Research Center for Alcoholic Liver and Pancreatic Diseases and Department of Medicine, University of California, Los Angeles and Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California 90073
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Treatments for pancreatitis are
limited. Activation of transcription factor NF-B, a key regulator of
inflammatory molecule expression, is an early event in experimental
pancreatitis and correlates with the inflammatory response. We report
here that curcumin, a natural phytochemical known to inhibit NF-
B
and activator protein (AP)-1, another important proinflammatory
transcription factor, ameliorates pancreatitis in two rat models. In
both cerulein pancreatitis and pancreatitis induced by a combination of
ethanol diet and low-dose CCK, curcumin improved the severity of the
disease as measured by a number of parameters (histology, serum
amylase, pancreatic trypsin, and neutrophil infiltration). Curcumin
markedly inhibited NF-
B and AP-1 activation, assessed by DNA binding
and degradation of inhibitory I
B proteins, and the induction of
mRNAs for cytokines IL-6 and TNF-
, the chemokine KC, and inducible nitric oxide synthase in pancreas. Curcumin also blocked
CCK-induced NF-
B and AP-1 activation in isolated pancreatic acini.
Our findings indicate that blocking key signals of the inflammatory
response ameliorates pancreatitis in both ethanol and nonethanol
models. They suggest that curcumin, which is currently in clinical
trials for cancer prevention, may be useful for treatment of pancreatitis.
nuclear factor-B; activator protein-1; cerulein; cholecystokinin; chemokines; N-acetylcysteine
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ALTHOUGH THE COMPLETE
MOLECULAR mechanism of pancreatitis has not been established,
there is a substantial body of evidence suggesting a critical role for
the inflammatory response in this disease (4, 31, 40).
Recent results from our group and others (13, 14, 17, 20, 22, 25,
31, 47) indicate that the inflammatory response is initiated by
injured pancreatic acinar cells that produce inflammatory mediators,
such as cytokines (e.g., TNF-) and adhesion molecules (e.g.,
ICAM-1), ultimately leading to systemic complications. Blocking the
activation of these mediators by using various approaches ameliorated
pancreatitis in different experimental models (4, 9, 11, 13, 14, 17, 20, 25, 31, 47).
Further evidence suggests that the inflammatory response of pancreatitis mediates, at least in part, the parenchymal injury. That is, inhibition of cytokines and chemokines, removal of neutrophils from the experimental animal, or blockade of neutrophil infiltration into the pancreas by genetic deletion of ICAM-1 all lead to attenuation of the increases in serum amylase and lipase, microscopic evidence of pancreatic parenchymal damage, and intrapancreatic activation of trypsin (9, 11, 13, 14, 17, 19, 20, 25, 31, 37, 47).
Considering the central importance of the inflammatory response in
pancreatitis, therapeutic strategies should be aimed at the key steps
leading to this response. Because of the multitude of cytokines,
chemokines, and other inflammatory molecules involved in this disease
process, one can argue that the most effective strategy should target
upstream "master regulators" of these molecules. Recent data
(8, 11, 20, 22, 23, 25, 34, 38, 41, 42, 47) suggest that
one such key regulator is NF-B.
NF-B comprises a family of transcription factors regulating the
inflammatory, immune, and cell death responses (1, 12, 36, 45,
46). NF-
B is composed of hetero- or homodimers of the Rel
family proteins. In unstimulated cells, NF-
B is kept inactive in the
cytoplasm by association with the inhibitory (I
B) proteins. On
activation, I
Bs are phosphorylated by specific IKK kinases and are
rapidly degraded via proteasome-involving pathways. NF-
B then
translocates into the nucleus and activates the expression of a
multitude of genes that have
B-binding sites in their
promoters/enhancers.
NF-B activation, at least in part, regulates the expression of
cytokines (TNF-
, IL-6), chemokines (IL-8, KC, Mob-1), and other
inflammatory molecules such as ICAM-1 and the inducible nitric oxide
synthase (iNOS), all of which are upregulated in pancreatitis (4,
9, 11, 13, 14, 17, 20, 25, 31, 34, 42, 47). Recent data link the
inflammatory response to NF-
B in several experimental models of
pancreatitis (8, 11, 20, 23, 25, 34, 38, 41, 42, 47). In
particular, NF-
B activation in acinar cells is one of the very early
events in pancreatitis induced by high-dose cerulein, an analog of CCK, a widely used and well-characterized model (20, 23, 41). CCK and cerulein also activate NF-
B in isolated pancreatic acinar cells (16, 20-22, 47).
Furthermore, we recently found upregulation of NF-B and inflammatory
molecule expression in a model of pancreatitis in which an ethanol diet
sensitizes rats to pancreatitis induced by low-dose CCK (EtOH + CCK model) (34). The importance of these findings is that
they provide data on the mechanism of ethanol pancreatitis, which have
been unavailable because of the lack of animal models of
alcohol-mediated pancreatitis. Whether the inflammatory response plays
a causative role in this model of acute alcoholic pancreatitis, similar
to what has been established in nonethanol models, remains to be elucidated.
