Influence of Dimerization of Apocynin on Its Effects in Experimental Colitis

Article abstract of DOI:10.1021/acs.jafc.7b00872

Apocynin has been widely used as an inhibitor of the nicotinamide adenine dinucleotide phosphate oxidase (NADPH-oxidase) system and shows promise as an anti-inflammatory drug. Diapocynin, the dimeric product generated by the oxidation of apocynin in the presence of myeloperoxidase (MPO), is supposed to be its active form. In this study, diapocynin has been chemically synthesized and its activity on several inflammatory mediators in LPS-stimulated RAW 264.7 macrophages and its anti-inflammatory effect on ulcerative colitis induced by dextran sodium sulfate (DSS) in mice analyzed. We found that diapocynin showed higher inhibitory activity than apocynin. The dimer reduced ROS production, TNF-α, IL-6, and IL-1β levels and inhibited iNOS and COX-2 expression as well as decreased NO and PGE₂ production induced in LPS-stimulated RAW 264.7 cells. The anti-inflammatory molecular mechanism of diapocynin was associated with the suppression of NF-κB activation. However, these results were not paralleled by in vivo studies. Oral administration of apocynin and diapocynin (100 mg/kg) three times a week exhibited similar protections against experimental inflammatory bowel disease induced by DSS; therefore, apocynin should not be considered a prodrug. However, it should be taken into account that the dimer is more potent because its dose (0.3 mmol/kg) is half that of apocynin.

Full text of DOI:10.1021/acs.jafc.7b00872

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Article
"Influence of dimerization of apocynin on its effects in experimental colitis"
Marta Marin, Clotilde Gimeno, Rosa M Giner, Jose Luis Rios, Salvador Mañez, and M.C. Recio
J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 09 May 2017
Downloaded from http://pubs.acs.org on May 11, 2017
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Journal of Agricultural and Food Chemistry
FeSO4·7H2O
Na2S2O8
Apocynin (1)
Diapocynin (2)
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"Influence of Dimerization of Apocynin on its E ffects in Experimental C olitis"
M. Marín, C. Gimeno, R.M. Giner, J.L. Ríos, S. Máñez, M.C. Recio*
Departament de Farmacologia, Facultat de Fàrmacia, University of Valencia.
Avda. Vicent Andrés Estellés, s/n, 46100 BurjassotꢀValencia (Spain)
*Adress corresponding author: Departament de Farmacologia, Facultat de Fàrmacia,
University of Valencia. Avda. Vicent Andrés Estellés, s/n, 46100 BurjassotꢀValencia
(Spain)
Electronic mail: maria.c.recio@uv.es
Fax number: +34 963544942
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ABSTRACT
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Apocynin has been widely used as an inhibitor of nicotinamide adenine
dinucleotide phosphatase oxidase (NADPHꢀoxidase) system and shows promise as an
antiꢀinflammatory drug. Diapocynin, the dimeric product generated by the oxidation of
apocynin in presence of myeloperoxidase (MPO), is supposed to be its active form. In
this study, diapocynin has been chemically synthesized and analyzed its activity on
several inflammatory mediators in LPSꢀstimulated RAW264.7 macrophages and its
antiꢀinflammatory effect on ulcerative colitis induced by dextran sodium sulfate (DSS)
in mice. We found that diapocynin showed higher inhibitory activity than apocynin.
The dimer reduced ROS production, TNFꢀα, ILꢀ6 and ILꢀ1β levels and inhibited iNOS
and COXꢀ2 expression as well as decreased NO and PGE2 production induced in LPSꢀ
stimulated RAW264.7 cells. The antiꢀinflammatory molecular mechanism of
diapocynin was associated with the suppression of NFꢀκB activation. However, these
results were not paralleled by in vivo studies. Orally administration of apocynin and
diapocynin (100 mg/kg) three times a week exhibited similar protection against
experimental inflammatory bowel disease induced by DSS, therefore apocynin should
not be considered a prodrug. However, it should be taken into account the dimer is more
effective since its dose (0.3 mmol/kg) is half of that of apocynin.
