Mitigation of the toxic effects of nitrite: Role and mechanism of isoleucine in mitigating mitochondrial DNA leakage-induced inflammation in grass carp (Ctenopharyngodon idella) under nitrite exposure.

The physiological and growth processes of fish are closely associated with their surrounding environment. This study investigated the role and underlying mechanisms of isoleucine (Ile) in alleviating mitochondrial DNA (mtDNA) leakage-induced inflammation in grass carp under nitrite exposure. Grass carp were fed six experimental diets containing different Ile levels (0.00, 3.00, 6.00, 9.00, 12.00 and 15.00 g/kg) for 9 weeks, followed by a 96-hour nitrite exposure trial. Ile supplementation mitigated the deterioration of blood parameters including glutamic oxaloacetic transaminase (GOT), glutamic alanine transaminase (GPT), glucose, cortisol and lactate dehydrogenase (LDH) induced by nitrite exposure. Additionally, Ile enhanced its transport to the liver and mitochondria, as well as increased metabolism of Ile in mitochondria. Histological analyses revealed that Ile mitigated nitrite exposure-induced liver damage and mitochondrial cristae disruption. Furthermore, Ile preserved the mitochondrial cristae homeostasis by upregulating key proteins involved in mitochondrial structure maintenance, while inhibiting mtDNA leakage. Mechanistically, Ile attenuated mtDNA leakage-induced inflammation under nitrite exposure associated with the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-Stimulator of Interferon Genes (STING) and NOD-like receptor thermal protein domain associated protein 3 (NLRP3) pathways. These findings highlight the protective role of Ile in reducing inflammation triggered by environmental nitrite exposure, offering new insights into aquatic toxicology, and determined that Ile concentration of 11.13 g/kg diet could be optimal for mitigating nitrite-induced stress in grass carp, providing a theoretical basis for formulating anti- nitrite stress diets in aquaculture.