The Effect of Cu2+ Exposure on the Nrf2 Signaling Pathway of Tilapia Hepatocyte, Base on Experiments In Vitro

Abstract

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.