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Keywords
Apocynin, diapocynin, ulcerative colitis, COXꢀ2, macrophages RAW264.7
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INTRODUCTION
Inflammatory bowel disease (IBD) involves a group of inflammatory chronic
disorder, which includes ulcerative colitis and Crohn's disease. It affects the
gastrointestinal tract, and at the present, both diseases have no cure, and only can be
treated with antiꢀinflammatory drugs or monoclonal antibodies targeting proꢀ
inflammatory cytokines.1 However, these drugs could causes severe side effects and
complications when are used during a longꢀterm why it is necessary to identify new and
safe compounds for preventing or treating IBD. Many patients turn to alternative
strategies including phytochemicals found in traditional medicinal plants or foods.
Some significant studies reported the role of dietary polyphenols in foods and botanicals
on IBD prevention and treatment. Of them, green tea polyphenols, curcumin, and
stilbenes, as well as phenolics from different berries are the principal polyphenols tested
in animal models of colitis.2,3 The inflammatory characteristics of IBD can be induced
by administration of dextran sulfate sodium (DSS) in rodents. Indeed, the administration
of different cycles and doses of DSS in drinking water, mimics the pathologic effects of
both acute and chronic colitis.4 DSS affects the epithelial barrier integrity giving rise to
an inflammatory immune response to gut microbiota, and in consequence,
administration of DSS induces inflammation through T helper (Th) responses (Th1,
Th2, and Th17).5 Usually, we employ DSSꢀinduced ulcerative colitis in order to study
the antiꢀinflammatory effect of phenolic compounds present in food and/or medicinal
plants such as cocoa polyphenols,6 ellagic acid,7 oleuropein8 or apocynin.9 Previous in
vivo studies have demonstrated the potential of apocynin, or acetovanillone, as a
possible antiꢀinflammatory drug in experimental arthritis,10 airway inflammation,11,12
and experimental Crohnʼs disease.13,14 In a previous study,9 we found that the
supplementation of mice diet with 2% apocynin improved the course of colonic
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inflammation induced by DSS, as indicated by the decrease of the disease activity index
(DAI). Moreover, in treated colon tissues, apocynin significantly reduced the activation
of both nuclear transcription factor kappa B (NFꢀκB) p65 and signal transducer and
activator of transcription (STAT3). Taking this into account, the protective mechanism
of apocynin may be associated not only with its ability to reduce ROS generation via
NADPH oxidase, but also with its antiꢀinflammatory activity.15
Several reports have shown in vitro that its dimer diapocynin appears to be the main
active metabolite.16,17 Kanegae et al.17 have shown the relevance of some apocynin
metabolites for its biological activities, and demonstrated that the effects of this drug are
not only due to an enzyme inhibition, but also to preꢀtranscriptional events.
Increasingly, however, there are numerous studies demonstrating the higher
pharmacological potency of diapocynin. Ismail et al.18 demonstrated the capacity of
diapocynin, but not of apocynin, to inhibit ROS generation in skeletal muscle cells. One
relevant difference for the effect of diapocynin on different targets is the higher
lipophilicity of diapocynin vs. apocynin (about 13 times) enabling it to cross
membranes freely.19 Recently, Jantaree et al.20 have evaluated the antimetastatic effect
of the food additive divanillin, the homodimer of vanillin, and their structurally related
compounds apocynin and diapocynin. The assay showed that the dimeric forms are
more potent than those of vanillin and apocynin. Another study21 described that the antiꢀ
inflammatory activity of diapocynin was independent of its ROS modulating capability,
and Ghosh et al.22 demonstrated that it has a good pharmacokinetic profile when
administered orally, due to its liposolubility. These authors also demonstrated the
neuroprotective effect of diapocynin in models of Parkinson’s disease. 22
More recently, the hypotensive and vasodilator effect of diapocynin in normotensive
Wistar rats has been reported.23 It has been found that the antioxidant capacity of
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diapocynin is higher than that of apocynin, and both drugs, apocynin and diapocynin,
induced hypotensive effect without changing the heart rate. In intact mesenteric rings
NOS participates of relaxation induced by apocynin or diapocynin and both compounds
increased NO and decreased ROS levels in endothelial cells. On the other hand,
treatment with apocynin in lipopolysaccharide (LPS)ꢀstimulated RAW264.7
macrophage cells reduces the proꢀinflammatory mediators and cytokine release and
inhibits the nuclear translocation of NFꢀκB, through the inhibition of MAP kinase
signaling pathways24. In this study, we have chemically synthesized diapocynin and
evaluated in vitro its effect on proꢀinflammatory mediators released from RAW 264.7
macrophages stimulated with LPS, an useful tool to study inflammation which is
characterized by the expression of inflammatory cytokines and other mediators in
response to LPS, including NO, PGs, COX and iNOS that play an important role in
immune dysregulation occurred in inflammatory disorders of the gastrointestinal tract.
