1Jack Bell Research Centre and 2Department of Pediatric Gastroenterology, Children and Women's Hospital, Vancouver, British Columbia, Canada V5Z 3P1.
Submitted 21 October 2002 ; accepted in final form 11 March 2003
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ABSTRACT |
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inflammatory bowel disease; nuclear factor-B; p38 MAPK
Several cytokines including TNF- and IL-1
, have been shown to
be upregulated in IBD and serve to amplify and perpetuate tissue damage.
Furthermore, chemokines are also upregulated, thus providing a continuous
signal for the influx of leukocytes
(35,
42). The production of
chemokines is normally dependent on the coordinated activation of a number of
signaling pathways that converge on the transcription factor NF-
B. This
is an inducible heterodimeric transcription factor composed most commonly of
the Rel A (p65) and NF-
B1 (p50) subunits. Much has been learned about
the activation of this molecule, and key steps in its activation by a variety
of stimuli are known to include the activation of IKK followed by the
phosphorylation and ubiquitin-mediated degradation of inhibitor
B
. This enables the Rel A subunit to translocate to the nucleus
and bind to the promoter region of genes and switch on their expression
(22,
45).
Regulation of NF-B function has been documented by several agents
used in the management of IBD, such as corticosteroids, sulfasalazine, and
5-aminosalicylates (5-ASA)
(51,
55). Furthermore, antisense
oligonucleotides directed against the p65 subunit have been shown to attenuate
disease activity in an animal model of colitis
(32). Recent work has shown
that dietary constituents such as curcumin, may also potently inhibit
NF-
B (44) and attenuate
proinflammatory molecule expression
(5,
6,
26). Curcumin is a component
of the spice turmeric (Curcuma longa) used in curries and mustard,
whose anti-inflammatory properties have been recognized for years
(38). These effects are
related, in part, to inhibition of the activities of the cyclooxygenase,
lipoxygenase, and NF-
B in several cell systems
(17). Furthermore, its role in
the attenuation of colonic cancer in animal models has also been established
(37).
Recognition that the stress-responsive signaling pathways also play a role
in the expression of proinflammatory molecules is of critical importance, for
a more complete understanding of inflammation
(48). In this regard it is
well known that the p38 MAPK can modulate a number of different steps in the
inflammatory cascade. These include production of -interferon by
lymphocytes, degranulation of neutrophils, as well as the expression of
cyclooxygenase, inhibitory nitric oxide synthase, IL-1, and TNF by monocytes
(4,
36,
39). Despite a recent report
concerning the potential role of p38 MAPK as a target in IBD
(16), its role in IBD
pathogenesis remains unclear.
Management of IBD involves the use of 5-ASA and immunosuppressives such as
corticosteroids and 6-mercaptopurine as well as its precursor azathioprine
(14). Novel agents such as
monoclonal antibodies against TNF- have been developed and demonstrate
clinical efficacy (46).
However, these agents are expensive and not without side effects.
Consequently, there is a need for alternative agents that may be equally or
more effective as well as being cheaper. It is interesting to note that both
curcumin and sulfasalazine target IKK molecules
(21,
53). However, the relevance of
inhibition of IKK by curcumin has never been tested in IBD. Here we show that
this compound has beneficial effects in a murine model of IBD, and may warrant
further scrutiny in human IBD.
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MATERIALS AND METHODS |
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Immunohistochemistry. Paraffin-embedded colonic tissue samples were dewaxed in xylene twice for 5 min, rehydrated in a series of ethanol (10070%) for 3 min each followed by rehydration in PBS for 30 min. After rehydration the endogenous peroxidase was blocked with 0.3% hydrogen peroxide followed by antigen retrieval by microwaving sections in citrate buffer pH 6.0 (10 mM Na citrate). After antigen retrieval, the sections were stained using the above-mentioned kit according to manufacturer's recommendations but with the following modifications. Sections were incubated with the primary antibody at 4°C overnight. The following antibodies were used at the indicated dilutions: p-38 (1:500), p-p38 (1:100). Sections were stained with Vectastain ABC elite kit and DAB secondary detection kit (Vector Laboratories). Phospho-p38 antibody was purchased from New England Biolabs (Mississauga, ON, Canada). Antibodies to p38 protein were purchased from Stressgen Biotechnologies (Victoria, BC, Canada). Each section had its own control using the secondary antibody only. Preimmune serum was initially used to ensure specificity of the signal with each of the antibodies.
