Structure-Activity Relationship of Curcumin: Role of the Methoxy Group in Anti-inflammatory and Anticolitis Effects of Curcumin

Article abstract of DOI:10.1021/acs.jafc.7b01792

Curcumin, a dietary compound from turmeric, has beneficial effects on inflammatory diseases such as inflammatory bowel disease. Most previous studies have focused on the structure-activity relationship of the thiol-reactive α,β-unsaturated carbonyl groups of curcumin, so little is known about the roles of methoxy groups in biological activities of curcumin. Here we synthesized a series of curcumin analogues with different substitution groups (R = H-, Br-, Cl-, F-, NO₂-, CH₃-, and OH-) to replace the methoxy group and evaluated their biological effects in vitro and in vivo. Curcumin, Cur-OH, and Cur-Br (25 μM) suppressed 74.91 ± 0.88, 77.75 ± 0.89, and 71.75 ± 0.90% of LPS-induced NO production, respectively (P < 0.05). Similarly, these compounds also decreased iNOS expression, COX-2 expression, and NF-κB signaling in RAW 264.7 macrophage cells (P < 0.05). However, other analogues, especially Cur-NO₂, were inactive (P > 0.05). In the dextran sulfate sodium (DSS)-induced colitis mouse model, the Cur-Br analogue also showed a beneficial effect the same as curcumin (P < 0.05), whereas the Cur-NO₂ analogue had no effect in the animal model (P > 0.05). Together, the analogues have dramatically different effects on inflammation, supporting that the substitution group on the methoxy position plays an important role in the anti-inflammatory effects of curcumin. The methoxy group is a potential structural candidate for modification to design curcumin-based drugs for inflammatory diseases.

Full text of DOI:10.1021/acs.jafc.7b01792

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Article
Structure and Activity Relationship of Curcumin: Role of Methoxy
Group in Anti-inflammatory and Anti-colitis Effects of Curcumin
Haixia Yang, Zheyuan Du, Weicang Wang, Mingyue Song, Katherine Z. Sanidad, Elvira
Sukamtoh, Jennifer Zheng, Li Tian, Hang Xiao, Zhenhua Liu, and Guodong Zhang
J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 17 May 2017
Downloaded from http://pubs.acs.org on May 19, 2017
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Structure and Activity Relationship of Curcumin: Role of Methoxy Group in Anti-
inflammatory and Anti-colitis Effects of Curcumin
a,b,#
a,#
a
a
a,c
Haixia Yang , Zheyuan Du , Weicang Wang , Mingyue Song , Katherine Sanidad , Elvira
a
a
a,d
a,c
c,e
a,c,*
Sukamtoh , Jennifer Zheng , Li Tian , Hang Xiao , Zhenhua Liu , and Guodong Zhang
a
Department of Food Science, University of Massachusetts, Amherst, MA, 01003
b
Department of Nutrition and Food Safety, College of Public Health, Xi’an Jiaotong University,
Xi’an, Shaanxi, China, 710061
c
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Molecular and Cellular Biology Graduate Program, University of Massachusetts, Amherst, MA
01003
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Anyang Institute of Technology, Anyang, Henan, China, 455000
e
Department of Nutrition, University of Massachusetts, Amherst, MA, 01003
#
H.Y. and Z.D. contributed equally to this work.
