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Introduction
Curcumin (Cur), the prominent yellow pigment in turmeric, is a widely used spice and food coloring agent with
anti-inflammatory and anti-cancer
properties[1]. Previoulsy published studies have reported that cur inhibits IL-12 production
from macrophages, and results in a reduced ability to induce
IFN-g and an increased ability to induce IL-4, which suggests
that cur may inhibit Th1 and enhance Th2 cytokines synthesis and may be therapeutic for Th1-mediated immune
disease[2,3]. However, a published study of atopic
asthmatics indicates that cur inhibited the expression of IL-4 and
IL-5, both of which are Th2 cytokines. From this study, it appears that cur has the ability to also inhibit
Th2[4].
Inflammatory bowel disease (IBD) comprises the 2 conditions, Crohn¡¯s disease (CD) and ulcerative colitis (UC) which are
characterized by chronic relapsing and remitting an inflammatory condition in the
colon[5]. Recently, various animal models
for IBD have been developed, and it has been revealed that the dysfunction of T cells plays an important role in the
pathogenesis of this disease[6]. Trinitrobenzene sulphonic acid (TNBS)-induced colitis model mimics human CD, which has
a high level of T help 1 (Th1) cytokines (IL-12,
IFN-g, TNF-a, IL-1), but low levels of Th2 cytokines
(IL-4, IL-5, IL-10)[7]. From these cytokines, it has been reported
that IL-12 plays a pivotal role in the pathogenesis of TNBS-induced
colitis[8].
Frontline drugs, such as dexamethasone (Dex, one of the most commonly used drugs in the control of acute inflammation),
used in the treatment of IBD, have varying efficacy from patient to patient. They are expensive and inevitably without side
effects. New agents, including traditional herb medicines such as Cur (used in most chronic inflammation), have been tried
in the treatment of IBD. Applying cur on colitis models has been
reported[9_11], but different conclusions have been reached
on the aspect of the expression of cytokines. Sugimoto
et al[10] reported that IL-4 could not be detected in all the experimental
animals in their study, but Ukil et
al[11] reported that IL-4 increased in the Cur-treated groups. Therefore, we were prompted
to investigate the expression of Th1 and Th2 cytokines in the Cur-treated colitis model. We also wanted to see if Cur and Dex,
two agents used in different phases of inflammation, have additive and/or synergistic effects on rats that receive the 2 agents
simultaneously.
Materials and methods
Animals and experimental protocols Specific pathogen-free Sprague-Dawley rats
(200-220 g) were obtained from the Laboratory Animal Central of Wuhan University (Wuhan, China) and kept in a room with a constant temperature of 23±2
oC and humidity around 50%_70% with a 12-h light-dark cycle. The rats were allowed to adapt to our laboratory environment for
1 week before the onset of the experiment, during which they had free access to standard rodent chow and tap water. Seventy
five rats were distributed to 5 groups randomly: control (Con) group (receiving ethanol only without therapy), TNBS group
(receiving TNBS without therapy), Cur group (30
mg·kg-1·d-1 Cur, ip), Dex group (2
mg·kg-1·d-1 Dex, ip), Cur+Dex group (30
mg·kg-1·d-1 Cur, ip+2
mg·kg-1·d-1 Dex, ip). Cur (=95.0%) and Dex were purchased from Sigma (St Lonis, MO, USA). The rats in
the Con and TNBS groups received the same volume of 5% ethanol (vehicle of Cur) everyday until the end of the experiment.
All the rats were checked daily for behavior and body weight.
Induction of colitis Colitis was induced by TNBS using a modification of the method described by Morris
et al[12]. Briefly, all the rats were were made to fast on d 1; on d 2, colitis was induced. Each rat was anesthetized with ether, and 100 mg/kg of
TNBS (Sigma, St Lonis, MO, USA; 10% TNBS in 50% ethanol; total volume 1.0 mL) was then instilled via a rubber catheter
inserted 8 cm into the colon via the anus. The rubber catheter was modified with numerous holes in the last 4 cm of its length.
The instillation procedure required only a few seconds to be completed, and the rats were maintained in a vertical position for
3 min to prevent solution leakage. The Con group rats received 50% ethanol of the same volume using the same technique.
On d 15, all the rats were killed. The experiments were approved by the institution¡¯s animal care and use committee.
Macroscopic and histological assessment of the severity of colitis
After rapid removal of the colon, the specimen was
flushed with ice cold PBS, cut open, and photographed with a Pentax camera (Pentax Co, Tokyo, Japan) before being
snap-frozen in liquid nitrogen and stored at -80
oC. Scores were assessed using the following damage scoring
system[12]: (0) no damage; (1) localized hyperaemia, but no ulcers; (2) linear ulcers with no significant inflammation; (3) linear ulcers with
inflammation at 1 site; (4) 2 or more sites of ulceration and/or inflammation; and (5) 2 or more major sites of inflammation and
ulceration or 1 major site of inflammation and ulceration extending more than 1 cm along the length of the colon.