Measurement of MPO activity. MPO activity was measured according to the method described by Wallace (52). Tissue was homogenized in hexadecyltrimethylammonium bromide in 50 mM potassium phosphate buffer. Aliquots were then added to O-dianisidine hydrochloride solution. Absorbance was read at 450 nm using a microplate reader.
Western analysis. Tissue was placed in homogenization buffer (in
mM: 20 MOPS, 50 -glycerophosphate, 5 EGTA, 1 DTT, 1 sodium vanadate, 1
PMSF, and 50 NaF) and sonicated for 15 s (x2) and centrifuged at 14,000
rpm for 15 min. The protein concentration in the supernatent was determined by
the Bradford assay (Bio-Rad, Missisauga, ON, Canada). Twenty-five micrograms
protein from each sample were resolved using 10% SDS-PAGE before transferring
to nitrocellulose membranes (Bio-Rad). The blots were blocked in 5% skim milk
in 20 mM Tris · HCl pH 7.4, 250 mM NaCl, 0.05% Tween 20 (TBST) for 1 h
before probing for 2 h using the appropriate primary antibody. The blots were
washed with TBST for 10 min three times, before being incubated with the
appropriate secondary antibody for 1 h. After three further washes in TBS-T,
they were developed using the enhanced chemiluminescence detection system
(Amersham, Montreal, PQ, Canada).
Isolation of RNA and PCR. Isolated colon was snap frozen in liquid
nitrogen. RNA was isolated using the TRIzol method (Life Technologies,
Burlington, ON, Canada). The purity of the RNA was determined by running 1
µg of RNA on a 1% agarose gel for 1.5 h at 75 V. One microgram of RNA was
reverse transcribed using 0.5 µg of oligo(dT)1218
(Amersham), 1 µl of 10 mM 2-deoxynucleotide 5'-triphosphate (dNTP), 2
µl of 0.1 M DTT, 40 units of RNA-guard (Amersham) in 1 x first-strand
buffer (Life Technologies) using 200 units of Moloney murine leukemia virus
RT, by incubating the reaction mixture for 50 min at 37°C. Two microliters
of cDNA were used in each subsequent PCR reaction. For each 50 µl PCR
reaction, 2 units of Thermus aquatias DNA polymerase (PE Biosystems,
Branchburg, New Jersey), 1x PCR Buffer (PE Biosystems), 10 pmol of each
primer, 1 µl of 10 mM dNTP, and 3 µl of 25 mM MgCl2 were
used. The PCR temperatures used were 94°C denaturing for 45 s, 56°C
annealing for 45 s, and 72°C extension for 1 min. Ten microliters aliquots
of the reaction were electrophoresed on a 1.5% agarose gel containing ethidium
bromide. Negative controls for cDNA synthesis were run without template, and
also without RT. Linearity of PCR reactions was determined in the range
between 20 and 30 cycles. Densitometry was performed by using Bio-Rad
Quantity-One software. The sizes of the PCR products were 563 and 851 bp for
IL-1 and GAPDH, respectively
(54). Primers: IL-1
forward: 5'-ATGGCAACTGTTCCTGAACTCAACT-3'; IL-1
reverse:
5'-CAGGACAGGTATAGATTCTTTCCTTT-3'; GAPDH forward:
5'-CGCTGCTGAGTATGTCGTGGAGTCT-3'; GAPDH reverse:
5'-GTTATTATGGGGGTCTGGGATGGAA-3'.
