Dietary supplementation of <i>Chlorella pyrenoidosa</i> attenuated non-alcoholic fatty liver disease <i>via</i> the NF-κB/ferroptosis axis

Non-alcoholic fatty liver disease (NAFLD) stemming from eating disorders represents a significant public health threat. The therapeutic potential of <i>Chlorella pyrenoidosa</i> (CP) extract has been documented in metabolic disorders, including diabetes and hepatitis. Given the huge challenges patients face in self-regulating their diets, we suggest that incorporating a modest amount of CP into meals, without altering the original diet, may offer therapeutic benefits for NAFLD patients. It is important to note that our focus was on the overall effects of CP, rather than on the specific components of the extract. We supplemented a high-fat diet (HFD) with CP for daily feeding of NAFLD mice and observed that CP attenuated hepatic lipid accumulation and inflammation in these mice. To elucidate the mechanism of action of CP <i>in vitro</i>, we extracted its active compounds using methanol and characterized them <i>via</i> LC-MS. We then employed network pharmacology approaches to identify and validate targets for NAFLD treatment using CP methanol extracts. CP functioned by decreasing levels of interleukin 6 (IL-6), tumor necrosis factor (TNF), nuclear factor kappa B1 (NFKB1/NF-κB1) mRNA, NF-κB1 and phosphorylated NF-κB1 (p-NF-κB1) protein in the hepatoblastoma cell line G2 (HepG2) treated with free fatty acids (FFAs), along with reducing p-NF-κB protein levels in mouse liver, indicating that CP regulated the NF-κB signaling pathway. We subsequently analyzed the hepatic metabolite profiles in mice and found that CP inhibited ferroptosis by modulating metabolites such as iron and glutamic acid. The levels of Fe²⁺, malondialdehyde (MDA), and reactive oxygen species (ROS) in CP-treated cells corroborated these findings. Further studies showed that CP upregulated the expression of solute carrier family 7 member 11 (SLC7A11) mRNA and SLC7A11 protein <i>in vitro</i>, thereby promoting intracellular glutamic acid, increasing cysteine levels, and enhancing glutathione (GSH) synthesis. Additionally, CP increased the expression of glutathione peroxidase 4 (GPX4) mRNA and GPX4 protein, and augmented peroxide-scavenging capacity. These findings indicate that CP, as a dietary supplement, inhibits NAFLD <i>via</i> the NF-κB/ferroptosis axis.