RESUMO
The complement system is pivotal in innate immune defense, with Complement 1qb (C1qb) playing a key role in recognizing immune complexes and initiating the classical pathway. In this research, we cloned the full-length cDNA of silver pomfret (Pampus argenteus) c1qb and demonstrated its role in mediating defense responses against Nocardia seriolae (N. seriolae) infection, which notably causes significant economic losses in the aquaculture industry. Our investigation revealed that N. seriolae infection led to tissue damage in fish bodies, as observed in tissue sections. Subsequent analysis of differential genes (DEGs) in the transcriptome highlighted genes linked to apoptosis and inflammation. Through experiments involving overexpression and interference of c1qb in vitro, we confirmed that c1qb could suppress N. seriolae-induced apoptosis and inflammation. Moreover, overexpression of c1qb hindered N. seriolae invasion, and the purified and replicated C1qb protein displayed antimicrobial properties. Additionally, our study unveiled that overexpression of c1qb might stimulate the expression of membrane attack complexes (MAC), potentially enhancing opsonization and antibacterial effects. In conclusion, our findings offer valuable insights into the immune antibacterial mechanisms of c1qb and contribute to the development of strategies for controlling N. seriolae.
Assuntos
Apoptose , Complemento C1q , Complexo de Ataque à Membrana do Sistema Complemento , Inflamação , Nocardia , Complemento C1q/metabolismo , Complemento C1q/genética , Apoptose/genética , Animais , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Inflamação/genética , Inflamação/metabolismo , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Nocardiose/imunologia , Nocardiose/microbiologia , Nocardiose/metabolismo , Nocardiose/genéticaRESUMO
Compensatory growth (CG) in fish is heavily influenced by nutrient metabolism. However, there are limited studies examining how nutrient metabolism is regulated during this process. For silver pomfret, an important commercial marine fish, it's crucial to establish effective starvation and re-feeding strategies to ensure good water quality and fast growth. To identify the complete compensatory growth model of silver pomfret, we conducted an experiment with a control group (normal feeding) and three starvation/re-feeding groups. We observed that the recovery of weight and condition factor in the 14-day starvation and 14-day re-feeding groups was significantly faster than other groups, indicating full compensatory growth. Thus, we selected this group for the next experiment. We performed untargeted metabolomics and transcriptome analysis of muscle tissue on Day 14, 21 and 28 (CG process), and examined the key regulatory genes of nutrient metabolism on Day 0, 7, 14, 21 and 28 (starvation and re-feeding process). Our data revealed that during starvation, silver pomfret first utilized carbohydrates and short-chain lipids, followed by proteins and long-chain lipids. After re-feeding, lipids accumulated first, resulting in rapid growth, followed by the recovery of protein content in muscle. During starvation, the expression of anabolic-related genes such as TER and CALR decreased, and catabolic-related genes such as TSC2 and MLYCD increased, promoting the AMPK pathway. During re-feeding, anabolic-related gene expression increased without AMPK inhibition. Our findings provide insights into the energy utilization strategies of fish and molecular regulation during compensatory growth in fish.
Assuntos
Proteínas Quinases Ativadas por AMP , Perfilação da Expressão Gênica , Animais , Músculos , LipídeosRESUMO
Toll-like receptors (TLRs) are vital pattern recognition receptors that play a critical role in the innate immune response against pathogenic attack. Among the bacteria commonly found in the culture process of silver pomfret, Photobacterium damselae subsp. Damselae (PDD, gram-negative) and Nocardia seriolae (NS, gram-positive), can cause large-scale mortality in this fish species. However, there is currently no research on the role of TLRs in mediating the immune response of silver pomfret to these two bacterial infections. Therefore, in this study, we identified nine PaTLRs family members, including several fish-specific TLRs (TLR14 and TLR21). Phylogenetic analysis revealed that these PaTLRs genes could be classified into five subfamilies, namely TLR1, TLR3, TLR5, TLR7, and TLR11, indicating their evolutionary conservation. To further explore the interactions of TLR genes with immune-related mediators, protein and protein interaction network (PPI) results were generated to explain the association of TLR genes with TNF receptor-associated factor 6 (TRAF6) and other relevant genes in the MyD88-dependent pathway and NF-κb signaling pathway. Subsequently, RT-qPCR was conducted to verify the expression patterns of the nine TLR genes in the gills, skin, kidney, liver, and spleen of healthy fish, with most of the TLRs showing high expression levels in the spleen. Following infection with PDD and NS, these PaTLRs exhibited different expression patterns in the spleen, with PaTLR2, PaTLR3, PaTLR5, PaTLR7, PaTLR9, and PaTLR14 being significantly up-regulated. Furthermore, when spleen cells were treated with bacterial compositions, the majority of PaTLRs expression was up-regulated in response to Lipopolysaccharide (LPS) and lipophosphorylcholic acid (LTA) treatment, except for PaTLR21. Finally, changes in the expression levels of TLR-interacting genes were also observed under the stimulation of bacteria and bacterial compositions. The results of this study provide a preliminary reference for further understanding the mechanism of the innate immune response of the TLR gene family in silver pomfret and offer theoretical support for addressing the disease problems encountered during large-scale fish breeding.