Real-time PCR for IL-1 was performed by using an ABI Prism 5700
Sequence Detection System (PE Applied Biosystems, Foster City, CA), as
previously described (28,
29). This relies on the
SYBRgreen I dye binding to the dsDNA directly in the reaction tube. The
software detects the threshold cycle number (CT) when signals reach
10-fold the standard deviation of the baseline. It has been previously
reported that the CT values are a quantitative measurement for the
mRNA being tested (13). Each
reaction contained 25 µl of 2 x SYBR Green Master Mix (containing 200
µM deoxyadenosine triphosphate, deoxyguanosine triphosphate, and
deoxycytidine triphosphate; 400 µM deoxyuridine triphosphate; 2.5 mmol/l
MgCl2; and 0.625 U AmpliTaq Gold DNA polymerase). Primers were used
at a final concentration of 200 nM, and 2 µl complementary DNA in a final
volume of 50 µl. IL-1
primers used were as described above, and actin
primers were as follows: forward 5'-CCAACCGCGAGAAGATGACC-3';
reverse 5'-GATCTTCATGAGGTAGTCAGT-3'. Reactions were incubated at
50°C for 2 min followed by 95°C for 15 min. The PCR parameters used
were 40 cycles of 15 s denaturation at 95°C followed by 1 min annealing at
65°C and 1 min at 72°C. All reactions were performed in triplicate.
Data analysis used sequence detection system software provided by the
manufacturer.
CT was calculated by subtracting the
CT for actin from the CT for IL-1
in each
sample.
Electromobility shift assay. This was done as previously described (23). Briefly, 5 µg of tissue lysate were preincubated in binding buffer (20 mM HEPES pH 7.9, 100 mM KCl, 10% glycerol, 1 mM DTT) and 1 µg of poly(dI-dC) (Amersham), for 15 min. Probe (20,000 counts/min) was then added, and the reaction mixture was incubated at room temperature for 30 min, and then resolved on a 5% nondenaturing polyacrylamide gel in 0.25x TBE at 200 V for 1.5 h. The gel was subsequently dried for 45 min before phosphoimaging analysis by using a Bio-Rad molecular imager FX (or alternatively exposed to film overnight at 80°C and then developed). For supershift or cold competitor reactions, the nuclear extract was preincubated with 1 µg of anti-p65 antibody (Calbiochem, San Diego, CA), or 100-fold excess of unlabeled probe with binding buffer and poly(dI-dC) for 30 min before adding the radiolabeled probe. Sequence of probe: 5'-AATTCGGTTACAAGGGACTTTCCGCTGA-3'; 3'-GCCAATGTTCCCTGAAAGGCGACTTCGA-5'.
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RESULTS |
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Disease severity was evaluated based on a macroscopic score. As the data indicate (Fig. 1B), there was a predictable increase in inflammation in response to the induction of colitis with DNB. This was significantly attenuated in those animals, which had been prefed curcumin-containing chow (P < 0.001). Notably there was no increase in basal inflammation due to the diet alone. Thus, from this data, it would appear that animals are able to tolerate curcumin at these concentrations and that there was a beneficial response observed in the attenuation of DNB-induced colitis.
Histological improvement in response to curcumin. The removed colons were sectioned, fixed, and stained with hematoxylin and eosin. As the representative sections show (Fig. 2), there is a reduction in the inflammatory cellular infiltrate, mucosal and muscle damage, as well as wall thickening, observed with pretreatment with curcumin before DNB-induced colitis. Figure 2, A and C represent cross sections through the intestinal lumen for DNB treatment, whereas Fig. 2, B and D demonstrate longitudinal sections. Fig. 2, E and G are the corresponding sections for curcumin pretreatment, and Fig. 2, F and H are the longitudinal sections for comparison. Curcumin alone did not result in any dramatic changes in the intestinal mucosa other than a mild increase in the lymphocytic infiltrate (data not shown). A time course study was performed to assess the characteristics of the curcumin-induced response. The data indicate that there is a significant reduction in the histological damage score at both days 2 and 4 postinduction of colitis. At 10 days postinduction of colitis, there appears to be a similar trend toward improvement; however, it is also apparent that the disease resolves spontaneously. This data support the macroscopic damage scores in validating an anti-inflammatory role for curcumin in this model of colitis.
