Exploring the novel benefits of leucine: Protecting nitrite-induced liver damage in sub-adult grass carp (Ctenopharyngodon idella) through regulating mitochondria quality control.

Leucine is an essential amino acid for fish. The ability of leucine to resist stress in fish has not been reported. Nitrite is a common pollutant in the aquatic environment. Therefore, we investigated the effects of dietary leucine on growth performance and nitrite-induced liver damage, mitochondrial dysfunction, autophagy, and apoptosis for sub-adult grass carp. A total of 450 grass carp (615.91 ± 1.15 g) were selected and randomly placed into 18 net cages. The leucine contents of the six diets were 2.91, 5.90, 8.92, 11.91, 14.93, and 17.92 g/kg, respectively. After a 9-week feeding trial, the nitrite exposure experiment was set up for 96 h. These results indicated that dietary leucine significantly promoted FW, WG, PWG, and SGR of sub-adult grass carp (P < 0.05). Appropriate levels of dietary leucine (11.91-17.92 g/kg) decreased the activities of serum parameters (glucose, cortisol, and methemoglobin contents, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, and lactate dehydrogenase), the contents of reactive oxygen species (ROS), nitric oxide (NO) and peroxynitrite (ONOO-). In addition, appropriate levels of dietary leucine (11.91-17.92 g/kg) increased the mRNA levels of mitochondrial biogenesis genes (PGC-1α, Nrf1/2, TFAM), fusion-related genes (Opa1, Mfn1/2) (P < 0.05), and decreased the mRNA levels of caspase 3, caspase 8, caspase 9, fission-related gene (Drp1), mitophagy-related genes (Pink1, Parkin) and autophagy-related genes (Beclin1, Ulk1, Atg5, Atg7, Atg12) (P < 0.05). Appropriate levels of dietary leucine (8.92-17.92 g/kg) also increased the protein levels of AMP-activated protein kinase (AMPK), prostacyclin (p62) and decreased the protein levels of protein light chain 3 (LC3), E3 ubiquitin ligase (Parkin), and Cytochrome c (Cytc). Appropriate levels of leucine (8.92-17.92 g/kg) could promote growth performance and alleviate nitrite-induced mitochondrial dysfunction, autophagy, apoptosis for sub-adult grass carp. Based on quadratic regression analysis of PWG and serum GPT activity, dietary leucine requirements of sub-adult grass carp were recommended to be 12.47 g/kg diet and 12.55 g/kg diet, respectively.

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.

Capturing Protein-Protein Interactions with Acidic Amino Acids Reactive Cross-Linkers.

Acidic residues (Asp and Glu) have a high prevalence on protein surfaces, but cross-linking reactions targeting these residues are limited. Existing methods either require high-concentration coupling reagents or have low structural compatibility. Here a previously reported "plant-and-cast" strategy is extended to develop heterobifunctional cross-linkers. These cross-linkers first react rapidly with Lys sidechains and then react with Asp and Glu sidechains, in a proximity-enhanced fashion. The cross-linking reaction proceeds at neutral pH and room temperature without coupling reagents. The efficiency and robustness of cross-linking using model proteins, ranging from small monomeric proteins to large protein complexes are demonstrated. Importantly, it is shown that this type of cross-linkers are efficient at identifying protein-protein interactions involving acidic domains. The Cross-linking mass spectrometry (XL-MS) study with p53 identified 87 putative binders of the C-terminal domain of p53. Among them, SARNP, ZRAB2, and WBP11 are shown to regulate the expression and alternative splicing of p53 target genes. Thus, these carboxylate-reactive cross-linkers will further expand the power of XL-MS in the analysis of protein structures and protein-protein interactions.

Cellular antioxidant mechanism of mannan-oligosaccharides involving in enhancing flesh quality in grass carp (Ctenopharyngodon idella).