An attractiveness of NF-B as a therapeutic target in pancreatitis is
that it is "silent" in normal pancreas (5, 20, 23, 41,
47). In addition, inhibiting NF-
B activation should alleviate
inflammation not only in the pancreas but also in distant organs
affected during pancreatitis (25, 38).
Currently (12, 46), there are no specific small-molecule
inhibitors of NF-B, the type of inhibitor that is most suitable for
use in acute pancreatitis. Most of the studies that attempted to
ameliorate the pancreatitis inflammatory response by inhibiting NF-
B
activation showed an attenuation of the disease (8, 11, 20, 23,
38, 42), with one notable exception (41). The contradictory results may have been caused by several factors, including differences in the inhibitors used and methods of their administration. Furthermore, an NF-
B inhibitor that works in vitro
may not be effective in vivo, as we found for the proteasomal inhibitor
MG-132 (this study, see DISCUSSION; also see Refs.
5 and 47).
Recent studies indicate that the bioflavonoid curcumin inhibits NF-B
activation in a number of cell types (6, 7, 24, 26, 32,
39). Curcumin is the pigment in turmeric (Curcuma longa) that gives the yellow color to curry dishes
(2). It has long been known for its anti-inflammatory and
cancer chemopreventive activities and is used both in vitro and in vivo
(2, 6, 24, 26, 32, 39). The molecular mechanism of NF-
B
inhibition by curcumin involves blockade of IKK activation and
subsequent I
B degradation (26, 32, 39). Of importance,
curcumin is not toxic and can be administered in large quantities
(2, 24).
Another advantage of curcumin is that it also inhibits activator
protein (AP)-1, a transcription factor that often acts in concert with
NF-B to regulate the expression of chemokines (e.g., IL-8) and other
inflammatory molecules (3, 28, 43). We showed that AP-1 is
activated in taurocholate-induced pancreatitis and in CCK-stimulated
pancreatic acinar cells (21, 42). AP-1 activation in the
cerulein and EtOH + CCK models of pancreatitis has not been
studied. The mechanism of AP-1 activation involves phosphorylation of
c-Jun, a component of the AP-1 complex, by the JNK kinase (28,
43). Curcumin inhibits JNK (7).
There is a growing interest in natural compounds for the treatment of various inflammatory disorders. In this study, we tested whether curcumin is effective for in vivo application in ethanol and nonethanol pancreatitis. We found that curcumin inhibited the inflammatory response and significantly ameliorated pancreatitis in both models.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cerulein-induced pancreatitis.
Care and handling of the animals were approved by the Animal Research
Committees of the VA Greater Los Angeles Healthcare System and
University of Southern California (USC) in accordance with the National
Institutes of Health guidelines. Cerulein (5 µg · kg1 · h
1) or vehicle
control was administered to male Sprague-Dawley rats (275-350 g;
Harlan, Madison, WI) by continuous 6-h intravenous infusion as
previously described (17, 19, 20, 47). Animals were
euthanized by CO2-induced asphyxiation, and the blood and pancreas were harvested for measurements.
Ethanol and CCK-induced pancreatitis.
Sprague-Dawley rats (400-500 g) received intragastric infusion of
ethanol-containing or control diet for 6 wk followed by a 6-h infusion
of a low dose (3,000 pmol · kg1 · h
1) of CCK-8.
Animals were euthanized by CO2-induced asphyxiation, and
the blood and pancreas were harvested for measurements. This EtOH + CCK model of pancreatitis has been described in detail (34). Because we previously characterized the effect of
ethanol feeding alone in this model, the present study focused on the effect of curcumin on measures of pancreatitis induced by the combined
EtOH + CCK treatment.
Preparation of dispersed pancreatic acini. Acini were prepared from rat pancreas using a collagenase digestion method (33) and then incubated at 37°C in 199 medium as previously described (5, 15, 17, 20, 47).
Curcumin administration.
Curcumin was dissolved in DMSO and administered to the animal by
intravenous infusion together with cerulein or CCK-8 at a dose of 35 mg · kg1 · h
1. Thus, during
the 6-h treatment, rats received a total of 200 mg/kg (~0.5 mmol/kg)
curcumin. In separate experiments, we tested that
administration of the vehicle or curcumin alone to control animals did
not have a significant effect on any parameter measured. In experiments
on isolated acini, curcumin was added into the cell suspension to a
final concentration of 100 µM.
Measures of pancreatitis. For histological evaluation, pancreatic tissue was fixed in 10% buffered formaldehyde, then embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined by light microscopy as described previously (17-19, 34). Serum amylase and lipase were measured by using a Hitachi 707 analyzer (Antech Diagnostics, Irvine, CA). Active trypsin was measured in pancreatic tissue homogenates as described previously (19, 20, 42) by using a specific substrate, Boc-Gln-Ala-Arg-AMC (29). Cleavage of this substrate by trypsin releases 7-amino-4-methylcoumarin (AMC), which emits fluorescence at 440 nm with excitation at 380 nm. Trypsin activity in each sample was determined using a standard curve for purified trypsin.