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Reduction in inflammatory markers by curcumin pretreatment. Myeloperoxidase activity is an established marker for inflammatory cell (mainly neutrophils) infiltration and activity in murine models of colitis and was thus examined. Data clearly indicate (Fig. 3A) a significant reduction in this parameter on day 5 postinduction of colitis (P = 0.005). This was accompanied by a reduction in the number of neutrophils within the histology samples. We counted an average of 8.4 ± 0.9 (means ± SE) cells/high power field in the DNB-treated animals, whereas there were only 4.33 ± 0.8 cells/high-power field in the curcumin pretreated animals (P = 0.0007). This compared with only 0.7 ± 0.21 cells/high-power field in the control (PBS treated) animals.
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Another potential mechanism whereby curcumin achieves its effects within
the intestine was then examined, expression of proinflammatory cytokines. The
message for one of the best-characterized cytokines in IBD, IL-1,
(11,
30) was determined by using
semiquantitative RT-PCR with GAPDH acting as an internal control
(54). The data show
(Fig. 3, B and
C) that there was a significant reduction in the
DNB-induced IL-1
message after pretreatment with curcumin. This finding
was validated by using real-time PCR, which revealed CT values of
31.67 ± 0.11, 29.26 ± 0.35, and 32.97 ± 0.34 for the
control, DNB-treated, and curcumin-pretreated DNB animal groups, respectively
(means ± SD). The corresponding
CT values were
obtained by subtracting the CT for
-actin from these values
and were 16.21, 14.06, and 17.38, respectively. Collectively, these findings
confirm an anti-inflammatory effect for curcumin in murine colitis.
Reduced NF-B DNA binding in vivo. The next step
was to determine whether or not curcumin altered NF-
B DNA binding,
because this has been widely documented to occur in various cell culture
models. Five micrograms aliquots of total protein lysate were incubated with
the probe and resolved on nondenaturing polyacrylamide gels. A representative
autoradiogram (Fig. 4)
indicates that there is minimal basal binding within the intestine, and that
this is dramatically increased on exposure of the animals to DNB (a control
using CaCo-2 cells stimulated with IL-1
is shown with the actual band
and a nonspecific band shown for comparison). The curcumin-treated control
animals show little change in this parameter. When animals were pretreated
with curcumin, however, there was a clear reduction in DNA binding, thus
verifying that curcumin does indeed inhibit NF-
B activation in the
colon in vivo. These findings indicate for the first time that curcumin is
able to impact on an important transcriptional mechanism in the
gastrointestinal tract.
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Curcumin attenuates activation of p38 MAPK. In previous work curcumin has been reported to influence MAPK signaling, in addition to its effects on IKK regulation (7, 21). Therefore, we chose to investigate this signaling nexus in the DNB model. For this work, we carried out a time course in which the expression and activation of the p38 MAPK were examined by Western analysis. We made use of phosphospecific MAPK antibodies, which correlate with the activation of the respective family members. The data indicate (Fig. 5M) that there is an early activation of p38 MAPK as well as p42/44 ERKs, however, whereas curcumin led to an activation of only p38 MAPK, DNB activated both MAPKs. Intriguingly, whereas the p38 MAPK is inhibited in the curcumin-treated group, the p44/42 ERK activity is unaffected. It should be emphasized that the control samples depicted were those treated with ethanol and not PBS. The latter animals exhibited lower levels of p38 MAPK activity (data not shown). Remarkably, p38 MAPK inhibition was also observed at the 48-h time point (Fig. 5N).
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Site-specific curcumin attenuation of p38 MAPK activation. Because a Western immunoblot gives no indication of the location of the observed changes in p38 MAPK activity, this component was investigated in tissue sections by using the same antibody. There was an impressive increase in the active p38 MAPK signal in the nuclei of epithelial cells (Fig. 5I, red arrows). Other structures such as the smooth muscle and inflammatory cells also revealed evidence of p38 MAPK activity (data not shown). When the curcumin pretreated DNB sections were examined, in direct agreement with the Western data, there was a dramatic reduction in the activity of p38 MAPK, especially at the level of the mucosa. This indicates that the stress response in the intestine likely involves the epithelial cells (directly or indirectly), and that at least some of the inflammation-attenuating effect of curcumin involves a reduction in p38 MAPK signaling through these structures.
