Abstract
BackgroundInflammation has been implicated in a variety of diseases associated with ageing, including cancer, cardiovascular, and neurologic diseases. We have recently established that the proteasome is a pivotal regulator of inflammation, which modulates the induction of inflammatory mediators such as TNF-α, IL-1, IL-6, and nitric oxide (NO) in response to a variety of stimuli. The present study was undertaken to identify non-toxic proteasome inhibitors with the expectation that these compounds could potentially suppress the production of inflammatory mediators in ageing humans, thereby decreasing the risk of developing ageing related diseases. We evaluated the capacity of various proteasome inhibitors to suppress TNF-α, NO and gene suppression of TNF-α, and iNOS mRNA, by LPS-stimulated macrophages from several sources. Further, we evaluated the mechanisms by which these agents suppress secretion of TNF-α, and NO production. Over the course of these studies, we measured the effects of various proteasome inhibitors on the RAW 264.7 cells, and peritoneal macrophages from four different strains of mice (C57BL/6, BALB/c, proteasome double subunits knockout LMP7/MECL-1-/-, and peroxisome proliferator-activated receptor-α,-/- (PPAR-α,-/-) knockout mice. We also directly measured the effect of these proteasome inhibitors on proteolytic activity of 20S rabbit muscle proteasomes.ResultsThere was significant reduction of chymotrypsin-like activity of the 20S rabbit muscle proteasomes with dexamethasone (31%), mevinolin (19%), δ-tocotrienol (28%), riboflavin (34%), and quercetin (45%; P < 0.05). Moreover, quercetin, riboflavin, and δ-tocotrienol also inhibited chymotrypsin-like, trypsin-like and post-glutamase activities in RAW 264.7 whole cells. These compounds also inhibited LPS-stimulated NO production and TNF-α, secretion, blocked the degradation of P-IκB protein, and decreased activation of NF-κB, in RAW 264.7 cells. All proteasome inhibitors tested also significantly inhibited NO production (30% to 60% reduction) by LPS-induced thioglycolate-elicited peritoneal macrophages derived from all four strains of mice. All five compounds also suppressed LPS-induced TNF-α, secretion by macrophages from C57BL/6 and BALB/c mice. TNF-α, secretion, however, was not suppressed by any of the three proteasome inhibitors tested (δ-tocotrienol, riboflavin, and quercetin) with LPS-induced macrophages from LMP7/MECL-1-/- and PPAR-α,-/- knockout mice. Results of gene expression studies for TNF-α, and iNOS were generally consistent with results obtained for TNF-α, protein and NO production observed with four strains of mice.ConclusionsResults of the current study demonstrate that δ-tocotrienol, riboflavin, and quercetin inhibit NO production by LPS-stimulated macrophages of all four strains of mice, and TNF-α, secretion only by LPS-stimulated macrophages of C57BL/6 and BALB/c mice. The mechanism for this inhibition appears to be decreased proteolytic degradation of P-IκB protein by the inhibited proteasome, resulting in decreased translocation of activated NF-κB to the nucleus, and depressed transcription of gene expression of TNF-α, and iNOS. Further, these naturally-occurring proteasome inhibitors tested appear to be relatively potent inhibitors of multiple proteasome subunits in inflammatory proteasomes. Consequently, these agents could potentially suppress the production of inflammatory mediators in ageing humans, thereby decreasing the risk of developing a variety of ageing related diseases.
Highlights
Inflammation has been implicated in a variety of diseases associated with ageing, including cancer, cardiovascular, and neurologic diseases
We found that the degree of inhibition of TNF-a secretion by simultaneous treatment with LPS and quercetin (74%), dexamethasone (66%), riboflavin (65%), δtocotrienol (16%), or mevinolin (11%) was comparable to the level of inhibition attained when macrophages were pre-treated with proteasome inhibitors
Dexamethasone, mevinolin, δ-tocotrienol, riboflavin and quercetin were all found to be potent inhibitors of chymotrypsin-like activity of 20S rabbit muscle proteasomes. δ-Tocotrienol, riboflavin, and quercetin inhibited chymotrypsin-like, trypsinlike and post-glutamase activities of the proteasomes in RAW 264.7 cells. These compounds blocked LPSstimulated secretion of TNF-a nitric oxide (NO) production, activation of NF-B, and degradation of P-IB in RAW 264.7 murine macrophages. These compounds suppressed TNF-a secretion and NO production by LPSstimulated peritoneal macrophages derived from C57BL/ 6 and BALB/c mice. δ-Tocotrienol, riboflavin, and quercetin blocked the LPS-stimulated production of NO and had no effect on the secretion of TNF-a in macrophages derived from LMP7/MECL-1-/- and PPAR-a-/- knockout mice
Summary
Inflammation has been implicated in a variety of diseases associated with ageing, including cancer, cardiovascular, and neurologic diseases. We have recently established that the proteasome is a pivotal regulator of inflammation, which modulates the induction of inflammatory mediators such as TNF-a IL-1, IL-6, and nitric oxide (NO) in response to a variety of stimuli. The present study was undertaken to identify non-toxic proteasome inhibitors with the expectation that these compounds could potentially suppress the production of inflammatory mediators in ageing humans, thereby decreasing the risk of developing ageing related diseases. We have recently reviewed the important role of proteasomes in inflammation and other macrophage functions, and hypothesized that inhibition of proteasome activity can suppress inflammatory responses that contribute to ageing [8]. Lactacystin is a synthetic compound that contains a b-lactone moiety, which is responsible for lactacystin’s capacity to block production of a number of pro-inflammatory cytokines by LPS-stimulated macrophages [7]. Proteasomal activities are tightly regulated, and naturally-occurring compounds (g-tocotrienol and δ-tocotrienol) are able to inhibit or activate these activities [9]
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have