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Tocotrienols inhibit lipopolysaccharide-inducedpro-inflammatory cytokines in macrophages offemale miceAsaf A Qureshi1*, Julia C Reis1, Christopher J Papasian1, David C Morrison1, Nilofer Qureshi1,2

Abstract

Background: Inflammation has been implicated in cardiovascular disease, and the important role of proteasomesin the development of inflammation and other macrophage functions has been demonstrated. Tocotrienols arepotent hypocholesterolemic agents that inhibit b-hydroxy-b-methylglutaryl coenzyme A reductase activity, which isdegraded via the ubiquitin-proteasome pathway. Our objective was to evaluate the effect of tocotrienols inreducing inflammation. Lipopolysaccharide (LPS) was used as a prototype for inflammation in murine RAW 264.7cells and BALB/c female mice.

Results: The present results clearly demonstrate that a-, g-, or δ-tocotrienol treatments inhibit the chymotrypsin-like activity of 20 S rabbit muscle proteasomes (> 50%; P < 0.05). Chymotrypsin, trypsin, and post-glutamaseactivities were decreased > 40% (P < 0.05) with low concentrations (< 80 μM), and then increased gradually withconcentrations of (80 - 640 μM) in RAW 264.7 whole cells. Tocotrienols showed 9 - 33% (P < 0.05) inhibitions inTNF-a secretion in LPS-stimulated RAW 264.7 cells. Results of experiments carried out in BALB/c micedemonstrated that serum levels of TNF-a after LPS treatment were also reduced (20 - 48%; P < 0.05) bytocotrienols with doses of 1 and 10 μg/kg, and a corresponding rise in serum levels of corticosterone (19 - 41%;P < 0.05) and adrenocorticotropic hormone (81 - 145%; P < 0.02) was observed at higher concentrations (40 μM).Maximal inhibition of LPS-induced TNF-a was obtained with δ-tocotrienol (10 μg/kg). Low concentrations ofδ-Tocotrienols (< 20 μM) blocked LPS-induced gene expression of TNF-a, IL-1b, IL-6 and iNOS (>40%), while higherconcentrations (40 μM) increased gene expression of the latter in peritoneal macrophages (prepared from BALB/cmice) as compared to control group.

Conclusions: These results represent a novel approach by using natural products, such as tocotrienols asproteasome modulators, which may lead to the development of new dietary supplements of tocotrienols forcardiovascular diseases, as well as others that are based on inflammation.

BackgroundLipopolysaccharide (LPS), which is expressed on theouter membrane of essentially all Gram-negative bac-teria, is a potent inducer of pro-inflammatory cytokines,including tumor necrosis factor-a (TNF-a interleukin-1b (IL-1b), IL-6, IL-8, arachidonic acid metabolites andnitric oxide [1]. LPS can also induce corticosteroid pro-duction by the host, which tends to suppress furtherproduction of pro-inflammatory cytokines. Some

conditions leading to dysregulated production of inflam-matory cytokines by the host can produce profoundalterations in metabolic, cardiovascular, immunological,haemostatic, and endocrine functions, which may ulti-mately lead to septic shock [1-3]. Less profound inflam-matory responses have also been implicated in thepathogenesis of atherosclerosis, cancer, stroke and dia-betes in human subjects [4-7].Proteasomes are essential for numerous physiological

processes, including signal transduction, transcriptionalactivation, cell cycle progression, and certain immunecell functions [8]. We have reported a potentially impor-tant central role for proteasomes in inflammation and

* Correspondence: [email protected] of Basic Medical Sciences, University of Missouri-Kansas City,2411 Holmes Street, Kansas City, MO 64108, USAFull list of author information is available at the end of the article

Qureshi et al. Lipids in Health and Disease 2010, 9:143http://www.lipidworld.com/content/9/1/143

© 2010 Qureshi et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.

