Curcumin attenuates ochratoxin A and hypoxia co-induced liver injury in grass carp (Ctenopharyngodon idella) by dual targeting endoplasmic reticulum stress and apoptosis via reducing ROS content.

BACKGROUND: Ochratoxin A (OTA) is a toxin widely found in aquafeed ingredients, and hypoxia is a common problem in fish farming. In practice, aquatic animals tend to be more sensitive to hypoxia while feeds are contaminated with OTA, but no studies exist in this area. This research investigated the multiple biotoxicities of OTA and hypoxia combined on the liver of grass carp and explored the mitigating effect of curcumin (CUR). METHODS: A total of 720 healthy juvenile grass carp (11.06 ± 0.05 g) were selected and assigned randomly to 4 experimental groups: control group (without OTA and CUR), 1.2 mg/kg OTA group, 400 mg/kg CUR group, and 1.2 mg/kg OTA + 400 mg/kg CUR group with three replicates each for 60 d. Subsequently, 32 fish were selected, divided into normoxia (18 fish) and hypoxia (18 fish) groups, and subjected to hypoxia stress for 96 h. RESULTS: CUR can attenuate histopathological damage caused by coming to OTA and hypoxia by reducing vacuolation and nuclear excursion. The alleviation of this damage was associated with the attenuation of apoptosis in the mitochondrial pathway by decreasing the expression of the pro-apoptotic proteins Caspase 3, 8, 9, Bax, and Apaf1 while increasing the expression of the anti-apoptotic protein Bcl-2, and attenuation of endoplasmic reticulum stress (ERS) by reducing Grp78 expression and chop levels. This may be attributed to the fact that the addition of CUR increased the levels of catalase (CAT) and glutathione reductase (GSH), increased antioxidant capacity, and ensured the proper functioning of respiratory chain complexes I and II, which in turn reduced the high production of reactive oxygen species (ROS), thus alleviating apoptosis and ERS. CONCLUSIONS: In conclusion, our data demonstrate the effectiveness of CUR in attenuating liver injury caused by the combination of OTA and hypoxia. This study confirms the feasibility and efficacy of adding natural products to mitigate toxic damage to aquatic animals.

NAT10 promotes vascular remodelling via mRNA ac4C acetylation.

BACKGROUND AND AIMS: Vascular smooth muscle cell (VSMC) phenotype switching is a pathological hallmark in various cardiovascular diseases. N4-acetylcytidine (ac4C) catalyzed by N-acetyltransferase 10 (NAT10) is well conserved in the enzymatic modification of ribonucleic acid (RNA). NAT10-mediated ac4C acetylation is involved in various physiological and pathological processes, including cardiac remodelling. However, the biological functions and underlying regulatory mechanisms of mRNA ac4C modifications in vascular diseases remain elusive. METHODS: By combining in-vitro and in-vivo vascular injury models, NAT10 was identified as a crucial protein involved in the promotion of post-injury neointima formation, as well as VSMC phenotype switching. The potential mechanisms of NAT10 in the vascular neointima formation were clarified by RNA sequence (RNA-seq), acetylated mRNA immunoprecipitation sequence (acRIP-seq), and RNA binding protein immunoprecipitation sequence (RIP-seq). RESULTS: NAT10 and ac4C modifications were upregulated in injured human and rodent arteries. Deletion of NAT10 in VSMCs effectively reduced post-injury neointima formation and VSMC phenotype switching. Further RNA-seq, RIP-seq, and acRIP-seq revealed that NAT10, by its ac4C modification, directly interacts with genes, including integrin-ß1 (ITGB1) and collagen type I alpha 2 chain (Col1a2) mRNAs. Taking ITGB1 as one example, it showed that NAT10-mediated ac4C consequently increased ITGB1 mRNA stability and its downstream focal adhesion kinase (FAK) signaling, directly influencing the proliferation of VSMCs and vascular remodelling. The regulation of NAT10 on the VSMC phenotype is of translational significance because the administration of Remodelin, a NAT10 inhibitor, effectively prevents neointima formation by suppressing VSMC proliferation and downregulating ITGB1 expression and deactivating its FAK signaling. CONCLUSIONS: This study reveals that NAT10 promotes vascular remodelling via mRNA ac4C acetylation, which may be a promising therapeutic target against vascular remodelling.

Establishing the link between D-mannose and juvenile grass carp (Ctenopharyngodon idella): Improved growth and intestinal structure associated with endoplasmic reticulum stress, mitophagy, and apical junctional complexes.