*
To whom correspondence should be addressed: Guodong Zhang, Department of Food Science,
University of Massachusetts, Amherst, MA, USA. Email: guodongzhang@umass.edu, Tel: 413ꢀ
4541014, Fax: 413ꢀ5451262
Running title: SAR of curcumin
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Abstract
Curcumin, a dietary compound from Turmeric, has beneficial effects on inflammatory diseases
such as inflammatory bowel disease. While most previous studies have focused on structureꢀ
activity relationship of the thiolꢀreactive α, βꢀunsaturated carbonyl groups of curcumin, little is
known about the roles of methoxy groups in biological activities of curcumin. Here we
synthesized a series of curcumin analogs with different substitution groups (R = Hꢀ, Brꢀ, Clꢀ, Fꢀ,
NO ꢀ, CH ꢀ, and OHꢀ) to replace the methoxy group, and evaluated their biological effects in
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vitro and in vivo. Curcumin, CurꢀOH, and CurꢀBr (25 ꢁM) suppressed 74.91 ± 0.88 %, 77.75 ±
0.89% and 71.75 ± 0.90% of LPSꢀinduced NO production, respectively (P < 0.05). Similarly,
these compounds also decreased iNOS expression, COXꢀ2 expression and NFꢀκB signaling in
RAW 264.7 macrophage cells (P < 0.05). However, other analogs especially CurꢀNO were
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inactive (P > 0.05). In dextran sulfate sodium (DSS)ꢀinduced colitis mice model, CurꢀBr analog
also showed beneficial effect the same as curcumin (P < 0.05), while CurꢀNO analog had no
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effect in the animal model (P > 0.05). Together, the analogs have dramatically different effects
on inflammation, supporting that the substitution group on the methoxy position plays an
important role in the antiꢀinflammatory effects of curcumin. The methoxy group is a potential
structural candidate to modify, in order to design curcuminꢀbased drugs for inflammatory
diseases.
Keywords: curcumin; inflammatory bowel disease; inflammation; dextran sulfate sodium;
structureꢀactivity relationship
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1. Introduction
Inflammation is a common feature of many human diseases, such as inflammatory bowel disease
(IBD), which is characterized by the presence of chronic inflammation in the small intestine and
colon tissue. IBD is a serious health problem in developed countries, affecting over 1 million
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people in the US and 2.5 million people in Europe . IBD severely impacts quality of life of
patients, with symptoms including abdominal pain, vomiting, diarrhea, and rectal bleeding. To
date, there is no cure of IBD, therefore, it is of critical importance to develop effective strategies
for IBD prevention or maintenance of IBD remission. Curcumin, a dietary compound from
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turmeric, has been extensively studied for its beneficial effects on inhibition of inflammation .
Many studies have shown that curcumin inhibits expression of proꢀinflammatory mediators and
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activation of the NFꢀκB signaling pathway . In terms of IBD, animal experiments have shown
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that curcumin protected mice from dextran sulfate sodium (DSS)ꢀinduced colitis
. In a
randomized, doubleꢀblind, multicenter, and placeboꢀcontrolled human clinical trial, dietary
intake of 2 grams/day curcumin has been shown to maintain remission of ulcerative colitis (UC)
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in UC patients on standard therapy . Together, these results suggest that curcumin is a
promising dietary compound to prevent or control IBD.
A better understanding of structureꢀactivity relationship (SAR) of curcumin could help us to
apprehend its molecular mechanism, and help to design better curcuminꢀbased drugs to reduce
the risks of inflammatory diseases. To date, most previous studies of curcumin have focused on
its conjugated heptadienedione moiety, which possesses two thiolꢀreactive α,βꢀunsaturated
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carbonyl groups
. This group could react with cysteine residues of cellular proteins, leading
to covalent modification of key proteins involved in cell signaling and subsequent of modulation
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of various biological responses
. Curcumin possesses two methoxy groups (ꢀOCH , see
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chemical structure of curcumin in Figure 1) in its aromatic rings, however, to date little is known
whether the methoxy group has any contribution to the antiꢀinflammatory effect of curcumin.
Since previous studies have shown that bisdemethoxy curcumin, in which the methoxy groups in
curcumin is replaced with hydrogens (ꢀH, see chemical structure in Figure 1), has reduced antiꢀ
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inflammatory, antiꢀcancer, and antiꢀoxidant effects
, we hypothesize that the methoxy group
may be important for the antiꢀinflammatory effect of curcumin. In order to better understand the
role of the methoxy group in the biological activity of curcumin, here we synthesized a series of
curcumin analogs with different substitution groups to replace the methoxy group, then evaluated
their biological effects on inflammatory responses and NFꢀκB signaling in macrophage cells, and
DSSꢀinduced colitis in animal models.