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other macrophage functions [8]. Proteasomes often existas 26 S multi-subunit complexes containing a 20 S pro-teolytic proteasome and a 19 S regulatory complex. Cor-respondingly, the 20 S proteasome is comprised of avariety of distinct protein subunits that account for thedifferent proteolytic activities of the 20 S proteasome.Several different exogenous inhibitors or activators ofproteasome function have been described, and theseinhibitors act by blocking, or activating, the proteolyticactivity of the individual protein subunits of the 20 Sproteasome.We, and others, have reported that tocotrienols inter-

fere with the formation of atherosclerotic plaque, andpossess hypocholesterolemic, antioxidant, anti-inflamma-tory, antithrombotic, and anti-proliferative (anticancer)properties [9-22]. Tocotrienols are naturally occurringcompounds containing a chroman ring and a farnesylatedunsaturated side-chain with analogs of a-, b-, g- andδ-type. These tocotrienols are minor constituents ofnatural vitamin E (predominantly a-tocopherol) whichhas a saturated side-chain attached to a chroman ring(Figure 1). Tocotrienols lower serum total- and LDL-cholesterol levels by inhibiting hepatic b-hydroxy-b-methylglutaryl coenzyme A (HMG-CoA) reductaseactivity through a post-transcriptional mechanism, whichinduces degradation of the reductase enzyme [19]. Anunsaturated side-chain is essential for inhibition of hepa-tic HMG-CoA reductase activity. On the other hand,tocopherols (vitamin E) are well known for their charac-teristic antioxidant activity, but they do not increasereductase degradation or lower serum total or LDL-cholesterol levels [10,16]. The positive effects of tocotrie-nols as hypocholesterolemic, antioxidant, and anticanceragents have been confirmed in animal systems and var-ious cell lines by many investigators [15-22].

Moreover, the far superior efficacy of tocotrienols ver-sus tocopherols (vitamin E) as antioxidants has beenestablished, and δ-tocotrienol is found to be the mostpotent among the known tocotrienols [10,17,18,22].Tocotrienols also show non-antioxidant properties invarious in vitro and in vivo models. Perhaps mostimportantly, tocotrienols interact with the mevalonatepathway leading to the lowering of cholesterol levels,the prevention of cell adhesion to endothelial cells, thesuppression of tumor cell growth, and glutamate-induced neurotoxicity [10-12,23,24].The present investigation was carried out to evaluate

the mechanisms by which tocotrienols inhibit inflamma-tion. Murine systems were chosen in the present study,because they are relatively resistant to the lethal effectsof endotoxin compared to rabbit and sheep [1,3,25].Recently, rodent models have been developed which arevery sensitive to bacterial endotoxin and thus, femalemice were chosen due to their increased sensitivity toLPS-induced inflammation. These experiments weredesigned to test our hypothesis that tocotrienol treat-ment modulates proteasomal activity, induces corticos-teroids synthesis, and blocks LPS-induced signalingpathways that contribute to the inflammatory process.

Materials and methodsReagentsHighly purified, deep rough chemotype LPS (Re LPS)from E. coli D31m4 was prepared as described by Qur-eshi et al. [26]. For tissue culture studies, Dulbecco’sModified Eagle Medium (DMEM), heat-inactivatedlow-endotoxin fetal bovine serum (FBS), and gentami-cin were all purchased from Cambrex (Walkersville,MD). Thioglycollate was purchased from Sigma,Aldrich (St. Louis, MO) and the RNeasy mini kit fromQIAGEN sciences (Germantown, MD). Substrates forthe chymotrypsin-like activity of the proteasome werepurchased from Calbiochem (La Jolla, CA). Tocotrienolrich fractions (TRF) of palm oil were provided byMalaysian Palm Oil Board, Kuala Lumpur, Malaysia(previously known as “Palm Oil Research Institute ofMalaysia” [PORIM], Kuala Lumpur, Malaysia). “Protea-some-Glo” assays kits for chymotrypsin-like activity(substrate: Suc LLVY-Glo, Succinyl-leucine-leucine-valine-tyrosine-aminoluciferin), trypsin-like activity(Z-LRR aminoluciferin, Z-leucine-arginine-arginine-aminoluciferin), and post-glutamase activity (post-acidic,substrate, ZnLPnLD-Glo, Z-norleucine-proline-norleu-cine-aspartate-aminoluciferin) of the proteasome werepurchased from Promega (Madison, WI). RAW 264.7 cells(TIB 71) were purchased from American Type CultureCollection (Manassas, VA), and 4-week-old BALB/cfemale mice were obtained from The Jackson Laboratory(Bar Harbor, ME).

Figure 1 Chemical structures of various isomers of tocopherolsand tocotrienols.


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Purification of a-tocopherol, a-, g-, and δ-tocotrienolsfrom TRF of palm oilThe individual components of tocotrienol rich fraction(TRF) of palm oil were purified as described recently[10]. The purity of a-tocopherol and individual toco-trienols were established by high pressure liquid chro-matography (HPLC) against their respective purestandards [10].