D-mannose, essential for protein glycosylation, has been reported to have immunomodulatory effects and to maintain intestinal flora homeostasis. In addition to evaluating growth performance, we examined the impact of D-mannose on the structure of epithelial cells and apical junction complexes in the animal intestine. All 1800 grass carp (16.20 ± 0.01 g) were randomly divided into six treatments with six replicates of 50 fish each and fed with six different levels of D-mannose (0.52, 1.75, 3.02, 4.28, 5.50 and 6.78 g/kg diet) for 70 d. The study revealed that D-mannose increased feed intake (P < 0.001) but did not affect the percent weight gain (PWG), special growth rate, and feed conversion ratio (P > 0.05). D-mannose supplementation at 1.75 g/kg increased crude protein content in fish and lipid production value (P < 0.05). D-mannose supplementation at 4.28 g/kg increased intestinal length, intestinal weight and fold height of grass carp compared to the control group (P < 0.05). This improvement may be attributed to the phosphomannose isomerase (PMI)-mediated enhancement of glycolysis. This study found that D-mannose supplementation at 4.28 or 3.02 g/kg reduced serum diamine oxidase activity or D-lactate content (P < 0.05) and improved cellular and intercellular structures for the first time. The improvement of cellular redox homeostasis involves alleviating endoplasmic reticulum (ER) stress through the inositol-requiring enzyme 1 (IRE1), RNA-dependent protein kinase-like ER kinase (PERK), and activating transcription factor 6 (ATF6) signaling pathways. The alleviation of ER stress may be linked to the phosphomannomutase (PMM)-mediated enhancement of protein glycosylation. In addition, ubiquitin-dependent [PTEN-induced putative kinase 1 (PINK1)/Parkin] and ubiquitin-independent [BCL2-interacting protein 3-like (BNIP3L), BCL2-interacting protein 3 (BNIP3), and FUN14 domain containing 1 (FUNDC1)] mitophagy may play a role in maintaining cellular redox homeostasis. The enhancement of intercellular structures includes enhancing tight junction and adherent junction structures, which may be closely associated with the small Rho GTPase protein (RhoA)/the Rho-associated protein kinase (ROCK) signaling pathway. In conclusion, D-mannose improved intestinal cellular redox homeostasis associated with ER stress and mitophagy pathways, and enhanced intercellular structures related to tight junctions and adherent junctions. Furthermore, quadratic regression analysis of the PWG and intestinal reactive oxygen species content indicated that the optimal addition level of D-mannose for juvenile grass carp was 4.61 and 4.59 g/kg, respectively.

4-Octyl itaconate attenuates renal tubular injury in db/db mice by activating Nrf2 and promoting PGC-1α-mediated mitochondrial biogenesis.

Objectives: The aim of this study was to investigate the mechanism of itaconate's potential effect in diabetic kidney disease.Methods: Renal immune responsive gene 1 (IRG1) levels were measured in db/db mice and streptozotocin (STZ) + high-fat diet (HFD)-induced diabetic mice. Irg1 knockout mice were generated. db/db mice were treated with 4-octyl itaconate (4-OI, 50 mg/kg), a derivative of itaconate, for 4 weeks. Renal function and morphological changes were investigated. Ultrastructural alterations were determined by transmission electron microscopy.Results: Renal IRG1 levels were reduced in two diabetic models. STZ+HFD-treated Irg1 knockout mice exhibited aggravated renal tubular injury and worsened renal function. Treatment with 4-OI lowered urinary albumin-to-creatinine ratio and blood urea nitrogen levels, and restored renal histological changes in db/db mice. It improved mitochondrial damage, increased expressions of peroxisome-proliferator-activated receptor γ coactivator-1α (PGC-1α) and mitochondrial transcription factor A (TFAM) in the renal cortex of db/db mice. These were confirmed in vitro; 4-OI improved high glucose-induced abnormal mitochondrial morphology and TFAM expression in HK-2 cells, effects that were inhibited by PGC-1α silencing. Moreover, 4-OI reduced the number of apoptotic cells in the renal cortex of db/db mice. Further study showed that 4-OI increased renal Nrf2 expression and decreased oxidative stress levels in db/db mice. In HK-2 cells, 4-OI decreased high glucose-induced mitochondrial ROS production, which was reversed by Nrf2 silencing. Nrf2 depletion also inhibited 4-OI-mediated regulation of PGC-1α, TFAM, and mitochondrial apoptotic protein expressions.Conclusions: 4-OI attenuates renal tubular injury in db/db mice by activating Nrf2 and promoting PGC-1α-mediated mitochondrial biogenesis.

