AbstractAimTo determine if a novel positive allosteric modulator of the γ‐aminobutyric acid type A (GABAA) receptor, the thioacrylamide‐derivative HK4, which does not penetrate the blood–brain barrier, protects human hepatocytes against lipotoxicity‐induced injury.Materials and MethodsAllosteric modulation of the GABAAreceptor by HK4 was determined by patch clamp in HEK‐293 cells, calcium influx in INS‐1E cells and by using the specific GABAAchannel 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‐κB subunit p65, protein disulphide isomerase (PDI) and poly‐ADP‐ribose polymerase‐1 (PARP‐1) was detected by Western blotting.ResultsPatch clamping, calcium influx measurements and apoptosis assays with the non‐competitive GABAAchannel blockers picrotoxin and TBPS proved HK4 as a selective positive allosteric modulator of the GABAAreceptor. In HepG2 cells, which expressed the main GABAAreceptor 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‐κB phosphorylation, reduced cleaved PARP‐1 expression and upregulated the endoplasmic reticulum (ER) chaperone PDI.ConclusionsHK4 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‐κB. It is suggested that thioacrylamide compounds represent an innovative pharmacological tool to treat or prevent non‐alcoholic steatohepatitis as first‐in‐class drugs.
