃₀ of the NEEO caused evident reductions in preadult survival, developmental time, fecundity, and oviposition period, as well as the population growth parameter net reproductive rate (R₀). The exposure to the NEEO increased catalase (CAT), glutathione S-transferase (GST), and superoxide dismutase (SOD) actions and inhibited α-esterase (α-NE), β-esterase (β-NE), and cytochrome P450 (CYP450) actions. Both the NEEO and EO inhibited acetylcholinesterase (AChE) and Na⁺/K⁺-ATPase, with higher inhibition in the NEEO group. Generally, S. hortensis NEEO enhanced toxicity, intensified physiological perturbations, and caused greater negative impacts on population growth parameters. Consequently, nanoemulsification of S. hortensis EO can be considered an effective method to strengthen the insecticidal potential of this natural agent. Full article

Tumour necrosis factor-alpha (TNF-α) disrupts bioenergetic homeostasis in skeletal muscle cells through the suppression of ion-dependent ATPase activities, mitochondrial depolarisation, and impairment of antioxidant defences. Carvacrol, a phenolic monoterpenoid constituent of thyme and oregano essential oil, has been shown to exert cytoprotective effects in TNF-α-challenged L6 rat myoblasts. The mechanistic basis of these effects, specifically the relationship between membrane-associated ATPase function, mitochondrial polarisation status, and transcriptional regulation of metabolic stress-response genes, has not been formally characterised. L6 rat myoblasts were exposed to TNF-α (10 ng/mL, 1 h), then treated with carvacrol (6.25 µg/mL, 24 h) in a post-inflammatory rescue paradigm. Cell viability (MTT), membrane integrity (LDH), ion-dependent ATPase activities (Na⁺/K⁺, Ca²⁺, Mg²⁺), antioxidant enzyme activities (catalase, SOD), mitochondrial membrane potential (Muse™ MitoPotential flow cytometry), and SIRT1/AMPK mRNA expression were quantified. TNF-α significantly suppressed Na⁺/K⁺, Ca²⁺, and Mg²⁺-dependent ATPase activities (all p < 0.001), consistent with impaired membrane-associated bioenergetic function. Post-TNF-α carvacrol treatment partially restored all three ATPase activities (p < 0.05) and reduced the proportion of mitochondrially depolarised cells from 31.65 ± 4.25% to 19.0 ± 2.6% (p < 0.05). LDH release, catalase activity, and SOD activity were also significantly modulated. At the transcriptional level, carvacrol increased SIRT1 mRNA by 1.6-fold and AMPK mRNA by 2.0-fold relative to TNF-α-treated cells. An integrative bioenergetic model is proposed in which carvacrol’s membrane-intercalating properties restore the phospholipid environment required for ATPase conformational cycling, attenuating the Ca²⁺ overload that drives mitochondrial permeability transition, and thereby partially preserving Δψm. Transcriptional upregulation of SIRT1 and AMPKα may represent an adaptive response to residual energetic stress. The mechanistic relationships among these endpoints and the causal contribution of SIRT1 and AMPK to observed bioenergetic changes require protein-level and pathway-specific experimental validation. Full article

Saline-alkaline water is increasingly used for aquaculture. Qihe crucian carp (Carassius carassius) is one of the major economic aquacultural species in China; however, the impact of saline-alkaline water on this fish remains unclear. In this study, a 60 d chronic alkalinity stress experiment was conducted to investigate the effects on the growth, physiology, and muscle quality of Qihe crucian carp. One hundred and eighty juvenile fish (31.03 ± 0.71 g) were divided into three treatments (three replicate tanks per treatment, 20 fish per tank): a control group (freshwater), a CA20 group (20 mmol/L), and a CA40 group (40 mmol/L). Despite no effect on survival, alkalinity stress significantly impaired growth performance in both the CA20 and CA40 groups, leading to reductions in final body weight, weight gain rate, and specific growth rate. Similarly, the feed efficiency decreased with increasing alkalinity. Compared to the control group, the alkalinity groups exhibited elevated serum Na⁺ levels and higher gill Na⁺/K⁺-ATPase activity, but reduced Ca²⁺/Mg²⁺-ATPase activity. For histology, chronic alkalinity stress induced several changes in the gills, including wider filaments, shortened lamellae, and the increase in interlamellar distance. Regarding muscle nutritional composition, the contents of crude protein and amino acids and the proportions of polyunsaturated fatty acids showed a downward trend with rising alkalinity. Additionally, alkalinity stress significantly decreased muscle fiber density and hardness. Taking into account both growth performance and muscle quality, the suggested aquacultural alkalinity should be controlled below 20 mmol/L for Qihe crucian carp. In conclusion, these findings supported Qihe crucian carp as a promising candidate species for saline-alkaline water aquaculture, providing a scientific basis for the utilization of this special water resource. Full article