α-Ketoglutarate promotes cardiomyocyte proliferation and heart regeneration after myocardial infarction.

The neonatal mammalian heart can regenerate following injury through cardiomyocyte proliferation but loses this potential by postnatal day 7. Stimulating adult cardiomyocytes to reenter the cell cycle remains unclear. Here we show that cardiomyocyte proliferation depends on its metabolic state. Given the connection between the tricarboxylic acid cycle and cell proliferation, we analyzed these metabolites in mouse hearts from postnatal day 0.5 to day 7 and found that α-ketoglutarate ranked highest among the decreased metabolites. Injection of α-ketoglutarate extended the window of cardiomyocyte proliferation during heart development and promoted heart regeneration after myocardial infarction by inducing adult cardiomyocyte proliferation. This was confirmed in Ogdh-siRNA-treated mice with increased α-ketoglutarate levels. Mechanistically, α-ketoglutarate decreases H3K27me3 deposition at the promoters of cell cycle genes in cardiomyocytes. Thus, α-ketoglutarate promotes cardiomyocyte proliferation through JMJD3-dependent demethylation, offering a potential approach for treating myocardial infarction.

Methionine deficiency inhibited pyroptosis in primary hepatocytes of grass carp (Ctenopharyngodon idella): possibly via activating the ROS-AMPK-autophagy axis.

BACKGROUND: Methionine (Met) is the only sulfur-containing amino acid among animal essential amino acids, and methionine deficiency (MD) causes tissue damage and cell death in animals. The common modes of cell death include apoptosis, autophagy, pyroptosis, necroptosis. However, the studies about the major modes of cell death caused by MD have not been reported, which worth further study. METHODS: Primary hepatocytes from grass carp were isolated and treated with different doses of Met (0, 0.5, 1, 1.5, 2, 2.5 mmol/L) to examine the expression of apoptosis, pyroptosis, autophagy and necroptosis-related proteins. Based on this, we subsequently modeled pyroptosis using lipopolysaccharides and nigericin sodium salt, then autophagy inhibitors chloroquine (CQ), AMP-activated protein kinase (AMPK) inhibitors compound C (CC) and reactive oxygen species (ROS) scavengers N-acetyl-L-cysteine (NAC) were further used to examine the expression of proteins related to pyroptosis, autophagy and AMPK pathway in MD-treated cells respectively. RESULTS: MD up-regulated B-cell lymphoma protein 2 (Bax), microtubule-associated protein 1 light chain 3 II (LC3 II), and down-regulated the protein expression levels of B-cell lymphoma-2 (Bcl-2), sequestosome 1 (p62), cleaved-caspase-1, cleaved-interleukin (IL)-1ß, and receptor-interacting protein kinase (RIP) 1 in hepatocytes, while it did not significantly affect RIP3. In addition, MD significantly increased the protein expression of liver kinase B1 (LKB1), p-AMPK, and Unc-51-like kinase 1 (ULK1) without significant effect on p-target of rapamycin. Subsequently, the use of CQ increased the protein expression of NOD-like receptor thermal protein domain associated protein 3 (NLRP3), cleaved-caspase-1, and cleaved-IL-1ß inhibited by MD; the use of CC significantly decreased the protein expression of MD-induced LC3 II and increased the protein expression of MD-suppressed p62; then the use of NAC decreased the MD-induced p-AMPK protein expression. CONCLUSION: MD promoted autophagy and apoptosis, but inhibited pyroptosis and necroptosis. MD inhibited pyroptosis may be related regarding the promotion of autophagy. MD activated AMPK by inducing ROS production which in turn promoted autophagy. These results could provide partial theoretical basis for the possible mechanisms of Met in ensuring the normal structure and function of animal organs. Furthermore, ferroptosis is closely related to redox states, it is worth investigating whether MD affects ferroptosis in hepatocytes.

Dietary curcumin alleviates intestinal damage induced by ochratoxin A in juvenile grass carp (Ctenopharyngodon idella): Necroptosis and inflammatory responses.

