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QNZ is a quinazoline-type NF-κB inhibitor and is one of the hot anti-inflammatory drug candidates in recent years. With its development and application, QNZ will inevitably enter the aquatic environment posing a threat to aquatic organisms. To investigate the potential toxicity of QNZ in the early life stages of the organism, this study exposed embryos of large-scale loach (Paramisgurnus dabryanus) to 0, 20, 40, 60, and 80 nM of QNZ. The hatching of embryos was significantly inhibited and hatching time was delayed. We explored the mechanism of hatching delay and failure. The results suggested that QNZ exposure reduced the number of hatching gland cells (HGCs) and hatching enzyme activity. Also, the frequency of spontaneous movements was inhibited by interfering with the expression of genes related to the cholinergic system and skeletal muscle development. Further, QNZ exposure induces a series of morphological changes (spine deformation, pericardial edema, tail deformation, and yolk sac edema) in embryos and newly-hatched larvae, and finally increased the deformity rate and mortality rate of newly-hatched larvae. The information presented in this study will provide a scientific basis for further studies into the potential toxicity of QNZ on aquatic organisms. [Display omitted] ● QNZ causes increased embryo mortality, hatching inhibition, ultimately abnormal larval development. ● QNZ inhibited the hatching of embryos through decreasing the number of HGCs and activity of hatching enzyme. ● QNZ affected the spontaneous movements of embryos by interference related systems, further inhibiting the hatching. [ABSTRACT FROM AUTHOR]

Hsp70 is the most strongly induced in response to various cellular stresses and a good candidate for investigating its role in tissue injury. We firstly cloned full-length cDNA of hsp70 from Paramisgurnus dabryanus ( PdHsp70 ) by RACE method (GenBank: KP402408.1 ). Then regeneration and inflammation of fin were established by amputation and scratch respectively. Quantitative RT-PCR detected the PdHsp70 began to increase rapidly its expression at 1 days post amputation (dpa) and reached the peak at 2 dpa during fin regeneration. Its expression was also up-regulated at 2 days post scratch (dps) of inflammation but still significant weaker in comparison with it in regenerated fin at 2 dpa. Next, immunohistochemistry analysis of PdHsp70 showed that PdHsp70 located mainly in the deeper epidermis of regenerated fin and was stronger than its expression in the scratched inflammatory fin which was involved in whole epidermal. SDS-PAGE and Western blotting confirmed that the PdHsp70 protein expressed efficiently in Escherichia coli BL21. These findings have implied that PdHsp70 are implicated in different regulation of regeneration and inflammation in response to injury stimulation. During the regeneration, it is involved in the formation of wound epidermis by mediating cellular protection whereas it can modulate inflammatory by activating the innate immune response. [ABSTRACT FROM AUTHOR]

Abstract: Heat shock protein 60 (Hsp60) is a kind of highly conserved immunogenic molecule involved in a wide range of biochemical processes in response to external stressors. Its multifunction in regulating immune responses and modulating signal pathway interests us in investigating its role in fin regeneration that has become an excellent and interesting model for studying the molecular basis of morphogenesis. We firstly clarified basical process and crucial period of caudal fins regeneration in Paramisgurnus dabryanus by histological analysis. Then we cloned full-length cDNA of hsp60 from P. dabryanus (designated as PdHsp60) by RACE method. The cDNA contains a 124 bp 5′UTR, a 1731 bp open reading frame (ORF) encoding 576 amino acids and a 510bp 3′UTR (Accession no.: KF544774). The phylogenetic tree shows that the PdHsp60 fits within the hsp60 clade. And quantitative RT-PCR detected the PdHsp60 began to increase rapidly its expression at 1 dpa and reached its peak at 2 dpa. Next, spatial distribution analysis of PdHsp60 in fins showed that PdHsp60 located mainly in the deeper lay of regenerated epidermis when PdHsp60 expressed most. After the PdHsp60 had been cloned into the pET-32a vector, SDS-PAGE and Western blotting analysis confirmed that the PdHsp60 protein was efficiently expressed in Escherichia coli BL21. These findings have revealed that PdHsp60, a highly conserved gene related to the innate immune system and stress response during vertebrate evolution, is involved in response to wounding stimulation—in the formation of wound epidermis which occurs as the first phase of fin regeneration after fin amputation in caudal fin regeneration. [Copyright &y& Elsevier]