For measurement of neutrophil infiltration, pancreatic tissue was immunostained for neutrophils as described previously (17, 19, 34, 37, 42). In brief, 8-µm-thick serial cryostat sections were cut and mounted on glass slides. The tissue sections were then stained with FITC-labeled rabbit anti-rat polymorphonuclear leukocyte antibody (Accurate, Westbury, NY). The number of infiltrating neutrophils was obtained by counting the neutrophils at 40× magnification in an average of 50 fields covering at least 1,000 acinar cells. For each animal, neutrophil numbers were expressed as a percentage of acinar cells.Measurement of pancreatic secretion in vivo. Pancreatic secretion in rats was measured as previously described (19). Briefly, biliopancreatic secretion was allowed to drain freely for 30 min before initiation of the experiment. Then, secretion was collected in tubes in 10-min fractions to determine the basal amylase output, after which 0.25 µg/kg cerulein was given as an intravenous bolus and collection continued for 40 min in a similar manner. Collection volumes were measured by weight, and the amylase output was calculated by determining amylase concentration in the collected fractions. The mean amylase output obtained from two consecutive 10-min fractions after the stabilization period was taken as the basal secretion and considered as 100%. The results were expressed as percent increase over basal amylase output 40 min after cerulein or vehicle infusion.
Amylase secretion by isolated acinar cells. Secretion was measured spectrophotometrically using Phadebas amylase kit (Pharmacia Diagnostics) as previously described (15). Values for amylase secretion were expressed as ratios between the amount of amylase released into the extracellular medium and the total cellular amylase determined by permeabilizing cells with 0.1% SDS in 10 mM phosphate buffer (pH 7.8).
Measurement of LDH release. Acinar cell necrosis was determined by the release of lactate dehydrogenase (LDH) into the incubation medium, as described previously (15-17). LDH activity in the extracellular medium was measured using LDH detection ELISA kit (Roche Diagnostics, Indianapolis, IN).
Preparation of nuclear and cytosolic extracts.
Nuclear protein extracts were prepared as described previously
(5, 17, 20, 34, 42, 47). Briefly, samples of pancreatic tissue or isolated acini were rinsed in ice-cold PBS and homogenized in
the hypotonic buffer A (20) supplemented with 1 mM PMSF, 1 mM DTT, and 5 µg/ml each of protease inhibitors pepstatin, leupeptin, chymostatin, antipain, and aprotinin. The nonionic detergent Igepal CA-630 was added to a final concentration of 0.3-0.4% (vol/vol) after incubation on ice for 20-25 min, followed by an additional incubation on ice for 1-2 min. The crude nuclear pellet was
collected by microcentrifugation for 30 s. The supernatant
(cytosolic protein) was removed, and the nuclear pellet was resuspended
in the high-salt buffer C (20) containing 1 mM DTT, 1 mM
PMSF, and the protease inhibitor cocktail described above. After
nuclear membranes were rotated at 4°C for up to 1 h, they were
pelleted by microcentrifugation for 10 min, and the clear supernatant
(nuclear extract) was aliquoted and stored at 80°C. Protein
concentration in the nuclear extract was determined by the Bio-Rad
protein assay (Bio-Rad Laboratories, Hercules, CA).
EMSA.
EMSA was performed as described previously (5, 17, 20, 34, 42,
47). Aliquots of nuclear extracts with equal amounts of protein
(2-10 mg) were mixed in 20-µl reactions with a buffer containing
(in mM) 10 HEPES (pH 7.8), 50 KCl, 0.1 EDTA, and 1 DTT with 10%
(vol/vol) glycerol and 3 µg poly(dI-dC). Binding reactions were
started by the addition of ~60,000 counts/min of 32P-labeled DNA probe and were allowed to proceed for
20-25 min at room temperature. The oligonucleotide probe
for NF-B, 5'-GCAGAGGGGACTTTCCGAGA, containing a
consensus
B binding site (underlined), or the AP-1 oligo probe,
5'-GGCTTGATGAGTCAGCCGGAA, containing phorbol
ester-responsive element (TRE; underlined), was annealed with the
complementary strands and end-labeled using Klenow DNA polymerase I
(Stratagene, La Jolla, CA). Samples were electrophoresed at room
temperature in 0.5 × TBE buffer (1 × TBE: 89 mM Tris base,
89 mM boric acid, 2 mM EDTA) on a nondenaturing 4.5% polyacrylamide
gel at 200 V. Gels were dried and directly analyzed in the
PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Measurement of IB
and I
B
levels.
I
B
and I
B
levels were measured in cytosolic protein
extracts from pancreatic tissue as described previously
(20) by Western blot analysis using polyclonal antibodies
sc-371 and sc-945, respectively, from Santa Cruz Biotechnology (Santa
Cruz, CA).