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DISCUSSION |
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Precisely how curcumin achieves its effects is not clear. It has been shown
to possess free radical scavenging (antioxidant) properties in addition to its
known effects on the activation of NF-B. Its in vivo effects may well
rely on a complementation of these two and other activities. Previous work has
documented the beneficial effects of compounds that quench free radicals, such
as the membrane-permeable radical scavenger tempol in experimental colitis
(9). It is well established
that generation of free radicals can effect the activation of multiple
intracellular signaling pathways
(27). Inhibition of this
process would be expected to correlate with a reduction in the expression of
key inflammatory molecules; indeed, we show that there is a reduction of the
expression of the proinflammatory cytokine, IL-1
. Moreover, curcumin has
not only been shown to inhibit the proliferation and effect apoptosis of T
lymphocytes (43), but also to
inhibit the production of IL-12 by LPS-stimulated macrophages
(24). Its other properties
include inhibition of TNF-induced adhesion molecule expression on endothelial
cells (26), as well as
TPA-induced lipoxygenase and cyclooxygenase activities in mouse epidermis
(17). Thus its effects are
likely to occur at multiple sites in vivo, and further work will be required
to correlate which of these are important for attenuation of colitis.
Previous work has clearly indicated an involvement of the MAPK in the
inflammatory responses of epithelial cells
(15,
21). More recent work has
demonstrated that use of p38 MAPK inhibitors can be effective for human IBD
(16). Moreover, the
involvement of p38 MAPK in human IBD has also been addressed
(50). Thus, whereas it was
anticipated that the inflammatory response may entail an activation of the p38
MAPK in the infiltrating immune cells, it was unexpected and surprising to
observe such prominent activity within the other tissue components. This was
most obvious in the epithelial cell nuclei and to a lesser extent within the
smooth muscle. The exact role that the activated p38 MAPKs play at these sites
remains unclear and will require further investigation, but it may involve
secretion of chemokines, neuropeptides, trophic factors, or possibly even
reflect a response to cell damage
(8,
12). Significantly, it has
been shown that histone phosphorylation on the promoters of certain
proinflammatory genes, is regulated by p38 MAPK and enhances NF-B
recruitment (40). With
specific reference to epithelial cells, we have shown a role for p38 MAPK
(using pharmacological inhibitors and transfection of the dominant negative
construct) in regulation of IL-1
-induced IL-8 promoter activity and IL-8
gene expression (35a). Other
mechanisms may also be involved in regulation of IL-8 in intestinal epithelial
cells at the posttranscriptional level
(20).
Curcumin alone also influences p38 MAPK activation, as seen in the Western immunoblot, as well as on immunohistochemistry (Fig. 5). This will merit further investigation and is not dissimilar to the activation that has been documented with the anti-TNF antibody previously reported (50). This duality of function of p38 MAPK, i.e., activation in response to the disease and its treatment, is important and may offer an explanation of why specific inhibition using SB-203580 was not beneficial in a murine model of colitis (47). The authors of this work indicated that inhibition of IL-10 production by T regulatory cells in the lamina propria may be a potential mechanism (25).
Although curcumin has been shown to be safe up to levels as high as 10% (100,000 ppm), we have shown an effect at a concentration as low as 0.25%. This dose was well tolerated and we observed no reduction in dietary intake and, consequently, weight in these animals. Further work will help to clarify the optimal dose for this and other models of IBD. Additionally, comparison with other standard agents such as 5-ASA and sulfasalazine, which are compounds effective only in mild and moderate disease, will be of obvious importance. Curcumin may prove to be a cheap, well-tolerated, and effective therapy.
In conclusion, we report an intriguing immunomodulatory effect for a dietary component that has for generations been regarded as a potent anti-inflammatory within many eastern civilizations. It is equally intriguing that the same agent is a potent antineoplastic agent. It is proposed that it may hold promise for the treatment of IBD in humans.
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ACKNOWLEDGMENTS |
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This study was supported by funds from the Crohn's and Colitis Foundation of Canada (to B. Salh and K. Jacobson) and the Canadian Society for Intestinal Research and the Geraldine Dow Foundation
Present address of A. Gómez-Muñoz: Dept. of Biochemistry and Molecular Biology, Faculty of Science, University of the Basque Country, PO Box 644, 48080 Bilbao, Spain.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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