Dietary Supplementation of Chlorella pyrenoidosa Attenuated Non-alcoholic Fatty Liver Disease via NF-κB/Ferroptosis Axis

Abstract

Non-alcoholic fatty liver disease (NAFLD) stemming from eating disorders represents a significant public health threat. The therapeutic potential of Chlorella pyrenoidosa (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 specific components of the extract. We supplemented a high-fat diet (HFD) with CP for daily feeding to mice and observed that CP attenuated hepatic lipid accumulation and inflammation in NAFLD mice. To elucidate the mechanism of action of CP in vitro, we extracted its active compounds using methanol and characterized them via LC-MS. We then employed network pharmacology approaches to identify and validate targets for NAFLD treatment using CP methanol extracts. CP had a function in decreasing interleukin 6 (IL6), tumor necrosis factor (TNF), nuclear factor kappa B 1 (NFKB1/ NF-κB1) mRNA levels, NF-κB1 and phosphorylated NF-κB1 (p-NF-κB1) protein in hepatoblastoma cell line G2 (HepG2) treated by free fatty acids (FFA), along with the reduction in p-NF-κB proteins in the 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 in vitro, 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 via the NF-κB/Ferroptosis axis.