Effects of various tocols (tocopherols + tocotrienols) onthe chymotrypsin-like activity of 20 S rabbit muscleproteasomesProteasomal activities of the 20 S rabbit muscle protea-somes (0.4 μg/mL) were assayed with synthetic peptidesubstrates in 0.02 M Tris-HCl buffer (pH 7.2). The sub-strate used for the chymotrypsin-like activity was 100 μMof succinyl-Leu-Leu-Val-Tyr-amino-methyl-coumarin.Fluorescence was measured (absorption at 360 nm andemission at 460 nm) using an FLX 800 microplate fluor-escence reader (Bio-Tek Instruments, Winooski, VT).

Cell culture and maintenanceThe RAW 264.7 cells or mouse peritoneal macrophageswere maintained in DMEM supplemented with 10%heat inactivated fetal bovine serum (FBS) and 10 mggentamicin (in 500 mL) at 37°C in a humidified atmo-sphere with 5% CO2 as described previously [27]. Cellswere cultured in 6-well plates as described in thelegends to the figures.

Effects of δ-tocotrienol (concentrations of 10 - 640 μM)after 60 min treatment on different proteasomal activities(chymotrypsin-like, trypsin-like, and postglutamase) inRAW 264.7 whole cellsIn order to check the comparative inhibitory effect oftocols, only the most potent δ-tocotrienol was tested onthe chymotrypsin-like, trypsin-like, and post-glutamaseactivities of the proteasome using RAW 264.7 wholecells; the following experiment was carried out. RAW264.7 cells (10 × 104 cells/100 μl/well) were added inwhite plates 96-well, Fisher, 0877126), followed by theaddition of various concentrations of δ-tocotrienol (10,20, 40, 80, 160, 320, or 640 μM in 100 μL; dissolved in0.2% dimethyl sulfoxide (DMSO). The mixtures wereincubated at 37°C in an incubator at 5% CO2 for 60 min.After incubation period, the cells in the 96-well plateswere taken out 20 min prior to the addition of Caspase-Glo reagent (brought to room temperature before addi-tion to the wells). Caspase-Glo reagent (100 μL) wasadded to each well to a total volume of 200 μL/well (trisbuffer, pH 7.5; 0.02 M). The plate were covered with aplate sealer, removed from light and incubated at roomtemp for 10 min. The relative luminescence units (RLU)of assays were read with a Promega Plate Luminometer.

The chymotrypsin-like, trypsin-like, or post-glutamaseactivities were quantitated by measuring luminescenceafter stimulation of RAW 264.7 whole cells with variousdoses of δ-tocotrienol in a Luminometer (Promega),according to the directions of manufacturer.

Effects of various tocols (a-tocopherol, a-tocotrienol,g-tocotrienol or δ-tocotrienol) on the secretion of TNF-ain LPS-stimulated RAW 264.7 cellsThe levels of TNF-a in RAW 264.7 cell culture superna-tants were determined after treatment with LPS (1 ng/mL) and various doses (4, 8, 16 μM) of a-tocopherol,a-tocotrienol, g-tocotrienol or δ-tocotrienol by Quanti-kine M ELISA kit (R&D System, Minneapolis, MN)according to manufacturer’s instructions. The lowerlimit of detection for TNF-a in this method is approxi-mately, 5.0 pg/mL [28,29]. The TNF-a levels in mouse’sserum or thioglycollate-elicited peritoneal macrophageswere also quantified by using the same method [28,29].

Effects of a-, g-, and δ-tocotrienols on release of LPS-induced TNF-a in serum of 6-wk-old female BALB/c miceAll mice used in this study received humane care incompliance with the principles of laboratory animalcare formulated by the National Society of HealthGuide for the Care and Use of Laboratory Animals(DHHS Publication No. [NIH] 85 - 23, revised 1985).The experimental procedures involving animals werereviewed and approved by the Institutional AnimalCare and Use Committee of UMKC, Medical School,MO. Female BALB/c mice were acclimatized to thenew environment for fourteen days and were fedadlibitum regular commercial mice diet and had freeaccess to water throughout the experiment. A 12 hlight and 12 h dark cycle was maintained during feed-ing period. All samples were dissolved in 5% triethyla-mine solution.Seven groups of 4-wk-old female BALB/c mice (3/

group) were acclimatized (for two wks), and then injectedeither with saline (two control groups), dexamethazone orvarious doses (2.5, 5.0, and 10.0 μg/kg body weight) of a-tocopherol, a-tocotrienol, g-tocotrienol, or δ-tocotrienol.One h later, all mice were injected intraperitoneally withpure LPS (E. coli D31m4; 10 ng/mouse). After two h, allmice were sacrificed, serum was collected, and the levelsof TNF-a were quantified using a radioimmunoassay kitaccording to the manufacturer’s directions [28,29].