2. Materials and Methods
2.1 Chemicals
Curcumin and its analogs (>99% purity, assessed by HPLC and 1H NMR analysis) were
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synthesized according to the method as we described previously . Mouse macrophage RAW
264.7 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, SigmaꢀAldrich,
St. Louis, MO) supplemented with 10% fetal bovine serum (FBS, Corning, NY) and 1%
streptomycinꢀpenicillin under 5% CO atmosphere at 37 °C.
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2.2 Griess Assay to Measure Nitric Oxide (NO) Production
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RAW 264.7 macrophage cells were treated with 25 ꢁM different compounds and 2 µg/mL LPS
(SigmaꢀAldrich) and/or test compound in DMEM medium for 24 h, then concentration of nitrate
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in the cell culture medium was analyzed by Griess assay, as we described .
2.3 Immunoblotting
Cellular proteins were extracted with RIPA lysis buffer (Boston BioProducts, Ashland, MA)
containing 50 mM TrisꢀHCl, 150 mM NaCl, 1% NPꢀ40, 0.5% sodium deoxycholate, 0.1% SDS,
5 mM EDTA and 1 mM EGTA, with protease inhibitor cocktail (Boston BioProducts, Ashland,
MA). Protein concentrations were determined using the BCA protein assay kit according to the
manufacturer’s instruction (Thermo Scientific, Waltham, MA). The samples were resolved by
SDSꢀPAGE and transferred to nitrocellulose membrane (BioꢀRad, Hercules, CA) using transfer
system (BioꢀRad) at 80 V for 70 min. The membrane was blocked in 5% milk buffer for 1 h, and
then incubated with antibodies against phosphoꢀIKK, IKK, IκBα, COXꢀ2, iNOS and βꢀactin
(Cell Signaling Technology, Danvers, MA) at 4 °C overnight. All the primary and secondary
antibodies were diluted in Trisꢀbuffered saline with 0.5% Tween 20 (TBST) solution containing
5% BSA with ratio of 1:1000. The membrane was then probed with LIꢀCOR IRDye 800CW goat
antiꢀrabbit and IRDye 680RD goat antiꢀmouse secondary antibodies, and detected using the
Odyssey imaging system (LIꢀCOR, Lincoln, NE). Quantification of immunoblotting was
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performed using Image Studio Lite Software (LIꢀCOR).
2.4 DSSꢀinduced Colitis in C57BL/6 Mice
C57BL/6 male mice (age = 6 weeks) were preꢀtreated with 100 mg/kg/day curcumin or its
synthetic analogs, which were dissolved in 50 µL of 100% DMSO, or 50 µL DMSO vehicle, by
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intraperitoneal (i.p.) injection for 3 days using isoflurane anesthesia, then treated with 1.5% DSS
in the drinking water for 9 days. During the DSS treatment, the mice were continued with i.p.
injection of curcumin or its analogs. The animal experiment was conducted in accordance with
the protocols approved by the Institutional Animal Care and Use Committee of University of
Massachusetts Amherst.
2.5 Flow Cytometry Analysis of Immune Cells in Colon Tissues
At the end of the animal experiment, the colon tissues were dissected, washed with cold PBS,
and digested with Hank’sꢀbalanced salt solution (HBSS) supplemented with 1 mM DTT, 5 mM
EDTA for two hours at 4 °C. The released cells were filtered through 70 ꢁm cell sorters (BD
Biosciences, San Jose, CA) to obtain single cell suspension, which were stained with APCꢀ
conjugated antiꢀmouse CD11c antibody, FITCꢀconjugated antiꢀmouse CD45 antibody, PE/Cy7ꢀ
conjugated antiꢀmouse Lyꢀ6G/Lyꢀ6C (Grꢀ1) antibody, PerCP/Cy5.5ꢀconjugated antiꢀmouse
F4/80 antibody, and isotype control antibody (BioLegend, San Diego, CA). The stained cells
were analyzed using BD LSRFortessa™ cell analyzer (BD Biosciences) and data were analyzed
using FlowJo software.
