Figure 1. Effects of CAPE on IbeA+ E. coli K1-induced NF-κB activation and pathogenicities in vitro and in vivo.

(A) IbeA+ E. coli K1 induced NF-κB activation in HBMECs was suppressed by CAPE. HBMECs were incubated with or without the NF-κB inhibitor CAPE (25 µM) for 30 min before stimulation with E44 or ZD1 (10⁷/mL). IKK α/β phosphorylation (p-IKK α/β) in cytoplasmic fractions and NF-κB (p65) in nuclear fractions was examined after 2 h of stimulation with E. coli strains. The β-actin in both fractions was detected as internal loading controls. CON, control without E. coli stimulation. (B–C) Effects of CAPE (0–25 µM) on IbeA+ E. coli K1 penetration and PMN transmigration across HBMECs were examined. HBMECs were incubated with various concentrations of CAPE for 1 h before the invasion and PMN transmigration assays. (B). E. coli (10⁷ CFU) were added to the HBMEC monolayers after CAPE treatment. Invasion assays were carried out as described in the Materials and Methods. (C) The CAPE-pretreated HBMECs were stimulated with E. coli (10⁶ CFU) in the lower chamber for 2 h and incubated with PMN (10⁶) in the upper chamber at 37°C for another 4 h. All assays were performed in triplicates. Results for invasion are expressed as relative invasion compared to the positive control without drug treatment (100%). Results for PMNT are expressed as the percentage of leukocyte transmigration of the total added. Both the invasion and PMNT assays were done with E44 (black column) and ZD1 (white column). E. coli meningitis was induced in neonatal mice with or without CAPE treatment (n = 5) as described in Methods and Materials. (D) Recruitment of PMN into the CSF; (E) Flux of albumin into the CNS; and (F) Levels of soluble NF-κB (p65) in CSF. The significant differences with regard to the controls without CAPE treatment were marked by asterisks (*P<0.05; **P<0.01).