Effects of a-, g-, and δ-tocotrienols on the induction ofcorticosterone and adrenocorticotropic hormone (ACTH)in serum of LPS-stimulated 6-wk-old female BALB/c miceTo further probe the anti-inflammatory effects of varioustocotrienols in mice, we queried whether tocotrienolswould also induce the serum levels of corticosterone and


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adrenocorticotropic hormone (ACTH) after LPS treat-ment of mice as reported recently [3]. The levels ofserum (used from the above experiment) corticosteroneand ACTH were estimated according to publishedmethod [1,3].

Isolation of thioglycollate-elicited peritoneal macrophagesfrom BALB/c mice, total cellular RNA isolation and RT-PCRTen 4-wk-old female BALB/c mice were acclimatized tothe new environment for fourteen days, and thioglycol-late-elicited peritoneal macrophages were prepared asdescribed previously [29]. Macrophages (2 × 105) weretreated with either pure LPS (10 ng/treatment), LPS +a-tocopherol (25, 50, or 100 μM), LPS + δ-tocotrienol(10, 20, or 40 μM). All the samples were dissolved in0.2% dimethyl sulfoxide (DMSO). The assay mixtureswere incubated at room temperature for 4 h and thencentrifuged at 2,000 rpm for 20 min. The supernatantswere removed and concentrations of TNF-a were deter-mined using Quantikine M ELISA kit (R&D System,Minneapolis, MN) according to the manufacturer’sdirections [29]. The total RNA was isolated from eachpellet with RNeasy mini kit according to the manufac-turer’s instructions. To check the purity of the totalRNA, Reverse transcriptase polymerase chain reaction(RT-PCR) was conducted using a 1-step kit (Qiagen,Chatsworth, CA) according to the manufacturer’sinstructions.

Detection of cell viabilityViability of peritoneal macrophages treated with andwithout LPS plus dexamethasone (positive control),a-tocopherol or various doses of δ-tocotrienol wasdetermined by trypan blue dye exclusion or a quantita-tive colorimetric assay with 3-(4,5)-dimethylthiozol-2,5-diphenyltetrazolium bromide (MTT) as described pre-viously [30].

Statistical analysesThe analyses demonstrated the affect of various isomersof tocols (a-tocopherol or a-, g-, δ-tocotrienols) withinthe groups. Stat View software (version 4.01, AbacusConcepts, Berkeley, CA) was used for the analyses oftreatment-mediated effects as compared to the controlgroup. Treatment-mediated differences in variousinflammatory markers variables were evaluated using atwo-way ANOVA, and when F test indicated a signifi-cant effect, the differences between the means were ana-lyzed by a Fisher’s protected least significant and leastdifference test. Data were reported as means ± SD intext and Tables. The level for statistical significancelevel was established at 5% (P < 0.05).

ResultsThe results of the present study on the inhibition ofinflammation by tocotrienols are presented in the samein two different formats. For each figure, ‘A’ shows theraw values for each of the treatments and controls, and‘B’ shows the percent change compared to controls.

Inhibition of chymotrypsin-like activity of 20 S rabbitmuscle proteasomes by various tocols (tocopherols +tocotrienols)We have previously shown that the proteasome is a cen-tral regulator of inflammation; we therefore queriedwhether tocotrienols also affect proteasome activity, thusaffecting the induction of cytokines. The results of thisstudy reveals that inhibition of chymotrypsin-like activityof the 20 S rabbit muscle proteasomes was dose-dependent between 4 μM and 16 μM for a-tocotrienol(6% - 36%), g-tocotrienol (12% - 32%), and δ-tocotrienol(51% - 55%) as compared to control groups (Figure 2A&2B). An insignificant reduction (5%) in 20 S pro-teasomes activity was observed with a-tocopherol(Figure 2A &2B). These studies further suggest thatδ-tocotrienol is the most effective isomer of the tocotrie-nols for inhibiting the chymotrypsin-like activity of rabbitmuscle proteasomes.