BACKGROUND: Deterioration of flesh quality has bad effects on consumer satisfaction. Therefore, effects of safe mannan-oligosaccharides (MOS) on flesh quality of grass carp (Ctenopharyngodon idella) muscle were studied. A total of 540 healthy fish (215.85 ± 0.30 g) were randomly divided into six groups and fed six separate diets with graded levels of MOS (0, 200, 400, 600, 800 and 1000 mg kg-1 ) for 60 days. This study aimed at investigating the benefits of dietary MOS on flesh quality (fatty acids, amino acids and physicochemical properties) and the protection mechanism regarding antioxidant status. RESULTS: Optimal MOS could improve tenderness (27.4%), pH (5.5%) while decreasing cooking loss (16.6%) to enhance flesh quality. Meanwhile, optimal MOS improved flavor inosine 5'-monophosphate (IMP) of 11.8%, sweetness and umami-associated amino acid, healthy unsaturated fatty acid (UFA) of 14.9% and n-3 polyunsaturated fatty acids (n-3 PUFAs) especially C20:5n-3 (15.8%) and C22:6n-3 (38.3%). Furthermore, the mechanism that MOS affected pH, tenderness and cooking loss could be partly explained by the reduced lactate, cathepsin and oxidation, respectively. The enhanced flesh quality was also associated with enhanced antioxidant ability concerning improving antioxidant enzymes activities and the corresponding transcriptional levels, which were regulated through NF-E2-related factor 2 (Nrf2) and target of rapamycin (TOR) signaling. Based on pH24h , cooking loss, shear force and DHA (docosahexaenoic acid, C22:6n-3), optimal MOS levels for grass carp were estimated to be 442.75, 539.53, 594.73 and 539.53 mg kg-1 , respectively. CONCLUSION: Dietary MOS is a promising alternative strategy to improve flesh quality of fish muscle. © 2022 Society of Chemical Industry.

Glycoproteins as diagnostic and prognostic biomarkers for neurodegenerative diseases: A glycoproteomic approach.

Neurodegenerative diseases (NDs) are incurable and can develop progressively debilitating disorders, including dementia and ataxias. Alzheimer's disease and Parkinson's disease are the most common NDs that mainly affect the elderly people. There is an urgent need to develop new diagnostic tools so that patients can be accurately stratified at an early stage. As a common post-translational modification, protein glycosylation plays a key role in physiological and pathological processes. The abnormal changes in glycosylation are associated with the altered biological pathways in NDs. The pathogenesis-related proteins, like amyloid-ß and microtubule-associated protein tau, have altered glycosylation. Importantly, specific glycosylation changes in cerebrospinal fluid, blood and urine are valuable for revealing neurodegeneration in the early stages. This review describes the emerging biomarkers based on glycoproteomics in NDs, highlighting the potential applications of glycoprotein biomarkers in the early detection of diseases, monitoring of the disease progression, and measurement of the therapeutic responses. The mass spectrometry-based strategies for characterizing glycoprotein biomarkers are also introduced.

Elastic Net-Based Identification of a Multigene Combination Predicting the Survival of Patients with Cervical Cancer.

BACKGROUND The objective of the present study was to identify prognostication biomarkers in patients with cervical cancer. MATERIAL AND METHODS Survival related genes were identified in The Cancer Genome Atlas (TCGA) cervical cancer study, and they were included into an elastic net regularized Cox proportional hazards regression model (CoxPH). The genes that their coefficients that were not zero were combined to build a prognostication combination. The prognostication performance of the multigene combination was evaluated and validated using Kaplan-Meier curve and univariate and multivariable CoxPH model. Meanwhile, a nomogram was built to translate the multigene combination into clinical application. RESULTS There were 37 survival related genes identified, 9 of which were integrated to build a multigene combination. The area under the curve (AUC) of receiver operating characteristic (ROC) curve at 1-year, 3-year, 5-year, and 7-year in the training set were 0.757, 0.744, 0.799, and 0.854, respectively, and the multigene combination could stratify patients into significantly different prognostic groups (hazard ratio [HR]=0.2223, log-rank P<0.0001). Meanwhile, the corresponding AUCs in the test set was 0.767, 0.721, 0.735, and 0.703, respectively, and the multigene combination could classify patients into different risk groups (HR=0.3793, log-rank P=0.0021). The multigene combination could stratify patients with early stage and advanced stage into significantly different survival groups in the training set and test set. The prognostication performance of the multigene combination was better compared with 3 existing prognostic signatures. Finally, a multigene containing nomogram was developed. CONCLUSIONS We developed a multigene combination which could be treated as an independent prognostic factor in cervical cancer and be translated into clinical application.

Emerging regulatory roles of noncoding RNAs induced by bisphenol a (BPA) and its alternatives in human diseases.

