The differential impact of iron on ferroptosis, oxidative stress, and inflammatory reaction in head-kidney macrophages of yellow catfish (Pelteobagrus fulvidraco) with and without ammonia stress.

Ammonia toxicity in fish is closely related to ferroptosis, oxidative stress, and inflammatory responses. Iron is an essential trace element that plays a key role in many biological processes for cells and organisms, including ferroptosis, oxidative stress response, and inflammation. This study aimed to investigate the effect of iron on indicators of fish exposed to ammonia, specifically on the three aspects mentioned above. The head kidney macrophages of yellow catfish were randomly assigned to one of four groups: CON (normal control), AM (0.046 mg L-1 total ammonia nitrogen), Fe (20 µg mL-1 FeSO4), and Fe + AM (20 µg mL-1 FeSO4, 0.046 mg L-1 total ammonia nitrogen). The cells were pretreated with FeSO4 for 6 h followed by ammonia for 24 h. The study found that iron supplementation led to an excessive accumulation of iron and ROS in macrophages, but it did not strongly induce ferroptosis, oxidative stress, or inflammatory responses. This was supported by a decrease in T-AOC, and the downregulation of SOD, as well as an increase in GSH levels and the upregulation of TFR1, CAT and Nrf2. Furthermore, the mRNA expression of HIF-1, p53 and the anti-inflammatory M2 macrophage marker Arg-1 were upregulated. The results also showed that iron supplementation increased the progression of some macrophages from early apoptosis to late apoptotic cells. However, the combined treatment of iron and ammonia resulted in a stronger intracellular ferroptosis, oxidative stress, and inflammatory reaction compared to either treatment alone. Additionally, there was a noticeable increase in necrotic cells in the Fe + AM and AM groups. These findings indicate that the biological functions of iron in macrophages of fish may vary inconsistently in the presence or absence of ammonia stress.

The interaction between lipid and vitamin D3 impacts lipid metabolism and innate immunity in Chinese mitten crabs Eriocheir sinensis.

As a fat-soluble vitamin, vitamin D3 relies on fat to perform its biological function, affecting lipid metabolism and innate immunity. This study used different percentages of lipid and vitamin D3 diets to evaluate the synergistic effects on the growth, lipid metabolism and immunity of juvenile Eriocheir sinensis (5.83 ± 0.01 g) for 56 days, including low lipid (LL, 1.5%) and normal lipid (NL, 7.5%) and three levels of vitamin D3: low (LVD, 0 IU/kg), medium (MVD, 9000 IU/kg) and high (HVD, 27,000, IU/kg). The synergistic effect of lipid and vitamin D3 was not significant on growth but significant on ash content, total protein, hepatopancreas lipid content, hemolymph 1α,25-hydroxy vitamin D3 [1α,25(OH)2D3] content, hepatopancreas lipolysis and synthesis genes. Crabs fed normal lipid (7.5%) and medium vitamin D3 (9000 IU/kg) had the highest hepatopancreas index, hemolymph 1α,25(OH)2D3 content, antibacterial ability, immune-related genes and hepatopancreatic lipid synthesis genes expression, but down-regulated the lipolysis genes expression. In contrast, crabs fed diets with low lipid percentage (1.5%) had low growth performance, hemolymph 1α,25(OH)2D3, mRNA levels of lipid synthesis genes, antibacterial ability and immune-related gene expression. At the 1.5% lipid level, excessive or insufficient vitamin D3 supplementation led to the obstruction of ash and protein deposition, reduced growth and molting, aggravated the reduction in antioxidant capacity, hindered antimicrobial peptide gene expression and reduced innate immunity, and resulted in abnormal lipid accumulation and the risk of oxidative stress. This study suggests that diets' lipid and vitamin D3 percentage can enhance antioxidant capacity, lipid metabolism and innate immunity in E. sinensis. A low lipid diet can cause growth retardation, reduce antioxidant capacity and innate immunity, and enhance lipid metabolism disorder.

New insights into the influence of myo-inositol on carbohydrate metabolism during osmoregulation in Nile tilapia (Oreochromis niloticus).