For histological analyses, tissues were fixed in 10% paraformaldehyde in phosphate-buffered saline formalin and
paraffin-embedded tissue sections were stained with hematoxylin and eosin (H-E) using the standard techniques.
Assessment of myeloperoxidase activity Myelopero-xidase (MPO) activity was assessed as a marker of neutrophil
infiltration. The tissue was thawed, weighed and homogenized; the homogenate was then centrifuged and the pellet was
again homogenized in PBS, containing 0.5% hexadecyl-trimethylammonium bromide (HETAB) and 10 mmol/L
ethylenediamine tetraacetic acid (EDTA). The homogenate was subjected to 3 cycles of freezing/thawing and brief sonication. A sample
of homogenate was added to the reaction volume. The mixture was incubated at 37
oC for 5 min and the reaction was started
by the addition of H2O2. The complete reaction mixture was incubated for exactly 3 min at 37
oC and terminated by the sequential addition of catalase and sodium acetate. The changes in absorbance at 655 nm were measured with a
spectrophotometer. The results were quantified as U/mg protein.
Real-time quantitative reverse-transcription polymerase chain reaction
Total RNA were isolated using TRIzol Reagent (Invitrogen). Two micrograms of total RNA were reverse-transcribed into complementary deoxyribonucleic acids (cDNA) by
Superscript II reverse transcriptase (Invitrogen) according to the manufacturer¡¯s instructions. Real-time PCR was performed
using the SYBR green PCR kit (Qiagen), according to the manufacturer¡¯s instructions. Annealing temperature was optimized
to create a 1-peak melting curve and the productions of PCR were checked by agarose gel electrophoresis for a single band
of the expected size. The abundance of each mRNA was detected normalized to that of GAPDH mRNA. The sequences of all
the primers used in this study are shown in Table 1.
Splenocyte isolation and flow cytometry
The isolation of splenocytes and flow cytometry (FCM) were performed using
the methods described by Maisel et
al[13]. Briefly, the spleens were excised aseptically 15 min after rats were killed and placed
in RPMI-1640. Single cell suspensions were then prepared. The tissue was passed through a 70-µm-pore mesh and washed
with RPMI-1640. Samples were spun and supernatants were aspirated. Erythrocytes were lysed by ammonium chloride (BD
Bioscience, San Diego, CA, USA). Cells were resuspended in supplemented
RPMI-1640 and counted on a hemocytometer in
trypan blue to ensure viability. After being stimulated with PMA, ionomycin and monensin (GolgiStop), the cells were
stained with Cy-Chrome-labeled anti-rat CD4 antibody (BD PharMingen). The fixation and membrane permeabilization were
performed with FACSTM Perm 2 (BD) according to the manufacturer¡¯s instructions. Intracellular cytokines were stained with
FITC-labeled IFN-g or PE-labeled IL-4 monoclonal antibodies (BD Pharmingen). The
IFN-g- and IL-4-producing CD4+ T cells were analyzed with
FACSTM Calibur (BD, PharMingen).
Enzyme-linked immunosorbent analysis
(ELISA) Before the rats were killed, 1.5 mL of blood was collected from each rat
and centrifuged immediately to remove the blood cells.
Concentrations of IFN-g and IL-4 in the animal sera were estimated by
commercially available ELISA kits (R&D), according to the manufacturer¡¯s instruction. All samples were
assayed in duplicate. The proportions of IFN-g/IL-4 were
then calculated.
Statistics Data are expressed as mean±standard error of the mean (SEM). The statistical significance was evaluated by
one-way ANOVA followed by least significant difference (LSD) or Dunnett¡¯s test.
A P-value less than 0.05 was considered statistically significant. In the experiment involving histology, the figures shown are representative of at least 3 experiments
performed on different days.
Results
Cur therapy improves body weight recovery
The Con group showed transient and slight body weight loss and recovered
quickly (Figure 1A). Administration of TNBS caused a dramatic decrease of body weight in all the groups. At the end of the
experiment, no difference was observed between the Con and Cur groups in body weight, but there was a significant
difference between the 2 groups and the other 3 groups. No difference was observed between the Dex and Cur+Dex groups,
but they differed from the TNBS group.