Bisphenols (BPs), including BPA, BPF, BPS, and BPAF, are synthetic phenolic organic compounds and endocrine-disrupting chemicals. These organics have been broadly utilized to produce epoxy resins, polycarbonate plastics, and other products. Mounting evidence has shown that BPs, especially BPA, may enter into the human body and participate in the development of human diseases mediated by nuclear hormone receptors. Moreover, BPA may negatively affect human health at the epigenetic level through processes such as DNA methylation and histone acetylation. Recent studies have demonstrated that, as part of epigenetics, noncoding RNAs (ncRNAs), including microRNAs (miRNAs), long noncoding RNAs (lncRNAs), circular RNAs (circRNAs), and small nucleolar RNAs (snoRNAs), have vital impacts on BP-related diseases, such as reproductive system diseases, nervous system diseases, digestive system diseases, endocrine system diseases, and other diseases. Moreover, based on the bioinformatic analysis, changes in ncRNAs may be relevant to normal activities and functions and BP-induced diseases. Thus, we conducted a meta-analysis to identify more promising ncRNAs as biomarkers and therapeutic targets for BP exposure and relevant human diseases. In this review, we summarize the regulatory functions of ncRNAs induced by BPs in human diseases and latent molecular mechanisms, as well as identify prospective biomarkers and therapeutic targets for BP exposure and upper diseases.

Tea polyphenols protect against Flavobacterium columnare-induced gill injury via suppression of oxidative stress, inflammation, and apoptosis in grass carp.

Flavobacterium columnare (F. columnare) is one of the deadliest fish pathogens causing bacterial gill rot disease in various freshwater fish species globally. Tea polyphenols (TPs) are an inexpensive product extracted from tea that have received much attention as a feed additive in aquaculture. The current study was designed to investigate the underlying mechanisms and protective effects of dietary TPs against F. columnare-induced gill injury via suppression of oxidative stress, apoptosis, and inflammation in grass carp. TPs were not supplemented to the diet (control) and were supplemented at 40, 80, 120, 160 or 200 mg/kg diet. The feeding experiment was carried out for 60 days, followed by a 3-Day F. columnare challenge test. The results showed that 120 mg/kg TPs in the diet exerted the following five protective effects in fish gill: (1) control gill-rot disease and improved histopathology, (2) inhibit excessive apoptosis, (3) enhance the activity of antioxidant enzymes and upregulate related gene expression via the Nrf2/Keap1 pathway, (4) increase the activity of immune enzymes, And (5) mediate inflammatory cytokine gene expression via the JAK/STAT3 pathway. Taken together, dietary supplementation with TPs is a compelling approach to protect the gill function of fish against F. columnare.

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.

Exploring the dietary strategies of phenylalanine: Improving muscle nutraceutical quality as well as muscle glycogen and protein deposition in adult grass carp (Ctenopharyngodon idella).

Muscle is the main edible part of bony fish. The purpose of this study was to investigate the influences of phenylalanine (Phe) on muscle quality, amino acid composition, fatty acid composition, glucose metabolism, and protein deposition in adult grass carp. The diets at 2.30, 4.63, 7.51, 10.97, 13.53, and 17.07 g/kg Phe levels were fed for 9 weeks. The results manifested that Phe (10.97-13.53 g/kg) increased the pH of the fillets and decreased muscle cooking loss and lactic acid content; Phe (7.51-17.07 g/kg) improved the composition of the fillets in terms of flavor (free) amino acids, bound amino acids (especially EAA), and fatty acids (especially EPA and DHA); Phe (7.51-13.53 g/kg) increased muscle glycogen content (possibly related to the AMPK signaling pathway) and muscle protein deposition (possibly related to IGF-1/4EBP1/TOR and AKT/FOXOs signaling pathways). In conclusion, a diet with appropriate Phe levels could improve fillet quality.

Aflatoxin B1 decreased flesh flavor and inhibited muscle development in grass carp (Ctenopharyngodon idella).