A two-factor (2 × 3) orthogonal test was conducted to investigate the effects of dietary myo-inositol (MI) on the osmoregulation and carbohydrate metabolism of euryhaline fish tilapia (Oreochromis niloticus) under sustained hypertonic stress (20 practical salinity units [psu]). 6 diets containing either normal carbohydrate (NC, 30%) or high carbohydrate (HC, 45%) levels, with 3 levels (0, 400 and 1,200 mg/kg diet) of MI, respectively, were fed to 540 fish under 20 psu for 8 weeks. Dietary MI supplementation significantly improved growth performance and crude protein content of whole fish, and decreased the content of crude lipid of whole fish (P < 0.05). Curled, disordered gill lamella and cracked gill filament cartilage were observed in the gill of fish fed diets without MI supplementation. The ion transport capacity in gill was significantly improved in the 1,200 mg/kg MI supplementation groups compared with the 0 mg/kg MI groups (P < 0.05). Moreover, the contents of Na+, K+, Cl- in serum were markedly reduced with the dietary MI supplementation (P < 0.05). The fish fed 1,200 mg/kg MI supplementation had the highest MI content in the gills and the lowest MI content in the serum (P < 0.05). Additionally, the fish fed with 1,200 mg/kg MI supplementation had the highest MI synthesis capacity in gills and brain (P < 0.05). Dietary MI markedly promoted the ability of carbohydrate metabolism in liver (P < 0.05). Moreover, fish in the 1,200 mg/kg MI groups had the highest antioxidant capacity (P < 0.05). This study indicated that high dietary carbohydrate would intensify stress, and impair the ability of osmoregulation in tilapia under a long-term hypersaline exposure. The supplementation of MI at 1,200 mg/kg in the high carbohydrate diet could promote carbohydrate utilization and improve the osmoregulation capacity of tilapia under long-term hypertonic stress.

Dietary gamma-aminobutyric acid (GABA) improves non-specific immunity and alleviates lipopolysaccharide (LPS)-induced immune overresponse in juvenile Chinese mitten crab (Eriocheir sinensis).

Inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is an immunomodulator to inhibit immune-mediated pro-inflammatory response and has been used to treat various immune-related diseases in mammals. However, the immunoregulatory effect of GABA in crustaceans has not been reported. This study evaluates the regulatory effect of dietary GABA supplementation on the innate immune status and immunoregulatory potential in lipopolysaccharide (LPS)-induced immune response in juvenile Eriocheir sinensis. Juvenile crabs were fed with six diets supplemented with graded GABA levels (0, 40, 80, 160, 320 and 640 mg/kg dry matter) for 8 weeks and then 24 h LPS challenge test was carried out. The results showed that dietary GABA supplementation significantly decreased mortality at 4 and 8 weeks. Moreover, the hemocyanin content, acid phosphatase, and alkaline phosphatase activities significantly increased in the crabs fed GABA supplementation compared with the control. On the contrary, the alanine aminotransferase and alanine aminotransferase activities in serum decreased significantly in the GABA supplementation groups compared with the control. Similarly, superoxide dismutase activity, glutathione content, and the transcriptional expression of the antioxidant-related genes and immune-related genes were significantly higher in the GABA supplementation groups than in the control. In addition, the mRNA expressions of anti-lipopolysaccharide factors (ALF 1, ALF 2, ALF 3) and inflammatory signaling pathways related genes (TLR, Myd88, Relish, LITAF, P38-MAPK, ADAM17) were significantly up-regulated in LPS stimulation groups compared with PBS treatment. Meanwhile, pro-apoptosis-related genes' mRNA expressions were significantly up-regulated, and anti-apoptosis-related genes were significantly down-regulated under LPS stimulation compared with PBS treatment. However, GABA pretreatment effectively alleviated LPS-induced immune overresponse and apoptosis. Therefore, this study demonstrates that dietary GABA supplementation could be used as an immunomodulator to improve the non-specific immunity and antioxidant capacity and alleviate the immune-mediated immune overresponse of juvenile E. sinensis.

Influences of dietary vitamin D3 on growth, antioxidant capacity, immunity and molting of Chinese mitten crab (Eriocheir sinensis) larvae.