Metabolic syndrome (MeS) is a multifactorial disorder characterized by insulin resistance, dyslipidemia, hypertension, and obesity, and oxidative stress plays a key role in tissue damage in this syndrome. This study aimed to investigate this role in Na⁺/K⁺-ATPase (NKA) expression in skeletal muscle and to evaluate the effects of quercetin. A high-sucrose-diet-induced MeS model was established in Wistar albino rats (n = 32), and skeletal muscle tissues were analyzed. Biochemical parameters were measured, including aspartate aminotransferase (AST), lactate dehydrogenase (LDH), total antioxidant status (TAS), total oxidant status (TOS), superoxide dismutase (SOD), and malondialdehyde (MDA). In addition, thioredoxin-1 (TRX1) and NKA protein expression levels were evaluated using Western blot analysis. In the MeS group, AST, TAS, TRX1, and NKA expression significantly decreased, while LDH, TOS, SOD, and MDA levels increased, indicating disrupted redox balance, elevated oxidative stress, and impaired antioxidant defense. Increased MDA and TOS levels reflected enhanced lipid peroxidation, whereas decreased TAS and TRX1 suggested reduced antioxidant capacity. Elevated SOD activity may indicate a compensatory response to excessive reactive oxygen species (ROS). The reduction in NKA expression may contribute to impaired ion transport and potential skeletal muscle dysfunction. Quercetin administration improved oxidative stress markers and partially restored NKA expression. These findings suggest that oxidative stress contributes to NKA dysfunction in MeS, and quercetin may have therapeutic potential by modulating oxidative stress and preserving enzyme function. Full article

Pomegranate peel is a food industry waste rich in polyphenols. To date, its effect in alleviating fatigue remains unclear. This study aimed to characterize the chemical composition of pomegranate peel polyphenols (PPPs), evaluate its antioxidant and anti-fatigue capacities, and investigate the underlying mechanism. In the current study, twenty main compounds, primarily flavonoids, phenolic acids, and anthocyanins, were identified from PPPs using LC-MS/MS. In H₂O₂-induced HepG2 cells, PPPs promoted cellular repair and reduced the production of intracellular malondialdehyde (MDA) and reactive oxygen species (ROS) via enhancing the activity of antioxidant enzymes (SOD, CAT, and GSH-Px). In the endurance swimming-induced fatigue mice model, PPPs prolonged mice exhaustion times, reduced accumulation of fatigue-related metabolites (BUN, LA, BA, LDH and CK), and alleviated liver and muscle tissue damage. Mechanistically, PPPs mitigated oxidative stress via activation of the Keap1/Nrf2 pathway, leading to increased expression of hemeoxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1). Furthermore, PPPs stimulated energy metabolism by activating the AMPK/PGC-1α/PPAR-α pathway, promoting mitochondrial biogenesis, enhancing glycogen storage, increasing ATPase activity (Na⁺-K⁺-ATPase, Ca²⁺-Mg²⁺-ATPase, and T-ATPase) and accelerating lipid β-oxidation. These findings suggest that PPPs is a promising anti-fatigue supplement and could be further utilized in the nutritional industry. Full article