Assuntos
Doenças dos Peixes , Perciformes , Animais , Filogenia , Receptores Toll-Like , Photobacterium , Imunidade Inata/genéticaRESUMO
We cultured silver pomfret for 20 days, decreasing water temperature from 18 to 8 â, and sampled muscle every 5 days. Muscle fiber degeneration and apoptosis began to increase at 13 â detected by HE and TUNEL staining. Further analysis of transcriptome revealed that several apoptosis-related pathways were highly enriched by differentially expressed genes (DEGs). We analyzed 10 DEGs from these pathways by RT-qPCR during the temperature-decreasing process. JNK1, PIDD, CytC, Casp 3, and GADD45 were up-regulated after 15 and 20 days, while DUSP3, JNK2, and PARP genes were down-regulated after 15 and 20 days. DUSP5 was up-regulated from 10 to 20 days, and C-JUN was up-regulated after 20 days. We analyzed apoptosis in PaM cells under different temperatures (26 â, 23 â, 20 â, 17 â, and 14 â). The cell viability significantly declined from 14 to 20 â; the TUNEL and IHC results showed that the apoptosis signal increased with the temperature dropping, especially in 17 â and 14 â; DUSP5, JNK1, CytC, C-JUN, Casp 3, and GADD45 were up-regulated at 17 â and 14 â, and PIDD was up-regulated at 20 â, 17 â, and 14 â. DUSP3 was up-regulated at 20 â but down-regulated at 17 â and 14 â, and PARP was down-regulated at 17 â and 14 â. JNK2 was up-regulated at 20 â but down-regulated at 17 â and 14 â. Our results suggest that DUSP could help inhibit apoptosis in the initial stage of cold stress, but low temperature could down-regulate it and up-regulate JNK-C-JUN, inducing apoptosis in a later stage. These data provide a basis for the study of the response mechanism of fish to cold.
Assuntos
Sistema de Sinalização das MAP Quinases , Proteína Quinase 8 Ativada por Mitógeno , Animais , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/genética , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Proteína Quinase 9 Ativada por Mitógeno/farmacologia , Fosforilação , Resposta ao Choque Frio , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , ApoptoseRESUMO
Copper pollution might have a negative effect on collagen metabolism in fish. To test this hypothesis, we exposed an important economical fish, silver pomfret (Pampus argenteus), to three concentrations of Cu2+ for up to 21 days to simulate natural exposure to copper. With increasing copper exposure concentration and time, hematoxylin and eosin staining and picrosirius red staining revealed extensive vacuolization, cell necrosis, and tissue structure destruction, and a change of type and abnormal accumulation of collagen in the liver, intestine, and muscle tissues. To further study the mechanism of collagen metabolism disorder caused by copper exposure, we cloned and analyzed a key collagen metabolism regulation gene, timp, of silver pomfret. The full-length timp2b cDNA was 1035 bp with an open reading frame of 663 bp, encoding a protein of 220 amino acids. Copper treatment significantly increased the expression of akts, erks, and fgfs genes and decreased the mRNA and protein expression of Timp2b and MMPs. Finally, we constructed a silver pomfret muscle cell line (PaM) for the first time and used PaM Cu2+ exposure models (450 µM Cu2+ exposure for 9 h) to examine regulation function of the timp2b-mmps system. We knocked down or overexpressed timp2b in the model, and found that downregulation of mmps expression and upregulation of akt/erk/fgf were further aggravated in the timp2b- group (subjected to RNA interference), whereas some recovery was achieved in the timp2b+ group (overexpression). These results indicated that long-term excessive copper exposure can lead to tissue damage and abnormal collagen metabolism in fish, which might be caused by the alteration of akt/erk/fgf expression, which disrupts the effects of the timp2b-mmps system on extracellular matrix balance. The present study assessed the impact of copper on the collagen of fish and clarified its regulatory mechanism, providing a basis for toxicity of copper pollution study.