Abstract: The aim of this study was to investigate the protective effect and mechanism of action of hesperidin (HDN) on high-fat feeding-induced hepatic oxidative stress injury in mice. In this study, 18 male C57BL/6 (body weight 20-23 g) mice were randomly divided into a control group, a high-fat diet (HFD) group, and a high-fat diet + hesperidin (HFD+HDN) group (300 mg·kg^-1) of six mice each. The mice in the control group were fed with basal diet (10% fat, 70% carbohydrate, 20% protein); mice in the HFD group were fed with high-fat diet (60% fat, 20% carbohydrate, 20% protein); and mice in the HFD+HDN group were fed with high-fat diet while HDN was administered by gavage at 300 mg·kg^-1 per day. After 16 weeks, mice were anaesthetised by intraperitoneal injection of 3% sodium pentobarbital (60 mg·kg^-1) and then blood was collected from the eyeballs. After blood collection, mice were decapitated and dissected for liver tissues. The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which are indicators of hepatic function, were detected using kits in the blood; The levels of malondialdehyde (MDA), activities of total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), and the level of total antioxidant capacity (T-AOC), which are indicators of oxidative stress, were detected using the kits in the liver tissues;The liver tissues were also collected for transcriptomic sequencing to screen differentially expressed genes between the HFD and HFD+HDN group for KEGG pathway enrichment analysis, with P<0.05 as the threshold for significant enrichment, and the most significant signalling pathway affected by HDN intervention was screened accordingly. Genes with significant changes from the screened signalling pathway were selected to validate the transcriptomics results by qRT-PCR and protein immunoblotting methods, the relative content of mtDNA and the relative expression of the mitochondrial outer membrane protein(TOMM20), as well as the liver ATP content were detected. The results showed that, compared to the HFD group, the HDN intervention improved high-fat feeding-induced liver damage, significantly reduced ALT and AST activity in the blood of mice (P<0.01); Significantly reduced liver MDA levels in mice (P<0.01); Significantly increased T-AOC, T-SOD and GSH-Px levels (P<0.05); Compared to the HFD group, the HFD+HDN group of which 889 genes were up-regulated (P<0.05) and 405 genes were down-regulated (P<0.05); KEGG enrichment analysis screened the oxidative phosphorylation pathway as the most significantly up-regulated signaling pathway (P<0.000 1), Compared with the HFD group, the relative mRNA expression of Cox8b, Cox6a2, Gm10231, mt-Atp8, mt-Nd4l, Gm11237, Ndufb8, and Ndufb10 were significantly up-regulated in the liver tissues after the HDN intervention (P<0.05), and the relative mRNA expression of Atp6v0d2, Cox6c2 were significantly down-regulated (P<0.05). Protein expression of Cox6a2, Ndufb8, Ndufb10 were significantly increased (P<0.05), and the expression of Atp6v0d2d protein expression were significantly decreased (P<0.01), which was consistent with the transcriptomics results; And the relative expression, relative mtDNA content, and ATP content of TOMM20 were significantly higher after HDN intervention (P<0.05) compared with that of the HFD group. These results indicated that HDN reduces high-fat feeding-induced oxidative stress in mouse liver by modulating the oxidative phosphorylation pathway.

Key words: hesperidin, high-fat, transcriptome sequencing, KEGG enrichment analysis, oxidative phosphorylation pathway, oxidative stress