Cytokine mRNA detection by RT-PCR. The procedures were as we described previously (5, 17, 20, 34, 42, 47). Briefly, total RNA was obtained from pancreatic tissue with TRI reagent (Molecular Research Center, Cincinnati, OH), and its quality was verified by ethidium bromide staining of rRNA bands on a denaturing agarose gel. RNA was reverse transcribed with the SuperScript II preamplification kit (GIBCO-BRL, Rockville, MD) and subjected to PCR with rat gene-specific, intron-spanning primers described previously (5, 17, 20, 34, 42, 47). Target sequences were amplified at 56°C using the same amount of cDNA for all primer sets. The RT-PCR products were all of expected size, and their identity was confirmed by direct sequencing. Negative controls were performed by omitting the RT step or cDNA template from the PCR amplification. The cycle number was adjusted between 22 (for the housekeeping ARP gene) and 36 cycles (for iNOS) to yield visible products within the linear amplification range. Resulting RT-PCR products were run on agarose gel and visualized by staining with ethidium bromide.
Measurement of caspase-3 activation. Caspase activity was measured as previously described (15). Briefly, samples of pancreatic tissue were rinsed with ice-cold PBS and homogenized in a lysis buffer containing 150 mM NaCl, 50 mM Tris · HCl (pH 7.5), 0.5% Igepal CA-630, and 0.5 mM EDTA. Lysates were centrifuged for 10 min at 16,000 g, and the supernatants were collected. Caspase activity was determined at 37°C in a buffer containing 25 mM HEPES (pH 7.5), 10% sucrose, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 10 mM DTT, and 20 µM Ac-Asp-Glu-Val-Asp-AMC (Ac-DEVD-AMC), a specific fluorogenic substrate for measurement of caspase-3-like activity. Cleavage of this substrate releases AMC that emits a fluorescent signal with excitation at 380 nm and emission at 440 nm. Fluorescence was calibrated by using a standard curve for AMC. The data were expressed as moles of AMC per minute per milligram of protein.
Statistical analysis of the data. Data analysis was done by using two-tailed Student's t-test. A P value <0.05 was considered statistically significant.
Materials.
Cerulein was obtained from American Peptide (Sunnyvale, CA); CCK-8 was
from Peninsula Laboratories (Belmont, CA); Ac-DEVD-AMC was from AnaSpec
(San Jose, CA); Boc-Gln-Ala-Arg-AMC was from Bachem (Torrance, CA);
[-32P]dCTP (3,000 Ci/mmol) was from ICN
Pharmaceuticals (Costa Mesa, CA); poly[d(I-C)] was from Boehringer
Mannheim (Indianapolis, IN); and curcumin and all other chemicals were
from Sigma Chemical (St. Louis, MO).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effects of curcumin on measures of pancreatitis.
The results in Figs. 1-4 show the
effects of curcumin on measures of pancreatitis in the two
experimental models. Both in ethanol and nonethanol pancreatitis,
curcumin significantly inhibited the increases in serum amylase and
lipase (Fig. 1). Note that, as described previously (34),
the increase in serum amylase in pancreatitic animals is less in the
EtOH + CCK model than in the cerulein model, whereas the reverse
is true for serum lipase levels.
|
|
|
|
Effects of curcumin on transcription factor activation and
inflammatory molecules' expression.
Previously, our laboratory (20) demonstrated and
characterized in detail NF-B activation in rat cerulein
pancreatitis. The results in Fig.
5 show the effects of curcumin on NF-
B
DNA binding activity in the pancreas in this model. Virtually no
NF-
B activity was detected in control (vehicle-infused) rats treated either without or with curcumin (Fig. 5A). Cerulein caused
severalfold NF-
B activation, which was greatly attenuated by the
curcumin treatment (Fig. 5, A and C).
|
|
|
|
Effects of curcumin on caspase-3 activity in pancreas.
In addition to its role in inflammation, NF-B activation is
generally known to inhibit apoptosis (1, 45, 46).
NF-
B inhibits apoptosis by inhibiting activation of
caspases, the key mediators of apoptosis (1, 10,
44). We and others (18, 27) showed that the death
of parenchymal cells in pancreatitis occurs via both necrosis and
apoptosis and, furthermore, that the severity of pancreatitis
was less in models with increased apoptosis.
|
Effects of curcumin on CCK-induced NF-B and AP-1 activation in
isolated pancreatic acinar cells.
To confirm that the effects of curcumin occur in the pancreatic acinar
cell, we measured the effects of curcumin on the activation of NF-
B
and AP-1 in vitro in CCK-stimulated pancreatic acini. Both NF-
B and
AP-1 were activated by 0.1 µM CCK-8 in the acinar cells, and this
activation was abolished by curcumin (Fig.
