Project description:Background: Yellow catfish (Pelteobagrus fulvidraco) is one of the important aquaculture species in China. In recent years, due to the high breeding density, the increasing frequency of feeding, and the excessive addition of feed fat, the excessive deposition of body fat in cultured yellow catfish has become more frequent. MicroRNAs (miRNAs) are an important gene expression regulatory signal molecule that regulates liver fat synthesis and transport and play an important role in fat deposition. However, there is a little research on the mechanism of fatty liver caused by excessive deposition of liver fat. Results: After 60 days of high-fat stress, the growth and feed conversion rate of hybrid yellow catfish (Pelteobagrus fuIvidraco♀×P. vachelli♂) were significantly inhibited, and hepatosomatic index, viscerosomatic index, hepatic triglyceride and cholesterol, and red lipid droplets in liver tissues were increased. Through high-throughput sequencing, we constructed miRNA libraries of high-fat stress at 60d, identified 346 conserved miRNAs and 410 novel miRNAs, among which 13 differentially expressed miRNAs were screened between high-fat diet group and normal-fat diet group. Also, we constructed mRNA transcriptome libraries after high-fat stress. Potential target genes for differentially expressed miRNAs were identified by bioinformatics analysis. Seven miRNA-mRNA pairs were screened. The expression of differential miRNA and mRNA and potential binding sites were analyzed by qRT-PCR and dual luciferase assay. Hybrid yellow catfish could promote the oxidative degradation of liver glucose, reduce fatty acid peroxidation, regulate antioxidant enzyme activity and response of immune and inflammatory to relieve fat deposition and liver stress. Conclusions: The disorders of fat metabolism in liver not only result in feed wastage, increase metabolic burden of yellow catfish, but also cause immune function damage, resulting in a variety of nutritional diseases. The development of this study is to understand the molecular mechanism of hepatic fat deposition in yellow catfish. It has important biological significance for improving protection of liver against stress and healthy culture.

Project description:Hypoxia is a critical environmental stressor that affects fish survival and physiological metabolism. Extracellular vesicles (EVs) mediate intercellular signal communication and perform vital functions in the stress response of aquatic organisms; however, the functional regulatory mechanisms of EVs under hypoxia in fish remain unelucidated. In this study, we optimized enzymatic digestion and combined with ultracentrifugation and high-precision iodixanol density gradient centrifugation, high-purity hepatic EVs were efficiently isolated from Pelteobagrus vachelli, which were internalized by primary hepatocytes successfully. Integrative analyses of the EVs miRNAome and hepatic transcriptome were performed to construct a miRNA-mRNA negative regulatory network, identifying metabolic reprogramming, HIF-1 signaling, cellular homeostasis and stress response as core pathways in hypoxic adaptation. In vivo functional validation revealed that hypoxia-induced EVs (Hypo-EVs) reduced the critical asphyxiation point, enhanced blood oxygen-carrying capacity, and alleviated hypoxia-induced hepatic oxidative damage in a dose-dependent manner, thereby significantly improving the hypoxia tolerance of P. vachelli. Mechanistic studies revealed that miR-135a is the core functional molecule within Hypo-EVs, and its expression was significantly downregulated under hypoxia. Overexpression of miR-135a reversed the hypoxic protective capacity of Hypo-EVs, whereas inhibition of miR-135a significantly enhanced hypoxia tolerance. Dual-luciferase and in vivo validation confirmed that miR-135a directly targets the 3'-UTRs of the glycolysis/gluconeogenesis key genes hk1 and pck2, negatively regulates glucose metabolism at the post-transcriptional level and mediates hypoxia adaptation through metabolic reprogramming. Collectively, this study clarifies the molecular mechanism by which the Hypo-EVs/miR-135a/hk1/pck2 signaling axis regulates hypoxia tolerance in P. vachellii. These findings provide theoretical references for research on fish hypoxic response mechanisms and stress-resilient aquaculture regulation.

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