sodium benzoate, a metabolite of cinnamon and a food ... · sodium benzoate, a metabolite of ......

Sodium Benzoate, a Metabolite of Cinnamon and a FoodAdditive, Reduces Microglial and Astroglial InflammatoryResponses1

Saurav Brahmachari, Arundhati Jana, and Kalipada Pahan2

Upon activation, microglia and astrocytes produce a number of proinflammatory molecules that participate in the pathophysiologyof several neurodegenerative disorders. This study explores the anti-inflammatory property of cinnamon metabolite sodiumbenzoate (NaB) in microglia and astrocytes. NaB, but not sodium formate, was found to inhibit LPS-induced expression ofinducible NO synthase (iNOS), proinflammatory cytokines (TNF-� and IL-1�) and surface markers (CD11b, CD11c, and CD68)in mouse microglia. Similarly, NaB also inhibited fibrillar amyloid � (A�)-, prion peptide-, double-stranded RNA (polyinosinic-polycytidylic acid)-, HIV-1 Tat-, 1-methyl-4-phenylpyridinium�-, IL-1�-, and IL-12 p402-induced microglial expression of iNOS.In addition to microglia, NaB also suppressed the expression of iNOS in mouse peritoneal macrophages and primary humanastrocytes. Inhibition of NF-�B activation by NaB suggests that NaB exerts its anti-inflammatory effect through the inhibition ofNF-�B. Although NaB reduced the level of cholesterol in vivo in mice, reversal of the inhibitory effect of NaB on iNOS expression,and NF-�B activation by hydroxymethylglutaryl-CoA, mevalonate, and farnesyl pyrophosphate, but not cholesterol and ubiqui-none, suggests that depletion of intermediates, but not end products, of the mevalonate pathway is involved in the anti-inflammatory effect of NaB. Furthermore, we demonstrate that an inhibitor of p21ras farnesyl protein transferase suppressed theexpression of iNOS, that activation of p21ras alone was sufficient to induce the expression of iNOS, and that NaB suppressed theactivation of p21ras in microglia. These results highlight a novel anti-inflammatory role of NaB via modulation of the mevalonatepathway and p21ras. The Journal of Immunology, 2009, 183: 5917–5927.

A ctivation of glial cells (microglia and astroglia) has beenimplicated in the pathogenesis of a variety of neurode-generative diseases, including Alzheimer’s disease (AD),3

Parkinson’s disease, Creutzfeld-Jacob disease, HIV-associated de-mentia (HAD), stroke, and multiple sclerosis (MS) (1, 2). It hasbeen found that activated microglia and astroglia accumulate atsites of injury or plaques in neurodegenerative CNS (1–7). Al-though activated microglia scavenge dead cells from the CNS andsecrete different neurotrophic factors for neuronal survival, it isbelieved that severe activation causes various autoimmune re-sponses leading to neuronal death and brain injury (1–7). Duringactivation, microglia and astroglia express various genes related toinflammation, such as proinflammatory cytokines, proinflamma-

tory enzymes, and proinflammatory adhesion molecules (1–9).Therefore, characterization of signaling pathways required for theactivation of glial cells is an active area of investigation sincecompounds capable of antagonizing such signaling steps may havetherapeutic effect in neurodegenerative disorders.

Cinnamon contains three major compounds (cinnamaldehyde,cinnamyl acetate and cinnamyl alcohol), which are converted intocinnamic acid by oxidation and hydrolysis, respectively. In theliver, this cinnamic acid is �-oxidized to benzoate (10) that existsas sodium salt (sodium benzoate; NaB) or benzoyl-CoA. It hasbeen reported that minor amount of NaB is also excreted in theurine of humans (11, 12). NaB is of medical importance because itis a component of Ucephan, a Food and Drug Administration(FDA)-approved drug used in the treatment for hepatic metabolicdefects associated with hyperammonemia such as urea cycle dis-order in children (13, 14). It is also widely used as a preservativein broad range of foods and cosmetic products (15). It is nontoxicand can be administered as a solution in drinking water. It has beenreported that 2% solution of NaB in drinking water is safe forlifelong treatment in mice without any noticeable side effects (16).Because Ayurvedic as well as Yunani medicines have been usingcinnamon as vital medicines for inflammatory diseases like arthri-tis for centuries, we were prompted to test the effect of NaB on theexpression of proinflammatory molecules in glial cells.

Here we provide the first evidence that NaB attenuates the ex-pression of inducible NO synthase (iNOS) and proinflammatorycytokines in microglia, astrocytes, and macrophages. AlthoughNaB reduced the level of cholesterol in vivo in mice, it was not thecause behind the anti-inflammatory activity of NaB. Alternatively,hydroxymethylglutaryl-CoA (HMG-CoA), mevalonate, and farne-sylpyrophosphate reversed NaB-mediated inhibition of iNOS, in-dicating the involvement of intermediates, but not the end product,of the mevalonate pathway in the anti-inflammatory effect of NaB.

Department of Neurological Sciences, Rush University Medical Center, Chicago,IL 60612

Received for publication October 6, 2008. Accepted for publication August 20, 2009.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This study was supported by grants from the National Institutes of Health(NS39940), Alzheimer’s Association (IIRG-07-58684), and Michael J. Fox Founda-tion for Parkinson’s Research.2 Address correspondence and reprint requests to Dr. Kalipada Pahan, Department ofNeurological Sciences, Rush University Medical Center, 1735 West Harrison Street,Suite 320, Chicago, IL 60612. E-mail address: [email protected] Abbreviations used in this paper: AD, Alzheimer’s disease; HAD, HIV-associateddementia; MS, multiple sclerosis; NaB, sodium benzoate; FDA, Food and Drug Ad-ministration; iNOS, inducible NO synthase; HMG, CoA, hydroxymethylglutaryl-CoA; NaFO, sodium formate; poly(IC), polyinosinic-polycytidylic acid; MPP,1-methyl-4-phenylpyridinium; p402, p40 homodimer; GFAP, glial fibrillary acidicprotein; PBST, PBS-Tween 20; A�, amyloid �; PrP, prion peptide; wtNBD, wild-typeIKK� NEMO-binding domain peptide; FPT inhibitor II, inhibitor of p21ras farnesylprotein transferase; FPP, farnesyl pyrophosphate; IKK-�, I�B kinase-�.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

The Journal of Immunology

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0803336

Consistently, inhibition of the expression of iNOS and the produc-tion of NO by farnesylpyrophosphate transferase inhibitor, sup-pression of p21ras activation by NaB and induction of iNOS by theactivated p21ras alone suggest that NaB exerts its anti-inflammatoryeffect in glial cells via modulating farnesylation and activation ofp21ras. Our findings raise a possibility that NaB, a component of aprescribed drug for human urea cycle disorder and a widely used foodpreservative, may find further application in neuroinflammatory andneurodegenerative disorders.