We have also assessed the antiꢀinflammatory activity of diapocynin in an in vivo model
of ulcerative colitis induced by DSS and compared its effects with those of apocynin.
The results support the hypothesis that diapocynin induces greater antiꢀinflammatory
effect than apocynin on inflammation.
MATERIALS AND METHODS
General Experimental Procedures. All chemicals, biochemical reagents and
peroxidaseꢀconjugated secondary antibodies were purchased from SigmaꢀAldrich (St.
Louis, MO, USA). The reagents and antibodies were purchased from: DSS (36ꢀ50 kD)
from MP Biomedical (Santa Ana, CA, USA), cell culture media and supplements from
Gibco (Life Technologies, Langley, VA, USA), antibodies recognizing iNOS, COXꢀ2,
GAPDH, p65, pꢀIκBꢀα, pꢀSTAT3 and βꢀactin from Millipore Corporation (Billerica,
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MA, USA). ELISA kits for cytokines were purchase from eBioscience (San Diego, CA,
USA). PGE2 levels were quantified using an EIA obtained from Cayman Chemicals
(San Diego, CA, USA).
Synthesis of Diapocynin. Diapocynin was synthesized by the protocol previously
described by Wang.25 Briefly, apocynin (4.0 g) was added to 400 mL of deionized water
and solved with the help of heat and stir. Once dissolved, a sulfate radical is generated
as oxidizing agent after the addition of ferrous sulfate heptahydrate (0.3 g) and sodium
persulfate (3.2 g). The brown precipitate was separated by filtration after cooling and
dissolved in 50 mL of 3M NH4OH and then reꢀprecipitated with 30 mL of 6 M HCl.
Diapocynin was filtered, washed with boiling water and boiling methanol and dried in
1
vacuum. Compound was identified by H NMR and MS, and data compared with
previously described in literature.25
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Cell Culture and Sample Treatment. RAW 264.7 macrophages (ECACC, Salisbury,
UK) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented
with 10% fetal calf serum, penicillin (100 U/mL), and streptomycin (100 ꢀg/mL) in a
humidified 5% CO2 atmosphere. Apocynin and diapocynin were dissolved in a mixture
of dimethyl sulphoxide (DMSO)/phosphateꢀbuffered saline (PBS, pH 7.4, 1:4 v/v) or
DMSO, respectively, to obtain 100 mM stock solution. Different concentrations of the
compounds were freshly prepared in complete medium just before addition to the cell
culture. Cells were cultured for 24 h (approximately 80 to 90% confluence) and then
treated either with vehicle (0.4% DMSO) or with various concentrations of products
under study, followed by stimulation with 1 ꢁg/mL of LPS for the indicated time. Dose
response curves of diapocynin (25, 50, 100 and 150 ꢀM) were used to determine the
50% inhibitory concentration (IC50) values for ROS, NO and PGE2 production caused
by LPSꢀstimulated cells.
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Cell Viability Assay. It was assessed by the MTT reduction assay.26 RAW 264.7 cells
were treated with apocynin or diapocynin (500‒100 ꢀM), and 24 h later absorbance was
TM
read at 490 nm using a microplate spectrophotometer (Victor
X3, 2030 Multilabel
Reader, Perkin Elmer, Italy). Cell viability was expressed in absolute absorbance
readings; a decrease in absorbance indicated a reduction in cell viability.
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Intracellular ROS Activity. Intracellular ROS production was measured using the nonꢀ
polar DCFHꢀDA dye.27 Cells were treated with apocynin or diapocynin (100 and 400
µM, final concentration) for 1 h and then stimulated with LPS (1µg/mL) for 24 h.
Fluorescence intensity (485 nm/535 nm, ex/em) was measured using a fluorescence
spectrophotometer (Victor TM X3, 2030 Perkin Elmer, Italy).
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Determination of NO, PGE2 and Pro-inflammatory Cytokine Production in Cells.
