Suppression of nitric oxide induction and pro-inflammatory cytokines by novel proteasome inhibitors in various experimental models

Background 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-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. Results There 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. Conclusions Results 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.

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.

Background
Modern industrialized societies are experiencing great increases in many age-related diseases such as diabetes, cardiovascular, neurodegenerative diseases, and certain types of cancer. Although numerous factors undoubtedly contribute to this trend, significant evidence implicates nitric oxide (NO), and inflammation, in the pathogenesis of several of these age-related diseases [1]. A number of studies, using experimental animal models, have demonstrated that senescence is accompanied by increases in production of NO in response to a variety of microbial products. For example, lipopolysaccharide (LPS)-induced macrophages from 22 and 32 month old CBA/CA mice to produce approximately 5 fold and 15 fold more NO, respectively, than LPS-stimulated macrophages from young (2-month-old) CBA/CA mice [2]. Through further exploration of innate inflammatory responses we have learned that the kinetics of NO production and TNF-α secretion differ in LPS-stimulated murine macrophages, that induction of these inflammatory products are regulated by two independent signaling pathways, and that cytoplasmic proteasomes are key regulators of LPS-induced inflammatory responses in macrophages [3][4][5][6][7].
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]. Many of our earlier experiments designed to delineate the role of proteasomes in innate inflammatory responses utilized lactacystin, a potent proteasome inhibitor [7]. Lactacystin is a synthetic compound that contains a β-lactone moiety, which is responsible for lactacystin's capacity to block production of a number of pro-inflammatory cytokines by LPS-stimulated macrophages [7]. Unfortunately, lactacystin is very expensive and toxic even at micromolar levels so, although it has been quite useful for in vitro experimentation, it is not suitable for clinical use [7]. As reported recently, proteasomal activities are tightly regulated, and naturally-occurring compounds (γ-tocotrienol and δ-tocotrienol) are able to inhibit or activate these activities [9].
Consequently, we sought to identify other, non-toxic proteasome inhibitors with anti-inflammatory properties. Specifically, we have been evaluating a number of relatively inexpensive, commercially available naturallyoccurring, synthetic, and FDA approved compounds for their capacity to inhibit proteasome activity, and the production of nitric oxide, certain pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), and the iNOS enzyme. As part of this pursuit, we recently reported that two important inflammatory markers associated with ageing, TNF-α and NO, were effectively decreased in chickens whose diets were supplemented with a variety of naturally-occurring, synthetic or FDA approved compounds such as δ-tocotrienol, quercetin, riboflavin, (-) Corey lactone, amiloride, and dexamethasone [10]. As described earlier, NO production increases during ageing process [2], which could be due to a diminished activation of NF-signaling [11,12]. It was suggested that above mentioned compounds may also block the activation of NF-, thus resulting lowering of serum TNF-α and NO levels in chickens [10]. The important role played by NF-in various biological functions has been described [13].

Reagents
Highly purified, deep rough chemotype LPS (Re LPS) from E. coli D31m4 was prepared as described [4].  and dexamethasone were purchased from Sigma-Aldrich Chemical Co (St. Louis, MO, USA). Quercetin was purchased from Alfa Aesar (Johnson Matthey Co. Lancastor, UK). The 50% purified δ-tocotrienol fraction from annatto seeds was received as a gift from American River (Boston, MA, USA).
Purification of δ-tocotrienol from 50% purified fraction of annatto seeds The δ-tocotrienol was purified from 50% purified fraction of annatto seeds, as described previously [16]. The purity of δ-tocotrienol was established by high pressure liquid chromatography (HPLC) against its standard, as reported earlier [16].

Cell culture and maintenance
The RAW 264.7 cells or mouse thioglycolate-elicited peritoneal macrophages were maintained in DMEM supplemented with 10% heat inactivated FBS and 10 mg/mL gentamicin at 37°C in a humidified atmosphere with 5% CO 2 , as described previously [3]. Cells were cultured in 6-well plates as described in the Tables or legends to the Figures.
Mice used in this study received humane care in compliance with the principles of laboratory animal care formulated by the National Society of Health Guide for the "Care and Use of Laboratory Animals" by the US National Society of Health (NIH Publication No 85-23, revised 1996).
The experimental procedures involving animals were reviewed and approved by the "Institutional Animal Care and Use Committee of UMKC", Medical School, MO. All 6-week-old female mice (n = 20) were acclimatized to the new environment for 14 days before beginning experimentation. The mice were fed ad libitum regular commercial mouse diet and had free access to water throughout the experiment. A 12 h light and 12 h dark cycle was maintained during feeding period.
