Inhibition Of Hsp90 Is Involved In Suppression Inflammatory Mediator Release By Luteolin In LPS Challenged RAW264.7 Cells

Over-infection of bacterium induce uncontrolled inflammatory response, resulting in septic shock, systemic inflammatory response syndrome (SIRS) and further develop multiple organ dysfunction syndrome (MODS) and multiple organ failure (MOF). Lipopolysaccharide (LPS) also named endotoxin, a principal component of the outer membrane of gram-negative bacteria, can activate a variety of the mammalian cell types, including monocytes/macrophages and endothelial cells. Monocytes/ macrophages are prominent cells in chronic inflammation and express various cytokines and pro-inflammatory molecules such as chemokine, growth factors, interleukin, tumor necrosis factor in arthritis, atherosclerosis, and periodontal disease and other various inflammatory disease.Luteolin is one of the most widely found flavones in frequently consumed fruits and vegetables as well as in some medicinal plants. Luteolin is a low molecular weight polyphenolic compound composed of a three-ring structure with various substitutions, one of which is unsaturated ring. Luteolin has antioxidant, anticancer and anti-inflammatory activities.In the present study, we explored the suppressive activity of luteolin toward lipopolysaccharide(LPS)-induced inflammatory mediators expression in mouse macrophages RAW264.7 cells and the underlying molecular mechanisms. Here we report a novel mechanism of Luteolin that it can attenuate pro-inflammatory cytokine releasing through preventing the activity of HSP90. The 90-kDa Heat Shock Protein (HSP90) is one of the most abundant cytosolic proteins in eukaryotes. It normally functions as a molecular chaperone to participate in folding newly synthesized proteins, refolding denatured proteins after stress and regulating stability and activity of client proteins. Up to now, over one hundred HSP90 client proteins have been found such as transcription factors, protein kinases and polymerases, in which Akt, IRAK1, c-Jun and iNOS are known client proteins of Hsp90.Our results showed that luteolin may bind to HSP90 and dose and time dependently reduced the level of Hsp90 known client protein such as Akt, IRAK1 and c-Jun in RAW264.7 cells but has no obvious affect on the mRNA levels and this decrease can be recovered by overexpression of HSP90 but not HSP90(1-401aa). We also show that Lutolin shortens the half-life of Akt from 12h to 6h and destabilize the protein by an 60% reduction in its expression. We hypothesize that Luteolin maybe a potential Hsp90 inhibitor which inhibit its chaperone activity and then the immature client proteins are targeted for degradation by proteasome, ultimately a lot of biologic activities such as transcriptional regulation and signal transduction were influenced.Further findings show that Luteolin inhibite MAPKs signaling pathways particularly the phosphorylation of JNK and simultaneously induce the loss of c-Jun. Lower activation of JNK obviously brings down phosphorylation of c-Jun and Jun-2 luciferase activity and then alter the total protein activity of c-Jun in RAW264.7 cells. c-Jun is a significant component protein of the important nucleolus transcription factor AP-1, which is widely involved in transcriptional regulation and signal transduction in cells.On the other hand, incubation of Luteolin before or after LPS can decrease iNOS, COX-2 expression and subsequently inhibit tumor necrosis factor alpha (TNF-a) and nitric oxide (NO) synthesis in RAW264.7 macrophage-like cells. These data suggest that the Luteolin prevents LPS-induced excessive production of pro-inflammatory factors and plays an anti-inflammatory role in response to LPS.Taken together, all our evidences indicated that luteolin has two sides of functions exhibiting a favorable anti-inflammotory effects. On one aspect, luteolin blocked MAPK signaling pathways and weakened the expression of inflammation cytokines so that it plays an anti-inflammatory role in response to LPS. Another interesting finding in this study is luteolin abrogating chaperone activities of Hsp90, leading to degrade critical molecules in inflammatory related pathways. Above all, these results provide a new insight for analysing the potential effects of luteolin in therapeutic application for treating inflammatory disorders.