Atrazine induces necroptosis by miR-181-5p targeting inflammation and glycometabolism in carp lymphocytes

Atrazine (ATR) causes environmental problems and damages the health of fish and aquatic animals. MicroRNAs (miRNAs) play important roles in immune regulation. However, the immunotoxicity mechanism of ATR in fish lymphocytes and the role of miRNA in this process remain unclear. To further study these mechanisms, spleen lymphocytes were exposed to 20, 40 and 60 mu g/ml ATR for 18 h. Fluorescence staining and flow cytometry showed that the number of necrotic lymphocytes increased after ATR exposure. Compared with the control group, the mRNA expression of miR-181-5p was inhibited and the mRNA levels of TNF-alpha and HK2 were increased after ATR exposure. Additionally, the NF-kappa B inflammatory pathway and the levels of glycometabolism-related genes were upregulated. These results suggest that ATR induces inflammation and elevates glycometabolism in lymphocytes. We further found that the mRNA levels of receptor-interacting serine-threonine kinase 1 (RIP1), receptor-interacting serine-threonine kinase 3 (RIP3), mixed lineage kinase domain-like pseudokinase (MLKL), cylindromatosis (CYLD) and Fas-Associated protein with Death Domain (FADD) and the protein levels of RIP3 and MLKL. in the treatment groups were significantly increased compared to those in control group, suggesting that ATR causes lymphocyte necroptosis. We conclude that miR-181-5p plays a key role in necroptosis in carp lymphocytes exposed to ATR by downregulating the expression of HK and TNF-alpha, which increases the level of glycometabolism and induces the inflammatory response, respectively.