Effects of quercetin, riboflavin, and δ-tocotrienol (concentrations of 5 μM-40 μM) after 60 min treatment of different proteasomal activities (chymotrypsin-like, trypsin-like, post-glutamase) in RAW 264.7 whole cells The comparative inhibitory effect of quercetin, riboflavin, and δ-tocotrienol on the chymotrypsin-like, trypsinlike, and post-glutamase activities of proteasome using RAW 264.7 whole cells were carried out essentially as reported recently [9]. Briefly RAW 264.7 cells (10 × 10 3 cells/100 μL/well) were added in white plates (96-well, Fisher, 0877126), followed by the addition of various concentrations of quercetin, riboflavin, or δ-tocotrienol (5, 10, 20, or 40 μM in 100 μL; dissolved in 0.4% dimethyl sulfoxide (DMSO). The mixtures were incubated at 37°C in an incubator at 5% CO 2 for 60 min. After incubation period, the cells in the 96-well plates were taken out 20 min prior to the addition of Caspase-Glo reagent (brought to room temperature before addition to the wells). Caspase-Glo reagent (100 μL) was added to each well to a total volume of 200 μL/well (tris buffer, pH 7.5; 0.02 M). The plate were covered with a plate sealer and incubated at room temp for 30 min. The relative luminescence units (RLU) of assays were read in a Promega Plate Luminometer. The chymotrypsin-like, trypsin-like, or post-glutamase activities were quantitated by measuring luminescence after stimulation of RAW 264.7 whole cells with various concentration of each compound in a Luminometer (Promega, Madison, Wisconsin USA), according to the directions of manufacturer.
The levels of TNF-a in supernatants were measured by Quantikine M ELISA kit (R&D System, Minneapolis, MN, USA) according to manufacturer's instructions. The lower limit of detection for TNF-a in this method is approximately, 5.0 pg/mL [4,7].
The levels of NO were determined by measuring the amount of nitrite, a stable metabolic product of nitric oxide, as previously reported [17]. The assay mixture contained medium (100 μL) plus Griess reagent (100 μL), and absorption was measured at 570 nm using a "Microplate Reader" (MR 5000; Dynatech Labs, Inc. USA). The amount of nitrite was determined by comparison of unknowns using a NaNO2 standard curve. The NO detection limit was 0.20 nM [17].

LPS-induced thioglycolate-elicited peritoneal macrophages
The procedures described above are identical to those utilized for peritoneal macrophages except that thioglycolate-elicited peritoneal macrophages were adhered to the bottom of 100 mm tissue culture plates (1 × 10 7 cells/well in 1.0 mL media) for 4 h, the supernatants were removed, and cells were washed extensively with medium three times. The cells were cultured overnight in fresh media after the final wash. After overnight incubation at 37°C, the cells were treated with various proteasome inhibitors and LPS as described above. Viability of peritoneal macrophages treated with various inhibitors plus LPS were also determined by trypan blue dye exclusion or a quantitative colorimetric assay with 3-(4,5)-dimethylthiozol-2,5-diphenyl-tetrazolium bromide (MTT) as described previously [4,7].
Effects of various compounds on the inhibition of NF-B in LPS-stimulated RAW 264.7 cells RAW 264.7 cells were pretreated with dexamethasone, mevinolin, α -tocopherol, δ-tocotrienol, riboflavin, or quercetin for 1 h, then treated with LPS (10 ng/well; 400 μL) for 4 h. Nuclear protein was extracted and the NF-activation was measured using "electronic mobility shift assay" (EMSA) according to the reported procedure [18]. Nuclear protein was extracted following the recommendation of the manufacturer (QIAGEN Sciences; Germantown, MD, USA) [19]. NF-B was measured in nuclear extracts with the respective ELISA-based commercial kits (NF-p65). Nuclear protein (5 μg), was added to each well coated with an oligonucleotide containing the consensus binding site for NF-incubated for I h. Activated NF-was detected after 1 h with primary antibody, i.e. Anti-NF-, which specifically recognizes an epitope (p65) accessible only when the factor is activated and bound to its target DNA. A secondary anti-IgG horseradish peroxidase conjugate allows detection of the activated NF-by a colorimetric reaction. Absorption was read within 5 min at 450 nm with a reference wavelength of 655 nm [19].