This study investigates the effects of vitamin D3 (VD3) on growth performance, antioxidant capacity, immunity and molting of larval Chinese mitten crab Eriocheir sinensis. A total of 6,000 larvae (7.52 ± 0.10 mg) were fed with six isonitrogenous and isolipidic experimental diets with different levels of dietary VD3 (0, 3000, 6000, 9000, 12000 and 36000 IU/kg) respectively for 23 days. The highest survival and molting frequency were found in crabs fed 6000 IU/kg VD3. Weight gain, specific growth rate, and carapace growth significantly increased in crabs fed 3000 and 6000 IU/kg VD3 compared to the control. Broken-line analysis of molting frequency, weight gain and specific growth rate against dietary VD3 levels indicates that the optimal VD3 requirement for larval crabs is 4825-5918 IU/kg. The highest whole-body VD3 content occurred in the 12000 IU/kg VD3 group, and the 25-dihydroxy VD3 content decreased with the increase of dietary VD3. The malonaldehyde content was lower than the control. Moreover, the superoxide dismutase activity, glutathione peroxidase and total antioxidant capacity of crab fed 6000 IU/kg VD3 were significantly higher than in control. Crabs fed 9000 IU/kg showed the highest survival after 120 h of salinity stress, and the relative mRNA expressions indicate vitamin D receptor (VDR) is the important regulatory element in molting and innate immunity. The molting-related gene expressions showed that the response of crab to salinity was self-protective. This study would contribute to a new understanding of the molecular basis underlying molting and innate immunity regulation by vitamin D3 in E. sinensis.

Alleviation of the Adverse Effect of Dietary Carbohydrate by Supplementation of Myo-Inositol to the Diet of Nile Tilapia (Oreochromis niloticus).

This study investigated the effect of dietary myo-inositol (MI) on alleviating the adverse effect of the high carbohydrate diet in Nile tilapia (Oreochromis niloticus). Six diets contained either low carbohydrate (LC 30%) or high carbohydrate (HC 45%) with three levels of MI supplementation (0, 400 and 1200 mg/kg diet) to each level of the carbohydrate diet. After an 8-week trial, the fish fed 400 mg/kg MI under HC levels had the highest weight gain and fatness, but the fish fed 1200 mg/kg MI had the lowest hepatosomatic index, visceral index and crude lipid in the HC group. The diet of 1200 mg/kg MI significantly decreased triglyceride content in the serum and liver compared with those fed the MI supplemented diets regardless of carbohydrate levels. Dietary MI decreased triglyceride accumulation in the liver irrespective of carbohydrate levels. The content of malondialdehyde decreased with increasing dietary MI at both carbohydrate levels. Fish fed 1200 mg/kg MI had the highest glutathione peroxidase, superoxide dismutase, aspartate aminotransferase and glutamic-pyruvic transaminase activities. The HC diet increased the mRNA expression of key genes involved in lipid synthesis (DGAT, SREBP, FAS) in the fish fed the diet without MI supplementation. Dietary MI significantly under expressed fatty acid synthetase in fish fed the HC diets. Moreover, the mRNA expression of genes related to lipid catabolism (CPT, ATGL, PPAR-α) was significantly up-regulated with the increase of dietary MI levels despite dietary carbohydrate levels. The gene expressions of gluconeogenesis, glycolysis and MI biosynthesis were significantly down-regulated, while the expression of the pentose phosphate pathway was up-regulated with the increase of MI levels. This study indicates that HC diets can interrupt normal lipid metabolism and tend to form a fatty liver in fish. Dietary MI supplement can alleviate lipid accumulation in the liver by diverging some glucose metabolism into the pentose phosphate pathway and enhance the antioxidant capacity in O. niloticus.

Effects of dietary T-2 toxin on gut health and gut microbiota composition of the juvenile Chinese mitten crab (Eriocheir sinensis).