Impact of δ-tocotrienol (concentrations of 10 - 640 μM)on different proteasomal active sites (chymotrypsin-like,trypsin-like, and post-glutamase) in RAW 264.7 wholecells after 60 min treatmentThe effects of δ-tocotrienol on the chymotrypsin-like,trypsin-like, and post-glutamase activities of protea-somes were also carried out in RAW 264.7 murinemacrophages. The chymotrypsin-like activities decreasedto 46% of controls with 40 μM δ-tocotrienol treatmentof cells and then increased gradually as shown inFigure 3. A similar trend was observed with the trypsin-like activity and post-glutamase activities in response toδ-tocotrienol (Figure 3).

Inhibition of the secretion of TNF-a in LPS-stimulatedRAW 264.7 cells by various tocols (a-tocopherol,a-tocotrienol, g-tocotrienol or δ-tocotrienol)The effects of tocotrienols on the secretion of TNF-awith, and without, LPS-stimulation in RAW 264.7 cellswas investigated. A significant reduction of secretion ofTNF-a, 9% - 33% (P < 0.05) was observed with tocotrie-nols in LPS-stimulated RAW 264.7 cells in a dose-dependent manner (Figure 4A &4B). Tocotrienols didnot inhibit TNF-a secretion in the absence of LPS sti-mulation (data not shown in the figure). Cell viabilityfor all above experiments was 90% - 95%.


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Figure 2 Effects of various tocol treatments on the chymotrypsin-like activity of 20 S rabbit muscle proteasomes. Chymotrypsin-likeactivity was carried out by using synthetic dipeptide substrate III (Suc-Leu-Leu-Val-Tyr-AMC) in buffer pH 7.5, 0.02 M. Rabbit muscle proteasomeswere used. The compounds were dissolved in 2% dimethyl sulfoxide (DMSO). Fluorescence absorption was measured at excitation = 360 nmand emission = 460 nm, A = values; B = percentages.


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a-, g-, and δ-Tocotrienols reduced serum TNF-a levels inLPS-stimulated 6-wk-old BALB/c miceTo further probe the anti-inflammatory effects of var-ious tocols in mice, we queried whether dietary supple-mentation with tocotrienols would also block LPS-induced TNF-a secretion in mice. A dose-dependentinhibition (20% to 48%; P < 0.05) of LPS-induced serumTNF-a levels was observed with a-, g-, and δ-tocotrie-nols as compared to control diets (Figure 5A &5B). Thereduction with a-tocopherol (5% - 9%) was insignificantas compared to control or tocotrienol treatments (Figure5A &5B). These results also suggest that δ-tocotrienol isthe most effective isomer in reducing LPS-inducedTNF-a secretion in mice.

a-, g-, and δ-Tocotrienols induce corticosterone in serumof 6-wk-old BALB/c miceWe have previously shown that LPS treatment of miceinduces high endogenous corticosterone levels, and thatcorticosterone levels are inversely correlated with TNF-

a serum concentrations [9]. Therefore, to furtherexplore mechanisms responsible for the anti-inflamma-tory effects of tocotrienols, we queried whether tocolsincrease corticosterone levels produced in response toLPS. For this experiment BALB/c mice were injected i.p.with tocotrienols. A significant (P < 0.02) rise in LPS-induced serum corticosterone (19%, 31%, and 41%)levels was induced by treatment with a-, g-, or δ-toco-trienols as compared to the control group (Figure 6A&6B). These results suggest that the decrease in thesynthesis of TNF-a could be due to the rise inthe endogenous corticosteroids, which may modulatethe synthesis of inflammatory cytokines [3].

a-, g-, and δ-Tocotrienols induce adrenocorticotropichormone in serum of 6-wk-old BALB/c miceThe impact of a-tocopherol and various tocotrienols onthe levels of adrenocorticotropic hormone was alsodetermined in the serum obtained from the experiment

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Chymotrypsin-like Trypsin-like Post-glutamaseFigure 3 Effects of δ-tocotrienol (concentration 10 μM - 640 μM) on proteasomal active sites, chymotrypsin-like, trypsin-like and post-glutamase, after 60 min treatment of RAW 264.7 whole cells. Proteasome-Glo (Promega) chymotrypsin-like, trypsin-like, and post-glutamasecell based assays were carried out. 10,000 cells were plated per well in 100 μL of media in a 96-well white colored plate. Cells were allowed toadhere to plates for 2 h prior to testing. At the start of the test, media or 0.4% DMSO + media (used as control = CD), or δ-tocotrienol(concentrations 10 μM - 640 μM) dissolved in 0.4% DMSO were added to each well for a duration of 1 h. The mixtures in the plates wereassayed according to Promega Protocol. Plates were read with a “Promega Luminometer” according to the directions of manufacturer, whichgive relative luminescence units (RLV) values.


