Abstract

Heat stress (HS) triggers ferroptosis-associated liver injury in piglets • HS disrupts redox homeostasis and mitochondrial integrity in the liver • Time-restricted feeding (TRF) reverses ferroptotic signatures induced by HS • NR1D1 transcriptionally represses key ferroptosis-defense genes under HS • TRF restores NR1D1 promoter occupancy to limit hepatic ferroptosis Background Heat stress (HS) disturbs hepatic metabolic homeostasis and redox balance, compromising antioxidant capacity and mitochondrial function. Ferroptosis, a lipid peroxidation-associated form of regulated cell death, has been implicated in redox-sensitive metabolic disorders. Whether nutritional timing influences HS-associated ferroptotic responses in the liver remains unclear. This study examined the effects of time-restricted feeding (TRF) on hepatic redox regulation and ferroptosis-related molecular pathways under chronic heat exposure. Methods Piglets were exposed to cyclic heat challenge (35°C, 4 h/day) for 65 days with or without TRF intervention. Hepatic oxidative status, mitochondrial function, and ferroptosis-associated gene and protein expression were evaluated using biochemical assays, transcriptomic analysis, RT-qPCR, western blotting, and chromatin immunoprecipitation. Results Chronic heat exposure was associated with decreased hepatic glutathione levels, increased lipid peroxidation, reduced antioxidant enzyme activities, impaired mitochondrial ultrastructure, and lower ATP production ( P < 0.05). Transcriptomic profiling revealed coordinated changes in ferroptosis-related genes, including increased TFRC, ACSL4 , and HMOX1 expression and decreased GPX4, SLC7A11 , and NFE2L2 expression ( P < 0.05). Heat exposure was accompanied by elevated NR1D1 expression and altered promoter occupancy at redox-related genes. TRF partially restored antioxidant status, improved mitochondrial integrity, attenuated ferroptosis-associated transcriptional alterations, and modulated NR1D1 expressio