Down-regulation of EPHX2 gene transcription by Sp1 under high-glucose conditions.

sEH (soluble epoxide hydrolase), which is encoded by the EPHX2 gene, regulates the actions of bioactive lipids, EETs (epoxyeicosatrienoic acids). Previously, we found that high-glucose-induced oxidative stress suppressed sEH levels in a hepatocarcinoma cell line (Hep3B) and sEH was decreased in streptozotocin-induced diabetic mice in vivo. In the present study, we investigated the regulatory mechanisms underlying EPHX2 transcriptional suppression under high-glucose conditions. The decrease in sEH was prevented by an Sp1 (specificity protein 1) inhibitor, mithramycin A, and overexpression or knockdown of Sp1 revealed that Sp1 suppressively regulated sEH expression, in contrast with the general role of Sp1 on transcriptional activation. In addition, we found that AP2α (activating protein 2α) promoted EPHX2 transcription. The nuclear transport of Sp1, but not that of AP2α, was increased under high glucose concomitantly with the decrease in sEH. Within the EPHX2 promoter -56/+32, five Sp1-binding sites were identified, and the mutation of each of these sites showed that the first one (SP1_1) was important in both suppression by Sp1 and activation by AP2α. Furthermore, overexpression of Sp1 diminished the binding of AP2α by DNA-affinity precipitation assay and ChIP, suggesting competition between Sp1 and AP2α on the EPHX2 promoter. These findings provide novel insights into the role of Sp1 in transcriptional suppression, which may be applicable to the transcriptional regulation of other genes.

Keishi-bukuryo-gan preserves the endothelium dependent relaxation of thoracic aorta in cholesterol-fed rabbit by limiting superoxide generation.

Formerly, we have reported that keishi-bukuryo-gan prevents the progression of atherosclerosis in cholesterol-fed rabbits and inhibits the free radical-induced RBC haemolysis in rats. The present study was performed to investigate how keishi-bukuryo-gan (KBG) inhibits the early stage of atherosclerosis. Plasma lipid concentration and hydroxyl radical generation during respiratory burst in neutrophils were evaluated at the start and end of the study. The protective effect of KBG against endothelium disorder due to hypercholesterolaemia was examined. Twelve male Japanese white rabbits (2 kg body weight) were divided into two groups. Group A (n = 6) was fed standard rabbit chow containing 1% cholesterol for 4 weeks. Group B (n = 6) was fed standard rabbit chow containing 1% cholesterol and 1% KBG for 4 weeks. In the plasma lipid concentration, only the lipid peroxide concentration of group A was significantly higher than that of group B. At the end of the study, DMPO-OH, the spin-trapped adduct of hydroxyl radicals generated by neutrophils, was increased in both groups, and this increase was marked in group B. Endothelium-dependent vasodilatation by acetylcholine increased significantly in group B compared with group A. Thus, KBG protects the vascular endothelium function by its antioxidative effect and by inhibiting the release of free radicals from neutrophils in vivo.