Metabolomic analysis of the effect of tocotrienol-rich fraction on rat hepatic changes during ageing

Abstract

Ageing is a major contributor to metabolic dysfunction and chronic liver diseases. As the liver is a metabolic hub regulating energy metabolism, detoxification, and redox homeostasis, age-related hepatic alterations have health-related consequences. Ageing is associated with progressive structural and metabolic changes in the liver, contributing to oxidative stress and inflammation. Thus, understanding the mechanisms underlying liver ageing is essential for developing strategies to preserve metabolic health during ageing. TRF has been reported to possess antioxidant and anti-inflammatory properties, to modulate energy metabolism, and to counteract age-associated hepatic oxidative and metabolic disturbances. However, the relationship between oxidative stress, inflammation, histological alterations, and metabolic pathway changes during liver ageing, as well as the potential modulatory role of TRF, remain unclear. Metabolomics enables comprehensive profiling of small-molecule metabolites, facilitating the identification of age-related pathway-level alterations. This method provides insight into how TRF may modify age-related metabolic abnormalities and complements the biochemical and histological evaluations. Nevertheless, this platform has not been extensively applied in this context. Therefore, this study aimed to characterise age- related changes in hepatic oxidative stress, inflammatory markers, histology, and metabolic pathways, and to determine whether TRF modulates these alterations using an untargeted metabolomics approach. Sprague-Dawley rats aged 3, 9, and 21 months were assigned to control and treatment groups, receiving either palm olein or TRF supplementation. Liver tissues were analysed using untargeted metabolomics via LC- MS/MS, along with histological evaluation and biochemical assessments of antioxidant enzymes (SOD, CAT), inflammatory markers (IL-6, TNF-alpha), and lipid peroxidation markers (MDA). SOD activity declined significantly with age in both the control and TRF-treated old groups (p<0.05), suggesting reduced antioxidant defence with increasing age, whereas no significant changes were observed in CAT, IL-6, or MDA levels. TNF-a was significantly higher in the old TRF-treated group than in the young TRF- treated group. Histologically, TRF-treated livers displayed both milder fibrotic-like alterations and inflammatory cell infiltration than control livers. Metabolomics revealed age-related alterations in taurine and hypotaurine metabolism, pyrimidine metabolism (uridine and uracil) and TCA cycle components (malate and glutamate). These findings suggest that TRF might modulate age-related liver changes in morphology and metabolism, despite a persistent decline in antioxidant activity. It provides insight into key metabolic pathways involved, offering a foundation for future therapeutic strategies targeting liver ageing.