2.6 H&E Staining of Colon Tissues
Part of the dissected colon tissues were fixed in 4% formalin, gradually incubated with 15%
sucrose and 30% sucrose at 4 °C, then embedded in optimal cutting temperature compound
(OCT, Fisher Healthcare, Houston, TX) and frozen for cryosectioning. The OCTꢀfixed tissue
specimens were sliced into 5 ꢁmꢀthick sections, and airꢀdried for one hour. The sections were
mounted on glass slides and stained with H&E staining and examined with a light microscopy.
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2.7 Data Statistics
All data are expressed as the mean ± standard error of the mean (SEM). Group comparisons were
carried out using oneꢀway analysis of variance or Student’s tꢀtest. P values less than 0.05 were
considered statistically significant.
3. Results
3.1 Chemical Synthesis of Curcumin and Its Analogs
To study the roles of methoxy group in the biological activities of curcumin, we chemically
synthesized a series of curcumin analogs with different substitution groups on the methoxy
position (see chemical synthesis method and chemical structures in Figure 1). The synthetic
analogs (abbreviated as CurꢀR) included seven compounds (R = ꢀOH, ꢀBr, ꢀH, ꢀF, ꢀCH , ꢀCl, and
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ꢀNO ). The purities of the synthesized compounds were checked by HPLC, and their chemical
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structures were validated by NMR and MS analysis.
3.2 Effects of Curcumin and Its Analogs on LPSꢀinduced NO Production in Macrophage Cells
To study the potential roles of Rꢀ group in the antiꢀinflammatory activities of curcumin, we first
compared the effects of curcumin and its synthetic analogs on LPSꢀinduced NO production in
RAW 264.7 macrophage cells. Consistent with previous studies, treatment with curcumin
suppressed 74.5% of LPSꢀinduced NO production compared with the group treated with LPS
alone (P < 0.05, Figure 2A). In comparison to curcumin, the synthetic analogs at the same dose
showed different potencies. CurꢀOH and CurꢀBr decreased 77.8% and 71.8% of LPSꢀinduced
NO production, respectively (P < 0.05), and the CurꢀNO analog was inactive to inhibit LPSꢀ
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induced NO production (P > 0.05), supporting that the Rꢀ group plays a critical role in the antiꢀ
inflammatory effect of curcumin in vitro.
3.3 Effects of Curcumin and Its Analogs on LPSꢀinduced iNOS and COXꢀ2 Expressions in
Macrophage Cells
After we found that curcumin and its analogs had different potencies to inhibit LPSꢀinduced NO
production, we studied their comparative effects on LPSꢀinduced expressions of iNOS and COXꢀ
2, which are two important proteins involved in inflammation (Figure 2B-2D). Consistent with
the results we observed above, curcumin and its synthetic analogs had different potencies to
suppress expression of these two proteins. For iNOS expression, curcumin, CurꢀOH, and CurꢀBr
inhibited LPSꢀinduced iNOS expression (P < 0.05 vs. LPS alone). CurꢀH, CurꢀF, CurꢀCl, and
CurꢀNO were also active but with reduced potencies (P < 0.05 vs. LPS alone, and P < 0.05 vs.
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curcumin). For COXꢀ2 expression, curcumin, CurꢀOH, and CurꢀBr at the tested dose suppressed
LPSꢀinduced COXꢀ2 expression (P < 0.05 vs. LPS alone), while the other analogs were inactive
(P > 0.05 vs. LPS alone). Together, these results further support that the Rꢀ group plays a critical
role in the antiꢀinflammatory effect of curcumin in vitro.