Metabolic dysfunction, a hallmark of diet-induced obesity (DIO), is increasingly attributed to alterations in intestinal nutrient and electrolyte transport. Yet the mechanisms that drive obesity-associated functional alterations of intestinal transporters remain incompletely understood. In this context, the effects of a high-fat diet (HFD) induced obesity on sodium-dependent glucose co-transporter 1 (SGLT1), Na⁺/H⁺ exchanger 3 (NHE3), and Cl⁻/HCO₃⁻ exchangers (DRA/PAT1), the primary glucose, sodium, and chloride absorptive pathways in mice small intestinal villus cells, were investigated. SGLT1 activity significantly increased in intact villus cells and brush border membrane vesicles (BBMV) from HFD-fed mice. Kinetic analysis demonstrated reduced Km without a change in Vmax, indicating enhanced transporter affinity. Notably, SGLT1 mRNA and protein expression, including BBM localization, were unchanged. Basolateral Na⁺/K⁺-ATPase activity was decreased, excluding enhanced Na⁺ gradient generation as the mechanism for SGLT1 stimulation. In contrast, DRA/PAT1 activity was significantly increased in HFD-fed mice, and kinetic studies revealed elevated Vmax without a change in Km, indicating increased transport capacity. DRA/PAT1 mRNA, total protein, and BBM expression were all significantly elevated. NHE3 activity and expression remained unchanged. These findings demonstrate that DIO enhances intestinal glucose absorption by increasing SGLT1 affinity and chloride absorption by upregulating DRA/PAT1 transcription. These transporter-specific alterations may amplify nutrient absorption and contribute to metabolic dysregulation in obesity. Full article

Microcystin-LR (MC-LR) is a potent hepatotoxin that has been shown to cause liver damage even at doses lower than the established Low Observable Adverse Effect Level (LOAEL) of 200 μg/kg in animal models. We have previously observed that low-dose exposure to MC-LR in animals with diet-induced Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) and subsequent treatment with antioxidants like N-acetylcysteine (NAC) and the Na⁺/K⁺ ATPase-Src kinase inhibitor pNaKtide significantly alleviated hepatic infiltration of immune cells, downregulated markers of inflammation and hepatotoxicity, increased the breakdown of the toxin molecule, and restored phase I and II drug metabolism pathways, including the glutathione pathway. Because the liver is composed of heterogeneous cell types, this study aimed to determine the specific role of hepatocytes in the uptake and metabolism of MC-LR, especially in the setting of MASLD. To address this, we used two well-established hepatocyte cell lines—AML-12 murine hepatocytes and human Hep3B hepatocytes. Preliminary dose comparison studies with AML-12 cells showed that MC-LR at 10 μM concentration showed a significant upregulation in the genetic expression of the markers of hepatotoxicity—OSMR (p ≤ 0.01) and SerpinE (p ≤ 0.0001)—in comparison to Vehicle. Treatment with pNaKtide (1 µM) and/or NAC (10 mM) in the presence of MC-LR significantly reduced the expression of both OSMR (p ≤ 0.0001) and SerpinE (p ≤ 0.01 and p ≤ 0.0001, respectively). To model steatotic hepatocytes characteristic of the MASLD phenotype, Hep3B hepatocytes were first treated with 500 µM of oleic acid (OA) before exposing them to the toxin in the presence and absence of antioxidants. MC-LR exposure, induced markers of inflammation and hepatotoxicity to be elevated significantly in the presence of OA as compared to MC-LR exposure alone. This elevation of the genetic markers of inflammation and hepatotoxicity was significantly attenuated on treatment with pNaKtide (1 µM) and NAC (10 mM). Quantification of human SERPINE1 (PAI1) and 8-OHdG, a stable marker of oxidative stress, in the spent media of Hep3B cells corroborated the trends observed in the genetic markers of hepatotoxicity. These observations support the central role that hepatocytes play in the uptake and metabolism of MC-LR, which is complicated by the presence of MASLD-like conditions and can help in the development of future therapeutic strategies. Full article

Background and Objectives: The objective of this study is to evaluate whether deferoxamine modulates cell biological properties, such as proliferation and wound closure of porcine corneal endothelial cells (CECs) in vitro, and whether the treatment of CECs with deferoxamine results in an enhanced expression of vascular endothelial growth factor (VEGF). Materials and Methods: Corneal endothelial cells were extracted from porcine globes within 24 h postmortem. Immunohistochemistry for the endothelial Na⁺/K⁺-ATPase was performed to confirm the cells’ endothelial origin. To assess CEC viability and proliferation, a water-soluble tetrazolium salt (WST-1) and 5-bromo-2′-deoxyuridine (BrdU) assay were performed. Corneal endothelial wound closure was evaluated using a wound closure assay. VEGF mRNA expression was evaluated using real-time polymerase chain reaction (rt-PCR). Results: The extracted corneal endothelial cells showed a typical hexagonal morphology with Na⁺/K⁺-ATPase staining of the cell membrane. The treatment with 200 µM deferoxamine significantly increased CEC viability to 121 ± 24% compared to the control group (p = 0.0024). Corneal endothelial cell proliferation did not show any significant changes under the treatment with deferoxamine (p > 0.05). Both 100 µM and 200 µM deferoxamine led to a significantly smaller remaining wound area of 82.4 ± 6.7% and 78.7 ± 6.2% (p < 0.0001) in comparison to the control group after 24 h of treatment in the wound closure assay. Treatment with 200 µM deferoxamine significantly induced VEGF mRNA expression to 1.67- ± 0.57-fold from 1.00- ± 0.03-fold in the control group (p = 0.0006). Conclusions: Deferoxamine effectively enhances corneal endothelial cell viability and wound healing associated with an overexpression of VEGF. Thus, deferoxamine is a potent modulator of cell biological properties of corneal endothelial cells and maintains their integrity in vitro. Full article