10). Of note, curcumin inhibited not
only the CCK-induced activation of these transcription factors but also
their "basal" binding activity in isolated acinar cells, which was
more pronounced for AP-1 (Fig. 10). Our laboratory (5)
previously reported that such transcription factor activation occurs in
pancreatic acinar cells in the process of their isolation from tissue.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
On the basis of the idea that the activation of proinflammatory
transcription factors, such as NF-B, in pancreas plays a key role in
the inflammatory response of pancreatitis, in this study we sought to
determine whether an inhibitor of this activation could ameliorate
pancreatitis across different experimental models, including
alcohol-mediated pancreatitis. We found that curcumin improved the
severity of pancreatitis in both ethanol and nonethanol models. The
results suggest that the beneficial effects of curcumin are due to its
ability to inhibit activation of NF-
B and AP-1 and the resulting
inflammatory response. In particular, the effects of curcumin on
NF-
B and AP-1 led to inhibition of pancreatic expression of
cytokines/chemokines IL-6, TNF-
, and KC and the proinflammatory
enzyme iNOS in both models.
The molecular mechanism of curcumin action has not been completely
elucidated (2, 6, 7, 24, 26, 30, 32, 35, 39). However,
inhibition of NF-B and AP-1 is the only common upstream mechanism by
which curcumin inhibits gene expression of the above-mentioned and
other inflammatory molecules such as ICAM-1 (26) and
cyclooxygenase-2 (24). Of note, curcumin does not inhibit
other transcription factors, e.g., Sp1 (39). The evidence
on cell lines suggests that curcumin inhibits NF-
B and AP-1
activation upstream of IKK and JNK kinases, probably at the level of
MEKK-1 kinase (7, 26, 32, 39). Likewise, our finding that
curcumin prevented I
B degradation in models of pancreatitis suggests
that in the pancreas it also acts at one or more steps upstream of
I
B degradation.
As demonstrated by recent studies in experimental models, attenuation
of the inflammatory response in pancreatitis either by removing
inflammatory cells, preventing neutrophil access to the pancreatic
parenchyma, or blocking cytokine action leads to an improvement in the
severity of parenchymal injury in pancreatitis (4, 9, 13, 14, 17,
19, 20, 25, 31, 37, 47). Thus the effects of curcumin to inhibit
NF-B and AP-1 activation resulted in the improvement in the measures
of parenchymal injury (e.g., histological changes, increases in serum
amylase and lipase, and acinar cell vacuolization) as well as the
inflammatory response. Of note, in both models, curcumin significantly
attenuated the pathological intrapancreatic trypsin activation, which,
as was recently shown (19), could result from the decrease
in neutrophil infiltration.
The effect of curcumin occurs, at least in part, in the acinar cell, as
demonstrated by the experiments on pancreatic acini in vitro. Previous
work from our and other groups (5, 14, 17, 20-22, 31, 42,
47) indicates that, at the initiation of pancreatitis, the
acinar cell is a source of increased inflammatory molecule expression
through the activation of NF-B and AP-1.
Of importance, the amelioration of both cerulein and EtOH + CCK models of pancreatitis by curcumin suggests that the same mechanisms mediate the development of the disease in both models. Thus the inflammatory response in the ethanol model involves similar mechanisms and plays a similarly critical role to that in nonethanol pancreatitis.
There are several advantages of using curcumin to ameliorate pancreatitis. Curcumin is not toxic and can be given in large quantities (2, 24). Although most of the studies with curcumin were on cell lines, it has been applied in vivo (2, 6, 24). Furthermore, recent data (24, 32) indicate that the effects of curcumin administration are produced by curcumin itself and not by hydrogenated products of its metabolism.
Curcumin is one of the few pharmacological NF-B inhibitors available
for in vivo applications (12, 46). The two other NF-
B
inhibitors we tried appeared less promising. Although the proteasomal
inhibitor MG-132 greatly inhibited NF-
B activation in vitro in
isolated acinar cells (5, 47), it worsened pancreatitis in
the two models under study and even increased NF-
B activation in the
pancreas (data not shown). This may be due to the fact that proteasomes
have diverse functions in the organism.
N-acetylcysteine improved cerulein pancreatitis in rats and mice (8, 20) and also pancreatitis induced in rats by taurocholate infusion (42). However, we found that the same N-acetylcysteine treatment did not ameliorate the EtOH + CCK pancreatitis (data not shown). Because N-acetylcysteine is an antioxidant, this finding indicates that the amelioration of pancreatitis with curcumin that we observed cannot be explained solely by curcumin's antioxidant properties.
Another reason why curcumin was more effective could be that it
inhibited both NF-B and AP-1, whereas N-acetylcysteine
inhibited NF-
B but not AP-1 activation in pancreas (data not shown).