With the exception of faint infiltration of inflammatory cells, no pathological changes were observed in the Con group
(Figure 1B). Necrosis of epithelium, distortion of crypts, destruction of glands and infiltration of inflammatory cells were
observed in the TNBS group (Figure 1C). Treatment with Cur was found to decrease the necrosis of epithelium, and the
infiltration of inflammatory cells and granulation could be seen (Figure 1D). Dex was found to dramatically decrease the
necrosis of epithelium cells and the infiltration of inflammatory cells. However, it had little effect on the formation of
granulation; edema existed in most cases (Figure 1E). The effects of Cur+Dex on histological images were similar to that of the
Dex group.
Cur therapy decreases macroscopic scores and MPO activity
The different treatment effects on macroscopic scores in
TNBS-induced colitis were evaluated (Table 2). The macroscopic score of the Cur group was significantly lower than that of
the TNBS group (P<0.01). The Dex group also had decreased macroscopic scores. The combined therapeutic effects of
Cur+Dex on macroscopic scores were similar to that of Dex alone. MPO activity significantly
(P<0.01) increased in the TNBS group. Both Cur and/or Dex was found to significantly reduce its activity.
Cur therapy decreased Th1 and increased Th2 cytokines in colon mucosa
Relative mRNA abundance of Th1 cytokines in the TNBS group were elevated (10_50 fold) over the Con group (Table 3), however, that of Th2 cytokines decreased. The
Cur-treated group suppressed the expression of Th1 cytokines efficiently, which was significantly high in the Con group.
Th2 cytokines all increased after the treatment with Cur. Treatment with Dex was also found to decrease the expression of
Th1 cytokines. However, it seemed that Dex could not increase Th2 cytokines. The effects of Cur+Dex on the expression of
cytokines were similar to that of Dex.
Cur therapy decreased IFN-g and increased IL-4 in splenocytes
The frequencies of IFN-g-producing
CD4+ T cells increased, IL-4-producing
CD4+ T cells decreased and the proportion increased dramatically after colitis induced by TNBS
(Table 4). Cur decreased the frequencies of
IFN-g-producing CD4+ T cells and the proportion and increased
IL-4-producing CD4+ T cells. Frequencies of
IFN-g-producing CD4+ T cells decreased in the Dex-treated group, frequencies of
IL-4-producing CD4+ T cells increased slightly but show no difference from the TNBS group. The proportion decreased in the Dex-treated
group, but was higher than that of the Con group. The effect of Cur+Dex was similar to that of Dex.
Cur therapy decreased IFN-g and increased IL-4 in circulation
Compared to the Con group, the concentrations of IFN-
g increased and IL-4 decreased, thus, the proportion of
IFN-g/IL-4 increased significantly in the TNBS group (Table
5). Treatment with Cur could dramaticly decrease the concentration of
IFN-g, increase that of IL-4 and decrease the proportion
significantly (P<0.01). Dex and Cur+Dex could decrease
IFN-g but did not show the same effects on IL-4 and the proportion.
Discussion
The results of the present study indicate that Cur could (1) reduce body weight loss in colitis induced by TNBS; (2)
decrease macroscopic scores and MPO activity; (3) decrease the mRNA of Th1 cytokines; and (4) increase the mRNA of Th2
cytokines and decrease the proportion of IFN-g/IL-4. Dex exerted similar effects on the first 3 findings, but not on the last one.
To control the precise dosage and to compare it with Dex, which usually administered by injection, Cur and Dex were given
to animals separately or combined intra-peritoneally. Both Cur and Dex increased body weight, but it was much slower than reported in the findings of Sugimoto
et al[10]. The reason for this might be because (1) mice and rats may have different sensitivity and endurance to TNBS; and (2)
the different route of medication, especially if it is intraperitoneal as it was in our experiment, was traumatic to the animal.
However, the concurrent conclusion is that treatment with Cur is capable of increasing the body weight in TNBS-induced
colitis.
Administration of TNBS resulted in hyperaemia, edema, ulceration (some break through the whole wall) and adhesion. All
of these changes were expressed as the elevation of the macroscopic score. MPO is an enzyme found in neutrophils, and its
activity in the colon is linearly related to neutrophil infiltration. After colitis was induced by TNBS, the macroscopic score
and MPO activity increased. Treatment with cur was found to decrease the macroscopic score, MPO activity and ameliorate
histological images. Dex and Cur+Dex showed similar effects.
TNBS-induced colitis is an experimental model of Th1-like gut
inflammation[7]. IL-12, the inducer of Th1 cytokines
and an inhibitor of Th2 cytokines, plays an important role in TNBS-induced
colitis[8,14,15]. Inhibiting IL-12 has been shown to
prevent development and block the progression of Th1-mediated disease in experimental models of
autoimmunity[16,17]. Our results, which are consistent with previously published
reports[2,3,10,17], indicate that Cur could inhibit the production
of IL-12 mRNA in colon mucosa, sequentially IFN-g,
IL-1b, TNF-a decreased and IL-4 increased in TNBS-induced colitis.