Degradation of P-IB, protein in LPS-stimulated RAW 264.7 cells (Western blots analyses) RAW 264.7 cells (1 × 10 6 cells/500 μL/well) were treated with dexamethasone, mevinolin, α,-tocopherol, δ-tocotrienol, riboflavin, or quercetin for 1 h, then stimulated with LPS (10 ng/well; 400 μL) for 36 h. Macrophages were washed with phosphate-buffered saline, and cytoplasmic extracts were prepared using cell extraction buffer (Biosource, Camarillo, California, USA) supplemented with a protease inhibitor cocktail, containing phenylmethylsulfonyl fluoride and phosphatase inhibitors, according to the manufacturer's directions [20]. Protein concentrations were measured with BCA protein assay kits, and Western blots analyses were used to measure IB-α, (Santa Cruz). Each well of the gel was loaded with 40 μg of protein and gels were electrophoresed at a constant 150 V 1 × Tris glycine buffer for 50 minutes. Proteins in gels were transferred into the "Immobilon Transfer Membranes" (IPVH 15150; Millipore, Bedford, Mass, USA) using the semidry transfer cell and, after appropriate antibody treatments, the bands were visualized with an enhanced chemiluminescence detection kit (Pierce) as described previously [7,20].
After 4 h, assay mixtures were centrifuged at 2,000 rpm for 20 min. Cells were harvested, and total cellular RNA was extracted from each pellet with RNeasy mini kit (QIAGEN Sciences; Germantown, MD, USA) according to the instructions of the manufacturer. The RNA of each treatment was transcribed and resulting data was amplified and analyzed by real-time polymerase chain reaction (RT-PCR) to quantitate gene expression of TNF-α, and iNOS by using 1-step RT-PCR kit (QIA-GEN, Chatsworth, CA, USA; Southern blots analyses) according to the manufacturer's instructions [6,7,21].

Statistical Analyses
Stat View software (version 4.01, Abacus Concepts, Berkeley, CA) was used for the analyses of treatmentmediated effects as compared to control group. Treatment-mediated differences were detected with a twoway ANOVA, and when the F test indicated a significant effect, differences between the means were analyzed by a Fisher's protected least significant difference test. Data were reported as means ± SD in text and Tables. The statistical significance level was set at 5% (P < 0.05).

Effects of various compounds on the chymotrypsin activity of 20S rabbit muscle proteasomes
The anti-inflammatory effects of δ-tocotrienol, quercetin, riboflavin, (-) Corey lactone, amiloride and dexamethasone, as determined by effects on serum levels of TNF-α, and nitric oxide in chickens, have been reported recently [10]. In order to determine whether these compounds modulate proteasome activity, chymotrypsin-like activity of 20S rabbit muscle proteasomes was measured after treatment with these compounds at concentrations ranging from 5 μM to 320 μM, and compared to activity of vehicle treated controls. Results of this study revealed dose dependent inhibition of chymotrypsin-like activity of 20S rabbit muscle proteasomes between 5 μM and 40 μM for mevinolin and δ-tocotrienol, and between 5 μM to 320 μM for dexamethasone, quercetin, and riboflavin ( Table 1). The inhibitory effects of mevinolin and δtocotrienol were reversed at higher concentrations between 80 μM and 320 μM (Table 1). (-) Corey lactone and amiloride-HCL induced dose-dependent (5 μM to 320 μM) increases of chymotrypsin-like activity of 20S rabbit muscle proteasomes, whereas α,-tocopherol (vitamin E) had no effect on at the concentrations tested ( Table 1).
According to recent reports, the 20S proteasomes contain three distinct subunits (X, Y, and Z), with well  characterized protease sites; subunits × (β5), Y (β1), and Z (β2) display chymotrypsin-like, trypsin-like, and postglutamase actitivities, respectively [22]. In order to confirm that these naturally-occurring compounds (quercetin, riboflavin, and δ-tocotrienol) act as proteasome inhibitors, we measured their effects on chymotrypsinlike, trypsin-like and post-glutamase proteasome activities. These enzymatic activities in RAW 264.7 cells were inhibited in a dose-dependent manner at concentrations between 5 μM and 40 μM ( Table 2). Quercetin and δ-tocotrienol caused 50% inhibition of chymotrypsin-like activity with 40 μM concentration compared to control, whereas, riboflavin resulted only moderate inhibition of 14% with this dose (Table 2). Quercetin was the most potent inhibitor of trypsin-like (62%) and postglutamase (41%) activities compared to riboflavin and δtocotrienol treatments (Table 2), thus confirming our earlier results that these compounds modulate inhibition as proteasome inhibitors [10].