AP-1 activation may be necessary for upregulation of some cytokines (e.g., IL-8 and related CXC chemokines; Refs. 3,
43) that are believed to play an important role in the
inflammatory response of both human and experimental pancreatitis
(4, 20, 31). The results of the present study demonstrate
a marked activation of pancreatic AP-1 in the cerulein and EtOH + CCK pancreatitis. On the basis of DNA binding activity measurements,
the extent of AP-1 activation in both models was even greater than that
of NF-
B. We also reported AP-1 activation in taurocholate-induced pancreatitis (42).
The observed caspase-3 stimulation by curcumin suggests that curcumin
treatment may lead to an increase in apoptosis. This may be a
result of NF-B inhibition by curcumin, because in many situations
NF-
B plays an antiapoptotic role (1, 46). Caspase-3 is a key "executionary" caspase, activation of which leads to apoptosis (44). On the basis of the results from
our group and others, it has been speculated that an increase in
apoptosis alleviates the severity of experimental pancreatitis
(18, 27). We recently reported (15) that
CCK-8 stimulates caspase-3 activation with concomitant
apoptosis in isolated pancreatic acinar cells.
There is an increasing interest in the use of natural products to modulate inflammatory disorders. Our data show that curcumin ameliorates pancreatitis in two experimental models by inhibiting the inflammatory response. The results suggest a potential therapeutic role for curcumin, which is currently in clinical trials for cancer prevention, for the treatment of pancreatitis.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank F. Wong and A. Baycher for help with measurements of pancreatic neutrophil infiltration and Y. Jung for help with the preparation of this manuscript.
![]() |
FOOTNOTES |
---|
The intragastric ethanol feeding in the EtOH + CCK model was provided by the Animal Core of the Research Center for Alcoholic Liver and Pancreatic Diseases (H. Tsukamoto, Director).
This study was supported by the USC-Univ. of California Los Angeles (UCLA) Research Center for Alcoholic Liver and Pancreatic Diseases (Grant P50-A-11999 from the National Institutes of Health), National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-59508 (to S. J. Pandol), and the UCLA-CURE Digestive Diseases Research Center PFS grant (to I. Gukovsky).
Address for reprint requests and other correspondence: I. Gukovsky, UCLA/VA Greater Los Angeles Healthcare System, West Los Angeles Center, Bldg. 258, Rm. 340, 11301 Wilshire Blvd., Los Angeles, CA 90073 (E-mail: igukovsk{at}ucla.edu).
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.
10.1152/ajpgi.00138.2002
Received 9 April 2002; accepted in final form 1 October 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aggarwal, BB.
Apoptosis and nuclear factor-B: a tale of association and dissociation.
Biochem Pharmacol
60:
1033-1039,
2000[ISI][Medline].
2.
Ammon, HP,
and
Wahl MA.
Pharmacology of Curcuma longa.
Planta Med
57:
1-7,
1991[ISI][Medline].
3.
Ben Baruch, A,
Michiel DF,
and
Oppenheim JJ.
Signals and receptors involved in recruitment of inflammatory cells.
J Biol Chem
270:
11703-11706,
1995
4.
Bhatia, M,
Brady M,
Shokuhi S,
Christmas S,
Neoptolemos JP,
and
Slavin J.
Inflammatory mediators in acute pancreatitis.
J Pathol
190:
117-125,
2000[ISI][Medline].
5.
Blinman, TA,
Gukovsky I,
Mouria M,
Zaninovic V,
Livingston E,
Pandol SJ,
and
Gukovskaya AS.
Activation of pancreatic acinar cells on isolation from tissue: cytokine upregulation via p38 MAP kinase.
Am J Physiol Cell Physiol
279:
C1993-C2003,
2000
6.
Chan, MM,
Huang HI,
Fenton MR,
and
Fong D.
In vivo inhibition of nitric oxide synthase gene expression by curcumin, a cancer preventive natural product with anti-inflammatory properties.
Biochem Pharmacol
55:
1955-1962,
1998[ISI][Medline].
7.
Chen, YR,
and
Tan TH.
Inhibition of the c-Jun N-terminal kinase (JNK) signaling pathway by curcumin.
Oncogene
17:
173-178,
1998[ISI][Medline].
8.
Demols, A,
Van Laethem JL,
Quertinmont E,
Legros F,
Louis H,
Le Moine O,
and
Deviere J.
N-acetylcysteine decreases severity of acute pancreatitis in mice.
Pancreas
20:
161-169,
2000[ISI][Medline].
9.
Denham, W,
and
Norman J.
The potential role of therapeutic cytokine manipulation in acute pancreatitis.
Surg Clin North Am
79:
767-781,
1999[ISI][Medline].
10.
Deveraux, QL,
and
Reed JC.
IAP family proteins-suppressors of apoptosis.
Genes Dev
13:
239-252,
1999
11.
Dunn, JA,
Li C,
Ha T,
Kao RL,
and
Browder W.
Therapeutic modification of nuclear factor-B binding activity and tumor necrosis factor-
gene expression during acute biliary pancreatitis.
Am J Surg
63:
1036-1044,
1997.
12.
Epinat, JC,
and
Gilmore TD.
Diverse agents act at multiple levels to inhibit the Rel/NF-B signal transduction pathway.
Oncogene
18:
6896-6909,
1999[ISI][Medline].
