miR-199-5p mediates the regulation of autophagy by targeting mTOR signaling and involvement in ammonia detoxification under ammonia stress in yellow catfish (Pelteobagrus fulvidraco)

Ammonia is a widely distributed hazardous aquatic pollutant that poses a threat to the development of aquatic organisms. A two-stage study was carried out to the response of yellow catfish to ammonia stress. At stage 1, yellow catfish were subjected to 0 mg/L total ammonia nitrogen (T-AN, CON group) and 125 mg/L T-AN (AM group) for 96 h, respectively. At stage 2, yellow catfish in AM group were transferred to water containing 0 mg/L T-AN for a 48-h recovery experiment (AMR group). Ammonia exposure significantly increases serum ammonia (SA), total cholesterol (T-CHO), triglycerides (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels, while decreases glucose (Glu) level. Only SA, GLU, TCHO, and AST returned to normal levels in AMR group, while extensive hepatocyte necrosis and vacuolization persisted. The results showed that the liver possesses a certain degree of ammonia-lowering capacity, yet ammonia-induced liver damage is irreversible. Ammonia stress-induced 486 down-regulated and 18 up-regulated microRNAs (miRNAs). Functional enrichment analysis of the top 10 miRNAs highlighted significant effects of ammonia on the "mTOR signaling pathway" and "autophagy-animals". Furthermore, the significant negative correlation between the expression levels of miR-199-5p and mTOR suggests that mTOR as a potential target gene of miR-199-5p. The levels of LC3B, autophagosomes (AP), and autolysosomes (AL) in the AM group decreased significantly after 24 h of ammonia stress but returned to baseline after 96 h. Additionally, increased numbers of AP and AL were observed in the AMR group, suggesting that hepatic autophagy is initially suppressed under ammonia stress, followed by spontaneous recovery. This study also found that the expression levels of urea and glutamine synthesis-related genes (cps 1; arg; otc; ass; asl, and gs) significantly increased in the AM and AMR groups. Additionally, the contents of urea and glutamine in the liver was significantly elevated in the AM group. This indicates a simultaneous occurrence of urea and glutamine synthesis with the activation of autophagy during ammonia detoxification. The miR-199-5p/mTOR axis may regulate liver autophagy, potentially contributing to ammonia detoxification. The study has provided novel targets for enhancing ammonia tolerance in fish through nutritional regulation.