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Copper is a common component of industrial heavy metal waste and a major component of some fish medicines, which can cause oxidative stress and damage the health of farmed fish. The Nrf2 signaling pathway is an important pathway related to the oxidative stress on vertebrates. Exploring the effect of copper on the Nrf2 signaling pathway in fish hepatocytes would help improve the understanding of the molecular mechanism of antioxidants in fish hepatocytes and provide theoretical data for relevant toxicological research. Adult tilapia were cultured under properly controlled conditions for two weeks to adapt to laboratory culture conditions. Primary tilapia hepatocytes were obtained by cell culture. MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was used to detect the effect of copper ions on the viability of tilapia hepatocytes. The lipid peroxidation level (MDA) and antioxidant ability of tilapia hepatocytes (SOD and CAT activity) were detected. Quantitative PCR (qPCR) was used to detect the differential expression of each gene (Nrf2, Keap1a, Keap1b, CuZnSOD, MnSOD, HO-1, and GSTA) in the Nrf2 signaling pathway. The results suggested that after treatment with 100 μM copper ions for 4 h, 8 h, and 24 h, the viability of hepatocytes significantly decreased (p < 0.05). LDH and MDA after 8 h and 24 h treatment were significantly higher than those in the control group (p < 0.05). CAT activity significantly decreased after 4 h (p < 0.05), and SOD activity significantly decreased after 8 h and 24 h (p < 0.05). The results of qPCR showed that the expression of MnSOD significantly increased after a treatment with copper ions for 4 h, and the expression of Nrf2, Keap1a, CuZnSOD, HO-1 as well as GSTA significantly increased after a treatment with copper ions for 8 h, compared with the control group (p < 0.05). After being treated with copper ions for 24 h, the expression of Nrf2 and CuZnSOD significantly increased compared with the control group (p < 0.05). There was no significant difference in the expression of Keap1b or CAT at each time point. In conclusion, with copper ions exposure, the viability of tilapia hepatocytes was reduced, causing lipid peroxidation, a reduction in the antioxidant capacity of cells, the activation of the Nrf2 signaling pathway, and the increase in the expression of most genes in this pathway, which are defensive responses of hepatocytes to oxidative stress caused by copper ions. This study can provide theoretical data for related toxicological research.

Summer outbreaks of the hepatobiliary syndrome in fish impose a heavy burden on aquaculture in China. Curcumin is a polyphenol with antioxidant activity that has been used to protect the health of fish livers, but the mechanism underlying its protective effect is unclear. In this study, an in vitro model of hepatocyte oxidative damage in Oreochromis niloticus was established using H2O2. Treatment with 5 mM H2O2 for 2.5 h markedly reduced cell viability and antioxidant activity and elevated lactate dehydrogenase (LDH) activity, indicating conditions that can be used to establish an oxidative stress model. Under H2O2 stress, curcumin pretreatment significantly maintained cell viability, reduced malondialdehyde (MDA) levels, and increased superoxide dismutase (SOD) activity. RNA-seq results showed that acute H2O2 treatment resulted in minor changes in gene expression, whereas curcumin changed the expression profile and affected cytochrome P450 (Cyp 450), glutathione (GSH) metabolism, and the peroxisome proliferator-activated receptor (PPAR) signaling pathway. Several critical antioxidant defense signaling pathways were identified, and altered expression was confirmed by q-PCR. These results indicate that curcumin might upregulate PPAR expression by increasing Cyp2J2 expression. Further experiments showed that curcumin can upregulate the Nrf2-Keap1 signaling pathway at the transcriptional level, and this upregulation can induce downstream defense genes, including glutamate cysteine ligase catalytic subunit(GCLC) and glutamate cysteine ligase modifier subunit (GCLM), and thereby promote GSH synthesis and the expression of related antioxidases. This study might shed light on the effects of curcumin on the prevention and alleviation of liver diseases in fish.

Curcumin is a polyphenol with antioxidant activity that has been used to protect the health of fish livers. Our previous studies about comparative transcriptome have shown that curcumin can enhance the Nrf2-Keap1 signaling pathway and induce downstream anti-stress genes to maintain cell viability. However, the possible role of miRNAs in the protective mechanism of curcumin is not understood. In this study, the tilapia hepatocyte H2O2 stress model was used, and the miRNA expression profile for four groups (control group, curcumin group, H2O2 group, and protection group) were established by high-throughput sequencing. In our results, 278–333 types of Oreochromis niloticus miRNAs, 309–543 types of conserved miRNAs, and 535–746 types of novel miRNAs were identified in different samples. Differentially expressed miRNAs were identified by comparing miRNA expression profiles among the four groups. The expression levels were confirmed by q-PCR. The target genes of these differentially expressed miRNAs were predicted, and their functional annotations were enriched by GO and KEGG analysis, which revealed that many target genes were involved in “response to stimulus” and “antioxidant activity” in each pair of groups. Several miRNAs related to oxidative stress showed differential expression. For example, in the H2O2 group, the expression of miR-122 was decreased, and the expression of miR-21 and miR-489 increased significantly. In the curcumin group, the expression of miR-153b was decreased, and the expression of miR-200a and miR-29 was increased significantly. miR-153b, miR-200a, and miR-29 may be involved in the regulation of the Nrf2-Keap1 signaling pathway by curcumin. This work might provide insights into the molecular mechanisms of miRNA regulation of curcumin on the prevention and alleviation of liver diseases in fish.