In nature, aflatoxins, especially aflatoxin B1 (AFB1), are the common mycotoxins, which cause serious health problems for humans and animals. This paper aimed to study the effects of AFB1 on flesh flavor and muscle development of grass carp (Ctenopharyngodon idella) and its mechanism. There were 1440 individual fish in total, with 6 treatments and each treatment replicated 3 times. The 6 treatments were fed a control diet with different doses of AFB1 (0.04, 29.48, 58.66, 85.94, 110.43 and 146.92 µg/kg diet) for 60 d. AFB1 increased myofiber diameter, as well as decreased myofiber density of grass carp muscle (P < 0.05). The contents of free amino acid decreased gradually (P < 0.05) as dietary AFB1 increased in the muscle of grass carp. The levels of reactive oxygen species, malonaldehyde and protein carbonyl (PC) were increased (P < 0.05) with the dietary AFB1 increased. The levels of antioxidant enzyme (glutathione peroxidase, glutathione, glutathione reductase, total antioxidant capacity, anti-superoxide anion, and anti-hydroxyl radical) were decreased (P < 0.05) with the dietary AFB1 increased. In addition, dietary AFB1 decreased the content of collagen, and downregulated the mRNA and protein levels of transforming growth factor-ß (TGF-ß)/Smads signaling pathway in grass carp muscle (P < 0.05). The mRNA and protein levels of myogenic regulatory factors were downregulated in grass carp muscle (P < 0.05). Furthermore, the activities of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) were increased (P < 0.05), and the protein levels of phosphorylate-38 mitogen-activated protein kinase (p-p38MAPK), phosphorylate-c-Jun N-terminal kinase, urokinase-type plasminogen activator (uPA), MMP-2 and MMP-9 were upregulated (P < 0.05), but collagen Ⅰ, laminin ß1 and fibronectin were downregulated (P < 0.05) with the dietary AFB1 increased in the muscle of grass carp. Based on the results of this study, we can draw the following conclusion: dietary AFB1 might damage flesh flavor and inhibit the muscle development through MAPK/uPA/MMP/extracellular matrix (ECM) signaling pathway in grass carp. Moreover, the recommended safe limit of AFB1 in feed is no more than 26.77 µg/kg diet according to the PC levels in grass carp muscle.

Dietary cinnamaldehyde improves muscle protein content by promoting muscle fiber growth via PTP1B/IGF1/PI3K/AKTs-TOR/FOXO3a signaling pathway in grass carp (Ctenopharyngodon idella).

Flesh quality is evaluated according to nutritional value and sensory quality. Cinnamaldehyde (CIN) improves mammalian meat quality, but research relating this to aquaculture is scarce. In this study, five doses of CIN (0, 36, 72, 108, 144 mg/kg diet) were fed to grass carp (Ctenopharyngodon idella) for 60 days. The results show that CIN supplementation increased nutritional value by increasing crude protein content. CIN also improved the sensory quality by increasing the pH and collagen content, decreasing shear force, lactate, and cooking loss. These changes may be related to changes in muscle fiber growth by increasing myofiber diameter. The increased myofiber diameter induced by CIN is associated with TOR mRNA and protein levels, and down-regulated FOXO3a mRNA levels, which might be associated with PTP1B/IGF1/PI3K/AKTs-TOR/FOXO3a signaling. Based on muscle crude protein content, optimal CIN supplementation dosage was 88.01 mg/kg.

A new insight on copper: Promotion of collagen synthesis and myofiber growth and development in juvenile grass carp (Ctenopharyngodon idella).

Copper (Cu) is a trace element, essential for fish growth. In the current study, in addition to growth performance, we first explored the effects of Cu on collagen synthesis and myofiber growth and development in juvenile grass carp (Ctenopharyngodon idella). A total of 1080 fish (11.16 ± 0.01 g) were randomly divided into 6 treatments (3 replicates per treatment) to receive five doses of organic Cu, which were Cu citrate (CuCit) at 0.99 (basal diet), 2.19, 4.06, 6.15, and 8.07 mg/kg, and one dose of inorganic Cu (CuSO4·5H2O at 3.15 mg/kg), for 9 weeks. The results showed appropriate Cu level (4.06 mg/kg) enhanced growth performance, improved nutritional Cu status, and downregulated Cu-transporting ATPase 1 mRNA levels in the hepatopancreas, intestine, and muscle of juvenile grass carp. Meanwhile, collagen content in fish muscle was increased after Cu intake, which was probably due to the following pathways: (1) activating CTGF/TGF-ß1/Smads signaling pathway to regulate collagen transcription; (2) upregulating of La ribonucleoprotein domain family 6 (LARP6) mRNA levels to regulate translation initiation; (3) increasing proline hydroxylase, lysine hydroxylase, and lysine oxidase activities to regulate posttranslational modifications. In addition, optimal Cu group increased myofiber diameters and the frequency of myofibers with diameter >50 µm, which might be associated with upregulation of cyclin B, cyclin D, cyclin E, proliferating cell nuclear antigen, myogenic determining factor (MyoD), myogenic factor 5, myogenin (MyoG), myogenic regulatory factor 4 and myosin heavy chain (MyHC) and downregulation of myostatin mRNA levels, increasing protein levels of MyoD, MyoG and MyHC in fish muscle. Finally, based on percentage weight gain (PWG), serum ceruloplasmin (Cp) activity and collagen content in fish muscle, Cu requirements were determined as 4.74, 4.37 and 4.62 mg/kg diet (CuCit as Cu source) of juvenile grass carp, respectively. Based on PWG and Cp activity, compared to CuSO4·5H2O, the efficacy of CuCit were 131.80% and 115.38%, respectively. Our findings provide new insights into Cu supplementation to promote muscle growth in fish, and help improve the overall productivity of aquaculture.