The RAW 264.7 cells were seeded at a density of 2 × 105 cells/well in 96ꢀwellꢀplates,
incubated for 1 h in the presence or absence of various concentrations of apocynin or
diapocynin (100 and 400 µM), and stimulated with LPS (1 ꢁg/mL) for 24 h. Nitrite
levels in culture media, that presumed to reflect NO levels, were determined using the
Griess reagent. Fresh culture medium was used as the blank in all experiments. Nitrites
in samples were measured at 540 nm after comparison with the serial dilution standard
curve of sodium nitrite.
The levels of PGE2, TNFꢀα, ILꢀ1β and ILꢀ6 were determined using the commercially
available kits, according to the manufacturer’s specifications. Cytokines content was
expressed as pg/mL per mg of protein.
Cytosolic and Nuclear Protein Extraction. RAW 264.7 macrophages were treated
with various concentrations of apocynin or diapocynin, and subsequently stimulated
with LPS (1 µg/mL) for determined times. Protein fractions of cytoplasm and nucleus
were extracted with the aid of the Proteojet kit (Fermentas, Thermo Scientific,
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Waltham, MA, USA), according to the manufacturer’s instructions, and their
concentration determined by Bradford’s method.28
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Animals. All protocols with animal were approved by the Institutional Ethics
Committee of the University of Valencia, Spain (108A1233134999884, 28ꢀ01ꢀ2009)
(2015/VSC/PEA/00023) and were carried out in accordance with the current guidelines
for the care and ethical treatment of laboratory animals. BALB/c mice (20ꢀ25 g),
obtained from Harlan Interfauna Iberica (Barcelona, Spain), were used to conduct DSSꢀ
induced acute colitis. All animals were maintained under a 12ꢀh light/dark cycle at 22
°C and 60% humidity for one week previous the experiments and feed with a standard
diet for mice and water ad libitum.
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Induction of Acute DSS Colitis and Treatment. Administration of 3% DSS (w/v)
solution induces an acute ulcerative colitis, supplied in fresh tap water for one week ad
libitum as previously was reported by Marín et al.9 BALB/c mice were randomly
distributed in four groups (n=6). Blank (mice received fresh tap water), control (mice
received 3% DSS in fresh tap water), DSS + apocynin (mice received 3% DSS in fresh
tap water and treatment with apocynin) and DSS + diapocynin (mice received 3% DSS
in fresh tap water and treatment with diapocynin). Both compounds (100 mg/kg) were
suspended in 0.1 M NaOH solution (0.2 mL) and administered by oral gavage on days
1, 3 and 5 from DSS feeding was initiated.
Variations in weight and water consumption were measured every day for one week.
The animals were sacrificed by means of cervical dislocation on day 8, the abdomen
was dissected, and the colon was removed. Samples from colon (three of each) were
sectioned longitudinally along the main axis and rolled from distal to mid, and then
were included in a solution of 4% formaldehyde for histological analysis. The rest of the
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colons were rinsed with cold PBS, blotted dry, weighed and measured in length.
Immediately the samples were frozen in liquid nitrogen and stored at −80 °C until use.
Histological analysis. Colon samples fixed in formaldehyde were embedded in
paraffin, sectioned longitudinally (5 µm), stained with hematoxylinꢀeosin, and eight
randomly selected fields in each section were observed at 100× and 200×, and evaluated
by an expert pathologist in a blinded mode.
Myeloperoxidase (MPO) activity assay. MPO activity in colon samples was
determined as previously described by Marín et al.9 using a Labsystems Multiskan EX
reader set at 630 nm. MPO activity was expressed as grams of enzyme required to
convert 1 ꢁmol of H2O2 to water in 1 min, per grams of wet weight of wet tissue.
Measurement of IL-6 in tissue extracts. Tissue from colons were cultured in an iceꢀ
cold extracting buffer (1:5 v/v), consisting of 0.1% Igepal CAꢀ630 nonionic detergent
(Sigma, St. Louis, MO, USA) in PBS with added protease inhibitor cocktail (Complete
Mini; Roche Applied Science, Indianapolis, Ind., USA). After homogenization, samples
were centrifuged at 12,000 ×g at 4 °C for 20 min, supernatants were collected and
stored at –80 °C until use. The concentration of ILꢀ6 in the colon tissue culture was
measured with an ELISA kit (eBioscience).