Dex was found to inhibit IL-12 and also other Th1 cytokines. The effect of Cur+Dex was similar to that of Dex.
IL-4 is the inducer and the production of Th2 cells. After colitis was induced by TNBS, it decreased. Treatment with
Cur could increase its level, which is consistent with the report of Ukil
et al[11], but different from the findings in the study
by Sugimoto et al[10]. Different durations of treatment with
Cur may have resulted in the different findings in our study and
that of Sugimoto et al. In their study, the animals were treated for only 1 week, but in our experiment all the animals
received Cur for 2 weeks. However, the differences between the study of Sugimoto
et al and that of Ukil et al should be
explored in future. Kobayashi et
al[4] reported on the contrary that Cur inhibited IL-4 and improved IL-2 (Th1 cytokine) in
atopic asthmatics. The reason for this may be because their experiment focused on allergic diseases in which Th2
cytokines increased and Th1 cytokines decreased, whereas ours focused on Th1-mediated inflammatory diseases. These
different results indicate that Cur might exert its pharmacological effects by different mechanisms. IL-10 also increased
after the treatment with Cur. Dex induced the expression of IL-4 , slightly to the level of the con group, even though it did
not show differences from that of TNBS group. These results are consistent with the report of DeKruyff
et al[18]. Combined Cur with Dex did not show additive or synergistic effect on the expression of Th2 cytokines.
The proportion of IFN-g/IL-4 is reported as an important factor in regulating the shift of
Th1/Th2[19]. The relative abundance of mRNA in colon mucosa is a local index of Th1/Th2. To determine the whole body balance of Th1/Th2, the
intracellular concentrations of IFN-g and IL-4 in splenocytes and in circulation were detected. The results in splenocytes
and in circulation were similar. Increased IFN-g and the proportion and decreased IL-4 were observed in the TNBS group.
Treatment with Cur was found to significantly decrease
IFN-g and increase IL-4, thus the proportion decreased markedly
to the level of the Con group. Treatment with Dex decreased
IFN-g significantly and slightly increased IL-4; the proportion
also decreased, although it was higher than that of the Con group. Contrary to our results, Xie
et al[20] reported that Dex decreased IL-4 and increased
IFN-g, thus the proportion of IFN-g/IL-4 increased. The possible reason for this difference
may be because that experiment, similar to that of Kobayashi
et al[4], focused on the allergic asthmatics model, different
dosages were administered (0.5
mg·kg-1·d-1
vs 2
mg·kg-1·d-1), and the course of treatment differed (6 d
vs 14 d). Cur+Dex showed similar effects as Dex alone. All of these results were similar to the results of the colon mucosa.
Our unpublished data indicates that treatment with Cur can significantly increase the concentration of prostaglandin
E2 (PGE2) in colon mucosa, but Dex decreases it.
PGE2, participated in epithelium repairing, has been implicated in the
enhancement of Th2 responses by inhibiting the production of Th1
cytokines[21]. Thus, changes of cytokines in this
experiment suggest that Cur regulates the shift from Th1 to Th2, possibly by inhibiting Th1 cytokines directly or indirectly
by increasing PGE2 or by the two pathways simult- aneously.
To investigate if Cur, the traditional Chinese medicine, shows additive and/or synergistic effects with
Dex, the most commonly-used Western medicine, some rats were given the 2 agents simultaneously. Our results indicate that they do
not show any additive and/or synergistic effects. The reason may be because of the long length of time (2 weeks) and
high-dosage (2
mg·kg-1·d-1) delivery of Dex which resulted in the over-suppression of the immune system. Another reason
may be that Cur and Dex acted on some common signal pathways. In addition to its anti-inflammation effects, Cur has been
reported to have chemo-preventive characteristics in animal models of colon
cancer[22,23]. One important clinical issue in
the management of patients with long term IBD is a high risk for development of dysplasia and neoplasia. This manifests
our belief that in acute phases of IBD, Dex is powerful, but in chronic phases, Cur may be more useful, not only in the
control of inflammation and minimal side effects, but also in chemo-preventive activity.
In conclusion, we provide evidence that both Cur and Dex can attenuate TNBS-induced colitis. We also provide
evidence that Cur can regulate the shift from Th1 to Th2 in TNBS-induced colitis. Even though it has previously been
reported to suppress Th1 and improve Th2
cytokines[24], we found that Dex can also inhibit Th1 cytokines, but its effect
on improving Th2 cytokines is not as powerful as Cur. Combined Cur and Dex did not show additive and/or synergistic
effects. Thus, Cur may be a new agent in the clinical in the treatment of IBD.
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