Effects of various proteasome inhibitors on production of TNF-a, and NO by LPS-stimulated RAW 264.7 cells
The anti-inflammatory properties of dexamethasone (10 μM), mevinolin (20 μM), δ-tocotrienol (10 μM), riboflavin (40 μM), quercetin (40 μM), and α,-tocopherol (100 μM) were explored by pre-treating RAW 264.7 macrophage-like cells for 1 h with each of these compounds followed by stimulation with LPS and measurement of TNF-α, or nitrite in culture supernatants 4 h and 36 h, respectively, after LPS stimulation. Quercetin (76%), dexamethasone (70%), and riboflavin (68%) significantly inhibited LPS-induced secretion of TNF-α, whereas, δtocotrienol and mevinolin produced only moderate reductions of 19% and 17%, respectively as compared to controls ( Figure 3). Our previous studies with LPS-stimulated macrophages demonstrated that lactacystin, which inhibits primarily chymotrypsin-like activity of the proteasome, did not significantly inhibit TNF-α, production (22). In order to suppress TNF-α, secretion by LPS-stimulated macrophages, both the chymotrypsin-like and trypsin-like enzymatic proteasomal activity of the proteasome had to be inhibited (22). Thus, in the current experiments (Figure 3), the capacity of the proteasome inhibitors to inhibit TNF-α, secretion by LPSstimulated macrophages further supports the conclusion that these inhibitors suppress both chymotrypsin-like and trypsin-like activities of the proteasome. We also tested the ability of proteasome inhibitors to suppress TNF-α, secretion by LPS-stimulated macrophages under conditions in which macrophages were treated simultaneously with proteasome inhibitors and LPS. We found that the degree of inhibition of TNF-α, secretion by simultaneous treatment with LPS and quercetin (74%), dexamethasone (66%), riboflavin (65%), δtocotrienol (16%), or mevinolin (11%) (data not presented) was comparable to the level of inhibition attained when macrophages were pre-treated with proteasome inhibitors. Consequently, further studies were carried out only under pre-treatment condition.
When testing the ability of proteasome inhibitors to block production of NO by LPS-stimulated RAW 264.7 cells, we found that dexamethasone (76%) and quercetin (46%) were the most efficient inhibitors, while mevinolin (34%), δ-tocotrienol (28%), and riboflavin (14%) blocked the production of NO only modestly ( Figure 4). α,-Tocopherol had no significant effect on the levels of nitric oxide (Figure 4).
Effects of various proteasome inhibitors on the inhibition of NF-B in LPS-stimulated RAW 264.7 cells As described earlier, serum NO levels increase during ageing, and one potential explanation involves diminished regulation of NF-B signaling with age [11,12]. Cytoplasmic NF-B is usually bound to its inhibitor, IB, and maintained in an inactive state. NF-B becomes activated when IB is phosphorylated, ubiquitinated, and degraded by the proteasome. NF-B then migrates to the nucleus and binds to promoter sites of a variety of pro-inflammatory genes including TNF-α, and iNOS [13]. We hypothesized that increased NO levels, associated with ageing, may be attributable to increased degradation of IB by ageing proteasomes, with resultant NF-B activation. Consequently, we tested the ability of various proteasome inhibitors (dexamethasone, mevinolin, δ-tocotrienol, riboflavin, and quercetin) to inhibit activation of NF-B, specifically using EMSA assays to detect NF-B in nuclear extracts of LPS stimulated RAW-264.7 cells. Significant inhibition (P < 0.02) of LPS-induced NF-B activation, compared to medium controls, was achieved with δ-tocotrienol (56%), mevinolin (52%), quercetin (46%), dexamethasone (42%), and riboflavin (34%), while α,-tocopherol (vitamin E) had no effect ( Figure 5). Thus, the capacity of each of these proteasome inhibitors to suppress LPS-induced NF-B activation could account for their ability to inhibit TNF-α, secretion, and NO production.