Preparation of Cytosolic Fractions in Tissue Extracts. Protein extraction from
cytosolic fractions was performed as previously described by Andújar et al.29 In brief,
tissues from colon samples were homogenized for 1 min with a polytron tissue
homogenizer (Kinematica AG, Lucerne, Switzerland) in ice cold buffer. Then, 0.5%
Nonidet NPꢀ40 solution was added and incubated on ice with gentle shaking for 45 min.
The membrane fraction was precipitated by centrifugation at 106 ×g for 5 min and the
cytosolic fraction (supernatant) stored at −80 °C until use.
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Western Blot Analysis. Proteins (25 ꢁg of each fraction) were separated by 10%
sodium dodecyl sulfateꢀpolyacrylamide gel electrophoresis and transferred onto
polyvinylidenedifluoride membranes (Amersham HybondꢀP, GE Healthcare, Little
Chalfont, UK). After hybridation with primary antibodies, blots were subsequently
incubated with horseradish peroxidase (HRP)ꢀconjugated secondary antibody. To prove
equal loading, the blots were analyzed for βꢀactin, GADPH or PARPꢀ1 expression. The
immunoreactive bands were visualized with an enhanced chemiluminiscence system
(Millipore Corporation). The images were acquired with the image analysis system
LASꢀ3000 mini (Fujifilm Corp., Tokyo, Japan). The density of bands was measured
with a Multi Gauge V3.0 software package (Fujifilm). Data from the control group were
taken as a reference and assigned the value 100, relative percentages of the other groups
were then calculated and represented graphically.
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Statistical analysis
All results are expressed as arithmetic mean ± standard error of the mean (S.E.M). Data
were evaluated with Graph Pad Prisms version 5.01 Software (La Jolla, San Diego,
CA). Differences among means were tested for statistical significance using oneꢀway or
two way analysis of variance (ANOVA), followed by post hoc significance Dunnett’s tꢀ
test or Bonferroni test, respectively.
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RESULTS AND DISCUSSION
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Diapocynin Decreased NO/PGE2 Production and iNOS/COX-2 Expression in LPS-
Stimulated RAW 264.7 Cells
RAW 264.7 murine macrophages stimulated with LPS are considered an appropriate
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model in which to identify molecular targets of antiꢀinflammatory agents. In the
inflammatory processes that occur upon LPS stimulation, the proꢀinflammatory
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mediators NO and PGE2 are generated by iNOS and COXꢀ2, respectively.29 Therefore,
after demonstrating that neither apocynin nor diapocynin at 400 ꢁM exhibited any
cytotoxicity on RAW 264.7 cells in the 3ꢀ[4,5ꢀdimethylthiazolꢀ2ꢀyl]ꢀ2,5ꢀdiphenylꢀ
tetrazolium bromide (MTT) test (data not shown), we examined the influence of
treatment with both compounds on the production of NO and PGE2 as well as on the
expression of iNOS and COXꢀ2 induced by LPS exposure. We found that diapocynin
was more effective than apocynin in inhibiting NO production, with a 50% inhibitory
concentration (IC50) of 80 µM (coefficient of determination: r2 = 0.86, p < 0.01) (Figure
2c). According to our findings, the highest dose of diapocynin strongly reduced iNOS
expression by 88% (Figure 2a). In addition, treatment with diapocynin demonstrated a
significant inhibitory effect on COXꢀ2 expression (Figure 2b) as well as on PGE2
production, with an IC50 of 56 µM (r2 = 0.99, p < 0.01) (Figure 2d). However, the
effects of apocynin on NO production and iNOS expression were controversial. We
have shown that apocynin inhibited NO production at the highest dose of 400 µM
(Figure 2c), although the iNOS expression in treated cells was not reduced, even it was
not significative increased. In a previous study30 was described that in vitro treated cells
with apocynin at 1 mM reduced NO production, iNOS expression and NFꢀκB
activation, but in vivo the intranasal application of apocynin did not exert any
significant effect on these inflammatory mediators. Riganti et al.31 observed in mouse
glial cells treated with different concentrations of apocynin, an increased synthesis of
NO and this was associated with increased expression of iNOS mRNA, increased
translocation of NFꢀκB to the nucleus and decreased intracellular level of IκB.