Materials and MethodsReagents

NaB, sodium formate (NaFO), LPS (Escherichia coli), polyinosinic-poly-cytidylic acid (poly(IC)), and 1-methyl-4-phenylpyridinium (MPP)� werepurchased from Sigma-Aldrich. FBS, HBSS, trypsin, and DMEM/F-12were from Mediatech. HIV-1 coat protein gp120 (expressed in Chinesehamster ovary cells; strain HIV-1 MN) was obtained from U.S. Biological.Prion peptides and human A� peptides 1–42 were obtained from BachemBioscience. Recombinant mouse IL-1� and IL-12 p40 homodimer (p402) wereobtained from R&D Systems. 125I-Labeled protein A and [�-32P]dCTP (3000Ci/mmol) were purchased from PerkinElmer Life Sciences. Pravastatin andAbs against human and mouse iNOS were purchased from Calbiochem.

Isolation of primary mouse microglia

Microglia were isolated from mixed glial cultures according to the proce-dure of Giulian and Baker (17). Briefly, on days 7–9, the mixed glialcultures were washed three times with DMEM/F-12 and shaken at 240 rpmfor 2 h at 37°C on a rotary shaker. The floating cells were washed andseeded on to plastic tissue culture flasks and incubated at 37°C for 1 h. Theattached cells were seeded onto new plates for further studies. From 90 to95% of this preparation was found to be positive for Mac-1 surface Ag.Mouse BV-2 microglial cells (gift from Dr. Virginia Bocchini, Universityof Perugia, Perugia, Italy) were also maintained and induced as indicatedabove.

Isolation of primary human astroglia

Primary human astroglia were prepared as described by us in many studies(18–20). All of the experimental protocols were reviewed and approved bythe Institutional Review Board of the Rush University Medical Center.Briefly, 11- to 17-wk-old fetal brains obtained from the Human Embryol-ogy Laboratory (University of Washington, Seattle, WA) were dissociatedby trituration and trypsinization. On the ninth day, these mixed glial cul-tures were placed on a rotary shaker at 240 rpm at 37°C for 2 h to removeloosely attached microglia. Then on the 11th day, flasks were shaken againat 190 rpm at 37°C for 18 h to remove oligodendroglia. The attached cellsremaining were primarily astrocytes. These cells were trypsinized and sub-cultured in complete medium at 37°C with 5% CO2 in air to yield moreviable and healthy cells (18–20). More than 98% of these cells obtained bythis method were positive for glial fibrillary acidic protein (GFAP), amarker for astrocytes.

Isolation of mouse peritoneal macrophages

Resident macrophages were obtained from mouse by peritoneal lavagewith sterile RPMI 1640 containing 1% FBS and 100 �g/ml gentamicin asdescribed earlier (21).

Assay for NO synthesis

Synthesis of NO was determined by assay of culture supernatant fornitrite, a stable reaction product of NO with molecular oxygen, usingGriess reagent (21–24), except that nitrate in culture supernatants wasfirst reduced to nitrite with nitrate reductase and NADPH (Sigma-Aldrich) for 1 h at 37°C before the assay. Protein was measured by theprocedure of Bradford (25).

Immunoblot analysis for iNOS

Immunoblot analysis for iNOS was conducted as described earlier (22, 23,26). Briefly, cells homogenates were electrophoresed, proteins were trans-ferred onto a nitrocellulose membrane, and the iNOS band was visualizedby immunoblotting with Abs against human iNOS.

Northern blot analysis

Analysis was conducted as described earlier (21, 26). Briefly, total RNAwas isolated using Ultraspec-II RNA reagent (Biotecx Laboratories) ac-

cording to the manufacturer’s protocol. For Northern blot analyses, 20 �gof total RNA were electrophoresed on 1.2% denaturing formaldehyde-aga-rose gels, electrotransferred to Hybond-nylon membrane (Amersham Bio-sciences) and hybridized at 68°C with 32P-labeled cDNA probe. After hy-bridization, filters were washed two or three times in solution I (2� SSC,0.05% SDS) for 1 h at room temperature followed by solution II (0.1�SSC, 0.1% SDS) at 50°C for another hour. The membranes were then driedand exposed to x-ray films (Eastman Kodak). The same amount of RNAwas hybridized with probe for GAPDH.

Semiquantitative RT-PCR analysis

To remove any contaminating genomic DNA, total RNA was digested withDNase. Semiquantitative RT-PCR was conducted as described earlier (27,28) using a RT-PCR kit from Clontech. Briefly, 1 �g of total RNA wasreverse transcribed using oligo(dT)12–18 as primer and Moloney murineleukemia virus reverse transcriptase (Clontech). The resulting cDNA wasappropriately diluted and then cDNA amplified. Amplified products wereelectrophoresed on a 1.8% agarose gels and visualized by ethidium bro-mide staining.

iNOS: sense, 5�-CCCTTCCGAAGTTTCTGGCAGCAGC-3�; antisense,5�-GGCTGTCAGAGCCTCGTGGCTTTGG-3�. IL-1�: sense, 5�-CTCCATGAGCTTTGTACAAGG-3�; antisense, 5�-TGCTGATGTACCAGTTGGGG-3�. IL-6: sense, 5�-GACAACTTTGGCATTGTGG-3�; antisense, 5�-ATGCAGGGATGATGTTCTG-3�. TLR4: sense, 5�-CAGAAATTCCTGCAGTGGGT-3�; antisense, 5�-GTGAAGGCAGAGGTGAAAGC-3�. TLR3: sense, 5�-ACAACGTAGCTGACTGCAGC-3�; antisense, 5�-GAGTTCTGTCAGGTTCGTGC-3�. B7-1: sense, 5�-AAGTACCTGGGCCGCACGAGC-3�;antisense, 5�-GCCACACACCATCCGGGAATG-3�. B7-2: sense, 5�-GGGCCTGGTCCTTTCAGACCG-3�; antisense, 5�-CCTCTGACACGTGAGCATCTC-3�. CD11b: sense, 5�-CAGATCAACAATGTGACCGTATGGG-3�; antisense, 5�-CATCATGTCCTTGTACTGCCGCTTG-3�. MHC-II: sense, 5�-AGCCTCTGTGGAGGTGAAGA-3�; antisense,5�-GGTTGACGAAGAAGCTGGTC-3�. CD11c: sense, 5�-CAGTCTGGCAGATGTGGCTA-3�; antisense, 5�-CACCTGCTCCTGACACTCAA-3�. CD68: sense, 5�-GACCTACATCAGAGCCCGAG-3�; anti-sense, 5�-AGAGGGGCTGGTAGGTTGAT-3�. GAPDH: sense, 5�-GGTGAAGGTCGGTGTGAACG-3�; antisense, 5�-TTGGCTCCACCCTTCAAGTG-3�.

Real-time PCR analysis

It was performed using the ABI-Prism7700 sequence detection system(Applied Biosystems) as described earlier (27, 28). The mRNA expressionsof respective genes were normalized to the level of GAPDH mRNA. Datawere processed by the ABI Sequence Detection System 1.6 software andanalyzed by ANOVA.

Assay of iNOS promoter-driven reporter activity

Cells plated at 50–60% confluence in six-well plates were cotransfectedwith 1 �g of phiNOS(7.2)Luc and 50 ng of pRL-TK (a plasmid encodingRenilla luciferase, used as transfection efficiency control; Promega) byLipofectAMINE Plus (Invitrogen) as described in several studies (20, 26).Twenty-four hours after transfection, cells were treated with different stim-uli for 12 h. Firefly and R. luciferase activities were obtained by analyzingtotal cell extract according to standard instructions provided in the DualLuciferase Kit (Promega) in a TD-20/20 Luminometer (Turner Designs).Relative luciferase activity of cell extracts was typically represented as theratio of firefly luciferase value to the R. luciferase value � 10�3.

