Ethyl caffeate alleviates NAFLD by modulating JNK/IRS-1/AKT signaling and the gut microbiota

Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease worldwide, yet no specific pharmacotherapy has been approved. Ethyl caffeate (EC), a natural phenolic compound with anti-inflammatory and antioxidant properties, has been suggested as a potential hepatoprotective agent, but its efficacy against NAFLD remains unexplored. Here, we comprehensively evaluated the effects of EC on NAFLD using network pharmacology prediction, in vivo high-fat diet (HFD)-fed mouse models, in vitro oleic acid (OA)-induced HepG2 cells, as well as 16S rRNA gene sequencing and untargeted metabolomics of cecal contents. EC dose-dependently improved metabolic disturbances in HFD-fed mice and OA-induced HepG2 cells, as evidenced by reduced body weight gain, liver index, serum transaminases, and hepatic triglyceride/cholesterol levels, alongside enhanced glucose tolerance and insulin sensitivity. Mechanistically, EC alleviated insulin resistance and lipid accumulation by inhibiting the aberrant phosphorylation of JNK and IRS-1, thereby restoring AKT activation, and concurrently suppressing the expression of the lipogenic transcription factor SREBP-1c and the inflammatory cytokine TNF-α. In parallel, EC remodeled the gut microbiota by decreasing the Firmicutes/Bacteroidota ratio, specifically enriching the beneficial genus Akkermansia, and modulating cecal metabolite profiles, particularly affecting bile acid, fatty acid, and amino acid metabolism pathways. These findings suggest that EC ameliorates NAFLD potentially through dual direct (hepatic insulin signaling) and indirect (gut-liver axis) mechanisms, highlighting its potential as a promising natural candidate for NAFLD therapy.