However, in our experimental conditions we have found that apocynin inhibited the NO
production at the higher concentration without affect to iNOS expression. This NO
reduction could be explained by a direct effect on the enzymatic activity of iNOS,30 or
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well, the NO released could react with the ROS induced by LPS generating
peroxinitrite.31
In response to LPS, macrophages rapidly induce the release of tumor necrosis factor
(TNF)ꢀα, which acts by inducing and perpetuating inflammatory autoimmune reactions
that activate both T cells and macrophages through the production of other cytokines
such as interleukin (IL)ꢀ6 and ILꢀ1β, along with adhesion molecules, which are
responsible for increasing the recruitment of leukocytes to the site of inflammation.32 In
our study, we observed that the levels of both TNFꢀα and ILꢀ6 released by LPSꢀ
stimulated RAW 264.7 cells decreased after incubation with apocynin or diapocynin,
but diapocynin was more effective than apocynin at 100 µM. In addition, in contrast to
apocynin, the dimer was also effective in inhibiting ILꢀ1β release by LPSꢀstimulated
macrophages (Table 1).
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Because most of these cytokines inhibited by apocynin and diapocynin are regulated
by NFꢀκB, it is possible that both compounds may target the transcription factor
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directly. To clarify this inhibitory mechanism on the NFꢀκB signaling pathway, we
assayed their effects on LPSꢀinduced translocation of p65 and IkBα phosphorylation.
Only diapocynin was found to reduce both LPSꢀinduced nuclear translocation of p65
(74% inhibition) (Figure 3a) as well as IκBα phosphorylation (56% inhibition) (Figure
3b). These results indicate that diapocynin blocks the activation factor by inhibiting the
phosphorylation of the inhibitor IκBα while also blocking the nuclear translocation of
the subunit p65. Although NFꢀκB is a central regulator of TNFꢀα and ILꢀ6 production,
apocynin could inhibited these cytokines through other molecules that are upꢀregulated
in LPSꢀactivated macrophages, such as the transcription factor cAMP response elementꢀ
binding protein (CREB), which may have a geneꢀspecific regulatory effect on the
production of these cytokines. In this way, Trevelin et al.33 have demonstrated in a
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luciferase reporter assay using RAW264.7ꢀLuc cells, that the treatment with apocynin
potentiated LPSꢀinduced NFꢀκB activation in a concentrationꢀdependent manner. In our
work, treatment with apocynin did not overactivated NFꢀκB, but neither had we
observed any significant inhibition of this transcriptional factor activation. Given this
result, it is obvious that inhibition of proꢀinflammatory cytokines production such as
TNFꢀα and ILꢀ6 should be mediated by additional mechanisms independent of NFꢀκB
signaling pathway.
Apocynin has previously been shown to inhibit production of ROS, either as an antiꢀ
oxidant and/or as an inhibitor of the p47phox component of NOX complexes.34 The effect
of apocynin and its dimer on ROS production in the present study was assessed with the
aid of the ROSꢀsensitive fluorescence indicator 2′,7′ꢀdichlodihydrorofluorescin diacetate
(DCFHꢀDA). As expected, LPS induced ROS generation relative to the untreated cells;
however, treatment with diapocynin significantly attenuated intracellular ROS
production, with an IC50 of 81 µM (r2= 0.98, p < 0.01). In contrast, apocynin had no
effect (Figure 4).
The absence of an effect for apocynin in in vitro studies may be due to the
environmental requirement that the apocynin form the dimer, which seems to be the
active form in the presence of ROS and MPO.35 Barbieri et al.36 found that the amount
of ROS induced by LPS in human monocytes was four times lower than that in
monocytes activated by 12ꢀOꢀtetradecanoylphorbolꢀ13 acetate (TPA) and zymosan
opsonizated, both of which are more effective factors in modulating the activation of
apocynin through peroxidase. The compound's lack of effect could thus be due to the
inability of LPS to induce the intense ROS generation necessary to form the dimer.
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This is the first study on the antiꢀinflammatory effect of diapocynin on LPSꢀinduced
proꢀinflammatory mediators in RAW264.7 cells, although another recent study has
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published24 the activity of apocynin in similar in vitro conditions. At the highest
concentration assayed (500 µM), the authors indicated that the antiꢀinflammatory
activity of apocynin occurred via the suppression of MAP kinase signaling pathways.
However, under our experimental conditions, apocynin showed no activity, while its
dimer diapocynin did exhibit antiꢀinflammatory effects.