The current study aims to investigate the effects of dietary T-2 toxin on the intestinal health and microflora in the juvenile Chinese mitten crab (Eriocheir sinensis) with an initial weight 2.00 ± 0.05 g. Juvenile crabs were fed with experimental diets supplemented with T-2 toxin at 0 (control), 0.6 (T1 group), 2.5 (T2 group) and 5.0 (T3 group) mg/kg diet for 8 weeks. Dietary T-2 toxin increased the malondialdehyde (MDA) content and the expression of Kelch-like ECH-associated protein 1 (keap1) gene while the expression of cap 'n' collar isoform C (CncC) decreased in the intestine. The activities of glutathione peroxidase (GSH-Px) and total anti-oxidation capacity (T-AOC) in the intestine increased only in the lower dose of dietary T-2. Dietary T-2 toxin significantly increased the mRNA expression of caspase-3, caspase-8, Bax and mitogen-activated protein kinase (MAPK) genes and the ratio of Bax to Bcl-2 accompanied with a reduction of Bcl-2 expression. Furthermore, T-2 toxin decreased the mRNA levels of antimicrobial peptides (AMPs), peritrophic membrane (PM1 and PM2) and immune regulated nuclear transcription factors (Toll-like receptor: TLR, myeloid differentiation primary response gene 88: Myd88, relish and lipopolysaccharide-induced TNF-α factor: LITAF). The richness and diversity of the gut microbiota were also affected by dietary T-2 toxin in T3 group. The similar dominant phyla in the intestine of the Chinese mitten crab in the control and T3 groups were found including Bacteroidetes, Firmicutes, Tenericutes and Proteobacteria. Moreover, the inclusion of dietary T-2 toxin of 4.6 mg/kg significantly decreased the richness of Bacteroidetes and increased the richness of Firmicutes, Tenericutes and Proteobacteria in the intestine. At the genus level, Dysgonomonas and Romboutsia were more abundant in T3 group than those in the control. However, the abundances of Candidatus Bacilloplasma, Chryseobacterium and Streptococcus in T3 group were lower than those in the control. This study indicates that T-2 toxin could cause oxidative damage and immunosuppression, increase apoptosis and disturb composition of microbiota in the intestine of Chinese mitten crab.

Inhibitory effects of quercetin on angiogenesis in larval zebrafish and human umbilical vein endothelial cells.

Angiogenesis plays an essential role in many physiological and pathological processes. Quercetin, a plant pigment and traditional Chinese medicinal herb, is an important flavonoid that has anti-cancer activity. However, the function of quercetin in blood vessel development in vivo and in vitro is still unclear. In this study, we investigated the anti-angiogenic activity of quercetin in zebrafish embryos and in human umbilical vein endothelial cells (HUVECs). Our results showed that quercetin disrupted the formation of intersegmental vessels, the dorsal aorta and the posterior cardinal vein in transgenic zebrafish embryos. In HUVECs, quercetin inhibited cell viability, the expression of vascular endothelial growth factor receptor 2 and tube formation in a dose-dependent manner. In inhibiting angiogenesis, quercetin was found to be involved in suppressing the extracellular signal-regulated kinase signaling pathway in vivo and in vitro. This study has shown that quercetin has potent anti-angiogenic activity and may be a candidate anti-cancer agent for future research.

Molecular cloning, characterization and mRNA expression of copper-binding protein hemocyanin subunit in Chinese mitten crab, Eriocheir sinensis.

Hemocyanin is a copper-binding protein and plays a crucial role in the physiological processes in crustacean. In this study, the cDNA encoding hemocyanin subunit from Chinese mitten crab Eriocheir sinensis (EsHc) was cloned by using EST analysis and rapid amplification of cDNA ends (RACE) approach. The full-length cDNA of EsHc was 2573 bp, consisting of a 5' untranslated region of 51 bp, a 3' untranslated region of 458 bp, and an open reading frame of 2064 bp. The deduced protein had 688 amino acid residues with molecular mass of 77,997.31 Da. Quantitative real-time RT-PCR analysis showed that the EsHc gene was expressed in haemocytes, hepatopancreas, muscles, gills, and intestines with the highest level of expression in the hepatopancreas and the lowest in the muscles. After Aeromonas hydrophila challenge, the relative expression level of EsHc in hemolymph was up-regulated at 3 h post-injection of bacteria followed by a gradual recovery from 12 to 24 h. In the second set of transcriptional studies, the mRNA expression patterns of EsHc in haemocytes and hepatopancreas were measured by quantitative real-time RT-PCR after the Chinese mitten crab were fed six diets containing different levels of copper (0, 10, 20, 40, 80 and 400 mg kg(-1)) for 8 weeks, respectively. The feeding trial showed that the expression levels of EsHc mRNA significantly increased at the copper levels of 20-40 mg kg(-1). This study implies that the expression levels of EsHc could be affected by dietary copper in the hepatopancreas and haemocytes, and hemocyanin may be potentially involved in the immune responses of the Chinese mitten crab.