Gut microbiota metabolite trimethylamine N-oxide promoted NAFLD progression by exacerbating intestinal barrier disruption and intrahepatic cellular imbalance

•Fecal TMAO levels are associated with NAFLD severity.•TMAO promotes the progression of NAFLD models in vitro and in vivo.•TMAO exacerbates intestinal barrier damage and inflammation at various levels.•TMAO promotes endothelial dysfunction and capillarization of LSECs.•TMAO promotes M1 macrophage while inhibiting M2 macrophage polarization. Non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease worldwide, with the gut microbiota and its metabolites are important regulators of its progression. Trimethylamine N-oxide (TMAO), a metabolite of the gut microbiota, has been closely associated with various metabolic diseases, but its relationship with NAFLD remains to be elucidated. In this study, we found that fecal TMAO levels correlated with NAFLD severity. Moreover, TMAO promoted lipid deposition in HepG2 fatty liver cells and exacerbated hepatic steatosis in NAFLD rats. In the colon, TMAO undermined the structure and function of the intestinal barrier at various levels, further activated the TLR4/MyD88/NF-κB pathway, and inhibited the WNT/β-catenin pathway. In the liver, TMAO induced endothelial dysfunction with capillarization of liver sinusoidal endothelial cells, while modulating macrophage polarization. In conclusion, our study suggests that gut microbiota metabolite TMAO promotes NAFLD progression by impairing the gut and liver and that targeting TMAO could be an alternative therapeutic strategy for NAFLD.