Oleoylethanolamide exerts anti-inflammatory effects on LPS-induced THP-1 cells by enhancing PPARα signaling and inhibiting the NF-κB and ERK1/2/AP-1/STAT3 pathways.

The present study aimed to examine the anti-inflammatory actions of oleoylethanolamide (OEA) in lipopolysaccharide (LPS)-induced THP-1 cells. The cells were stimulated with LPS (1 µg/ml) in the presence or absence of OEA (10, 20 and 40 µM). The pro-inflammatory cytokines were evaluated by qRT-PCR and ELISA. The THP-1 cells were transiently transfected with PPARα small-interfering RNA, and TLR4 activity was determined with a blocking test using anti-TLR4 antibody. Additionally, a special inhibitor was used to analyse the intracellular signaling pathway. OEA exerted a potent anti-inflammatory effect by reducing the production of pro-inflammatory cytokines and TLR4 expression, and by enhancing PPARα expression. The modulatory effects of OEA on LPS-induced inflammation depended on PPARα and TLR4. Importantly, OEA inhibited LPS-induced NF-κB activation, IκBα degradation, expression of AP-1, and the phosphorylation of ERK1/2 and STAT3. In summary, our results demonstrated that OEA exerts anti-inflammatory effects by enhancing PPARα signaling, inhibiting the TLR4-mediated NF-κB signaling pathway, and interfering with the ERK1/2-dependent signaling cascade (TLR4/ERK1/2/AP-1/STAT3), which suggests that OEA may be a therapeutic agent for inflammatory diseases.

Effects of fluid shear stress on the expression of Omi/HtrA2 in human umbilical vein endothelial cells.

To investigate the molecular mechanisms of laminar shear stress on the inhibition of apoptosis in endothelial cells, human umbilical vein endothelial cells (HUVECs) were starved in medium containing 2% fetal bovine serum (FBS) and treated with 15 dyne/cm2 shear stress. We confirmed that 15 dyne/cm2 shear stress inhibited the expression of Omi/HtrA2 at the mRNA and protein levels in cultured HUVECs. Furthermore, the release of Omi/HtrA2 from the mitochondria was induced by removal of basic fibroblast growth factor and decrease of FBS in the medium, while shear stress inhibited its release under the same conditions. These results suggest that downregulation of Omi/HtrA2 may contribute to the potent anti-atherosclerotic effect of shear stress by preventing endothelial cells from entering apoptosis.

Effects of Fluid Shear Stress on Expression of Smac/DIABLO in Human Umbilical Vein Endothelial Cells.

OBJECTIVE: To investigate the molecular mechanisms of laminar shear stress on inhibition of apoptosis in endothelial cells, human umbilical vein endothelial cells (HUVECs) were starved in medium containing 2% fetal bovine serum and 20 dyne/cm(2) shear stress. METHODS: HUVECs were subjected to shear stress or incubated in a static condition and then Smac/DIABLO expression was quantified by reverse-transcription polymerase chain reaction, real-time PCR, and western blot. The effect of shear stress on the migration of Smac/DIABLO proteins was detected by immunofluorescence microscopy. RESULTS: Results demonstrated that 20 dyne/cm(2) shear stress inhibited the expression of Smac/DIABLO at both the mRNA and protein levels in cultured HUVECs. Furthermore, release of Smac/DIABLO from mitochondria was induced by removal of basic fibroblast growth factor and decrease of fetal bovine serum in the medium, whereas shear stress inhibited its release under the same conditions. CONCLUSIONS: These results suggest that down-regulation of Smac/DIABLO may contribute to the potent antiatherosclerotic effect of shear stress by preventing endothelial cells from entering apoptosis.

Molecular genetic analysis of sexual rejection: roles of octopamine and its receptor OAMB in Drosophila courtship conditioning.

After Drosophila males are rejected by mated females, their subsequent courtship is inhibited even when encountering virgin females. Molecular mechanisms underlying courtship conditioning in the CNS are unclear. In this study, we find that tyramine ß hydroxylase (TßH) mutant males unable to synthesize octopamine (OA) showed impaired courtship conditioning, which could be rescued by transgenic TßH expression in the CNS. Inactivation of octopaminergic neurons mimicked the TßH mutant phenotype. Transient activation of octopaminergic neurons in males not only decreased their courtship of virgin females, but also produced courtship conditioning. Single cell analysis revealed projection of octopaminergic neurons to the mushroom bodies. Deletion of the OAMB gene encoding an OA receptor expressed in the mushroom bodies disrupted courtship conditioning. Inactivation of neurons expressing OAMB also eliminated courtship conditioning. OAMB neurons responded robustly to male-specific pheromone cis-vaccenyl acetate in a dose-dependent manner. Our results indicate that OA plays an important role in courtship conditioning through its OAMB receptor expressed in a specific neuronal subset of the mushroom bodies.

Inhibition of TNFα-induced adhesion molecule expression by (Z)-(S)-9-octadecenamide, N-(2-hydroxyethyl,1-methyl).

Inflammation is a primary event in atherogenesis. Oleoylethanolamide (OEA), a naturally occurring fatty-acid ethanolamide, lowers lipid levels in liver and blood through activation of the nuclear receptor, peroxisome proliferator-activated receptor-alpha (PPARα). We designed and synthesized (Z)-(S)-9-octadecenamide, N-(2-hydroxyethyl, 1-methyl) (OPA), an OEA analog. The present study investigated the effect of OPA on the expression of adhesion molecules in human umbilical vein endothelial cells (HUVEC). OPA inhibited expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) stimulated by Tumor Necrosis Factor-α (TNF-α) via activation of PPARα. This inhibition of VCAM-1 and ICAM-1 expression decreased adhesion of monocyte-like cells to stimulated endothelial cells. These results demonstrate that OPA may have anti-inflammatory properties. Our results thus provide new insights into possible future therapeutic approaches to the treatment of atherosclerosis.

Subdomain-specific collapse of denatured staphylococcal nuclease revealed by single molecule fluorescence resonance energy transfer measurements.

By using single molecule fluorescence resonance energy transfer (smFRET), the equilibrium denaturation of staphylococcal nuclease (SNase) induced by guanidinium hydrochloride (GdmCl) has been investigated. We have characterized the collapse of the denatured chain and its relation to structure formation. Two mutants, K28C/H124C and K28C/K97C, were constructed and labeled for monitoring the behaviors of the global molecule and the beta subdomain, respectively. For both the labeled mutants, only native and non-native conformations were observed, and the non-native conformations expanded with increasing GdmCl concentrations. The non-native chains of the two derivatives exhibited different changes of persistence length at higher GdmCl concentrations, suggesting a subdomain-specific collapse of the denatured state of SNase. This local chain specific collapse is likely to play a role in modulating the formation of early intermediate during protein folding.