Positive allosteric gamma-aminobutyric acid type A receptor modulation prevents lipotoxicity-induced injury in hepatocytes in vitro

Aim To determine if a novel positive allosteric modulator of the gamma-aminobutyric acid type A (GABA(A)) receptor, the thioacrylamide-derivative HK4, which does not penetrate the blood-brain barrier, protects human hepatocytes against lipotoxicity-induced injury. Materials and Methods Allosteric modulation of the GABA(A) receptor by HK4 was determined by patch clamp in HEK-293 cells, calcium influx in INS-1E cells and by using the specific GABA(A) channel blockers picrotoxin and tert-butylbicyclophosphorothionate (TBPS) in HepG2 cells. Apoptosis was analysed using caspase 3/7, terminal deoxynucleotidyl transferase-dUTP nick end labelling (TUNEL) and array assays in HepG2 cells and/or human primary hepatocytes. Phosphorylation of STAT3 and the NF-kappa B subunit p65, protein disulphide isomerase (PDI) and poly-ADP-ribose polymerase-1 (PARP-1) was detected by Western blotting. Results Patch clamping, calcium influx measurements and apoptosis assays with the non-competitive GABA(A) channel blockers picrotoxin and TBPS proved HK4 as a selective positive allosteric modulator of the GABA(A) receptor. In HepG2 cells, which expressed the main GABA(A) receptor subunits, HK4 prevented palmitate-induced apoptosis. This protective effect was mediated by downregulation of caspase 3/7 activity and was additionally verified by TUNEL assay. HK4 effectively prevented palmitate-induced apoptosis in human primary hepatocytes. HK4 reduced STAT3 and NF-kappa B phosphorylation, reduced cleaved PARP-1 expression and upregulated the endoplasmic reticulum (ER) chaperone PDI. Conclusions HK4 reduced lipotoxic-induced apoptosis by preventing inflammation, DNA damage and ER stress. We propose that the effect of HK4 is mediated by STAT3 and NF-kappa B. It is suggested that thioacrylamide compounds represent an innovative pharmacological tool to treat or prevent non-alcoholic steatohepatitis as first-in-class drugs.