This study explored the physiological responses and gene expression profiles of the Manila clam (Ruditapes philippinarum) and the hard clam (Mercenaria mercenaria) under heat and hyposaline stress. Experimental conditions involved increasing the temperature from 25 °C to 35 °C and decreasing salinity from 25 ppt to 15 ppt over a 6 h acclimation period, followed by 72 h exposure. Key physiological and immune indicators, including filtration rate, oxygen consumption rate, ammonia excretion rate, and the expression of related genes, were measured. Under heat stress, R. philippinarum exhibited higher filtration, oxygen consumption, and ammonia excretion rates than M. mercenaria at most sampling time points. The expression of fatty acid desaturase (Δ6FAD) and heat shock protein (HSP70) genes increased and then decreased for both species, whereas superoxide dismutase (Cu/Zn SOD) gene expression gradually decreased over time. Furthermore, the expression levels of all three genes were generally significantly higher in M. mercenaria compared to R. philippinarum. Under hyposaline stress, R. philippinarum exhibited significantly higher filtration, oxygen consumption, and ammonia excretion rates than M. mercenaria between 24 h and 72 h. Expression levels of the Na⁺-K⁺-ATPase (NKAα), HSP70, and Cu/Zn SOD genes remained higher in M. mercenaria compared to R. philippinarum. Overall, the present study indicates that M. mercenaria maintains relative stability and R. philippinarum exhibits greater physiological fluctuation under both heat and hyposaline stress. This study highlights bivalve species-specific responses to environmental stressors and provides valuable insights for aquaculture planning and ecological management in different environmental regions, particularly in the context of global climate change. Full article

Na⁺,K⁺-ATPase possesses a highly conserved glycine (G358 in the α3 isoform) that—together with a nearby isoleucine (I363 in α3)—is targeted by mutations causing some of the most severe neurological phenotypes of the clinical spectrum of α3-Na⁺,K⁺-ATPase mutations. The disease mutations α3-G358V and α3-I363N affect Na⁺ and K⁺ transport to an extent incompatible with cell growth. However, alanine replacement of the corresponding glycine G363 in the α1 isoform is compatible with cell growth, allowing the effects on Na⁺,K⁺-ATPase function to be addressed using enzymatic assays on plasma membranes isolated from transfected cells. Occlusion of Na⁺ appears to be defective in mutant G363A, resulting in a reduced rate of phosphorylation from ATP. Furthermore, the mutation displaces the major conformational equilibrium of Na⁺,K⁺-ATPase such that the K⁺-occluded state is destabilized and occluded K⁺ is released faster, thereby leading to accumulation of a non-productive state without bound Na⁺ or K⁺. The critical function of the glycine can be ascribed to a strategic location at the bending point between an α helix and a β strand, where it connects the catalytic ATP hydrolysis site in the cytoplasmic P domain with the ion-binding region in the membrane and coordinates important intramolecular domain movements during the Na⁺,K⁺-ATPase transport cycle. Full article