Preparation of nuclear extracts and EMSA

Nuclear extracts were prepared from microglial cells as described previ-ously (7, 19, 21). Nuclear extracts were used for EMSA using 32P-end-labeled double-stranded (NF-�B; 5�-AGTTGAGGGGACTTTCCCAGGC-3�; Promega) oligonucleotides. Double-stranded mutated (NF-�B, 5�-AGTTGAGGCGACTTTCCCAGGC-3�; Santa Cruz Biotechnology)oligonucleotides were used to verify the specificity of NF-�B bindingto DNA.

Assay of transcriptional activities of NF-�B

Cells plated at 70–80% confluence in 12-well plates were cotransfectedwith 0.25 �g of either PBIIX-Luc (an NF-�B-dependent reporter construct)and 12.5 ng of pRL-TK using LipofectAMINE Plus (22, 23). After 24 h oftransfection, cells were treated with different stimuli for 6 h. Firefly andR. luciferase activities were obtained as described above.

5918 INHIBITION OF INFLAMMATION BY CINNAMON METABOLITE

Assay of cytokines by ELISA

Microglial cells preincubated with NaB for 6 h were stimulated with LPS.After 24 h of stimulation, supernatants were collected to assay TNF-� andIL-1� by high-sensitivity ELISA kits (BD Biosciences).

Immunofluorescence analysis

It was performed as described earlier (19, 28). Briefly, coverslips contain-ing 100–200 cells/mm2 were fixed with 4% paraformaldehyde followed bytreatment with cold ethanol and two rinses in PBS. Samples were blockedwith 3% BSA in PBS-Tween 20 (PBST) for 30 min and incubated in PBSTcontaining 1% BSA and goat anti-CD11b (1/50), rabbit anti-iNOS (1/200),or goat anti-GFAP (1/50). After three washes in PBST (15 min each),slides were further incubated with Cy2 (Jackson ImmunoResearch Lab-oratories). For negative controls, a set of culture slides was incubatedunder similar conditions without the primary Abs. The samples weremounted and observed under a Bio-Rad MRC1024ES confocal laser-scanning microscope.

Assay of cholesterol in serum

Total cholesterol was quantified in serum by using an Amplex Red Cho-lesterol Assay kit from Invitrogen. Briefly, cholesterol was oxidized bycholesterol oxidase to yield H2O2, which then reacted with 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red). In the presence of HRP, this Am-plex Red-H2O2 complex produced highly fluorescent resorufin, which wasdetected by fluorometry.

Statistics

Statistical comparisons were made using one-way ANOVA followed byStudent’s t test.

ResultsNaB attenuates the expression of iNOS and proinflammatorycytokines in LPS-stimulated mouse microglia

Activated microglia are known to produce excessive amount ofNO having the potential of damaging neurons and oligodendro-cytes. Because cinnamon has been used as a natural supplement fornormal human health for centuries, we investigated the effect ofNaB, a major metabolite of cinnamon, on the expression of iNOSin microglia. LPS is a prototype inducer of various proinflamma-tory molecules in different cell types including mouse microglia.Therefore, BV-2 microglial cells preincubated with different dosesof NaB and sodium formate (NaFO), the negative control for NaB,for 6 h were stimulated with LPS under serum-free condition. Al-though at lower concentrations (50 and 100 �M), NaB was noteffective in inhibiting the production of NO (data not shown), athigher concentrations, NaB markedly suppressed LPS-inducedproduction of NO in microglial cells as evident from estimation oftotal NO (nitrite and nitrate; Fig. 1A). Significant inhibition ofLPS-induced NO production was observed at 500 �M NaB, andmaximum inhibition was noted at 1 mM or higher concentration(Fig. 1A). Alternatively, NaFO had no effect on LPS-induced pro-duction of NO (Fig. 1A). However, a 1- or 2-h preincubation ofmicroglial cells with NaB was not enough for this molecule toexhibit its inhibitory effect on LPS-induced NO production (datanot shown). To understand the mechanism further, we investigatedthe effect of NaB on the mRNA expression of iNOS in microglialcells. It is clearly evident from RT-PCR in Fig. 1B and real-timePCR in Fig. 1C that NaB, but not NaFO, inhibited LPS-inducedmRNA expression of iNOS in BV-2 microglial cells. It is wellknown that LPS requires TLR4 to transduce its signal and subse-quently to induce iNOS. Therefore, there is a possibility that NaBmight inhibit the expression of TLR4, and as a result decreasedexpression of iNOS is observed. To examine this possibility, weinvestigated the effect of NaB on the expression of TLR4. TheRT-PCR results in Fig. 1D show that NaB had no effect on theexpression of TLR4 in LPS-treated microglial cells, clearly sug-

gesting that NaB-mediated inhibition of iNOS is not due to anyinhibition of expression of TLR4.

Next, to investigate whether NaB suppresses the induction ofNO production in primary cells, we used peritoneal macrophagesand primary microglia. As evident from semiquantitative RT-PCRin Fig. 1E and quantitative PCR in Fig. 1F, NaB, but not NaFO,markedly suppressed the expression of iNOS mRNA in LPS-stim-ulated peritoneal macrophages. Similar to BV-2 microglial cells,NaB dose-dependently suppressed LPS-induced production of NOin primary mouse microglia (Fig. 1G). On the other hand, NaFOhad no effect on LPS-induced production of nitrite (Fig. 1G). MTTresults show that neither NaB nor NaFO was toxic to microglia atany of the concentrations tested (Fig. 1H), suggesting that the in-hibitory effect of NaB on microglial expression of iNOS was notdue to any change in cell viability.

In addition to producing NO, activated microglia also secretevarious proinflammatory cytokines. Therefore, we examined

FIGURE 1. Dose-dependent inhibition of LPS-induced NO productionby NaB, but not NaFO, in mouse microglia. Mouse BV-2 microglial cellswere treated with different concentrations of NaB and NaFO for 6 h fol-lowed by stimulation with LPS under serum-free condition. A, After 24 hof stimulation, concentrations of total NO (nitrate plus nitrite) were mea-sured in supernatants. After 5 h of stimulation, the expression of iNOSmRNA was monitored by semiquantitative RT-PCR (B) and real-time PCR(C). D, The expression of TLR4 was monitored by semiquantitative RT-PCR. Mouse peritoneal macrophages isolated by peritoneal lavage weretreated with NaB (1 mM) or NaFO (1 mM) for 5 h followed by stimulationwith LPS. After 6 h of stimulation, the mRNA expression of iNOS wasmonitored by semiquantitative RT-PCR (E) and real-time PCR (F). Pri-mary microglia isolated from 7- to 9-day-old mouse pups were treated withdifferent concentrations of NaB and NaFO for 6 h followed by stimulationwith LPS under serum-free conditions. After 24 h of stimulation, concen-tration of nitrite (G) was measured in supernatants. H, Cell viability wasassayed by MTT. Results are means � SD of three different experiments.b, p � 0.05 vs LPS; a, p � 0.001 vs LPS.

