In vitro hepatic steatosis model based on gut-liver-on-a-chip.

Hepatic steatosis, also known as fatty liver disease, occurs due to abnormal lipid accumulation in the liver. It has been known that gut absorption also plays an important role in the mechanism underlying hepatic steatosis. Conventional in vitro cell culture models have limitations in recapitulating the mechanisms of hepatic steatosis because it does not include the gut absorption process. Previously, we reported development of a microfluidic gut-liver chip that can recapitulate the gut absorption of fatty acids and subsequent lipid accumulation in liver cells. In this study, we performed a series of experiments to verify that our gut-liver chip reproduces various aspects of hepatic steatosis. The absorption of fatty acids was evaluated under various culture conditions. The anti-steatotic effect of turofexorate isopropyl (XL-335) and metformin was tested, and both drugs showed different action mechanisms. In addition, the oxidative stress induced by lipid absorption was evaluated. Our results demonstrate the potential of the gut-liver chip for use as a novel, physiologically realistic in vitro model to study fatty liver disease.

Three-tissue microphysiological system for studying inflammatory responses in gut-liver Axis.

The interaction between the gut and the liver, often known as the gut-liver axis, play crucial roles in modulating the body's responses to the xenobiotics as well as progression of diseases. Dysfunction of the axis can cause metabolic disorders as well as obesity, diabetes, and fatty liver disease. During the progression of such diseases, inflammatory responses involving the immune system also play an important part. In this study, we developed a three-tissue microphysiological system (MPS) that can accommodate three different cell types in separated compartments connected via fluidic channels in a microfluidic device. Using computational fluid dynamics, geometry of fluidic channels and flow conditions were optimized for seeding and culturing different cell types in the three-tissue MPS. Caco-2 (gut), RAW264.7 (immune), and HepG2 (liver) cells were seeded and cultured in the chip. Stimulation of the gut cells in the MPS with lipopolysaccharide (LPS) resulted in induction of inflammatory response and production of nitric oxide (NO) in all connected chambers. The anti-inflammatory effect of luteolin was demonstrated. Our study demonstrates that the three-tissue MPS can recapitulate the inflammatory responses involving the gut, liver and immune cells.

Angiotensin-cleaving catalysts: conversion of N-terminal aspartate to pyruvate through oxidative decarboxylation catalyzed by Co(III)cyclen.

To provide a firm basis for the new paradigm of drug discovery based on peptide-cleaving catalysts, oligopeptide-cleaving catalysts were searched for by using human angiotensin I (Ang-I) and angiotensin II (Ang-II) as the substrates. Catalyst candidates containing the Co(III) complex of cyclen as the catalytic center were prepared by multicomponent condensation reactions. From two types of chemical libraries containing about 3,600 catalyst candidates, two compounds [SS-Co(III)X and S-Co(III)Y] were selected as the most active catalysts. On incubation with SS-Co(III)X and S-Co(III)Y, both Ang-I and Ang-II were cleaved by oxidative decarboxylation instead of peptide hydrolysis: the N-terminal Asp residues of Ang-I and Ang-II were converted to pyruvate residues. Catalysts for oxidative decarboxylation of the N-terminal Asp residue contained in an oligopeptide are unprecedented in both biological and chemical systems. Detailed kinetics analysis suggested that Ang-I and Ang-II can be cleaved with half-lives much less than 1 h if the structures of the chelating ligands of the catalysts are further improved. The results indicated that the concept of the peptide-cleaving catalysts can be expanded to include oligopeptides as the targets and nonhydrolytic reactions as the means for cleavage.

Toward protein-cleaving catalytic drugs: artificial protease selective for myoglobin.

A protein-cleaving catalyst highly selective for a disease-related protein can be used as a catalytic drug. As the first protein-cleaving catalyst selective for a protein substrate, a catalyst for myoglobin (Mb) was designed by attaching the Cu(II) or Co(III) complex of cyclen to a binding site searched by a combinatorial method using peptide nucleic acid monomers as building units. Various linkers were inserted between the catalytic Co(III) center and the binding site of the Mb-cleaving catalyst. Kinetic data revealed catalytic turnover of the Mb cleavage by the Cu(II) or Co(III) complex. MALDI-TOF MS revealed cleavage of the polypeptide backbone of Mb at selected positions. N-Terminal sequencing of the cleavage products identified the cleavage site and provided evidence for the hydrolytic nature of the Mb cleavage. Various chelating ligands were tested as the ligand for the Co(III) center of the Mb-cleaving catalyst. Among the nine chelating ligands examined, only cyclen and its triaza-monooxo analogue manifested catalytic activity.

Protein-cleaving catalyst selective for protein substrate.

A protein-cleaving catalyst specific for a disease-related protein can be used as a catalytic drug. As the first protein-cleaving catalyst selective for a protein substrate, a catalyst for myoglobin was designed by attaching Cu(II) or Co(III) complex of cyclen to a binding site searched by a combinatorial method using peptide nucleic acid monomers as building units. [reaction: see text]