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Figure 4 Effects of various tocols on secretion of TNF-a in LPS-stimulated RAW 264. 7 cells. RAW 264.7 cells (500 μL) were adhered in wellsfor 2 h at room temperature. The cells were then treated with various concentrations of a-tocopherol, or a-, g-, or δ-tocotrienols (100 μL) for 1 h, thenthe wells were treated with LPS (1 ng/well; 400 μL) for 4 h. The supernatants were then transferred in glass vials and stored at -20°C for subsequentTNF-a assays. Cell viability was determined for all treatment and control groups. The experimental solution were prepared by dissolving highestconcentration amount of a-tocopherol or a-, g-, δ-tocotrienols in 1.0 mL DMSO = X. Solution × (50 μL) was mixed with 950 μL of media = Y. Theremaining required concentrations were prepared with Y (1:2 dilutions). The TNF-a assays were carried out using an ELISA kit, and kit control valuevaries 266 - 444 (287), A = values; B = percentages.


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Figure 5 Effect of various tocols on serum TNF-a levels in LPS-stimulated, 6-week-old female BALB/c mice. The BALB/c female micewere acclimatized for 2 wk. Three mice for each concentration were injected intraperitoneally (i.p.) with compounds suspended in 5%triethylamine solution (0.2 mL/mouse) one h before LPS challenge. Mice were bled 2 h later to collect serum. The serum samples were stored at-20°C to for subsequent TNF-a analysis by ELISA. Values in a column with a different superscript letter are significantly different at P < 0.05, A =values; B = percentages.


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described above. A corresponding significant (P < 0.02)dose-dependent rise in LPS-induced serum adrenocortico-tropic hormone levels was observed (81%, 118%, and145%) by treatment with a-, g-, or δ-tocotrienols, respec-tively, as compared with the control group (Figure 7A&7B). An insignificant rise in the levels of corticosterone(7%) and adrenocorticotropic hormone (14%) with a-toco-pherol was also observed (Figure 6B &7B). In summary,these results clearly indicate that δ-tocotrienol is mosteffective in inhibiting the serum levels of TNF-a and alsoin inducing the serum levels of corticosterone and adreno-corticotropic hormone as compared to a-, g-tocotrienolsand a-tocopherol, which were the least effective com-pounds as compared to their respective control groups.

δ-Tocotrienol inhibits LPS-induced TNF-a, IL-1b, IL-6, andiNOS gene expression in thioglycollate-elicited peritonealmacrophages of 6-wk-old BALB/c miceIn order to define the mechanism of action of tocotrienolswith respect to lowering inflammation, thioglycollate-eli-cited peritoneal macrophages were prepared from BALB/cfemale mice, and treated with a-tocopherol, or δ-tocotrie-nol 1 h before, or at the same time as LPS. After 4 h of thetreatment, total cellular RNA was extracted and reverse-transcribed, and gene analysis was performed by RT-PCRand southern blot analyses. The results of gene expressionstudies clearly demonstrate that δ-tocotrienol is mosteffective in blocking LPS-induced gene expression ofTNF-a Figure 8A &8B, IL-1b, IL-6 and iNOS at low dosesof 10 μM and 20 μM (Figure 9A &9B).There were significant (P < 0.02) reductions of TNF-a

(45%), IL-1b (63%), IL-6 (46%) and iNOS (32%) mRNAwith a dose of 20 μM compared to their respective con-trol values, and significant increases (40%, 17%, 26%,13%) in the levels of these mRNA’s, with a dose of 40 μMof δ-tocotrienol, as compared to their respective 20 μMcontrol values (Figure 8B &9B). These results are inagreement with earlier results of tocotrienols on theactivities of chymotrypsin-like, trypsin-like and post-glutamase in RAW 264.7 whole cells (Figure 3). Thea-tocopherol (doses of 25, 50 or 100 μM) did not haveany impact on the gene expression of TNF-a, IL-1b, IL-6and iNOS compared to their respective control values(Figure 8A, Figure 9B).