5919The Journal of Immunology

whether NaB was capable of suppressing the expression of proin-flammatory cytokines in BV-2 microglial cells. Similar to the in-hibition of iNOS, NaB dose-dependently inhibited the productionof TNF-� and IL-1� protein (Fig. 2A) and the expression ofTNF-� and IL-1� mRNA (Fig. 2, B and C). On the other hand,NaFO had no effect on the expression of these proinflammatorycytokines.

NaB inhibits A�-, prion peptide (PrP)-, dsRNA (poly(IC))-,HIV-1 gp120-, MPP�-, IL-1�-, and IL-12 p402-inducedexpression of iNOS in microglial cells

Activated microglia are considered to play an important role invarious pathological conditions associated with viral encephalop-athy, AD, MS, Parkinson’s disease, HAD, Creutzfeldt-Jakob dis-ease, etc. (1). Because NaB inhibited LPS-induced expression ofproinflammatory molecules in microglia, we were prompted to in-vestigate whether NaB was also capable of negating the expressionof iNOS in microglial cells stimulated with etiological reagents ofvarious neurological disorders. BV-2 microglial cells were chal-lenged with fibrillar A� peptides (etiological reagent for AD),fibrillar PrP peptides (etiological reagent for prion diseases),dsRNA in the form of poly(IC) (one of the etiological reagents forviral encephalopathy), HIV-1 gp120 (one of the etiological re-agents for HAD), IL-12 p402 (one of the etiological reagent forMS), and MPP� (Parkinsonian toxin). It is clearly evident fromFig. 3 that A� peptides (Fig. 3A), gp120 (Fig. 3B), MPP� (Fig.3C), PrP (Fig. 3D), poly(IC) (Fig. 3E), IL-12 p402 (Fig. 3F), andIL-1� (Fig. 3G) induced the expression of iNOS mRNA in micro-glial cells. However, NaB knocked down A�-, gp120-, MPP�-,PrP-, poly(IC)-, p402-, and IL-1b-induced microglial expression ofiNOS (Fig. 3). On the other hand, NaFO had no such inhibitoryeffect (Fig. 3) suggesting the specificity of the effect. We furtherexamined the effect of NaB on the expression of TLR3, the pro-totype receptor of poly(IC). It is clearly evident from the Fig. 3E

(middle) that NaB had no effect on the mRNA level of TLR3,indicating that inhibition of iNOS by NaB in poly(IC)-stimulatedcells is not due to any suppression of TLR3.

NaB inhibits the expression of microglial surface markers,MHC class II, and costimulatory molecules in LPS- orMPP�-stimulated mouse microglial cells

During severe activation, microglia not only secrete various neu-rotoxic molecules but also express different proteins and surfacemarkers. Among different surface markers, CD11b is the most po-tential one with immense biological significance (29–31). It is re-ported that in various neuroinflammatory diseases, the increasedCD11b expression corresponds to the severity of microglial acti-vation (1). We examined whether NaB could abrogate increasedexpression of various surface molecules in microglia. As expected,LPS increased the expression of CD11b, CD11c, and CD68 inBV-2 microglial cells (Fig. 4A). We also used MPP� (Parkinso-nian neurotoxin) to stimulate CD11b in microglia. Double-labelimmunofluorescence analysis of CD11b and iNOS in mouseprimary microglia shows that MPP� stimulation increased the ex-pression of CD11b and iNOS and that NaB attenuated LPS-me-diated CD11b and iNOS expression (Fig. 4B). We further exam-ined the surface expression of CD11b and CD68 by FACS. It isevident from Fig. 4 that NaB, but not NaFO, markedly inhibitedthe surface expression of CD11b (Fig. 4C) and CD68 (Fig. 4D) inLPS-stimulated microglial cells. Taken together, these studies sug-gest that NaB is capable of suppressing the activation of microglia.

FIGURE 2. NaB, but not NaFO, inhibits LPS-induced expression ofproinflammatory cytokines in mouse BV-2 microglial cells. Cells weretreated with different concentrations of NaB and NaFO for 6 h followed bystimulation with 1 �g/ml LPS under serum-free condition. A, After 24 h ofstimulation, concentrations of TNF-� and IL-1� were measured in super-natants by ELISA. Results are means � SD of three different experiments.b, p � 0.05 vs LPS-TNF; a, p � 0.001 vs LPS-TNF; c, p � 0.05 vsLPS-IL-1; d, p � 0.001 vs LPS-IL-1. After 5 h of stimulation, the mRNAexpression of TNF-� and IL-1� was monitored by semiquantitative RT-PCR (B) and real-time PCR (C). a, p � 0.001 vs LPS. FIGURE 3. Effect of NaB and NaFO on A�-, HIV-1 gp120-, MPP�-,

PrP-, poly(IC)-, IL-1� p402, and IL-12 p402-induced expression of iNOSin mouse BV-2 microglial cells. Cells preincubated with NaB (1 mM) orNaFO (1 mM) for 6 h were stimulated with 1 �M A�1–42 (A), 200 pg/mlgp120 (B), 1 �M MPP� (C), 1 �g/ml PrP (D), 100 �g/ml poly(IC) (E), 20ng/ml p402 (F), or 10 ng/ml IL-1� (G) under serum-free condition. After5 h of stimulation, the expression of iNOS mRNA (A–G) and TLR3 mRNA(E) was analyzed in microglial cells by semiquantitative RT-PCR. Resultsrepresent three independent experiments.


Activated microglia also express MHC class II and costimulatorymolecules like B7-1 and B7-2, which are critical for Ag presen-tation. As evident from Fig. 4A, LPS increased the expression ofclass-II MHC and the costimulatory molecules B7-1 and B7-2(Fig. 4A). However, NaB, but not NaFO, markedly suppressed theexpression of these activation markers and costimulatory mole-cules in microglial cells (Fig. 4A).

NaB inhibits the activation of NF-�B in mouse BV-2 microglialcells

LPS, proinflammatory cytokines (TNF-� and IL-1�) and otherstimuli (HIV-1 gp120, IL-12 p402, A�, PrP, etc.) are known toinduce iNOS expression via activation of NF-�B (19–24, 26, 27).Because NaB attenuated the expression of iNOS in mouse micro-glia, we examined the effect of NaB on the activation of NF-�B.Activation of NF-�B was monitored by both DNA binding andtranscriptional activity of NF-�B. As expected, treatment of BV-2microglial cells with LPS resulted in the induction of DNA-bind-ing activity of NF-�B (Fig. 5A). However, NaB inhibited LPS-induced DNA-binding activity of NF-�B in microglial cells (Fig.5A). We then tested the effect of NaB on LPS-induced transcrip-tional activity of NF-�B. Consistent to the effect of NaB on theDNA binding activity of NF-�B, NaB also suppressed NF-�B-dependent transcription of luciferase in a dose-dependent mannerin LPS-stimulated cells (Fig. 5B). NaFO had no effect on the LPS-induced transcriptional activity of NF-�B in microglial cells (Fig.5B). We next examined the effect of NaB on other proinflamma-tory stimuli-induced transcriptional activity of NF-�B. The wild-type I�B kinase-� (IKK-�) NEMO binding domain peptide(wtNBD), a specific inhibitor of induced NF-�B activation, wasused as a positive control. As expected, NaB markedly suppressed

NF-�B-dependent transcription of luciferase in microglial cellsstimulated by A�, IL-1�, gp120, PrP, and IL-12 p402 (Fig. 5,C–G). These results suggest that NaB attenuates the expression ofiNOS by suppressing the activation of NF-�B.

