Abstract
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.
Highlights
Nonalcoholic fatty liver disease (NAFLD), or as it has been more recently re-named “metabolic dysfunction-associated fatty liver disease (MAFLD)”[1], is the most frequent chronic hepatic disorder worldwide, affecting 20%-40% of the adult population in Western countries[2,3]
To investigate whether Caco-2 polarized monolayers are affected by endotoxin and/or fatty acids, we added in the quiescent medium of the apical chamber 100 ng/mL LPS and/or a combination of palmitic and oleic acid (PAOA) at the concentration of 0.25 mM, to recapitulate the clinical condition of NAFLD patients in which intestinal dysbiosis and excessive free fatty acids (FFAs) have been reported[10]
In keeping with the enhanced permeability, protein expressions of ZO1, claudin-1, and claudin2, tight-junctions associated proteins, were decreased (P < 0.0001, one-way analysis of variance (ANOVA); adjusted P < 0.001 for PAOA LPS vs. PAOA or LPS alone; Figure 2B-D), mirroring what occurs in NAFLD patients, in which gut permeability is increased according to the disruption of intercellular tight junctions[12,38]
Summary
Nonalcoholic fatty liver disease (NAFLD), or as it has been more recently re-named “metabolic dysfunction-associated fatty liver disease (MAFLD)”[1], is the most frequent chronic hepatic disorder worldwide, affecting 20%-40% of the adult population in Western countries[2,3]. The fine-tuned mechanisms which regulate the switching from fatty liver to NASH are still under study
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