Ochratoxin A (OTA) is one of the most common pollutants in aquatic feed. As a first line of defense, intestinal barriers could be utilized against OTA in order to prevent disorders. Natural product supplementation is one of the most popular strategies to alleviate toxicity induced by mycotoxins, but there is a lack of knowledge about how it functions in the teleost intestine. In this study, 720 juvenile grass carp of about 11 g were selected and four treatment groups (control group, OTA group, curcumin [Cur] group, and OTA + Cur group) were set up to conduct a 60-day growth test. After the test, the growth performance and intestinal health related indexes of grass carp were investigated. The addition of dietary Cur could have the following main results: (1) inhibit absorption and promote efflux transporters mRNA expression, reducing the residuals of OTA, (2) decrease oxidative stress by reducing oxidative damage and enhancing the expression of antioxidant enzymes, (3) promote mitochondrial fusion proteins to inhibit the expression of mitotic proteins and mitochondrial autophagy proteins and enhance mitochondrial function, (4) reduce necroptosis-related gene expression through inhibiting the tumor necrotic factor receptor-interacting protein kinase/mixed lineage kinase domain-like pathway, (5) reduce the expression of pro-inflammatory factors by inhibiting the Toll-like receptor 4/nuclear factor-κB signaling pathway to alleviate the intestinal inflammatory response. In summary, the results suggested that Cur could alleviate OTA-induced intestinal damage by enhancing antioxidant capacity and mitochondrial function as well as reducing necroptosis and inflammation in the grass carp intestine. This study provided a theoretical basis and production implications for dietary Cur that could improve growth performance and alleviate the intestinal damage induced by OTA in fish.

Myo-inositol: A potential game-changer in preventing gill cell death and alleviating "gill rot" in grass carp (Ctenopharyngodon idellus).

Increasing evidence shows the potential threat of gill rot in freshwater fish culture. F. columnare is wide-spread in aquatic environments, which can cause fish gill rot and result in high mortality and losses of fish. This study investigated the effects of myo-inositol (MI) on the proliferation, structural integrity, and different death modes of grass carp (Ctenopharyngodon idella) gill epithelial cells, as well as its possible mechanism. 30 mg/L MI up-regulated CCK8 OD value and the protein level of solute carrier family 5A 3 (SLC5A3), and down-regulated the reactive oxygen species (ROS) content in gill cells and lactate dehydrogenase (LDH) release in the culture medium (P < 0.05). MI up-regulated the protein level of Beclin1, the protein level and fluorescence expression of microtubule-associated protein light chain 3B (LC3B) and down-regulated the protein level of sequestosome-1 (SQSTM1, also called p62) (P < 0.05). MI down-regulated the protein levels of Cysteine aspartate protease-1 (caspase-1), Gasdermin E (GSDME) and Cleaved interleukin 1 beta (IL-1ß) (P < 0.05). MI up-regulated the protein level of caspase-8 (P < 0.05), but had no effect on apoptosis (P > 0.05). MI down-regulated the mRNA expressions and protein levels of tumor necrosis factor α (tnfα), TNF receptor 1 (tnfr1), receptor interacting protein 1 (ripk1), receptor interacting protein 3 (ripk3) and mixed lineage kinase domain-like protein (mlkl), and reduce the ratio of p-MLKL/MLKL (P < 0.05). The addition of MI or necrosulfonamide (NSA) alone, or the addition of MI after induction of necroptosis, significantly up-regulated the cell activity and the protein level of SLC5A3 in gill cells, and significantly reduced the LDH release in the culture medium and the intracellular ROS content, the number of necroptosis cells, the protein expression of TNFα, TNFR1 and RIPK1, and the ratio of p-RIPK3/RIPK3 and p-MLKL/MLKL (P < 0.05). It indicated MI induce autophagy may relate to Beclin1/LC3/p62 signaling pathway, inhibits pyroptosis may attribute to Caspase-1/GSDMD/IL-1ß signaling pathway, and inhibits necroptosis via MLKL signaling pathway. However, MI had no effect on apoptosis.

Dietary copper improves intestinal structural integrity in juvenile grass carp (Ctenopharyngodon idella) probably related to its increased intestinal antioxidant capacity and apical junction complex.

