Emodin suppresses lipopolysaccharide-induced pro-inflammatory responses and NF-κB activation by disrupting lipid rafts in CD14-negative endothelial cells.
OBJECTIVE
Emodin [1,3,8-trihydroxy-6-methylanthraquinone] has been reported to exhibit vascular anti-inflammatory properties. However, the corresponding mechanisms are not well understood. The present study was designed to explore the molecular target(s) of emodin in modifying lipopolysaccharide (LPS)-associated signal transduction pathways in endothelial cells.


METHODS
Cultured primary human umbilical vein endothelial cells (HUVECs; passages 3-5) were pre-incubated with emodin (1-50 µg·mL(-1) ). LPS-induced expression of pro-inflammatory cytokines [interleukin (IL)-1β, IL-6] and chemokines (IL-8; CCL2/MCP-1) were determined by reverse transcription-PCR and elisa. Nuclear factor-κB (NF-κB) activation, inhibitor of κB (IκB)α degradation and Toll-like receptor-4 (TLR-4) were detected by immunocytochemistry and Western blotting. Cholesterol depletion [by methyl β-cyclodextrin (MBCD), a specific cholesterol binding agent] and cholesterol replenishment were further used to investigate the roles of lipid rafts in activation of HUVECs.


RESULTS
Emodin inhibited, concentration-dependently, the expression of LPS-induced pro-inflammatory cytokines (IL-1β, IL-6) and chemokines (IL-8, CCL2) and, in parallel, inhibited NF-κB activation and IκBα degradation in HUVECs. However, emodin did not inhibit the NF-κB activation and IκBα degradation induced by IL-1β. The cholesterol binding agent, MBCD, inhibited LPS-induced NF-κB activation in passaged HUVECs [which also lack the LPS receptor, membrane CD14 (mCD14)], showing that lipid rafts played a key role in LPS signalling in mCD14-negative HUVECs. Moreover, emodin disrupted the formation of lipid rafts in cell membranes by depleting cholesterol.


CONCLUSIONS
Lipid rafts were crucial in facilitating inflammatory responses of mCD14-negative HUVECs to LPS. Emodin disrupted lipid rafts through depleting cholesterol and, consequently, inhibited inflammatory responses in endothelial cells.
Meng
Guoquan
G
Department of Biophysics, School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, Sichuan, China.
Liu
Yiyao
Y
Lou
Changchun
C
Yang
Hong
H
D050197:Atherosclerosis / Q000188:drug therapy; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D016207:Cytokines / Q000378:metabolism; D004642:Emodin / Q000494:pharmacology*; D042783:Endothelial Cells / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007249:Inflammation / Q000378:metabolism*; D018950:Lipopolysaccharide Receptors / Q000032:analysis*; D008070:Lipopolysaccharides / Q000008:administration & dosage / Q000276:immunology*; D021962:Membrane Microdomains / Q000378:metabolism*; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D047392:beta-Cyclodextrins / Q000378:metabolism / Q000494:pharmacology
D016207:Cytokines; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D016328:NF-kappa B; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D002784:Cholesterol; D004642:Emodin