NaB suppresses the expression of iNOS and GFAP in primaryhuman astrocytes

Next we examined whether NaB could suppress the expression ofiNOS in human brain cells. Astroglia are the major glial cells in theCNS, and astroglial activation also plays a role in various neuro-degenerative disorders. Therefore, we investigated the effect ofNaB on the expression of iNOS in primary human astroglia. Ear-lier, we found that IL-1� is the only cytokine that induces iNOS inprimary astroglia (20). Consistently, IL-1� induced the productionof nitrite (Fig. 6A) and the expression of iNOS protein (Fig. 6B) inprimary astroglia isolated from human fetal brains. NaB markedlyinhibited IL-1�-induced production of NO (Fig. 6A) and the ex-pression of iNOS protein (Fig. 6B) in human fetal astroglia.

To understand the effect of NaB on the transcription of theiNOS gene, primary human astrocytes were transfected withphiNOS(7.2)Luc, a construct containing the human iNOS promoterfused to the luciferase gene, and activation of this promoter was mea-sured after stimulating the cells with IL-1� in the presence or absenceof NaB. As we found earlier (20, 23), IL-1� induced iNOS pro-moter-driven luciferase activity by �4-fold (Fig. 6C). Consistentwith the effect of NaB on the expression of iNOS, NaB itself hadno effect on iNOS promoter-driven luciferase activity but it sig-nificantly inhibited iNOS promoter-driven luciferase activity in IL-1�-stimulated cells (Fig. 6C), suggesting that NaB inhibits IL-1�-induced production of NO and the expression of iNOS byinhibiting the activation of iNOS promoter.

FIGURE 4. NaB, but not NaFO,inhibits the expression of surfacemarkers, MHC class II, and costimu-latory molecules in activated micro-glia. BV-2 microglial cells weretreated with NaB and NaFO for 6 hfollowed by stimulation with 1 �g/mlLPS under serum-free conditions. A,After 24 h of stimulation, the expres-sions of CD11b, CD11c, CD68, B7-2,B7-1, and MHC class II were moni-tored by semiquantitative RT-PCR. B,Primary mouse microglia were treatedwith NaB followed by stimulationwith 1 �M MPP�. After 24 h of stim-ulation, levels of CD11b and iNOSwere monitored by double-label im-munofluorescence. Cells were incu-bated with appropriately dilutedFITC-conjugated CD11b (C) or CD68(D) followed by FACS. The percent-age of cells in the indicated quadranthas been mentioned. Results aremeans � SD of three different trials.


Increased expression of glial fibrillary acidic protein (GFAP)represents astroglial activation and gliosis during neurodegenera-tion. Because NaB decreased the expression of iNOS in humanastroglia, we investigated whether this drug was capable of inhib-iting the increased expression of GFAP in primary human astro-glia. As expected, IL-1� markedly increased the mRNA expres-sion of GFAP in astroglia (Fig. 6E). However, NaB, but not NaFO,suppressed IL-1�-induced astroglial mRNA expression of GFAP(Fig. 6E). Immunofluorescence analysis in Fig. 6D also indicatesthat IL-1� stimulation increased the level of GFAP and iNOScompared with control and that NaB markedly attenuated IL-1�-mediated up-regulation of GFAP and iNOS in human astrocytes.Western blot analysis in Fig. 6F further substantiates our findingsand also indicates that NaB has no additive effect when used incombination with wtNBD, suggesting that NaB-mediated inhibi-

tion of proinflammatory markers is solely due to the inhibitionof NF-�B.

Intermediates of the mevalonate pathway reverses the inhibitoryeffect of NaB on the expression of iNOS and the activation ofNF-�B in mouse BV-2 microglial cells

The requirement of at least 6 h of preincubation of cells with NaBto see its anti-inflammatory effect suggests that metabolite(s) sen-sitive to NaB may be involved in the process. Earlier, Pahan et al.

FIGURE 5. NaB attenuates activation of NF-�B in mouse BV-2 micro-glial cells. A, Cells were treated with different concentrations of NaB for6 h followed by stimulation with 1 �g/ml LPS under serum-free conditions.After 1 h of stimulation, the DNA-binding activity of NF-�B was moni-tored. B, Cells plated in 12-well plates were cotransfected with 0.25 �g ofPBIIX-Luc (an NF-�B-dependent reporter construct) and 12.5 ng of pRL-TK. Twenty-four hours after transfection, cells received different concen-trations of NaB and NaFO. After 6 h of incubation, cells were stimulatedwith LPS for 6 h under serum-free condition. Firefly (ff-Luc) and Renillaluciferase (r-Luc) activities were obtained by analyzing the total cell ex-tract. C–H, Twenty-four hours after transfection, cells received NaB (1mM); 1 h before stimulation, cells were treated with wtNBD (10 �M).After 6 h of incubation with NaB, cell were stimulated with LPS (1 �g/ml;C), IL-1� (10 ng/ml; D), PrP (1 �g/ml; E), A� (1 �M; F), gp120 (200pg/ml; G), or p402 (20 ng/ml; H). Firefly and Renilla luciferase activitieswere obtained by analyzing the total cell extract. Results are means � SDof three different experiments. a, p � 0.001 vs control; b, p � 0.05 vs LPS;c, p � 0.001 vs LPS.

FIGURE 6. NaB inhibits IL-1�-induced expression of iNOS and theactivation of human iNOS promoter in primary human astrocytes. Cellspretreated with different concentrations of NaB for 6 h were stimulatedwith 20 ng/ml IL-1�. After 48 h of stimulation, the level of nitrite wasmeasured by Griess reagent (A) and the level of iNOS protein was moni-tored by Western blot (B). Actin was run as control. a, p � 0.001 vs IL-1�.C, Cells plated at 70–80% confluence in 12-well plates were cotransfectedwith 0.25 �g of phiNOS(7.2)Luc and 12.5 ng of pRL-TK using the Lipo-fectamine-Plus (Invitrogen). Twenty-four hours after transfection, cells re-ceived NaB and NaFO. After 6 h of incubation, cells were stimulated withIL-1� (20 ng/ml) for 12 h under serum-free condition. Firefly (ff-Luc) andRenilla (r-Luc) luciferase activities were obtained by analyzing the totalcell extract. Data are means � SD of three different experiments. a, p �0.001 vs control; b, p � 0.001 vs IL-1�. Primary human astroglia weretreated with NaB (1 mM) and NaFO (1 mM) for 6 h followed by stimu-lation with IL-1� (20 ng/ml). After 24 h of stimulation, mRNA expressionof GFAP was examined by semiquantitative RT-PCR (E), and protein ex-pression of GFAP and iNOS was monitored by double-label immunoflu-orescence (D). Cells pretreated with appropriate concentrations of NaB orNaFO for 6 h or wtNBD (10 �M) alone or along with NaB for 1 h werestimulated with 20 ng/ml IL-1� under serum-free conditions. After 48 h ofstimulation, the level of iNOS and GFAP proteins was monitored by West-ern blot (F). �-Actin was run as control Results represent three independentexperiments.