This research evaluated the effects of copper (Cu) on intestinal antioxidant capacity and apical junctional complex (AJC) in juvenile grass carp. A total of 1080 healthy juvenile grass carp (11.16 ± 0.01 g) were fed six diets including different dosages of Cu, namely 0, 2, 4, 6, 8 mg/kg (Cu citrate [CuCit] as Cu source) and 3 mg/kg (CuSO4·5H2O as Cu source). The trial lasted for 9 weeks. The findings revealed that dietary optimal Cu supplementation (2.2 to 4.1 mg/kg) promoted intestinal growth, including intestinal length, intestinal length index, intestinal weight, and intestinal somatic index (P < 0.05). Furthermore, optimal Cu boosted the intestinal mucosal barrier in juvenile grass carp. On the one hand, optimal Cu reduced diamine oxidase and D-lactate levels in serum (P < 0.05), reduced levels of the oxidative damage indicators malondialdehyde, reactive oxygen species (ROS), protein carbonyl, superoxide dismutase (P < 0.05), and catalase mRNA levels were elevated (P < 0.05), thus boosting intestinal antioxidant capacity, the binding protein Keap1a/1b/Nrf2 signaling pathway might be involved. Optimal Cu had no impact on glutathione peroxidase 1b (GPx1b) gene expression (P > 0.05). On the other hand, optimal Cu increased intestinal tight junction (TJ) proteins (except for claudin 15b) and adherens junction (AJ) proteins (E-cadherin, α-catenin, ß-catenin, nectin and afadin) mRNA levels (P < 0.05), which could be connected to the signaling pathway formed by the Ras homolog gene family, member A (RhoA), Rho-associated kinase (ROCK), and myosin light chain kinase (MLCK). Finally, based on serum indicator D-lactate and intestinal oxidative damage index (ROS), Cu requirement (CuCit as Cu source) for juvenile grass carp from initial weight to final weight (from 11 to 173 g) was determined to be 4.14 and 4.12 mg/kg diet, respectively. This work may provide a theoretical foundation for identifying putative Cu regulation pathways on fish intestinal health.

Transcriptome Analysis of Myocardial Ischemic-Hypoxic Injury in Rats and Hypoxic H9C2 Cells.

AIMS: This study aimed to address inconsistencies in results between the H9C2 myocardial hypoxia (MH) cell line and myocardial infarction (MI) rat models used in MI research. We identified differentially expressed genes (DEGs) and underlying molecular mechanisms using RNA sequencing technology. METHODS: RNA sequencing was used to analyse DEGs in MI rat tissues and H9C2 cells exposed to hypoxia for 24 h. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were used to identify key biological processes and pathways. Weighted correlation network analysis [weighted gene co-expression network analysis (WGCNA)] was used to construct gene co-expression networks, and hub genes were compared with published MI datasets [Gene Expression Omnibus (GEO)] for target identification. RESULTS: GO analysis revealed enrichment of immune inflammation and mitochondrial respiration processes among 5139 DEGs in MI tissues and 2531 in H9C2 cells. KEGG analysis identified 537 overlapping genes associated with metabolism and oxidative stress pathways. Cross-analyses using the published GSE35088 and GSE47495 datasets identified 40 and 16 overlapping genes, respectively, with nine genes overlapping across all datasets and our models. WGCNA identified a key module in the MI model enriched for mRNA processing and protein binding. GO analysis revealed enrichment of mRNA processing, protein binding and mitochondrial respiratory chain complex I assembly in MI and H9C2 MH models. Five relevant hub genes were identified via a cross-analysis between the 92 hub genes that showed a common expression trend in both models. CONCLUSIONS: This study reveals both shared and distinct transcriptomic responses in the MI and H9C2 models, highlighting the importance of model selection for studying myocardial ischaemia and hypoxia.

Exploring lipin1 as a promising therapeutic target for the treatment of Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a progressive and devastating muscle disease, resulting from the absence of dystrophin. This leads to cell membrane instability, susceptibility to contraction-induced muscle damage, subsequent muscle degeneration, and eventually disability and early death of patients. Currently, there is no cure for DMD. Our recent studies identified that lipin1 plays a critical role in maintaining myofiber stability and integrity. However, lipin1 gene expression levels are dramatically reduced in the skeletal muscles of DMD patients and mdx mice. METHODS: To identify whether increased lipin1 expression could prevent dystrophic pathology, we employed unique muscle-specific mdx:lipin1 transgenic (mdx:lipin1Tg/0) mice in which lipin1 was restored in the dystrophic muscle of mdx mice, intramuscular gene delivery, as well as cell culture system. RESULTS: We found that increased lipin1 expression suppressed muscle degeneration and inflammation, reduced fibrosis, strengthened membrane integrity, and resulted in improved muscle contractile and lengthening force, and muscle performance in mdx:lipin1Tg/0 compared to mdx mice. To confirm the role of lipin1 in dystrophic muscle, we then administered AAV1-lipin1 via intramuscular injection in mdx mice. Consistently, lipin1 restoration inhibited myofiber necroptosis and lessened muscle degeneration. Using a cell culture system, we further found that differentiated primary mdx myoblasts had elevated expression levels of necroptotic markers and medium creatine kinase (CK), which could be a result of sarcolemmal damage. Most importantly, increased lipin1 expression levels in differentiated myoblasts from mdx:lipin1Tg/0 mice substantially inhibited the elevation of necroptotic markers and medium CK levels. CONCLUSIONS: Overall, our data suggest that lipin1 is a promising therapeutic target for the treatment of dystrophic muscles.