Myo-inositol (MI) is recognized as a potential stress regulator capable of alleviating abiotic stress. The objective of this study is to analyze the role of MI in the salt stress response of Daucus carota L. and its potential mechanisms. “Hongxin Qicun” carrot seedlings were subjected to five treatments: control; salt stress (50 mM NaCl); and salt stress combined with 50, 100, or 200 μM of MI. Through an integrated approach combining physiological assays, non-invasive micro-test technology (NMT), and gene expression profiling, we found that salt stress severely inhibited seedling growth, disrupted K⁺/Na⁺ homeostasis, and triggered excessive H₂O₂ accumulation. Exogenous MI application mitigated these salt-induced damages, with 100 μM MI exerting the optimal effect. MI enhanced Na⁺ efflux and reduced K⁺ efflux in carrot roots under salt stress. Inhibitor experiments indicated that MI-promoted Na⁺ efflux relies on active transport via the plasma membrane (PM) Na⁺/H⁺ antiporter system, and qRT-PCR analysis showed that this response was accompanied by the upregulation of DcSOS1. Furthermore, MI contributes to K⁺ homeostasis by synergistically modulating PM H⁺-ATPase and high-affinity potassium transporters. The established proton gradient helps reduce salt-induced K⁺ loss through depolarization-activated potassium channels and non-selective cation channels. MI treatment decreased electrolyte leakage, malondialdehyde content, and H₂O₂ accumulation by enhancing the activities of the plant antioxidant defense system. Meanwhile, MI upregulated the expression of myo-inositol oxygenase (DcMIOXs) genes, which may contribute to osmotic balance maintenance and facilitate ROS scavenging. In conclusion, exogenous MI alleviates salt-induced physiological disorders in Daucus carota L. by coordinately regulating K⁺/Na⁺ and ROS homeostasis, with 100 μM identified as the optimal concentration for this effect. Full article

Staphylococcus aureus is a major cause of skin and soft tissue infections, necessitating the development of new topical agents with rapid bactericidal activity and low resistance potential. Here, we evaluated the antibacterial activity of a pyrazolone copper complex (P-FAH-Cu-phen) against S. aureus, investigated its in vitro mode of action, and its assessed therapeutic efficacy in a murine model of S. aureus-infected skin trauma. P-FAH-Cu-phen exhibited potent bactericidal activity (minimum inhibitory concentration [MIC] 1.4 μg/mL; minimum bactericidal concentration [MBC] 2.8 μg/mL) and rapid killing (>91% eradication within 2.5 min), with no detectable MIC increase under the tested serial passaging conditions. Cell-envelope dysfunction was evidenced by increased supernatant alkaline phosphatase activity, elevated leakage of nucleic acids and proteins, and reduced membrane-associated Na⁺/K⁺- and Ca²⁺/Mg²⁺-ATPase activities. At sub-inhibitory concentrations, P-FAH-Cu-phen reduced haemolytic and coagulase activities, modulated virulence gene expression (sea, hla, agrA), and inhibited biofilm formation and biofilm-associated metabolic activity. In vivo, topical treatment accelerated wound closure and histopathological repair, increased hydroxyproline content, reduced bacterial burden, and lowered TNF-α and IL-10 levels in wound tissues. Collectively, P-FAH-Cu-phen shows multi-faceted anti-infective activity and exhibits further development as a topical candidate for S. aureus-infected skin wounds. Full article

Na,K-ATPase (NKA) is expressed as two isoforms in mouse sperm: the testis-specific Na,K-ATPase α4 (NKAα4) and the somatic Na,K-ATPase α1 (NKAα1). Currently, the role that NKAα4 and NKAα1 play in the sperm acrosome reaction (AR) is unknown, and is the subject of our investigation. We studied the following: (1) the differential sensitivity of NKAα4 and NKAα1 to inhibition by ouabain; (2) the effects of deleting NKAα4 in mice (using NKAα4-KO mouse). In sperm from wild type (WT) mice, inhibiting NKAα4 with a low concentration of ouabain reduced AR. Inhibiting NKAα1 with a higher ouabain concentration further reduced AR, indicating that both NKA isoforms are necessary for AR. Surprisingly, sperm from NKAα4-KO mice exhibited an abnormally high AR. This was not due to a lack of acrosome development during sperm differentiation, but rather from premature release of the acrosome after they were isolated from the epididymis. When WT and NKAα4-KO sperm were exposed to media with or without Na⁺, K⁺, and Ca²⁺, or with the ionophores nigericin, valinomycin, and A23187, they displayed abnormal AR, indicating that NKAα4-KO sperm are unable to control intracellular Na⁺, K⁺, and Ca²⁺ levels. NKAα4 directly maintains intracellular Na⁺ and K⁺ and indirectly influences Ca²⁺ levels. This provides the necessary ion environment for full development of sperm AR. Along with NKAα4, NKAα1 also contributes to sperm AR. Full article