# Ferulic Acid Protects Against LPS-Induced Sheep Hepatocytes Oxidative Damage via Activating the GSH-GPX4 Pathway and Inhibiting Lipid Metabolism-Mediated Ferroptosis

## TL;DR

Ferulic acid protects sheep liver cells from LPS-induced damage by reducing oxidative stress and preventing ferroptosis through the GSH-GPX4 pathway.

## Contribution

This study reveals a novel protective mechanism of ferulic acid against LPS-induced oxidative damage in sheep hepatocytes via the GSH-GPX4 pathway and lipid metabolism.

## Key findings

- Ferulic acid reduced MDA and LDH levels, indicating decreased oxidative damage in LPS-treated hepatocytes.
- Ferulic acid modulated lipid metabolism and glutathione pathways, inhibiting ferroptosis and restoring GSH levels.
- Transcriptomics and metabolomics confirmed that ferulic acid attenuates LPS-induced gene expression changes linked to ferroptosis.

## Abstract

Lipopolysaccharide (LPS) triggers oxidative damage in sheep hepatocytes, linked to ferroptosis. Ferulic acid (FA) is known for its antioxidative properties, but its protective role against LPS via ferroptosis regulation was unclear. The objective of this research is to explore the protective role of FA in mitigating LPS-induced oxidative stress in sheep hepatocytes. The experimental setup consisted of three groups: a control group, an LPS group treated with 10 µg/mL of LPS, and FA group that received both 10 µg/mL of LPS and 750 µg/mL of FA. We found that FA treatment decreased in contents of MDA and LDH. Metabolomics revealed that LPS affected glycerophospholipid metabolism, unsaturated fatty acids biosynthesis, ferroptosis, and arachidonic acid metabolism mainly by reducing the level of PUFAs and LPC in the hepatocyte supernatant, while FA affected glutathione metabolism by increasing L-cysteine, L-ornithine, L-glutamic acid, and L-glutamine. Moreover, transcriptomics demonstrated that the comparison of LPS and control groups were mainly enriched in arachidonic acid metabolism, glycerophospholipid metabolism, and ferroptosis, the comparison of FA and LPS groups was mainly enriched in glutathione metabolism. The results further confirmed the findings in the metabolomics and transcriptomics analyses, showing that LPS treatment upregulated the mRNA expression of ACSL4, LPCAT3, ALOX15, STEAP3, GPX4, GCLC, and GCL in hepatocytes, while reducing GSH and GR levels. In contrast, FA intervention attenuated LPS-induced iron overload by decreasing Fe2+ accumulation and suppressing the mRNA expression of ACSL4, LPCAT3, STEAP3, and ALOX15. Furthermore, FA enhanced the expression of GPX4, GCLC, GCLM, and restored GSH content in LPS-exposed hepatocytes. The above results demonstrated that the protective effect of FA on LPS-induced oxidative damage in the sheep hepatocytes was achieved by activating the GSH-GPX4 pathway and inhibiting lipid metabolism-mediated ferroptosis.