DiscussionIt is well established that LPS-induced TNF-a produc-tion requires activation of NF-�B and activation of pro-teasomal activity. The results presented heredemonstrate that tocotrienols block production of keypro-inflammatory cytokines induced by LPS. Wehypothesized that this blockade of LPS-induced inflam-mation by tocotrienols could be due to modulation ofproteasomal activity [31], since tocotrienols have been

shown to be effective inhibitors of b-hydroxy-b-methylglu-taryl coenzyme A (HMG-CoA) reductase, (the rate-limit-ing enzyme in cholesterol biosynthesis), and are verypotent antioxidant and hypocholesterolemic agents [12]. Ithas also been demonstrated that HMG-CoA reductase isdegraded via the ubiquitin-proteasome pathway [32,33].We have shown that tocotrienols can inhibit chymotryp-sin-like activity of 20 S rabbit muscle proteasome, and thatδ-tocotrienol specifically inhibits the chymotrypsin-like,trypsin-like and post-glutamase activities of the protea-some in a dose-dependent manner in RAW 264.7 wholecells at concentrations below 80 μM; at higher concentra-tions (80 - 640 μM) tocotrienole enhance these activitiesas has been previously reported for lactacystin [34,35].Tocotrienols also inhibit the LPS-stimulated secretion

of TNF-a in RAW 264.7 cells and in serum of BALB/cmice, and a corresponding significant (P < 0.02) rise wasobserved in the serum levels of corticosterone and adre-nocorticotropic hormone compared to controls. More-over, δ-Tocotrienol is effective in reducing LPS-inducedgene expression of TNF-a, IL-1b IL-6 and iNOS at lowconcentrations. Interestingly, at high concentrationsδ-Tocotrienol activates chymotrypsin-like, trypsin-like,and post-glutamase activities, and upregulates LPS-induced gene expression of TNF-a, IL-1b, IL-6 andiNOS. It seems that tocotrienols actually upregulates theproteasomal activity, and thus can upregulate LPS-stimulated transcription of several pro-inflammatorygenes at higher concentrations. Therefore, the capacity ofδ-tocotrienol to modulate inflammation may be attribu-table, in part, to inhibition and activation of the chymo-trypsin-like, trypsin-like and post-glutamase’ activities ofmouse macrophages.The present results also demonstrated that δ-tocotrie-

nol is the most effective tocol among the known, a- andg-tocotrienols for inhibition or induction of various cyto-kines involved in inflammation, whereas a-tocopheroldoes not play any role in inhibiting LPS-induced inflam-mation. This conclusion has been further confirmed in arecent study [32] which demonstrated that δ-tocotrienolis very effective in inducing ubiquitination and degrada-tion of HMG-CoA reductase. δ-tocotrienol also blocksthe processing of sterol regulatory element-binding pro-teins-2 (SREBPs-2) which, when activated, can enhancetranscription of genes encoding cholesterol biosyntheticenzymes, including HMG-CoA reductase [32]. On theother hand, tocopherols neither increase degradation ofreductase, nor decrease SREBP-2 processing [32].The unsaturated isoprenoid side-chain in tocotrienol

molecules is important for its biological activity and itwas suggested that this could stimulate binding ofHMG-CoA reductase to Insig (endoplasmic reticulummembrane protein). Alternatively, the side-chain mightallow improved penetration and distribution in cell


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membranes [32,33]. This may contribute to the differ-ences observed in the potency of tocotrienols versustocopherols in stimulating HMG-CoA reductase in vitroubiquitination [32,33] thus confirming our previous andpresent results showing that δ-tocotrienol (one methyl

group) is more potent than g-tocotrienol (two methylgroups) due to the number of methyl groups whichabolishes regulatory activity with respect to HMG-CoAreductase degradation and SREBP-2 processing[10,11,32].

342 342 342 342

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Figure 6 Effects of a-tocopherol, a-tocotrienol, g-tocotrienol and δ-tocotrienol on the induction of corticosterone in serum of LPS-stimulated 6-wk-old BALB/c female mice. Three mice for each concentration were injected i.p. with tocols in 5% triethylamine solution (0.2mL/mouse) one h prior to LPS challenge, after conditioning them for 14 days. Mice were bled after 2 h later to collect serum. Serumcorticosterone levels were determined according to a published procedure (3). Values in a column with a different superscript letters aresignificantly different at P < 0.05, A = values; B = percentages.