Dietary threonine improves muscle nutritional value and muscle hardness associated with collagen synthesis in grass carp (Ctenopharyngodon idella).

To further explain the improvement effect of threonine (Thr) on the fillet quality of fish, a 9-week feeding experiment was conducted. After feeding graded levels of Thr (2.38, 5.38, 8.38, 11.38, 14.38 and 17.38 g/kg), the compositions of fillet hydrolyzed amino acid and fatty acid, and the muscle hardness associated with collagen biosynthesis were mainly analyzed in grass carp (Ctenopharyngodon idella). The results showed that Thr increased the pH value, changed the amino acids and fatty acid composition of fillets, especially essential amino acid (EAA), C22:6n3 (DHA) and C20:5n3 (EPA). Furthermore, this study revealed for the first time that the improvement of muscle hardness by Thr was associated with collagen biosynthesis, and the TGF-ß1/Smads, LARP6a and Hsp47 regulate transcriptional processes, translation initiation and post-translational modifications in collagen biosynthesis, respectively. This study offered a basis for exploring the contribution of Thr in improving muscle quality in sub-adult grass carp.

New Insight on the Immune Modulation and Physical Barrier Protection Caused by Vitamin A in Fish Gills Infected With Flavobacterium columnare.

In this study, we have investigated the influence of vitamin A on gill barrier function of grass carp (Ctenopharyngodon idella) infected with Flavobacterium columnare. The fish were fed different concentrations of vitamin A diets for 10 weeks and then infected with F. columnare by immersion. We observed that optimal vitamin A significantly prevented gill rot morbidity in fish infected with F. columnare. Further investigations revealed that vitamin A boosted the gill immunity by increasing the contents of complements (C3 and C4), activities of acid phosphatase (ACP) and lysozyme, mRNAs of ß-defensin-1, liver-expressed antimicrobial peptide 2A and 2B (LEAP-2A and LEAP-2B), hepcidin, and anti-inflammatory cytokines like transforming growth factor ß1 (TGF-ß1), TGF-ß2, interleukin-10 (IL-10), and IL-11. It also enhanced the levels of various related signaling molecules including inhibitor protein κBα (IκBα), target of rapamycin (TOR), and ribosome protein S6 kinase 1 (S6K1) but downregulated the expression of pro-inflammatory cytokines including IL-1ß, IL-8, tumor necrosis factor α (TNF-α), and interferon γ2 (IFN-γ2) and related signaling molecules including nuclear factor κB p65 (NF-κB p65) (rather than NF-κB p52), IκB kinase ß (IKKß), IKKγ (rather than IKKα), eIF4E-binding protein 1 (4E-BP1), and 4E-BP2 mRNA levels in fish gills. In addition, dietary vitamin A markedly lowered the concentrations of reactive oxygen species (ROS), malondialdehyde (MDA), and protein carbonyl (PC), increased both the activities and mRNAs of copper/zinc superoxide dismutase (Cu/ZnSOD), MnSOD, glutathione transferases (GSTs), glutathione peroxidase (GPx), and glutathione reductase (GR) associated with upregulation of NF-E2-related factor 2 (Nrf2) mRNAs and downregulation of Kelch-like-ECH-associated protein (Keap1a) and Keap1b mRNAs. Moreover, vitamin A decreased the mRNAs of different apoptotic mediators [caspases 8, 9, 3 (rather than 7)] associated with downregulation of signaling molecule p38 mitogen-activated protein kinase (p38MAPK) mRNAs in fish gills. Besides, vitamin A promoted tight junction (TJ) complex mRNAs [including claudin-b, -c, -3, -7, -12, occludin, and zonula occludens-1 (ZO-1)] that have been linked to the downregulation of myosin light chain kinase (MLCK) signaling. Taken together, the current study demonstrated for the first time that vitamin A markedly enhanced gill health associated with immune modulation and physical barrier protection. Based on protecting fish against gill rot morbidity, ACP activity, and against lipid peroxidation, optimum vitamin A concentrations in on-growing grass carp (262-997 g) were found to be 1,991, 2,188, and 2,934 IU/kg diet, respectively.