Effects of various proteasome inhibitors on levels of P-IB in LPS-stimulated RAW 264.7 cells As mentioned above, phosphorylation of IB is a prerequisite to its degradation by the proteasome, with resultant liberation, and activation of NF-B. If the mechanism by which various proteasome inhibitors suppress activation of NF-B in LPS-stimulated RAW 264.7 cells involves a decreased capacity to degrade P-IB, levels of P-IB would be expected to increase in LPS-stimulated RAW-264.7 cells treated with proteasome inhibitors, compared to vehicle treated controls. Pretreatment of LPS-stimulated RAW 264.7 cells with quercetin, dexamethasone, mevinolin, riboflavin, and δ- Proteasome-Glo (Promega) chymotrypsin-Like, trypsin-Like, and postglutamase cell-based assays were used. 10,000 cells were plated per well (in 100 μL of media) in a 96-well white plate. Cells were allowed to adhere to plates for 2 h prior testing. 2 Each assay were carried out according to Promega Protocols, and plates were read with a Luminometer to give relative luminescence units (RLU) values. 3 Media + cells + 0.4% dimethyl sulfoxide (DMSO) was used as control. All concentrations of each compound contain 0.4% DMSO. 4 Percentages of control values are in parenthesis. *Similar values were observed for this range of reference [9] in Lipids in Health and Disease 2010, 9:143. tocotrienol resulted in significant increases of 90%, 70%, 58%, 50%, and 35% (P < 0.05), respectively, compared to vehicle treated controls ( Figure 6). Thus, the capacity of these proteasome inhibitors to suppress degradation of P-IB probably accounts for their ability to inhibit activation of NF-B, and secretion of TNF-α, and production of NO in LPS-stimulated RAW 264.7 cells.

Effects of various proteasome inhibitors on the secretion of TNF-a, by LPS-stimulated peritoneal macrophages from BALB/c mice
In order to confirm the results obtained above using cell cultures, we determined the effects of these proteasome inhibitors on LPS-induced responses of thioglycolate-elicited peritoneal macrophages from several strains of mice. Serial dilutions (5 μM to 640 μM) of dexamethasone, mevinolin, α,-tocopherol, δ-tocotrienol, riboflavin, and quercetin, were tested for their ability to inhibit TNF-α, secretion by thioglycolate-elicited peritoneal macrophages of 8-week-old female BALB/c mice stimulated with LPS (Table 3). Comparable to results obtained with RAW 264.7 cells, significant inhibition of TNF-α, secretion was attained with all agents tested except α,-tocopherol. Based on these results, all further experiments were carried out using the following concentrations of these agents: dexamethasone (10 μM); mevinolin (20 μM); δ-tocotrienol (10 μM); riboflavin (40 μM), and; quercetin (40 μM).
We previously reported that NO production by LPSstimulated peritoneal macrophages was markedly reduced in LMP7/MECL-1 -/knockout mice; TNF-α, production, in contrast, was not markedly affected by the LMP7/MECL-1 -/genotype [23]. We also demonstrated that in order to suppress TNF-α, production by LPS stimulated macrophages with proteasome inhibitors, both chymotrypsin-and trypsin-like proteasome activities must be suppressed [22]. Thus, the capacity of δtocotrienol, riboflavin, and quercetin to block TNF-α, production in C57BL/6 and BALB/c, but not in LMP7/ MECL-1 -/-, would be explained if these agents inhibited primarily the LMP2, LMP7 and MECL-1 subunits of mouse immunoproteasomes, with comparatively lower suppressive effects on X, Y, and Z subunits of constitutively expressed proteasomes. LPS-stimulation of macrophages from C57BL/6 and BALB/c would induce production of immunoproteasomes in which X, Y and Z subunits were partially replaced by LMP2, LMP7 and Then all the wells were challenged with LPS (10 ng/well; 400 μL) and incubated at 37°C 5% CO 2 for 36 h. Cells were washed with phosphate buffer saline, and cytoplasmic extracts were prepared using cell extraction buffer supplemented with phenylmethylsulfonyl fluoride and phosphatase inhibitors, according to the directions of manufacture [20]. Protein concentrations were measured with BCA protein assay kits, and P-IB-α, levels were measured by Western blots analyses [7,20]. Data are presented as the percent of P-IB compared to controls. The treatments 1-7 correspond to: 1. Control (media + cells + LPS [10 ng/mL] + 0.5% DMSO); 2. dexamethasone; 3. mevinolin; 4. α,-tocopherol; 5. δ-tocotrienol; 6. riboflavin; 7. quercetin-HCL. MECL-1 -/-, decreased TNF-α, production would occur if these latter subunits were potently suppressed by δtocotrienol, riboflavin, and quercetin. In contrast, the × and Z components of LPS-stimulated macrophages from LMP7/MECL-1 -/mice, could not be replaced by LMP7 and MECL-1 -/-. If the chymotrypsin-like or trypsin-like activities of × and Z proteasome subunits were comparatively resistant to the inhibitory effects of δ-tocotrienol, riboflavin, and quercetin, TNF-α, production by LPS-stimulated macrophages from LMP7/MECL-1 -/mice would be unaffected, since inhibition of both chymotrypsin-and trypsin-like proteasomal activities are required to suppress TNF-α, secretion.