(32) have demonstrated that intermediates of the mevalonate path-way play a role in the expression of iNOS and proinflammatorycytokines in glial cells. The end product of the mevalonate path-way is cholesterol; therefore, we investigated whether NaB hadany effect on the level of cholesterol in vivo in mice. After 7 daysof treatment, NaB reduced the level of cholesterol in serum of miceby �28%, and this reduction was comparable (�30%) with that bythe so-called cholesterol-lowering drug pravastatin (Fig. 7). Alter-natively, NaFO had no effect on serum level of cholesterol (Fig. 7),indicating the specificity of the effect. These results are importantbecause they suggest that NaB may be used to lower cholesterol inpatients with hypercholesterolemia.

Next we examined the role of different members of the meval-onate pathway in the anti-inflammatory effect of NaB. HMG-CoA,mevalonate, and farnesyl pyrophosphate abrogated the inhibitoryeffect of NaB on the expression of iNOS mRNA (Fig. 8A), theproduction of NO (Fig. 8B), and the activation of NF-�B (Fig. 8C)in microglial cells. Alternatively, cholesterol (the end product ofthe mevalonate pathway) and coenzyme Q (an unrelated lipid mol-ecule) had no effect on NaB-mediated inhibition of iNOS mRNA(Fig. 8A), NO production (Fig. 8B), and the activation of NF-�B(Fig. 8C). These results suggest that depletion of intermediaryproducts rather than end products of the mevalonate pathway isresponsible for the observed anti-inflammatory effect of NaB.

An inhibitor of p21ras farnesyl protein transferase (FPTinhibitor II) suppresses LPS-induced expression of iNOS mRNAand activation of NF-�B in mouse BV-2 microglial cells

Inhibition of LPS-induced expression of iNOS and activation ofNF-�B by NaB and its reversal by farnesyl pyrophosphate (FPP)and other upstream members of the mevalonate pathway (HMG-CoA and mevalonate), but not by the end product of the samepathway, suggest a possible involvement of the farnesylation re-action in the activation of NF-�B and the expression of iNOS.Because farnesylation is a necessary step for the activation ofp21ras, we examined the effect of FPT inhibitor II on LPS-inducedexpression of iNOS and activation of NF-�B in microglial cells.FPT inhibitor II selectively inhibits p21ras farnesyl protein trans-ferase with the IC50 of 75 nM. In whole cells, however, a higherconcentration (25–250 �M) of FPT inhibitor II is required to in-hibit farnesylation of p21ras by 90% (33). Therefore, BV-2 cellswere pretreated with 100, 200, and 300 �M FPT inhibitor II for 2 hfollowed by stimulation with LPS. It is clearly evident from Fig. 9that FPT inhibitor II dose-dependently suppressed LPS-inducedexpression of iNOS mRNA (Fig. 9A) and activation of NF-�B(Fig. 9B) in microglial cells. These results suggest that p21ras far-nesylation plays an important role in the activation of NF-�B andthe expression of iNOS in LPS-stimulated microglia.

To exclude any possibility that FPTI-II inhibits iNOS indepen-dent of NF-�B, we examined the effect of wtNBD and FPT inhib-itor II, alone or together, on the mRNA expression of iNOS. It isevident from Fig. 9C that FPT inhibitor II did not show any ad-ditive inhibitory effect on the expression of iNOS in wtNBD-treated cells, suggesting that inhibition of iNOS by FPT inhibitorII is purely due to the suppression of NF-�B activation. Further-more, inability of FPP to reverse the inhibitory effect of wtNBD oniNOS as evident from Fig. 9D clearly indicates that farnesylationof p21ras is an upstream event of NF-�B activation and thuswhether NF-�B is directly inhibited, inhibition of iNOS is notreversed by FPP. The semiquantitative RT-PCR studies in Fig. 9Eagain shows that NaB has no additive inhibitory effect on iNOSover wtNBD, further substantiating the fact that NaB-mediatedinhibition of iNOS in microglial cells solely involves suppressionof NF-�B activation. To further confirm, we investigated the effect

FIGURE 7. Effect of NaB on serum level of cholesterol in maleC57BL/6 mice. Mice (6–8 wk old) were treated with NaB (100 mg/kgbody weight), NaFO (100 mg/kg body weight), and pravastatin (1 mg/kgbody weight) separately via gavage for 7 days followed by quantificationof cholesterol in serum using a simple fluorometric method. Results rep-resent means � SD of five mice per group (n � 5). b, p � 0.05 vs control.

FIGURE 8. Intermediates of the mevalonate pathway negate the inhib-itory effect of NaB on the expression of iNOS and the activation of NF-�Bin mouse BV-2 microglial cells. Cells were treated with NaB in the pres-ence or absence of different concentrations of HMG-CoA, mevalonate,FPP, cholesterol, and ubiquinone for 6 h followed by stimulation with LPS.A, After 5 h of stimulation, the expression of iNOS mRNA was monitoredby RT-PCR. B, After 24 h of stimulation, the level of nitrite was measuredin supernatants using Griess reagent. Results are means � SD of threeindependent experiments. a, p � 0.001 vs LPS; b, p � 0.001 vs LPS�NaB.C, Cells were cotransfected with 0.25 �g of PBIIX-Luc and 12.5 ng ofpRL-TK. Twenty-four hours after transfection, cells were incubated withNaB in the presence or absence of HMG-CoA, mevalonate, FPP, choles-terol, and ubiquinone. After 6 h of incubation, cells were stimulated withLPS for 6 h followed by assay of firefly (ff-Luc) and Renilla (r-Luc) lu-ciferase activities. Results are means � SD of three different experiments.a, p � 0.001 vs LPS; b, p � 0.001 vs LPS plus NaB.


of NaB on the expression of IFN-�-induced expression of iNOS inBV2 microglial cells. IFN-� induces iNOS via activation ofSTATs, which is independent of NF-�B. It is evident from Fig. 9Fthat NaB had no significant effect on IFN-�-induced expression ofiNOS mRNA in microglial cells even at a dose as high as 2 mM.These results suggest that NaB is only able to inhibit iNOS ifinduced by activation of NF-�B and that the observed inhibition ofiNOS by NaB is purely due to suppression of NF-�B through thedepletion of mevalonate intermediates.

NaB suppresses the activation of p21ras in mouse BV-2microglial cells

Because p21ras farnesylation is involved in the activation ofNF-�B and the expression of iNOS and NaB-mediated inhibitionof these events were reversed by FPP, we examined the whetherNaB suppressed the activation of p21ras. Earlier, we have seenactivation of p21ras in human astroglioma cells within 2–4 min ofLPS stimulation (34). Therefore, at different times (2, 3, and 5 min)of stimulation by LPS, microglial cells were analyzed for the ac-tivation of p21ras. Although we did not see p21ras activation at 2min of LPS stimulation, marked activation was observed at 3 and5 min of stimulation (Fig. 10A). Therefore, we examined the effectof NaB on the activation of p21ras at 3 min of LPS stimulation. Itis clearly evident from Fig. 10B that NaB, but not NaFO, markedlyinhibited LPS-induced activation of p21ras in microglial cells.These results suggest that NaB attenuates the expression of proin-

flammatory molecules in glial cells probably by suppressing theactivation of p21ras.

