RhoA and NF-κB are involved in lipopolysaccharide-induced brain microvascular cell line hyperpermeability

The aim of this study was to investigate the signaling mechanisms surrounding changes in tight junction (TJ) and the permeability of brain microvascular cell lines induced by lipopolysaccharide (LPS).

To confirm that LPS induces endothelial barrier hyperpermeability by disrupting tight junction, Bend.3 cells were exposed to LPS, and changes in endothelial permeability (transendothelial electrical resistance (TEER) assay), F-actin dynamics (Rhodamine-Phalloidin staining) and tight junction protein expression (western blot or immunofluorescence) were monitored. Moreover, to ensure that both RhoA and NF-κB participated in the regulatory mechanisms, Bend.3 cells were transfected with n19RhoA and DNMu-IκBα plasmids, and the above experiments were repeated. To clarify the relationship between RhoA and NF-κB in the process, the activities of NF-κB (via luciferase reporter assays) and RhoA (via pull-down assays) were detected in transfected and untreated Bend.3 cells. Lastly, to investigate whether RhoA and NF-κB regulate MLC phosphorylation, we measured changes in myosin light chain (MLC) phosphorylation in untreated and transfected Bend.3 cells by western blot.

LPS caused RhoA and NF-κB activation, MLC phosphorylation, F-actin rearrangement, tight junction disruption and barrier dysfunction. These effects were suppressed by inhibitors of RhoA or NF-κB; inhibiting RhoA was more efficient. Inactivating RhoA prohibited LPS-induced NF-κB activation, but the inverse was not true.

LPS induces brain microvascular endothelial barrier hyperpermeability by disrupting TJs, in part through RhoA and NF-κB activation, in which RhoA is the positive upstream regulator for NF-κB.