The capacity of δ-tocotrienol, riboflavin, and quercetin to inhibit NO production by LPS-stimulated macrophages from LMP7/MECL-1 -/mice might be explained by the high sensitivity of NO production to modulation of immunoproteasome subunits [22,23]. Thus, we previously demonstrated that LPS-stimulated macrophages from LMP2 -/knockout mice produce substantially less NO than control littermates, and concluded from those experiments that inducible immunoproteasome subunits play a critical role in NO production [23]. Thus, NO production by LPS-stimulated macrophages from LMP7/MECL-1 -/mice is very low, therefore IFN-γ has to be added concurrently to observe NO production and the defect is reversed. The present inhibitors suppress the LPS-induced NO production in the presence of IFN-γ ( Figure 10A, B) Effect of various proteasome inhibitors on gene expression of TNF-a, and iNOS by LPS-stimulated thioglycolate-elicited peritoneal macrophages from C57BL/6, BALB/c, double knockout LMP7/MECL-1 -/-, and PPAR-a, -/knockout mice The experiments described above demonstrated that δtocotrienol, riboflavin, and quercetin inhibited secretion of TNF-α, by LPS-stimulated thioglycolate-elicited peritoneal macrophages from C57BL/6 and BALB/c, but not LMP7/MECL-1 -/or PPAR-α, -/knockout mice ( Figures 9A, B). In contrast, these compounds all inhibited NO production by LPS-stimulated thioglycolate-elicited peritoneal macrophages from all four strains of mice ( Figure 10A, B). In order to determine whether these changes resulted from alterations in transcription of the relevant genes, we measured the effect of various proteasome inhibitors on mRNA levels for TNF-α, and iNOS in LPS-stimulated thioglycolate-elicited peritoneal macrophages from 8-weekold female C57BL/6, BALB/c, double knockout LMP7/ MECL-1 -/-, and PPAR-α, -/mice. The concentrations of each inhibitor and condition were similar to those used in earlier experiments. Cells were treated with δtocotrienol (10 μM), riboflavin (40 μM), and quercetin (40 μM) for 1 h, then LPS (10 μL of 1.0 μg/mL) was added to each well and incubated for an additional 4 h. Total cellular RNA was then extracted and reversetranscribed, and gene analyses were carried out by RT-PCR and Southern blots analyses. Percentages of control values are in parenthesis. *Values from reference [10] in Lipids in Health and Disease 2011,10:39. Figure 7 Effects of various compounds on the secretion of TNF-a, in LPS-stimulated (pre-treatment) peritoneal macrophages of 8week-old C57BL/6 versus BALB/c female mice. Thioglycollate-elicited peritoneal macrophages were prepared from 8-week-old C57BL/6 and BALB/c (5 of each) female mice as described previously [6]. The macrophages of each mouse were adhered to the bottom of 12 well plates (1 × 10 7 cells/well in 1 mL media) for 4 h, the cells were washed with media three times. The cells were cultured overnight in the fresh media (500 μL) after the final wash. The cells were treated with 100 μL dissolved in 0.2% DMSO of dexamethasone, 10 μM; mevinolin, 20 μM; δ-tocotrienol, 10 μM; α,-tocopherol, 100 μM; riboflavin, 40 μM; or quercetin-HCL, 40 μM for I h, followed by stimulation with LPS (10 ng/mL) of each treatment. The assay mixtures were incubated at room temperature for 4 h, and the assay mixtures were centrifuged at 2,000 rpm for 20 min. The cells were then harvested, and the total cellular RNA was extracted from each pellet with RNeasy mini kit according to manufacturer's instructions [7,17]. The total secretion of TNF-α, of each inhibitor was estimated in the supernatants by radio-immunoassay (ELISA) kit according to the manufacture directions [7,17]. The cells viability were very good (> 95%) in all the treatments [4,7]. Data are means ± SD, n = 5 per treatment, and triplicate analyses of each sample. The treatments 1-7 correspond to: 1. Control (macrophages + LPS + 0.2% DMSO); 2. dexamethasone; 3. mevinolin; 4. α,-tocopherol; 5. δ-tocotrienol; 6. riboflavin; 7. Quercetin-HCL. Values in a column with a different superscript symbols are significantly different at P < 0.05, A = values, B = Percentages.