13.
Frossard, JL,
Saluja A,
Bhagat L,
Lee HS,
Bhatia M,
Hofbauer B,
and
Steer ML.
The role of intercellular adhesion molecule 1 and neutrophils in acute pancreatitis and pancreatitis-associated lung injury.
Gastroenterology
116:
694-701,
1999[ISI][Medline].
14.
Grady, T,
Liang P,
Ernst SA,
and
Logsdon CD.
Chemokine gene expression in rat pancreatic acinar cells is an early event associated with acute pancreatitis.
Gastroenterology
113:
1966-1975,
1997[ISI][Medline].
15.
Gukovskaya, AS,
Gukovsky I,
Jung Y,
Mouria M,
and
Pandol SJ.
Cholecystokinin induces caspase activation and mitochondrial dysfunction in pancreatic acinar cells. Roles in cell injury processes of pancreatitis.
J Biol Chem
277:
22595-22604,
2002
16.
Gukovskaya, AS,
Gukovsky I,
Mouria M,
and
Pandol SJ.
Cerulein stimulates apoptosis in pancreatic acinar cells (Abstract).
Gastroenterology
116:
A1128,
1999[ISI].
17.
Gukovskaya, AS,
Gukovsky I,
Zaninovic V,
Song M,
Sandoval D,
Gukovsky S,
and
Pandol SJ.
Pancreatic acinar cells produce, release, and respond to tumor necrosis factor-. Role in regulating cell death and pancreatitis.
J Clin Invest
100:
1853-1862,
1997
18.
Gukovskaya, AS,
Perkins P,
Zaninovic V,
Sandoval D,
Rutherford R,
Fitzsimmons T,
Pandol SJ,
and
Poucell-Hatton S.
Mechanisms of cell death after pancreatic duct obstruction in the opossum and the rat.
Gastroenterology
110:
875-884,
1996[ISI][Medline].
19.
Gukovskaya, AS,
Vaquero E,
Zaninovic V,
Gorelick FS,
Lusis AJ,
Brennan ML,
Holland S,
and
Pandol SJ.
Neutrophils and NADPH oxidase mediate intrapancreatic trypsin activation in murine experimental acute pancreatitis.
Gastroenterology
122:
974-984,
2002[ISI][Medline].
20.
Gukovsky, I,
Gukovskaya AS,
Blinman TA,
Zaninovic V,
and
Pandol SJ.
Early NF-B activation is associated with hormone-induced pancreatitis.
Am J Physiol Gastrointest Liver Physiol
275:
G1402-G1414,
1998
21.
Gukovsky, I,
Gukovskaya AS,
and
Pandol SJ.
Cerulein activates NF-kB and AP-1 in pancreatic acinar cells (Abstract).
Gastroenterology
114:
A465,
1998.
22.
Han, B,
and
Logsdon CD.
Cholecystokinin induction of mob-1 chemokine expression in pancreatic acinar cells requires NF-B activation.
Am J Physiol Cell Physiol
277:
C74-C82,
1999
23.
Hietaranta, AJ,
Singh VP,
Bhagat L,
van Acker GJ,
Song AM,
Mykoniatis A,
Steer ML,
and
Saluja AK.
Water immersion stress prevents caerulein-induced pancreatic acinar cell NF-kappaB activation by attenuating caerulein-induced intracellular Ca2+ changes.
J Biol Chem
276:
18742-18747,
2001
24.
Ireson, C,
Orr S,
Jones DJ,
Verschoyle R,
Lim CK,
Luo JL,
Howells L,
Plummer S,
Jukes R,
Williams M,
Steward WP,
and
Gescher A.
Characterization of metabolites of the chemopreventive agent curcumin in human and rat hepatocytes and in the rat in vivo, and evaluation of their ability to inhibit phorbol ester-induced prostaglandin E2 production.
Cancer Res
61:
1058-1064,
2001
25.
Jaffray, C,
Yang J,
Carter G,
Mendez C,
and
Norman J.
Pancreatic elastase activates pulmonary nuclear factor kappa B and inhibitory kappa B, mimicking pancreatitis-associated adult respiratory distress syndrome.
Surgery
128:
225-231,
2000[ISI][Medline].
26.
Jobin, C,
Bradham CA,
Russo MP,
Juma B,
Narula AS,
Brenner DA,
and
Sartor RB.
Curcumin blocks cytokine-mediated NF-B activation and proinflammatory gene expression by inhibiting inhibitory factor I-
B kinase activity.
J Immunol
163:
3474-3483,
1999
27.
Kaiser, AM,
Saluja AK,
Sengupta A,
Saluja M,
and
Steer ML.
Relationship between severity, necrosis, and apoptosis in five models of experimental acute pancreatitis.
Am J Physiol Cell Physiol
269:
C1295-C1304,
1995
28.
Karin, M,
Liu Z,
and
Zandi E.
AP-1 function and regulation.