3.4 Effects of Curcumin and Its Analogs on NFꢀκB Signaling Pathway in Macrophage Cells
Given the different potencies of curcumin and its synthetic analogs on production of proꢀ
inflammatory mediators and/or proteins, we next studied their comparative effects on NFꢀκB
signaling, which is among the most important signaling pathway involved in regulating
inflammation. Consistent with previous studies, curcumin suppressed LPSꢀinduced
phosphorylation of IKK and degradation of IκBα (P < 0.05 vs. LPS alone), demonstrating its
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inhibitory effect on NFꢀκB signaling. In comparison, curcumin analogs exerted different
potencies. The CurꢀOH and CurꢀBr analogs showed similar potency as curcumin to inhibit the
activation of NFꢀκB signaling in macrophage cells (P < 0.05 vs. LPS alone, and P > 0.05 vs.
curcumin), while the other analogs were inactive at the tested dose (P > 0.05, Figure 3).
Together, the results in Figure 2-3 showed an order of potency of curcumin, CurꢀOH, CurꢀBr >
CurꢀH, CurꢀF, CurꢀCH , CurꢀCl, > CurꢀNO in terms of inhibition of LPSꢀinduced inflammatory
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responses in macrophage cells. These results support that the Rꢀ group plays a critical role in the
antiꢀinflammatory effect of curcumin in macrophage cells.
3.5 Effects of Curcumin and Its Analogs on DSSꢀinduced Colon Inflammation in Mice
Finally, we studied the comparative effects of curucmin and its synthetic analogs on
inflammation in animal models, using a DSSꢀinduced colitis model in C57BL/6 mice. Among
synthesized curcumin analogs, our studies in macrophage cells showed that CurꢀBr analog was
highly active to suppress inflammation, while CurꢀNO analog was inactive in most of the assays
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(Figure 2-3). Therefore, we focused on these two compounds as representative examples of
curcumin analogs. Consistent with previous studies, curcumin protected mice from DSSꢀinduced
colitis, with reduced colonic infiltration of immune cells (leukocytes, neutrophils, and dendritic
cells, P < 0.05, see Figure 4) and crypt damage (see H&E staining of colon tissues in Figure 5).
CurꢀBr analog also showed beneficial effect in DSSꢀinduced colitis model (P < 0.05), while Curꢀ
NO analog had no effect in the animal experiment (P > 0.05, Figure 4-5). These results are
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largely in agreement with the data we observed in cell culture experiments that curcumin was
biologically active, while CurꢀNO analog had no antiꢀinflammatory effect (see Figure 2-3).
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Together, these results further demonstrate that replacing the substitution groups on the methoxy
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position could dramatically change the biological activities of curcumin, supporting a critical
importance of the methoxy group in the antiꢀinflammatory effect of curcumin.
4. Discussion
Curcumin is among the most promising dietary compound for prevention of inflammatory
disease such as IBD. A better understanding of the SAR of curcumin could help us to apprehend
its molecular mechanism, and design better curcuminꢀbased therapeutic drugs to treat or prevent
inflammatory diseases. While most previous studies have focused on the thiolꢀreactive α,βꢀ
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unsaturated carbonyl groups
, little is known about the roles of methoxy groups in the
biological activities of curcumin. In this study, we chemically synthesized a series of curcumin
analogs with different substitution groups on the methoxy position, and found that these analogs
have different antiꢀinflammatory effects in vitro and in vivo. Our results are in agreement with
previous studies which showed that compared with curcumin, bisdemethoxy curcumin has
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reduced antiꢀinflammatory and antiꢀcancer effects
. Together, these results support that that
the previously uncharacterized methoxy group plays a critical role in the antiꢀinflammatory
effects of curcumin.