Physicochemical property optimization and nutrient redistribution in the muscle of sub-adult grass carp (Ctenopharyngodon idella) by conjugated linoleic acid.

We studied the effects of conjugated linoleic acid (CLA) on the amount of nutrients, flavour substances, and healthcare fatty acids, the physicochemical properties, and the potential molecular mechanisms in the muscles of sub-adult grass carp (Ctenopharyngodon idella). Fish were fed graded levels of CLA (0.0, 3.1, 6.4, 9.6, 12.7, and 15.9 g/kg diets) for 60 days. Protein, glutamic acid, alanine, inosine monophosphate (IMP), eicosapentaenoic acid (EPA; 20:5n-3), docosahexaenoic acid (DHA; 22:6n-3), and total CLA contents (p < 0.05) increased in CLA 3.1 âˆ¼ 12.7, 6.4 âˆ¼ 9.6, 6.4 âˆ¼ 9.6, 6.4 âˆ¼ 15.9, 3.1 âˆ¼ 9.6, 3.1 âˆ¼ 9.6, and 3.1 âˆ¼ 15.9 g/kg diet, respectively (p < 0.05). In addition, optimal CLA significantly increased pH24, shear force, collagen content, and myofibre density in the muscle (P < 0.05); however, it decreased myofibre diameter (p < 0.05). We concluded that 6-9 g/kg CLA in the diet could improve the flesh quality of sub-adult grass carp.

Dietary mannan oligosaccharides strengthens intestinal immune barrier function via multipath cooperation during Aeromonas Hydrophila infection in grass carp (Ctenopharyngodon Idella).

In recent years, mannose oligosaccharide (MOS) as a functional additive is widely used in aquaculture, to enhance fish immunity. An evaluation of the effect of dietary MOS supplementation on the immune barrier function and related signaling molecules mechanism of grass carp (Ctenopharyngodon idella) was undertaken in the present study. Six diets with graded amounts of MOS supplementation (0, 200, 400, 600, 800, and 1000 mg/kg) were fed to 540 grass carp over 60 days. To examine the immune response and potential mechanisms of MOS supplementation on the intestine, a challenge test was conducted using injections of Aeromonas hydrophila for 14 days. Results of the study on the optimal supplementation with MOS were found as follows (1) MOS enhances immunity partly related to increasing antibacterial substances content and antimicrobial peptides expression; (2) MOS attenuates inflammatory response partly related to regulating the dynamic balance of intestinal inflammatory cytokines; (3) MOS regulates immune barrier function may partly be related to modulating TLRs/MyD88/NFκB and TOR/S6K1/4EBP signalling pathways. Finally, the current study concluded that MOS supplementation could improve fish intestinal immune barrier function under Aeromonas hydrophila infected conditions.

An emerging role of vitamin D3 in amino acid absorption in different intestinal segments of on-growing grass carp (Ctenopharyngodon idella).

Vitamin D3 (VD3), an essential nutrient for animals, has been demonstrated to stimulate the uptake of certain amino acids. However, the role of VD3 in the intestine, the primary site for digestion and absorption of nutrients, remains poorly characterized. Here, the grass carp (Ctenopharyngodon idella) was studied to assess the influence of different doses of VD3 (15.2, 364.3, 782.5, 1,167.9, 1,573.8, and 1,980.1 IU/kg) on growth performance, intestinal morphology, digestive absorption, amino acid transport, and potential signaling molecule levels in a feeding experiment. As a result, dietary VD3 improved growth performance, intestinal structure, and digestive and brush border enzyme activities. Additionally, most intestinal free amino acids and their transporters were upregulated after VD3 intake, except for Ala, Lys, Asp, Leu, solute carrier (SLC) 7A7, SLC1A5, and SLC1A3 mRNA in different segments, Leu and SLC6A14 mRNA in the proximal intestine, and SLC7A5 mRNA in the mid and distal intestine. In the crucial target of rapamycin (TOR) signal pathway of amino acid transport, the gene and protein expression of TOR, S6 kinase 1, and activating transcription factor 4 were elevated, whereas 4E-binding protein 1 was decreased, further suggesting an advanced amino acid absorption capacity in the fish due to VD3 supplementation. Based on percentage weight gain, feed efficiency, and trypsin activity, the VD3 requirements of on-growing grass carp were estimated to be 968.33, 1,005.00, and 1,166.67 IU/kg, respectively. Our findings provide novel recommendations for VD3 supplementation to promote digestion and absorption capacities of fish, contributing to the overall productivity of aquaculture.