Transient induction of actin cytoskeletal remodeling associated with dedifferentiation, proliferation, and redifferentiation stimulates cardiac regeneration.

The formation of new and functional cardiomyocytes requires a 3-step process: dedifferentiation, proliferation, and redifferentiation, but the critical genes required for efficient dedifferentiation, proliferation, and redifferentiation remain unknown. In our study, a circular trajectory using single-nucleus RNA sequencing of the pericentriolar material 1 positive (PCM1+) cardiomyocyte nuclei from hearts 1 and 3 days after surgery-induced myocardial infarction (MI) on postnatal Day 1 was reconstructed and demonstrated that actin remodeling contributed to the dedifferentiation, proliferation, and redifferentiation of cardiomyocytes after injury. We identified four top actin-remodeling regulators, namely Tmsb4x, Tmsb10, Dmd, and Ctnna3, which we collectively referred to as 2D2P. Transiently expressed changes of 2D2P, using a polycistronic non-integrating lentivirus driven by Tnnt2 (cardiac-specific troponin T) promoters (Tnnt2-2D2P-NIL), efficiently induced transiently proliferative activation and actin remodeling in postnatal Day 7 cardiomyocytes and adult hearts. Furthermore, the intramyocardial delivery of Tnnt2-2D2P-NIL resulted in a sustained improvement in cardiac function without ventricular dilatation, thickened septum, or fatal arrhythmia for at least 4 months. In conclusion, this study highlights the importance of actin remodeling in cardiac regeneration and provides a foundation for new gene-cocktail-therapy approaches to improve cardiac repair and treat heart failure using a novel transient and cardiomyocyte-specific viral construct.

Unveiling the emerging role of curcumin to alleviate ochratoxin A-induced muscle toxicity in grass carp (Ctenopharyngodon idella): in vitro and in vivo studies.

BACKGROUND: Ochratoxin A (OTA), a globally abundant and extremely hazardous pollutant, is a significant source of contamination in aquafeeds and is responsible for severe food pollution. The developmental toxicity of OTA and the potential relieving strategy of natural products remain unclear. This study screened the substance curcumin (Cur), which had the best effect in alleviating OTA inhibition of myoblast proliferation, from 96 natural products and investigated its effect and mechanism in reducing OTA myotoxicity in vivo and in vitro. METHODS: A total of 720 healthy juvenile grass carp, with an initial average body weight of 11.06 ± 0.05 g, were randomly assigned into 4 groups: the control group (without OTA and Cur), 1.2 mg/kg OTA group, 400 mg/kg Cur group, and 1.2 mg/kg OTA + 400 mg/kg Cur group. Each treatment consisted of 3 replicates (180 fish) for 60 d. RESULTS: Firstly, we cultured, purified, and identified myoblasts using the tissue block culture method. Through preliminary screening and re-screening of 96 substances, we examined cell proliferation-related indicators such as cell viability and ultimately found that Cur had the best effect. Secondly, Cur could alleviate OTA-inhibited myoblast differentiation and myofibrillar development-related proteins (MyoG and MYHC) in vivo and in vitro and improve the growth performance of grass carp. Then, Cur could also promote the expression of OTA-inhibited protein synthesis-related proteins (S6K1 and TOR), which was related to the activation of the AKT/TOR signaling pathway. Finally, Cur could downregulate the expression of OTA-enhanced protein degradation-related genes (murf1, foxo3a, and ub), which was related to the inhibition of the FoxO3a signaling pathway. CONCLUSIONS: In summary, our data demonstrated the effectiveness of Cur in alleviating OTA myotoxicity in vivo and in vitro. This study confirms the rapidity, feasibility, and effectiveness of establishing a natural product screening method targeting myoblasts to alleviate fungal toxin toxicity.

Aflatoxin B1 inhibited the development of primary myoblasts of grass carp (Ctenopharyngodon idella) by degrading extracellular matrix.