In LPS-stimulated macrophages from PPAR-α, -/knockout mice, the pattern of inhibition of mRNA production for TNF-α, and iNOS genes by proteasome inhibitors also varied markedly from that observed with either C57BL/6 or BALB/c mice, and more closely resembled the pattern obtained with LMP7/MECL-1 -/knockout mice. As with LMP7/MECL-1 -/mice, proteasome inhibitors did not affect TNF-α, mRNA levels ( Figure 14A). Treatment with quercetin markedly suppressed iNOS mRNA levels (77%); iNOS gene expression was inhibited less profoundly by δ-tocotrienol (20%) and riboflavin (14%) ( Figure 14B). In summary, results of mRNA expression studies were generally consistent with those of TNF-α, secretion and NO production, though the enhanced secretion of TNF-α, by LPSstimulated macrophages from PPAR-α, -/knockout mice treated with riboflavin and δ-tocotrienol was not explained by a corresponding increase in mRNA levels. Moreover, quercetin was found to be slightly better anti-inflammatory compound for the above mentioned markers than δ-tocotrienol and riboflavin.

Discussion
The primary objectives in the present study were to further evaluate several naturally-occurring proteasome inhibitors for their capacity to suppress inflammatory processes, and to define mechanisms responsible for these anti-inflammatory effects. Anti-inflammatory properties of the proteasome inhibitors were evaluated in macrophages derived from several sources (e.g. the RAW 264.7 cell line, and thioglycolate-elicited peritoneal macrophages from four strains of mice (C57BL/6, BALB/c, double knockout LMP7/MECL-1 -/and PPARα, -/-). As a result of these studies we have identified several naturally-occurring proteasome inhibitors that could potentially decrease levels of inflammatory cytokines and NO that may contribute to the development of diseases associated with ageing.
First, we demonstrated that dexamethasone, mevinolin, δ-tocotrienol, riboflavin and quercetin are potent inhibitors of chymotrypsin-like activity of 20S rabbit muscle proteasomes, and that (-) Corey lactone and amiloride enhanced this activity. We also demonstrated that dexamethasone, mevinolin, δ-tocotrienol, riboflavin and quercetin inhibited the secretion of TNF-α and NO production by LPS-stimulated RAW 264.7 macrophage like murine cell cultures. Further, levels of NF-within the nucleus were decreased, whereas cellular P-Ilevels were increased, by pre-treatment of LPS-stimulated RAW 264.7 cells with dexamethasone, mevinolin, δtocotrienol, riboflavin and quercetin.
NF-is maintained in an inactive state in the cytoplasm of cells when it is bound to I. LPS induces a series of events that results in phosphorylation and ubiquitination of Iwith subsequent degradation by the proteasome. These actions result in NF-activation, translocation to the nucleus, and increased transcription of several genes encoding pro-inflammatory cytokines. Consequently, the capacity of dexamethasone, mevinolin, δ-tocotrienol, riboflavin and quercetin to inhibit proteasome activity in conjunction with their capacity to increase cellular levels of P-Iand decrease nuclear translocation of NF-, suggests that the mechanism by which these agents suppress production of TNF-α, and NO involves decreased degradation of ubiquinated P-Iby the proteasome, resulting in depressed translocation of NF-to the nucleus. Therefore, ultimately, these proteasome inhibitors suppress production of TNF-α, and NO, and exert their anti-inflammatory effects by inhibiting NF-B, activation.
Our previously published studies strongly support the concept that proteasomes are key regulators of LPS-stimulated inflammatory signaling pathways (3,4,(6)(7)(8). Proteolytic activity of proteasomes is mediated by the 20S proteasomes, a hollow, cylindrical multi-protein complex consisting of three proteolytic subunits, X, Y, and Z, with chymotrypsin-like, trypsinlike, and post-glutamase activities, respectively. A variety of inflammatory stimuli induce alterations in newly assembled "immuno-proteasomes" in which X, Y and Z subunits are partially replaced by LMP7, LMP2, and MECL-1, respectively.
In the current study, the capacity of the investigated proteasome inhibitors to inhibit TNF-α, secretion by LPS-stimulated macrophages from several sources (i.e. the RAW 264.7 cell line, and peritoneal macrophages from C57BL/6 and BALB/c mice) supports the conclusion that these inhibitors suppress both chymotrypsinlike and trypsin-like activities of the proteasomes, since both of these activities must be suppressed in order to inhibit TNF-α, secretion. These conclusions were supported by our analysis of the effects of these inhibitors on proteolytic activity of the proteasome.