An Antioxidant Supplement Function Exploration: Rescue of Intestinal Structure Injury by Mannan Oligosaccharides after Aeromonas hydrophila Infection in Grass Carp (Ctenopharyngodon idella).

Mannan oligosaccharides (MOS) are a type of functional oligosaccharide which have received increased attention because of their beneficial effects on fish intestinal health. However, intestinal structural integrity is a necessary prerequisite for intestinal health. This study focused on exploring the protective effects of dietary MOS supplementation on the grass carp's (Ctenopharyngodon idella) intestinal structural integrity (including tight junction (TJ) and adherent junction (AJ)) and its related signalling molecule mechanism. A total of 540 grass carp (215.85 ± 0.30 g) were fed six diets containing graded levels of dietary MOS supplementation (0, 200, 400, 600, 800 and 1000 mg/kg) for 60 days. Subsequently, a challenge test was conducted by injection of Aeromonas hydrophila for 14 days. We used ELISA, spectrophotometry, transmission electron microscope, immunohistochemistry, qRT-PCR and Western blotting to determine the effect of dietary MOS supplementation on intestinal structural integrity and antioxidant capacity. The results revealed that dietary MOS supplementation protected the microvillus of the intestine; reduced serum diamine oxidase and d-lactate levels (p < 0.05); enhanced intestinal total antioxidant capacity (p < 0.01); up-regulated most intestinal TJ and AJ mRNA levels; and decreased GTP-RhoA protein levels (p < 0.01). In addition, we also found several interesting results suggesting that MOS supplementation has no effects on ZO-2 and Claudin-15b. Overall, these findings suggested that dietary MOS supplementation could protect intestinal ultrastructure, reduce intestinal mucosal permeability and maintain intestinal structural integrity via inhibiting MLCK and RhoA/ROCK signalling pathways.

Bioactive Small Molecule Enhances Skin Burn Wound Healing and Hair Follicle Regeneration by Activating PI3K/AKT Signaling Pathway: A Preclinical Evaluation in Animal Model.

Rational: A bioactive small molecule of precision medicine involves targeted therapies. Shikonin, a herbal extract, is an active small molecule that is traditionally used in wound healing for its anti-tumor and anti-inflammatory properties. Therefore, the present study aims to evaluate the anti-inflammatory role of shikonin in skin burn wound healing and hair follicle regeneration and to identify molecular signaling pathways that promote the regeneration. Method: A secondary skin burn model of mice was established by conventional method. The burn wound was externally treated with shikonin ointment and excipient treated mice were used as controls. Skin samples were taken on the day 3 and 7 after drug treatment and the dosage was unified in the experiments. The wound healing process was observed by histopathological and immunofluorescence (IF) staining. The proliferation of hair follicle cells in wound skin was tracked by 5-Ethynyl-2'-deoxyuridne (EdU) staining. The inflammatory factors at the wound healing site were quantified by polymerase chain reaction (qPCR). The PI3K/Akt, P65, Ki67 signaling proteins and Bax/BCL2 apoptosis proteins were studied by western blot analysis. The functionality of PI3K/Akt signaling pathway was tested using LY294002, an inhibitor of PI3K. Result: Shikonin treated mice group exhibited better and faster skin burn wound healing in comparison with the controls. The proliferation of new skin cells and hair follicle regeneration in the wound site of the shikonin treated group was more active. The recruitment of macrophages in shikonin treated group was inhibited inturn decreased the expression of inflammatory factors. However, LY294002 inhibited the shikonin-mediated PI3K/Akt signaling pathway and affected the wound healing process. Conclusion: In conclusion, this study strengthens the hypothesis that bioactive small molecule, shikonin, inhibits inflammation, promotes wound healing and has a significant protective effect on the deep hair follicles against burn skin injury by activating the PI3K/Akt signaling pathway.