According to the International Agency for Research on Cancer (IARC), aflatoxin B1 (AFB1) has been recognized as a major contaminant in food and animal feed and which is a common mycotoxin with high toxicity. Previous research has found that AFB1 inhibited zebrafish muscle development. However, the potential mechanism of AFB1 on fish muscle development is unknown, so it is necessary to conduct further investigation. In the present research, the primary myoblast of grass carp was used as a model, we treated myoblasts with AFB1 for 24 h. Our results found that 5 µM AFB1 significantly inhibited cell proliferation and migration (P < 0.05), and 10 µM AFB1 promoted lactate dehydrogenase (LDH) release (P < 0.05). Reactive oxygen species (ROS), protein carbonyl (PC) and malondialdehyde (MDA) levels were increased in 15, 5 and 10 µM AFB1 (P < 0.05), respectively. Catalase (CAT), glutathione peroxidase (GPx) and total superoxide dismutase (T-SOD) activities were decreased in 10, 10 and 15 µM AFB1 (P < 0.05), respectively. Furthermore, 15 µM AFB1 induced oxidative damage by Nrf2 pathway, also induced apoptosis in primary myoblast of grass carp. Meanwhile, 15 µM AFB1 decreased MyoD gene and protein expression (P < 0.05). Importantly, 15 µM AFB1 decreased the protein expression of collagen Ⅰ and fibronectin (P < 0.05), and increased the protein levels of urokinase plasminogen activator (uPA), matrix metalloproteinase 9 (MMP-9), matrix metalloproteinase 2 (MMP-2), and p38 mitogen-activated protein kinase (p38MAPK) (P < 0.05). As a result, our findings suggested that AFB1 damaged the cell morphology, induced oxidative damage and apoptosis, degraded ECM components, in turn inhibiting myoblast development by activating the p38MAPK/urokinase-type plasminogen activator (uPA)/matrix metalloproteinase (MMPs)/extracellular matrix (ECM) signaling pathway.

Declined flesh quality resulting from niacin deficiency is associated with elevated glycolysis and impaired mitochondrial homeostasis in grass carp (Ctenopharyngodon idella).

Energy metabolism exerts profound impacts on flesh quality. Niacin can be transformed into nicotinamide adenine dinucleotide (NAD), which is indispensable to energy metabolism. To investigate whether niacin deficiency could affect energy metabolism and flesh quality, six diets with graded levels of 0.49, 9.30, 21.30, 33.30, 45.30 and 57.30 mg/kg niacin were fed to grass carp (Ctenopharyngodon idella) for 63 days. The results showed that niacin deficiency declined flesh quality by changing amino acid and fatty acid profiles, decreasing shear force, increasing cooking loss and accelerating pH decline. The accelerated pH decline might be associated with enhanced glycolysis as evident by increased hexokinase (HK), pyruvate kinase (PK) and lactic dehydrogenase (LDH) activities, and mitochondrial dysfunction as evident by destroyed mitochondrial morphology, impaired respiratory chain complex I and antioxidant ability. Based on PWG and cooking loss, the niacin requirements for sub-adult grass carp were 31.95 mg/kg and 29.66 mg/kg diet, respectively.

Emerging role of vitamin D3 in alleviating intestinal structure injury caused by Aeromonas hydrophila in grass carp (Ctenopharyngodon idella).

Bacterial pathogens destroy the structural integrity of functional organs in fish, leading to severe challenges in the aquaculture industry. Vitamin D3 (VD3) prevents bacterial infections and strengthens immune system function via vitamin D receptor (VDR). However, the correlation between VD3/VDR and the structural integrity of functional organs remains unclarified. This study aimed to investigate the influence of VD3 supplementation on histological characteristics, apoptosis, and tight junction characteristics in fish intestine during pathogen infection. A total of 540 healthy grass carp (257.24 ± 0.63 g) were fed different levels of VD3 (15.2, 364.3, 782.5, 1,167.9, 1,573.8, and 1,980.1 IU/kg) for 70 d. Subsequently, fish were challenged with Aeromonas hydrophila, a pathogen that causes intestinal inflammation. Our present study demonstrated that optimal supplementation with VD3 (1) alleviated intestinal structural damage, and inhibited oxidative damage by reducing levels of oxidative stress biomarkers; (2) attenuated excessive apoptosis-related death receptor and mitochondrial pathway processes in relation to p38 mitogen-activated protein kinase signaling (P < 0.05); (3) enhanced tight junction protein expression by inhibiting myosin light chain kinase signaling (P < 0.05); and (4) elevated VDR isoform expression in fish intestine (P < 0.05). Overall, the results demonstrated that VD3 alleviates oxidative injury, apoptosis, and the destruction of tight junction protein under pathogenic infection, thereby strengthening pathogen defenses in the intestine. This finding supports the rationale for VD3 intervention as an essential practice in sustainable aquaculture.