The capacity of δ-tocotrienol, riboflavin, and quercetin to block TNF-α, secretion in C57BL/6 and BALB/c, but not in LMP7/MECL-1 -/mice, is an intriguing result. LPS-stimulation of macrophages from C57BL/6 and BALB/c mice would be expected to induce production of immunoproteasomes in which X, Y and Z subunits were partially replaced by LMP2, LMP7 and MECL-1 -/-. The × and Z components of LPS-stimulated macrophages from LMP7/MECL-1 -/mice, however, cannot be replaced by LMP7 and MECL-1 -/-. The macrophages from knockout mice when induced produce robust amounts of TNF-α,. Thus, the differential capacity of δtocotrienol, riboflavin, and quercetin to inhibit TNF-α, secretion by LPS-stimulated macrophages from C57BL/6 and BALB/c vs LMP7/MECL-1 -/knockout mice would appear to be attributable to a differential susceptibility of × and Z vs LMP7 and MECL-1 proteasomal subunits to inhibition by these inhibitors.
The finding that δ-tocotrienol, riboflavin, and quercetin inhibited NO production by LPS-stimulated macrophages from C57BL/6, BALB/c, LMP7/MECL-1 -/-, and PPAR-α, -/mice was also quite interesting; the LMP7/ MECL-1 -/genotype mice did not affect susceptibility to inhibition by these compounds. These macrophages from knockout mice do not induce very much NO, therefore IFN-γ had to be added along with LPS to induce NO in cells that have X, Y, Z, and LMP2 subunits [22].
PPAR-α, -/knockout mice have exaggerated inflammatory responses to a variety of stimuli, because activation of PPAR-α, leads to anti-inflammatory effects [24]. The mechanisms leading to these exaggerated inflammatory responses are not clearly understood, but are believed to be at least partially attributable to increased NFB activity [24,25]. Consequently, one would expect TNF-α, secretion by LPS-stimulated macrophages from PPARα, -/knockout mice to be highly up-regulated with respect to TNF-α, secretion and relatively resistant to inhibition by proteasome inhibitors that degrade IB, and decrease NFB activity. Therefore, we found that δtocotrienol, riboflavin, and quercetin failed to suppress TNF-α, secretion by LPS-stimulated macrophages from PPAR-α, -/knockout mice. In fact TNF-α, secretion was substantially enhanced by riboflavin and δ-tocotrienol. A clearer explanation of these results will be dependent on further elucidation of interactions between PPAR-α, and ubiquitin proteasome pathways [26].
Our present results demonstrate that δ-tocotrienol, riboflavin, and quercetin are naturally-occurring potent proteasome inhibitors for the inhibition of NO production tested in vitro. These results also confirms earlier report that γ-tocotrienol blocked LPS-stimulated activation of NF-B, and also blocked TNF-α, induced phosphorylation and degradation of IBα, through the inhibition of IBα, kinase activation [27]. Tocotrienols have been shown to modestly inhibit or activate the proteasomal activity depending on its concentrations (Tables 1, [9]). Therefore, blocking the proteasomal activity with low doses of tocotrienols could potentially reduce inflammatory responses, but at high doses of tocotrienols may cause apoptotic cell death in cancer [9,28]. Quercetin, on the other hand is a natural proteasome inhibitor that can affect several proteasomal activities.

Conclusions
The present study describes the possible mechanism of inhibition of NO production (released during ageing processes or acute and chronic disease) by naturallyoccurring proteasome inhibitors (quercetin, δ-tocotrienol, and riboflavin). 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 also inhibited chymotrypsin-like, trypsinlike and post-glutamase activities of the proteasomes in RAW 264.7 cells. These compounds also blocked LPSstimulated secretion of TNF-α, NO production, activation of NF-B, and degradation of P-IB in RAW 264.7 murine macrophages. Similarly, these compounds suppressed TNF-α, 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-α, in macrophages derived from LMP7/MECL-1 -/and PPAR-α, -/knockout mice. These results indicate that δ-tocotrienol, riboflavin, and quercetin treatments differentially inhibit the secretion of TNF-α, of LPS-stimulated macrophages derived from C57BL/6, BALB/c versus LMP7/MECL-1 -/and PPAR-α, -/knockout mice. Moreover, all the gene expression results of TNF-α, and iNOS genes are generally consistent with the results of protein levels of secretion of TNF-α, and production of NO observed with either C57BL/6, BALB/c, LMP7/MECL-1 -/-, or PPAR-α, -/mice. All these results indicate that the production of NO may be modulated via NF-B pathway, and δ-tocotrienol acts as a proteasome inhibitor at lower doses and as activator at higher doses (Table 1). These collective properties of δ-tocotrienol, riboflavin, and quercetin can be referred to as "novel proteasome inhibitors", which might be used to suppress the production of inflammatory mediators in ageing humans, thereby decreasing the risk of developing a variety of age-related diseases that appear to be at least partially attributable to aberrant control of inflammatory responses with increasing age.