Recreating gut-liver axis during NAFLD onset by using a Caco-2/HepG2 co-culture system

Nonalcoholic fatty liver disease (NAFLD) onset and its progression towards nonalcoholic steatohepatitis (NASH) features increased intestinal permeability and leaky gut, thereby favoring the escape of endotoxin [lipopolysaccharides (LPS)] from the gut to the liver. The aim of this study was to resemble the crosstalk between intestine and liver during NAFLD by using an in vitro model of co-culture system. Enterocytes (Caco-2) were seeded on Transwell filters (pore size: 0.4 μm) and cultured for 21 days to constitute a confluent monolayer, and then they were co-cultivated with hepatocytes (HepG2) for an additional 24 h. Caco-2 on the apical chamber were exposed to LPS and/or a mixture of palmitic and oleic acid (PAOA) for 24 h. FITC-4000 dextrans (FD4) permeability across Caco-2 monolayer was increased by the treatment of Caco-2 cells with PAOA and LPS, consistently with tight junction-associated proteins reduction. Caco-2 exposure to PAOA/LPS promoted ApoB, triglyceride (TG), and free fatty acid secretion in basolateral media. In turn, HepG2 co-cultured with Caco-2 exposed to LPS, PAOA, or both accumulated lipid droplets and increased intracellular TG content. Likewise, Caco-2 released pro-inflammatory cytokines in basolateral media. These events triggered endoplasmic reticulum (ER) and oxidative stress, enhancing reactive oxygen species (ROS), H2O2, aldehyde derivate production, and ROS-induced DNA damage in HepG2 cells. Hence, Caco-2/HepG2 co-culture system may faithfully reproduce the breach in the intestinal barrier integrity that occurs in NAFLD, thus resulting in the increased inflammatory response and ER and oxidative and stress, which promote the switch towards NASH.