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Figure 7 Effects of a-tocopherol, a-tocotrienol, g-tocotrienol and δ-tocotrienol on the induction of adrenocorticotropic hormone inserum of LPS-stimulated 6-wk-old BALB/c female mice. Three mice for each concentration were injected i. p. with various tocols in 5%triethylamine solution (0.2 mL/mouse) one hr prior to LPS challenge, after conditioning them for 14 days. Mice were bled after 2 h later tocollect serum. Serum adrenocorticotropic hormone levels were determined according to published procedure (3). Values in a column with adifferent superscript letters are significantly different at P < 0.05, A = values; B = percentages.


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Figure 8 Effects of a-tocopherol and δ-tocotrienol on the gene expression of TNF-a in LPS-stimulated peritoneal macrophages of6-week-old BALB/c female mice. The RT-PCR procedure is described in the experimental section. The ratios of relative optical density (TNF-a/b-actin) of each treatment for TNF-a was used to draw this figure, A = values; B = percentages.


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Figure 9 Effects of a-tocopherol and δ-tocotrienol on the gene expression of IL-1b, IL-6, and iNOS in LPS-stimulated peritonealmacrophages of 6-week-old BALB/c female mice. The RT-PCR procedure is described in the experimental section. The ratios of relativeoptical density (cytokines/b-actin) of each treatment for each marker was used to draw this figure, A = values; B = percentages.


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ConclusionsAlthough LPS is a potent inducer of inflammation viathe proteasome, it also activates an anti-inflammatoryresponse by inducing corticosteroid production. In sum-mary, δ-tocotrienol treatment has several anti-inflamma-tory effects that could be mechanistically analogous tothe modulation of proteasomal activity by lactacystin, awell established proteasome inhibitor that can eitherincrease or decrease proteasomal activity under differentconditions [34,35]. The results of our current study alsodemonstrate that tocotrienols increase levels of adreno-corticotropic hormone (ACTH) and corticosteroids pro-duced in response to LPS. The mechanisms by whichLPS plus tocotrienols stimulate the hypothalamus-pituitary-adrenal (HPA) axis and the sites of action arecurrently unclear [3]. Regardless of the mechanism,however, the capacity of tocotrienols to increase thehost corticosteroid response to LPS is likely to modulateinflammation. Other possible mechanisms for the anti-inflammatory properties of tocotrienols also include itssuperior antioxidant activity compared to a-tocopherol(vitamin E, [10,17,22]). All these latter mechanisms suchas corticosteroid induction and antioxidant activitiesmay be dependent on the proteasomal modulation bytocotrienols.

AbbreviationsACTH: adrenocorticotropic hormone; DMSO: dimethyl sulfoxide; FBS: fetalbovine serum; IL-1b: interleukin-1b; IL-6: interleukin-6; IL-8: interleukin-8;HMG-CoA: b-hydroxy- b-methylglutaryl coenzyme A; HPLC: high pressureliquid chromatography; iNOS: inducible nitric oxide synthase; LDL: lowdensity lipoprotein; LPS: lipopolysaccharide; MTT: 3-(4,5)-dimethyl-2,5-diphenyltetrazolium bromide; RNA: ribonucleic acid; RT-PCR: reverse-phasetranscriptase polymerase chain reaction; SREBP-2: sterol regulatory element-binding protein-2.; TCL: total cholesterol; Tocols: mixtures of a-, b-, g-, δ-tocopherols + a-, b-, g-, δ-tocotrienols; TNF-a: tumor necrosis factor-a; TRF:tocotrienol rich fraction.

AcknowledgementsThis study was supported in part by Advanced Medical Research (AMR) anda grant from the Malaysian Palm Oil Board, Kuala Lumpur, Malaysia(previously known as Palm Oil Research Institute of Malaysia [PORIM]) andNIH grant 50870 (NQ). The study was carried out under a FDA approved INDnumber 36906.

Author details1Departments of Basic Medical Sciences, University of Missouri-Kansas City,2411 Holmes Street, Kansas City, MO 64108, USA. 2Department ofPharmacology/Toxicology, School of Pharmacy, 2464 Charlotte Street, KansasCity, MO 64108, USA.

Authors’ contributionsJCR (graduate student) has carried out most of the experiments. All theauthors were involved in the design of the study. CJP edited themanuscript. All the authors have read and approved the final version.

Competing interestsThe authors declare that they have no competing interests.

Received: 29 October 2010 Accepted: 16 December 2010Published: 16 December 2010

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doi:10.1186/1476-511X-9-143Cite this article as: Qureshi et al.: Tocotrienols inhibitlipopolysaccharide-induced pro-inflammatory cytokines in macrophagesof female mice. Lipids in Health and Disease 2010 9:143.

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