Gut-microbiota-derived phenylacetylglutamine induces oxidative stress in endothelial cells via a NOX2-dependent pathway

Abstract Introduction The gut microbiota-derived metabolite phenylacetylglutamine (PAG) is clinically and mechanistically linked to cardiovascular disease (CVD)(1). Here, we aim to identify the effect of PAG on endothelial cells and elucidate the underlying molecular pathways. Methods We treated human aortic endothelial cells (HAEC) at a dose of 100 µM PAG (a concentration which can be observed in vivo) for 72h. As a positive control, cells were treated with H2O2 for 1h. For the determination of reactive oxygen species (ROS) formation, we used the CellROX Green assay with subsequent fluorescence microscopy. For signaling studies, protein expression levels of the NADPH oxidases NOX2 and NOX4 as well as the transcription factor NF-kB were determined by western blot analyses. Results Our studies showed that PAG treatment for 72h significantly increased the fluorescence intensity after CellROX Green staining (p<0.0001). Concomitantly, our immunoblotting results revealed that NOX2 (p<0.01) and NF-kB (p<0.001) protein levels were upregulated, whereas NOX4 remained unchanged in response to PAG. Conclusion We conclude that PAG-induced oxidative stress in endothelial cells is mediated by a NOX2 and NF-kB but not by a NOX4 dependent pathway. This study is a first step towards better understanding the molecular mechanisms of cellular responses to the gut microbiota-derived metabolite PAG.