Novel Perspective on Mechanism in Muscle Growth Inhibited by Ochratoxin A Associated with Ferroptosis: Model of Juvenile Grass Carp (Ctenopharyngodon idella) In Vivo and In Vitro Trials.

Ochratoxin A (OTA) is a common mycotoxin in food and feed that seriously harms human and animal health. This study investigated the effect of OTA on the muscle growth of juvenile grass carp (Ctenopharyngodon idella) and its possible mechanism in vitro. Our results have the following innovative findings: (1) Dietary OTA increased the expression of increasing phase I metabolic enzymes and absorbing transporters while reducing the expression of efflux transporters, thereby increasing their residue in muscles; (2) OTA inhibited the expressions of cell cycle and myogenic regulatory factors (MyoD, MyoG, and MyHC) and induced ferroptosis by decreasing the mRNA and protein expressions of FTH, TFR1, GPX4, and Nrf2 both in vivo and in vitro; and (3) the addition of DFO improved OTA-induced ferroptosis of grass carp primary myoblasts and promoted cell proliferation, while the addition of AKT improved OTA-inhibited myoblast differentiation and fusion, thus inhibiting muscle growth. Overall, this study provides a potential research target to further mitigate the myotoxicity of OTA.

Resveratrol improves diabetic kidney disease by modulating the gut microbiota-short chain fatty acids axis in db/db mice.

Diabetic kidney disease is associated with the dysbiosis of the gut microbiota and its metabolites. db/db mice were fed chow diet with or without 0.4% resveratrol for 12 weeks, after which the gut microbiota, faecal short-chain fatty acids (SCFAs), and renal fibrosis were analysed. Resveratrol ameliorated the progression of diabetic kidney disease and alleviated tubulointerstitial fibrosis. Further studies showed that gut microbiota dysbiosis was modulated by resveratrol, characterised by the expansion of SCFAs-producing bacteria Faecalibaculum and Lactobacillus, which increased the concentrations of SCFAs (especially acetic acid) in the faeces. Moreover, microbiota transplantation experiments found that alteration of the gut microbiota contributed to the prevention of diabetic kidney disease. Acetate treatment ameliorated proteinuria, glomerulosclerosis and tubulointerstitial fibrosis in db/db mice. Overall, resveratrol improved the progression of diabetic kidney disease by suppressing tubulointerstitial fibrosis, which may be involved, at least in part, in the regulation of the gut microbiota-SCFAs axis.

Dual-Functional Injectable Hydrogel for Osteoarthritis Treatments.

Osteoarthritis (OA) is a prevalent, chronic degenerative disease that affects people worldwide. It is characterized by the destruction of cartilage and inflammatory reactions. High levels of reactive oxygen species (ROS) cause oxidative stress, which damages lipids, proteins, and DNA, leading to cell damage and death. Furthermore, ROS also induces the production of inflammatory cytokines and cell chemotaxis, further worsening the inflammatory response and damaging cartilage resulted in limited movement. Herein, this work reports a dual-functional injectable hydrogel, which can help inhibit inflammation by scavenging ROS and provide lubrication to reduce wear and tear on the joints. To create the hydrogel, 3-aminophenylboronic acid modified hyaluronic acid is synthesized, then which is crosslinked with hydroxyl-containing polyvinyl alcohol (PVA) to construct a dual dynamic covalent crosslinked hydrogel oHA-PBA-PVA gel, Gel (HPP). The hydrogel mentioned here possesses a unique bond structure that allows it to be injected, self-heal, and provide lubrication. This innovative approach offers a new possibility for treating osteoarthritis by combining anti-inflammatory and lubrication effects.

Detection of trace phosphorus in water by plasma amplification laser-induced breakdown spectroscopy.

For monitoring the extent of eutrophication in water, phosphorus (P) was detected by laser-induced breakdown spectroscopy (LIBS). A plasma amplification method was proposed and the filtered aerosol was guided to interact with the collinear laser in conjunction with a nebulizer, cyclonic spray chamber, and quartz tube. With this method, the length of the plasma was amplified from 5.27∼8.73 to 17.58 mm. Moreover, the limit of detection (LoD) values of P in water improved from 6.13∼17.75 to 3.60 ppm. Furthermore, the average relative error (REAV) values reduced from 10.23∼23.84 to 6.17%. The root mean square error of cross-validation (RMSECV) values decreased from 16.68∼64.29 to 3.24 ppm. This demonstrated that plasma amplification LIBS could improve the quantitative analysis performance of LIBS detection of trace phosphorus in water.