Hypoxia caused by global climate change and human activities has become a growing concern eliciting serious effect and damages to aquatic animals. Hexagrammos otakii is usually a victim of hypoxia which caused by high density aquaculture and high nutrient input. The mechanism underlying ferroptosis regulation after hypoxia-stress in liver of H. otakii, however, remains elusive. For a duration of 15 days, expose the H. otakii to low concentrations of dissolved oxygen (3.4 ± 0.2 mg/L). Detecting alterations in the H. otakii liver tissue by chemical staining, immunohistochemistry, and electron microscopy. The expression variations of relevant genes in the liver of the H. otakii were simultaneously detected using Western blot and qPCR. A correlation analysis was performed between HIF-1α and iron ion expression in the liver of H. otakii following hypoxic stress. In this study, we conducted the whole ferroptosis integrated analysis of H. otakii under chronic hypoxic condition. Reactive oxygen species (ROS) are highly accumulated under the hypoxia treatment (Superoxide Dismutase, SOD; Catalase, CAT), and which results in a significantly enhanced of lipid peroxidation (Lipid Peroxidation, LPO; Malondialdehyde, MDA; Aminotransferase, AST; Alanine aminotransferase, ALT) in liver tissue. The HIF-1α signaling is activated to cope with the hypoxia stress through strategies including changing iron ion concentration (Fe³⁺ and TFR1) to breaking the oxidation balance (GSH and GSH-Px), and enhancing ferroptosis gene expression (GPX4). The expression of genes related to ferroptosis pathway (DMT1, FTH1, STEAP3, ACSL4, γ-GCS, SLC7A11) is significantly upregulated and associated to the expression of iron and HIF-1α. It is demonstrated that the HIF-1α/Fe³⁺/ROS/GPX4 axis is involved in promoting ferroptosis in fat greening hepatocytes following hypoxia-stress. Ultimately, our findings unveil a process by which hypoxic stress strongly encourages ferroptosis by triggering HIF-1α and boosting iron synthesis.

Introduction: Artemisinin (ART) is very common as a diet additive due to its immunoregulatory activities. Nonetheless, the immunoregulatory mechanism of ART in marine fish remains unknown. This study comprehensively examined the effects and explored the potential mechanism of ART ameliorating intestinal immune disease (IID) in fat greenlings (Hexagrammos otakii).Methods and results: The targets of ART were screened using the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database. Here, eight putative targets of ART were collected and identified with the Uniprot database, and 1419 IID-associated target proteins were filtered through the Drugbank, Genecards, OMIM, and PHARMGKB Databases. The results of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways point out that ART may have immunoprotective effects by regulating cellular responses to stress, hypoxia, inflammation, and vascular endothelial growth factor stimulus through the hypoxia-inducible factor 1 (HIF-1) signaling pathway. The findings of molecular docking indicated that ART contains one active ingredient and three cross-targets, which showed a kind combination with hypoxia-inducible factor 1-alpha (HIF1-a), transcription factor p65 (RELA), and vascular endothelial growth factor A (VEGF-A), respectively. Furthermore, an ART feeding model was established to assess the ART’s immunoprotect effect on the intestine of H.otakii in vivo. The D48 group showed smaller intestinal structural changes after being challenged by Edwardsiella tarda. The supplementation of ART to the diet improved total superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) and reduced the malondialdehyde (MDA) in intestine of H. otakii. The expression of transcription factor p65, HIF1-α, VEGF-A, cyclin D1, matrix metalloprotease 9 (MMP9), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6) was decreased after dietary ART in the intestinal of H. otakii.Discussion: The present results demonstrated that dietary ART improved antioxidants and immunity, optimized the intestinal structure, and increased resistance to E. tarda through the SOD2/nuclear-factor-kappa- B (NFkB)/HIF1-a/VEGF-A pathway in the intestinal tract of H.otakii. This study integrated pharmacological analysis and experimental validation and revealed the mechanism of ART on IID, which provides insight into the improvement of IID in H. otakii.

Fat greenling (Hexagrammos otakii) is a kind of economic fish that is widely consumed by human, and its intensive farming technology is making important progress. However, high-density farming may cause the occurrence of diseases in H. otakii. Cinnamaldehyde (CNE) is a new feed additive for aquatic animals and has a positive effect on disease resistance. In the study, dietary CNE was evaluated on the growth performance, digestion, immune response, and lipid metabolism of juvenile H. otakii ( g). Six experimental diets were formulated containing CNE at levels of 0, 200, 400, 600, 800, and 1000 mg/kg for 8 weeks. The percent weight gain (PWG), specific growth rate (SGR), survival (SR), and feeding rate (FR) were significantly increased by including CNE in fish diets regardless of the inclusion level ( ). The feed conversion ratio (FCR) was significantly decreased among the groups fed CNE supplemented diets ( ). A significant decrease in hepatosomatic index (HSI) was observed in fish fed 400 mg/kg-1000 mg/kg CNE compared to the control diet ( ). Fish-fed diets containing 400 mg/kg and 600 mg/kg CNE had a higher level of crude protein in muscles than the control diet ( ). Moreover, the activities of lipase (LPS) and pepsin (PEP) in the intestinal were markedly increased in juvenile H. otakii-fed dietary CNE ( ). Apparent digestibility coefficient (ADC) of dry matter, protein, and lipid was significantly increased with CNE supplement ( ). The activities of catalase (CAT) and acid phosphatase (ACP) in the liver were markedly enhanced by including CNE in juvenile H. otakii diets compared with the control ( ). The activities of superoxide dismutase (SOD) and alkaline phosphatase (AKP) in the liver were markedly enhanced in juvenile H. otakii treated with CNE supplements 400 mg/kg-1000 mg/kg ( ). Additionally, the levels of total protein (TP) in the serum were markedly increased by including CNE in juvenile H. otakii diets compared with the control ( ). In the CNE200, CNE400, and CNE600 groups, albumin (ALB) levels in the serum were markedly higher compared with that in the control ( ). In the CNE200 and CNE400 groups, the levels of immunoglobulin G (IgG) in the serum were significantly increased compared with that the control group ( ). The juvenile H. otakii-fed dietary CNE had lower triglycerides (TG) and total cholesterol (TCHO) levels in the serum than fish-fed CNE-free diets ( ). The gene expression of peroxisome proliferator-activated receptor alpha (PPAR-α), hormone-sensitive lipase (HSL), and carnitine O-palmitoyltransferase 1 (CPT1) in the liver was significantly increased by including CNE in fish diets regardless of the inclusion level ( ). However, fatty acid synthase (FAS), peroxisome proliferator-activated receptor gamma (PPAR-γ), and acetyl-CoA carboxylase alpha (ACCα) in the liver were markedly decreased with CNE supplements 400 mg/kg-1000 mg/kg ( ). The glucose-6-phosphate1-dehydrogenase (G6PD) gene expression levels in the liver were markedly decreased compared with the control (