Activation of p21ras alone is sufficient to induce the expressionof iNOS and the activation of NF-�B in mice BV-2 microglialcells

Our results that FPT inhibitor II inhibited microglial expression ofiNOS and that NaB suppressed microglial activation of p21ras andthe expression of iNOS prompted us to examine whether activationof p21ras alone was sufficient for the expression of iNOS in mi-croglial cells. Activation of p21ras in BV-2 glial cells was achievedby the expression of RasV12, a constitutively active mutant ofp21ras. It is clear from Fig. 11A that the expression of RasV12alone markedly induced the production of NO in microglial cellswhereas the expression of empty vector was unable to induce NOproduction. Inhibition of RasV12-induced production of NO byarginase, an enzyme that degrades the substrate (L-arginine) ofNOS, and N-methyl-L-arginine acetate, a competitive inhibitor ofNOS activity, suggests that the induction of NO production inRasV12-transfected cells is dependent on NOS-mediated argininemetabolism (data not shown). To understand the mechanism ofinduction of NO production, we analyzed the status of iNOS pro-tein and mRNA in RasV12-transfected cells. Western blot analysiswith Abs against murine macrophage iNOS and Northern blotanalysis for iNOS mRNA clearly showed that RasV12 induced theexpression of iNOS protein (Fig. 11B) and mRNA (Fig. 11C),suggesting that signal(s) provided by the activation of p21ras aloneis sufficient to induce the expression of iNOS.

Because the activation of NF-�B is important for the inductionof iNOS (19–23, 26–28), to understand the basis of iNOS expres-sion by RasV12, we also investigated whether Ras alone was suf-ficient for the activation of NF-�B. Activation of NF-�B was mon-itored by transcriptional activity of NF-�B using the expression ofluciferase from a reporter construct, PBIIX-Luc. Cells were co-transfected with PBIIX-Luc and RasV12 followed by incubation inserum-free medium. As evident from Fig. 11D, RasV12 markedlyinduced NF-�B-dependent transcription of luciferase, whereas theempty vector was unable to induce the transcriptional activity ofNF-�B.

FIGURE 9. Effect of FPT inhibitor II (FPTI II) on the expression ofiNOS and the activation of NF-�B in mouse BV-2 microglial cells. A, Cellstreated with different concentrations of FPT inhibitor II for 2 h were stim-ulated by LPS. After 5 h of stimulation, the expression of iNOS mRNAwas monitored by RT-PCR. B, Cells were cotransfected with 0.25 �g ofPBIIX-Luc and 12.5 ng of pRL-TK. Twenty-four hours after transfection,cells were incubated with different concentrations of FPT inhibitor II for2 h followed by stimulation with LPS. After 6 h of stimulation, activitiesof firefly (ff-Luc) and Renilla (r-Luc) luciferase were monitored. Resultsare mean � S.D. of three different experiments. a, p � 0.001 vs LPS; b,p � 0.05 vs LPS. C–E, Cells treated with appropriate concentrations ofFPT inhibitor II (200 �M) for 2 h (C), FPP (200 �M; D), or NaB (1 mM;E) alone for 6 h or along with wtNBD (10 �M; 1 h before stimulation)were stimulated by LPS. After 5 h of stimulation, the expression of iNOSmRNA was monitored by RT-PCR. F, Cells treated with different concen-trations of NaB for 6 h were stimulated by IFN-� (12.5 U/ml). After 5 h ofstimulation, the expression of iNOS mRNA was monitored by RT-PCR.The results represent three independent experiments.

FIGURE 10. Effect of NaB on the activation of p21ras in mouse BV-2microglial cells. A, Cells were stimulated with LPS under serum-free con-ditions. At different time points, activation of p21ras was monitored. B, Inanother set, cells pretreated with 1 mM NaB/NaFO for 6 h were challengedwith LPS under serum-free conditions followed by monitoring activationof p21ras at 3 min of stimulation. Results represent two independentexperiments.


DiscussionAlthough microglial activation has an important repairing functionthrough scavenging of unwanted bodies in the CNS and activationof astrocytes may have important beneficial effects in the recoveryof injured CNS by actively monitoring and controlling the extra-cellular water, pH, and ion homeostasis, once microglia and as-troglia are activated in the neurodegenerating microenvironment,activation always goes beyond control, and eventually detrimentaleffects of glial activation override its beneficial effects. Activatedglia produce NO, a number of proinflammatory cytokines, reactiveoxygen species, etc., in excessive amounts for a prolonged timeperiod that ultimately damage neurons and oligodendrocytes.Therefore, understanding mechanisms that regulate microglial andastroglial activation is an important area of investigation.

Cinnamon, the brown bark of the cinnamon tree, is a commonlyused spice and flavoring material for desert, candies, chocolate,etc. It has a long history as a medicine as well. Medieval physi-cians used cinnamon in medicines to treat a variety of disordersincluding arthritis, coughing, hoarseness, and sore throats. In ad-dition to containing manganese, dietary fiber, iron, and calcium,cinnamon contains three major compounds, cinnamaldehyde, cin-namyl acetate, and cinnamyl alcohol. After intake, these three ac-

tive compounds are converted into cinnamic acid by oxidation andhydrolysis, respectively. Then cinnamic acid is �-oxidized to ben-zoate in the liver. This benzoate exists as sodium salt (NaB) orbenzoyl-CoA. This NaB is a widely used food preservative due toits antimicrobial properties. Earlier, we have demonstrated thatNaB modifies T cells at multiple steps and protects experimentalallergic encephalomyelitis, an animal model of MS (35). Recently,one study by Cao et al. (36) demonstrates that cinnamon polyphe-nol extract increases the expression of tristetraprolin, an anti-inflammatory molecule, more rapidly than those of proinflamma-tory cytokines in macrophages. Several lines of evidence presentedin this study clearly support the conclusion that the cinnamon me-tabolite NaB attenuates the activation of mouse microglia and hu-man astroglia. Our conclusion is based on the following observa-tions. First, LPS, a prototype inducer of inflammation in many celltypes including microglia, induced the expression of iNOS andproinflammatory cytokines and up-regulated the expression of var-ious surface markers in mouse microglia. However, NaB attenu-ated LPS-induced expression of proinflammatory molecules with-out altering cell survival suggesting that this attenuation is not dueto any cell death. This inhibition was also specific as NaFO, acompound structurally similar to NaB but without having the ben-zene moiety, had no effect. Second, we extended the study beyondLPS and examined whether NaB was capable of suppressing mi-croglial expression of iNOS induced by other neurotoxins and eti-ological reagents of various neurodegenerative disorders. It is im-portant that NaB, but not NaFO, attenuated microglial expressionof iNOS mRNA induced by various neurotoxins such as A� (re-lated to AD), poly(IC) (related to viral neuropathy), HIV-1 gp120(related to HAD), MPP� (related to Parkinson’s disease), PrP (re-lated to prion disorders), IL-1� (related to neuroinflammation),and IL-12 p402 (related to MS). Third, IL-1� is critical for induc-ing iNOS and activating primary human astrocytes (20). NaB alsosuppressed the production of NO, the expression of iNOS, theactivation human iNOS promoter, and the up-regulation of GFAPin human astrocytes. Because these proinflammatory moleculeshave been implicated in the pathogenesis of demyelinating andneurodegenerative diseases, our results provide a potentially im-portant mechanism whereby cinnamon metabolite NaB may ame-liorate neural injury.

