Bidirectional fermentation of Tremella fuciformis and Acanthopanax trifoliatus alleviates lipid accumulation in LO2 cells.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a prevalent metabolic disorder with limited intervention. This study developed a bidirectional fermentation system combining Tremella fuciformis and Acanthopanax trifoliatus (TF-AT) to enhance metabolite biotransformation and bioactivity. TF-AT showed stronger lipid-lowering effects than the unfermented controls in the free fatty acid (FFA)-induced human normal hepatocyte cell line (LO2 cells). Network pharmacology and targeted metabolomics were used to identify key phenolic metabolites and potential signaling pathways. The TF-AT fermentation product was fractionated into free phenolic fractions (TF-AT-1-TF-AT-6) and a crude polysaccharide fraction (TF-AT-PS). In vitro assays were performed in FFA-induced LO2 cells to assess lipid accumulation, cell viability, triglyceride (TG), total cholesterol (TC), and mRNA expression of MAPK pathway-related genes. Ten phenolic metabolites, including quercetin, myricetin, and luteolin, were identified, and the MAPK pathway was predicted as a potential signaling pathway associated with TF-AT activity. TF-AT, TF-AT-1, and TF-AT-PS significantly improved the cell viability, reduced lipid droplets, and lowered TG and TC levels in FFA-induced LO2 cells, suggesting that bidirectional fermentation contributed to enhanced bioactivity. qPCR analysis showed that these effects were accompanied by downregulation of JNK1, ASK1, p38, ELK-1, and p53 mRNA, indicating the possible involvement of MAPK/JNK/p38-related transcriptional responses. Overall, TF-AT attenuated FFA-induced lipid accumulation in LO2 cells and may serve as a promising fermented multi-component functional ingredient for hepatic steatosis-related intervention, although further protein-level and in vivo validation is required.