BUSCADOR RECOLECTA

Ultra Performance Liquid Chromatography coupled with a high-resolution quadrupole-orbitrap mass spectrometry DATA

Cantharidin, a toxic monoterpene secreted by blister beetles, has long been used in traditional Chinese and modern medicine for its unique properties. However, despite its widespread use, its effects on fish have not been studied in depth. The aim of this study was to evaluate the potential therapeutic applications of cantharidin in fish by examining its antioxidant, hemoagglutinating, hemolytic and cytotoxic activities at different concentrations (0, 0.625, 1.25, 2.5, 5 and 10 μg mL-1) in three different cell lines. In addition, the study explored the bactericidal and bacteriostatic properties of cantharidin against various fish pathogenic bacteria. The results revealed that there were no significant differences in antioxidant, hemagglutinating or hemolytic activities between the different concentrations of cantharidin tested. However, the study found that cantharidin exhibited dose- and time-dependent cytotoxicity in sea bream (Sparus aurata) erythrocytes and in SAF-1, PLHC-1 and Hela cell lines, resulting in morphological changes indicative of apoptosis. Interestingly, the highest dose of cantharidin tested demonstrated potent bactericidal activity against four marine fish opportunistic bacteria, including Vibrio harveyi, V. anguillarum, Photobacterium damselae and Tenacibaculum maritimum, but no statistically significant changes in bacteriostatic activity were observed against any of the bacteria tested. Overall, these results provide valuable information on the potential therapeutic applications of cantharidin in fish aquaculture. Further research is needed to fully understand the mechanisms of action and to explore possible preventive uses of cantharidin in fish.

The role of hepcidins, antimicrobial peptides involved in iron metabolism, immunity, and inflammation is studied. First, gilthead seabream (Sparus aurata L.) head-kidney leucocytes (HKLs) were incubated with λ-carrageenin to study the expression of hepcidin and iron metabolism-related genes. The expression of most of the genes studied was up-regulated, whereas the ferroportin gene (slc40a) was down-regulated in HKLs incubated with λ-carrageenin. In the second part of the study, seabream specimens were injected with λ-carrageenin or buffer (control). The expression of the same genes was evaluated in the head kidney, liver, and skin at different time points after injection. The expression of Hamp1m, ferritin b and ferroportin genes (hamp1, fthb, and slc40a) was up-regulated in the head kidney of fish from the λ-carrageenin group, while the expression of Hamp2C and Hamp2E genes (hamp2.3 and hamp2.7) was down-regulated. In the liver, the expression of hamp1, ferritin a (ftha), slc40a, Hamp2J and Hamp2D (hamp2.5/6) genes was down-regulated in the λ-carrageenin group. In the skin, the expression of hamp1 and (Hamp2A Hamp2C) hamp2.1/3/4 genes was up-regulated in the λ-carrageenin group. A bioinformatic analysis was performed to predict the presence of transcription factor binding sites in the promoter region of hepcidins. Structural and physicochemical variations were found in the different mature hepcidin peptides. This study sheds light on the poorly understood roles of hepcidins in fish. The results provide insight into the regulatory mechanisms of inflammation in fish and could contribute to the development of new strategies for treat inflammation in farm animals.

Carrageenan, a high molecular weight sulphated polysaccharide, has been used in the screening of anti-inflammatory molecules in mammals. In this sense, Chiliadenus glutinosus (L.) Fourr., an endemic species of plant has been reported in the Spanish popular medicine due to their anti-inflammatory properties in humans. However, their properties have poorly been studied in fish. In this assay, different concentrations (0, 0.001, 0.01, 0.125, 0.25, 0.5 and 1 mg mL-1) of aqueous, ethanolic and methanolic extracts of this plant were elaborated, and the antioxidant, bactericidal (against four marine fish pathogen) and cytotoxic activity, as well as several immunological parameters in head-kidney leucocytes (HKLs) of gilthead seabream (Sparus aurata) were evaluated in a first experiment to determinate the extract with the best properties. Our results showed a dose-dependent antioxidant activity of each plant extract increasing with the concentration, a species- and extract-specific bactericidal activity, as well as a cytotoxic activity against the PLHC1 tumour cell line by the ethanolic and methanolic extracts. However, taking into account the cytotoxic effects of these two extracts on HKLs and the strong immunosuppressive effects on the immunological parameters assayed (peroxidase, respiratory burst activity and phagocytic capacity), a second experiment was developed by using only adjusted doses of the aqueous extract (0, 0.25 and 0.5 mg mL-1), in which immune parameters, cell morphology and inflammatory-related genes were studied in HKLs stimulated with λ-carrageenan. The present results offer a detailed view of the biological effects of C. glutinosus extracts, which could be interesting to the aquaculture sector due to their possible anti-inflammatory effects.

Cantharidin is a natural compound with known therapeutic applications in humans. However, its properties have hardly been studied in fish. The aim of this study was to investigate the in vitro effects of cantharidin on gilthead seabream (Sparus aurata) head kidney leucocytes (HKL) stimulated with λ-carrageenan. Thus, HKLs were incubated for 24 h with cantharidin (0, 2.5 and 5 μg mL-1) and λ-carrageenan (0 and 1,000 μg mL-1) and cell viability and morphology, immunological parameters and several selected genes related to inflammation and apoptosis were evaluated. The results showed that cantharidin only decreased HKL viability when incubated with the highest dose (5 μg mL-1) and in combination with λ-carrageenan. As for the immunological parameters studied, cantharidin increased the peroxidase activity of HKLs only when incubated in combination with λ-carrageenan, but inhibited the respiratory burst and phagocytic activities even after being previously increased by λ-carrageenan. Furthermore, cantharidin induced morphological changes in HKLs with apoptotic and vacuolization signs that were enhanced when incubated in combination with λ-carrageenan. Considering the analysis of gene expression in HKLs, although λ-carrageenan up-regulated the expression of the proinflammatory gene il1b, cantharidin down-regulated its expression in HKLs exposed to both λ-carrageenan and cantharidin. In addition, cantharidin up-regulated casp3 and casp9 expression, which was down-regulated in HKLs incubated with both molecules. This combination also up-regulated casp1 expression. The present results provide detailed insight into the immune-depressive and anti-inflammatory properties of cantharidin on immune cells, which could be of interest to the aquaculture sector.