The signaling events required for the transcription of iNOS andproinflammatory cytokines are becoming clear. Although manytranscription factors such as NF-�B, C/EBP�, AP-1, STAT, IRF-1,etc., play a role in the expression of various proinflammatory mol-ecules, activation of NF-�B seems essential for the transcription ofmost of the proinflammatory molecules (19–23, 26–28, 37, 38).Therefore, for a drug to exhibit an anti-inflammatory effect, it isalmost mandatory to attenuate the activation of NF-�B. Althoughwe did not see complete abrogation of NF-�B activation, NaBsignificantly decreased the activation of NF-�B in microglia. How-ever, it was unknown by which mechanisms NaB suppressed theactivation of NF-�B. p21ras, a membrane-associated small guaninenucleotide-binding protein, plays a central role in transmitting ex-tracellular signals within the cell and in controlling cellular pro-liferation and differentiation (39, 40). Here we present evidencethat NaB suppressed the activation of p21ras and thereby inhibitedthe activation of NF-�B and the expression iNOS in microglia. Ourconclusion is based on the following observations. First, NaB re-duced the level of cholesterol in vivo in mice at a level comparablewith pravastatin suggesting that this drug may be used to lowercholesterol in patients with hypercholesterolemia. However,HMG-CoA, mevalonate and farnesyl pyrophosphate, but not cho-lesterol, reversed the inhibitory effect of NaB on the expression ofiNOS and the activation of NF-�B, suggesting that depletion of

FIGURE 11. Activation of p21ras alone induces the expression of iNOSand the activation of NF-�B in mouse BV-2 microglial cells. Cells weretransfected with different concentrations of an empty vector and RasV12(constitutively active mutant of Ras). Twenty-four hours after transfection,cells were incubated in serum-free medium for 24 h followed by assay ofnitrite concentration in supernatant (A) and monitoring the level of iNOSprotein in cells by Western blot (B). Actin was run as control. a, p � 0.001vs IL-1�. C, Cells plated at 70–80% confluence in 12-well plates werecotransfected with 0.25 �g of phiNOS(7.2)Luc and 12.5 ng of pRL-TKusing the Lipofectamine-Plus (Invitrogen). Twenty-four hours after trans-fection, cells received NaB and NaFO. After 6 h of incubation, cells werestimulated with IL-1� (20 ng/ml) for 12 h under serum-free condition.Firefly (ff-Luc) and Renilla (r-Luc) luciferase activities were obtained byanalyzing the total cell extract as described in Materials and Methods. Dataare means � SD of three different experiments. a, p � 0.001 vs control; b,p � 0.001 vs IL-1�.


intermediates, but not end products, of the mevalonate pathway isinvolved in the anti-inflammatory effect of NaB. Second, FPT in-hibitor II, capable of inhibiting farnesylation of p21ras, inhibitedthe activation of NF-�B and the expression of iNOS. Third, NaB,but not NaFO, inhibited the activation of p21ras in LPS-stimulatedmicroglial cells. Fourth, activation of p21ras alone was sufficientfor the activation of NF-�B and the expression of iNOS in micro-glial cells underlining the importance p21ras activation in the ac-tivation of microglia.

The Ras proto-oncogene proteins, a family of GTP-binding pro-teins, function by binding to the cytoplasmic surface of the plasmamembrane. This membrane localization of p21ras involves preny-lation of cysteine in a CAAX motif present at the C terminus,proteolytic removal of AAX tripeptide, and then carboxymethyla-tion of the C-terminal cysteine (39). The activation of p21ras byreceptor tyrosine kinase occurs through conversion of the GDP-bound inactive form to the GTP-bound active form by Sos andGrb2 and then transduction of signal to downstream effector mol-ecules (40). The GTP-bound form is converted to the inactive formby the intrinsic GTPase activity, which is accelerated by GTPase-activating proteins (41). NaB preferentially attenuates farnesyla-tion of p21ras and thereby inhibits the signal transmission to thedownstream signaling molecules (42, 43). One such downstreamcandidate is Raf-1 (serine-threonine kinase). The p21ras interactsdirectly with Raf-1 and is believed to function by positioningRaf-1 at the plasma membrane in the vicinity of its activator, andtyrosine phosphorylation of Raf-1 seems to be essential for p21ras-induced activation of Raf-1 (42, 43). Raf-1, in turn, phosphorylatesand activates MEKs and ERKs (members of the MAPK cascade).Therefore, the observed inhibition of NF-�B activation and induc-tion of iNOS by NaB may be due to decrease and/or lack of signaltransmission from receptor tyrosine kinase to Raf/MAPK cascadevia p21ras.

There are several advantages of NaB over other proposed an-tineurodegenerative therapies. First, NaB is fairly nontoxic. Cin-namon has been widely used as flavoring material and spicethroughout the world for centuries. Cinnamon is metabolized toNaB. NaB is excreted through the urine, if in excess. NaB is anFDA-approved drug against urea cycle disorders in children. Sec-ond, NaB can be taken orally, the least painful route. Recently, wehave demonstrated that NaB treatment of mice with relapsing-re-mitting experimental allergic encephalomyelitis, an animal modelof MS, via drinking water suppressed the disease process of ex-perimental allergic encephalomyelitis (35). Third, NaB is veryeconomical compared with other existing antineurodegenerativetherapies. Fourth, although here we have not tested the premise,NaB as a lipophilic molecule is most likely able to diffuse throughthe blood-brain barrier. For example, glycine toxicity is a problemin urea cycle disorders. After treatment of patients with urea cycledisorders, NaB combines with glycine to produce hippurate, acompound that is readily excreted in the urine. Simultaneous se-rum and CSF sampling in those patients showed comparable levelsof NaB and hippurate in the CSF (13–15), suggesting that NaB iscapable of crossing the blood-brain barrier.

In summary, we have demonstrated that NaB (a metabolite ofcinnamon, commonly used food additive and a FDA-approveddrug for urea cycle disorders) inhibited glial activation of NF-�Band expression of iNOS and cytokines by modulating the meval-onate pathway and Ras activation. Because NaB suppressed themevalonate pathway, it was also able to lower cholesterol in vivoin mice at a level comparable with that of pravastatin, a choles-terol-lowering drug. These results highlight undiscovered proper-ties of NaB and indicate that this drug may be used for therapeutic

intervention in neurodegenerative disorders as primary or adjuncttherapy.

AcknowledgmentsWe thank Xiaojuan Liu for her help in transfection experiments.

DisclosuresThe authors have no financial conflict of interest.

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