In this study, we prepared seven analogs of curcumin, with different substitution groups on the
methoxy position (R = ꢀOH, ꢀBr, ꢀH, ꢀF, ꢀCH , ꢀCl, and ꢀNO ). These substitution groups have a
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wide range of electronꢀwithdrawing properties, with ꢀNO group being the most electronꢀ
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withdrawing, and ꢀOH group being the least electronꢀwithdrawing. We compared the effects of
curcumin and its synthesized analogs on inflammatory responses (LPSꢀinduced production of
NO, and expressions of iNOS and COXꢀ2) and NFꢀκB signaling (phosphorylation of IKK and
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degradation of IκBα) in macrophage cells. The results showed an overall order of potency of –
OCH , ꢀOH, ꢀBr > ꢀH, ꢀF, ꢀCH , ꢀCl, > ꢀNO . The DSSꢀinduced colitis assay in C57BL/6 mice
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also showed that curcumin and the CurꢀBr analog were active to protect mice from DSSꢀinduced
colitis, while the CurꢀNO analog had no effect, which is largely consistent with the order of
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potency we observed in cell culture models. By only changing the substitution groups on the
methoxy position, the biological activities of curcumin were dramatically changed, supporting
that the methoxy group plays a critical role in the biological activities of curcumin. For example,
in our experiments, curcumin potently inhibited inflammation in vitro and in vivo, while the Curꢀ
NO analog was inactive in all these assays. The CurꢀNO analog also possesses the thiolꢀ
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reactive α, βꢀunsaturated carbonyl moiety; therefore, our results seem to suggest that the α, βꢀ
unsaturated carbonyl moiety may not be the only structural component required for the
biological activities of curcumin, or the substitution groups on the methoxy position could
dramatically change the reactivity of the α, βꢀunsaturated carbonyl moiety.
It remains to determine the mechanism by which the analogs with different substitution groups
have varied potency of antiꢀinflammatory effects. One possibility is that changing the methoxy
groups may interfere the interaction of curcumin with cellular proteins, since previous studies
using molecular docking simulations have shown that the methoxy groups could be involved in
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protein binding
. Muthenna et al. showed that curcumin inhibited aldose reductase (ALR2) in
a nonꢀcompetitive manner, and molecular docking showed that the oxygen atom of the methoxy
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group may interact with Leu300 and Leu301 residues of ALR2 . Kaur et al. showed that the
oxygen atom of the methoxy group may interact with Thr132 and Asn165 residues of E. coli Ftsz,
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which is a key protein involved in cell division . Other studies have shown that the methoxy
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group may contribute to the interactions of curcumin with protein kinase C . While these results
were obtained from molecular docking simulations, there was no direct experimental evidence to
support the roles of methoxy group in protein binding. Based on these studies, it is likely that
replacing the methoxy group with other substitution groups could interfere protein binding and
modulate the biological responses of curcumin. For example, docking simulation studies showed
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that the oxygen atom of the methoxy group is involved in protein binding
, changing the
methoxy group (ꢀOCH ) to methyl group (ꢀCH ) could thus interfere protein binding and change
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the bioactivity of curcumin. Indeed, our results showed that the –CH analog has reduced antiꢀ
3
inflammatory effect than curcumin. Another possible mechanism is that changing the methoxy
groups may modulate the thiol reactivity of the conjugated heptadienedione chain, and therefore
change the potency of curcumin to covalently modify proteins. Further studies are needed to
better understand the molecular mechanisms of curcumin for suppressing inflammation.
A limitation of our research is that to study the effects of curcumin and its analogs on DSSꢀ
induced colitis in vivo, we used i.p. injection, not oral administration, to administer these
compounds into mice. We chose i.p. injection in our study, in part because it is difficult to
synthesize substantial curcumin analogs for oral administration. Despite previous studies
showing that the pharmacokinetic profiles of compounds administered intraperitoneally are
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similar to those seen through oral administration , there are limitations to study dietary
compounds through i.p. injection. Indeed, previous studies showed that oral administration of
curcumin leads to a poor absorption in the gastrointestinal tract, which is a major reason for the
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poor bioavailability of curcumin in vivo . Therefore, by using i.p. injection, it is difficult to
study whether curcumin analogs have varied gastrointestinal absorption. In addition, in our
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colitis assay, we only studied immune cell infiltration and crypt damage in colon tissues, but did
not measure the actions of curcumin and its analogs on specific proꢀinflammatory cytokines.