Curr Opin Cell Biol
9:
240-246,
1997[ISI][Medline].
29.
Kawabata, S,
Miura T,
Morita T,
Kato H,
Fujikawa K,
Iwanaga S,
Takada T,
Kimura T,
and
Sakakibara S.
Highly sensitive peptide-4-methylcoumaryl-7-amide substrates for blood-clotting proteases and trypsin.
Eur J Biochem
172:
17-25,
1988[Abstract].
30.
Logan-Smith, MJ,
East JM,
and
Lee AG.
Evidence for a global inhibitor-induced conformation change on the Ca(2+)-ATPase of sarcoplasmic reticulum from paired inhibitor studies.
Biochemistry
41:
2869-2875,
2002[ISI][Medline].
31.
Norman, J.
The role of cytokines in the pathogenesis of acute pancreatitis.
Am J Surg
175:
76-83,
1998[ISI][Medline].
32.
Pan, MH,
Lin-Shiau SY,
and
Lin JK.
Comparative studies on the suppression of nitric oxide synthase by curcumin and its hydrogenated metabolites through down-regulation of IB kinase and NF
B activation in macrophages.
Biochem Pharmacol
60:
1665-1676,
2000[ISI][Medline].
33.
Pandol, SJ,
Jensen RT,
and
Gardner JD.
Mechanism of [Tyr4]bombesin-induced desensitization in dispersed acini from guinea pig pancreas.
J Biol Chem
257:
12024-12029,
1982
34.
Pandol, SJ,
Periskic S,
Gukovsky I,
Zaninovic V,
Jung Y,
Zong Y,
Solomon TE,
Gukovskaya AS,
and
Tsukamoto H.
Ethanol diet increases the sensitivity of rats to pancreatitis induced by cholecystokinin octapeptide.
Gastroenterology
117:
706-716,
1999[ISI][Medline].
35.
Pollmann, C,
Huang X,
Mall J,
Bech-Otschir D,
Naumann M,
and
Dubiel W.
The constitutive photomorphogenesis 9 signalosome directs vascular endothelial growth factor production in tumor cells.
Cancer Res
61:
8416-8421,
2001
36.
Rothwarf DM and Karin M. (26 Oct 1999). The NF-B
activation pathway: a paradigm in information transfer from membrane
to nucleus [Online]. Science's STKE.
http://stke.sciencemag.org/cgi/content/full/sigtrans;1999/5/re1.
37.
Sandoval, D,
Gukovskaya AS,
Reavey P,
Gukovsky S,
Sisk A,
Braquet P,
Pandol SJ,
and
Poucell-Hatton S.
The role of neutrophils and platelet-activating factor in mediating experimental pancreatitis.
Gastroenterology
111:
1081-1091,
1996[ISI][Medline].
38.
Satoh, A,
Shimosegawa T,
Fujita M,
Kimura K,
Masamune A,
Koizumi M,
and
Toyota T.
Inhibition of nuclear factor-B activation improves the survival of rats with taurocholate pancreatitis.
Gut
44:
253-258,
1999
39.
Singh, S,
and
Aggarwal BB.
Activation of transcription factor NF-B is suppressed by curcumin (diferuloylmethane).
J Biol Chem
270:
24995-25000,
1995
40.
Steinberg, W,
and
Tenner S.
Acute pancreatitis.
N Engl J Med
330:
1198-1210,
1994
41.
Steinle, AU,
Weidenbach H,
Wagner M,
Adler G,
and
Schmid RM.
NF-B/Rel activation in cerulein pancreatitis.
Gastroenterology
116:
420-430,
1999[ISI][Medline].
42.
Vaquero, E,
Gukovsky I,
Zaninovic V,
Gukovskaya AS,
and
Pandol SJ.
Localized pancreatic NF-B activation and inflammatory response in taurocholate-induced pancreatitis.
Am J Physiol Gastrointest Liver Physiol
280:
G1197-G1208,
2001
43.
Wisdom, R.
AP-1: one switch for many signals.
Exp Cell Res
253:
180-185,
1999[ISI][Medline].
44.
Wolf, BB,
and
Green DR.
Suicidal tendencies: apoptotic cell death by caspase family proteinases.
J Biol Chem
274:
20049-20052,
1999
45.
Wulczyn, FG,
Krappmann D,
and
Scheidereit C.
The NF-B/Rel and I
B gene families: mediators of immune response and inflammation.
J Mol Med
74:
749-769,
1996[ISI][Medline].
46.
Yamamoto, Y,
and
Gaynor RB.
Therapeutic potential of inhibition of the NF-B pathway in the treatment of inflammation and cancer.
J Clin Invest
107:
135-142,
2001
47.
Zaninovic, V,
Gukovskaya AS,
Gukovsky I,
Mouria M,
and
Pandol SJ.
Cerulein upregulates ICAM-1 in pancreatic acinar cells, which mediates neutrophil adhesion to these cells.
Am J Physiol Gastrointest Liver Physiol
279:
G666-G676,
2000