Previous studies showed that curcumin reduced the levels of cytokines, such as ILꢀ1β and TNFꢀα,
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in animal models of colitis
. Considering the important roles of these cytokines in
inflammatory diseases, it would be important to characterize the effects of curcumin and its
analogs on colonic profiles of proꢀinflammatory cytokines.
In conclusion, here our SAR study showed that the previously unappreciated methoxy group
plays a critical role in the antiꢀinflammatory effects of curcumin. The methoxy group could be a
potential structural candidate to modify, in order to design better curcuminꢀbased drugs to treat
or prevent inflammatory disease such as IBD.
Abbreviations
IBD, inflammatory bowel disease; DSS, dextran sulfate sodium; UC, ulcerative colitis; SAR,
structureꢀactivity relationship; NO, nitric oxide; HPLC, high performance liquid chromatography;
NMR, nuclear magnetic resonance; MS, mass spectrometry; ALR2, aldose reductase
Acknowledgement
This work was supported by a new faculty startꢀup fund, Armstrong Fund of Science Award
from University of Massachusetts Amherst and USDA NIFA [grant number 2016ꢀ67017ꢀ24423].
Supporting Information
Materials and Methods; Characterization of synthesized curcumin and its analogs
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Figure Captions
Figure 1 Chemical synthesis of curcumin and its analogs. (A) Scheme to chemically prepare
curcumin and its analogs. (B) Chemical structures of curcumin and its analogs in this study.
Figure 2 Effects of curcumin and its analogs on LPSꢀinduced NO production and expression of
iNOS and COXꢀ2 in RAW 264.7 macrophage cells. RAW 264.7 cells were treated with
curcumin or its analogs (25 ꢁM) and LPS (2 ꢁg/mL) for 24 h. (A) Quantification of NO
production, the results are expressed as fold change of the negative control. (B) Representative
immunoblotting images of iNOS and COXꢀ2. (C-D) Quantifications of iNOS and COXꢀ2
expressions. The results are from three independent experiments. The results are mean ± SEM, *
P<0.05 vs. Ctrl, # P<0.05 vs. LPS alone.
Figure 3 Effects of curcumin and its analogs on NFꢀkB signaling in RAW 264.7 cells. RAW
264.7 cells were pretreated with curcumin or its analogs (25 ꢁM) for 1.5 h, then stimulated with
LPS (2 ꢁg/mL) for 20 min. (A) Representative immunoblotting images of phosphorꢀIKK, IKK,
and IκBα. (B-C) Quantifications of protein expressions. The results are from three independent
experiments. The results are mean ± SEM, * P<0.05 vs. Ctrl, # P<0.05 vs. LPS alone.
Figure 4 Effects of curcumin and its analogs on DSSꢀinduced colitis in C57BL/6 mice, as
assessed by immune cell infiltration into colon tissues. (A) total immune cells, (B) neutrophils,
(C) dendritic cells, and (D) macrophage cells. The results are mean ± SEM, * P<0.05 vs. Ctrl, n
= 5ꢀ6 mice per group.
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Figure 5 H&E staining of colon tissues demonstrates that curcumin and CurꢀBr analog protected
mice from DSSꢀinduced crypt damage, while CurꢀNO analog had no effect. (A-B) colon tissues
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from control group (mice treated with DSS and vehicle), (C-D) colon tissues from curcumin
group (mice treated with DSS and curcumin), (E-F) colon tissues from CurꢀBr group (mice
treated with DSS and CurꢀBr), and (G-H) colon tissues from CurꢀNO group (mice treated with
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curcumin and CurꢀNO2).
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