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Anti-inflammatory effect of Apocynin and Diapocynin in vivo on DSS-Induced
Acute Colitis
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The experimental model of DSSꢀinduced colitis in mice is considered of interest for
the study of both conventional and novel biological therapies currently used to treat IBD
patients.37 As commented above, diapocynin could potentially offer greater protection
than apocynin if the former is indeed the active metabolite of the latter as it would
remove the necessity of peroxidase involvement that might limit the generation of
diapocynin in vivo. We found that apocynin and diapocynin both reduced disease
severity when orally administered at 100 mg/kg three times a week and attenuated the
macroscopic and histopathological alterations induced in the colon by DSS. These
results are consistent with our previous research, which found apocynin to be an
effective treatment for DSSꢀinduced colitis9, although in that case it was supplemented
in the standard diet of the subject mice at a concentration of 2%.
Orally administered apocynin and diapocynin, 100 mg/kg each, did not affect the
viability of BALB/c mice (data not shown). Mice given 3% DSS in fresh tap water for a
week developed signs of severe acute colitis without mortality. Macromorphologic
observations of colon specimens after one week of DSS treatment revealed significantly
shortened colon lengths compared to nonꢀcolitic mice. Oral treatment with apocynin
significantly decreased this effect by 25%. However, after oral treatment with
diapocynin, only a slight decrease in colon shortening was observed (Figure 5b),
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although it was not statistically significant compared with the DSS group. The dose of
test compounds used was 100 mg/kg equivalent to 0.6 and 0.3 mmol/kg of apocynin and
diapocynin, respectively. It is to say, the effective dose of diapocynin is a half of the
dose of apocynin. So the in vivo results indicate that the dimer has an antiꢀinflammatory
higher than apocynin. Similar in vitro results have been observed for the dimers of
vanillin and apocynin20. Divanillin and diapocynin decrease the metastatic potential of
HepG2 liver cancer cells, by using a target similar to vanillin and apocynin, but with
greater potency.
On the other hand, it has been demonstrated38 that diapocynin bound to human
albumin about 100ꢀfold higher than apocynin, so the oral bioavailability of diapocynin
is lesser than that for apocynin, and causes smaller effect in vivo.
Histological examination of colon tissue (Figure 5a) revealed that all DSSꢀtreated
mice displayed crypt distortion, destruction of the epithelium, and increased
inflammatory cellular infiltration, mostly in the distal colon (Figure 5a; B). Treatment
with apocynin (Figure 5a; C) and diapocynin (Figure 5a; D) induced protection as
evidenced by a reduction in inflammatory cell infiltration and maintained the colonic
architecture.
The MPO activity is considered a marker of inflammatory response in damaged
tissues, including in our case the colonic mucosa, which is related to neutrophil
infiltration. In the present study, whereas increases in MPO were observed in the colon
tissue of DSSꢀtreated mice, treatment with apocynin and diapocynin significantly
decreased neutrophil accumulation in colon tissue, with reductions of 56% and 75%,
respectively, in comparison with DSS mice (Figure 5c). These results are positively
correlated with the reduced neutrophil infiltration observed in colon section tissue.
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ILꢀ6 is a proꢀinflammatory cytokine that plays a critical role in the development of
IBD.39 After one week, ILꢀ6 levels in colon tissue increased markedly in DSSꢀtreated
mice in comparison with the blank group. Both apocynin and diapocynin significantly
inhibited ILꢀ6 production by 69% and 65%, respectively, with respect to mice given
DSS alone (Figure 6a).
As a central downstream component of ILꢀ6 signaling, STAT3 has been implicated
in the development of colitis as well as in its evolution towards dysplasia and cancer.40
Since activation of this factor plays a relevant role in promoting inflammation in DSSꢀ
induced colitis,41 suppression of STAT3 activity may thus be necessary for the
reduction of the inflammatory response in the colon. In this study, we observed
increased expression of pꢀSTAT3Y705 in DSSꢀinduced colitis; however, it was strongly
downꢀregulated by up to 57% and 63% after administration of apocynin and diapocynin,
respectively (Figure 6b). Thus, both compounds were able to suppress the ILꢀ6/STAT3
pathway with similar levels of effectivity.
Various studies have demonstrated a relevant role of COXꢀ2 and iNOS for
prolonging the epithelial secretory and barrier dysfunction, which favor the colonic
inflammation in mice.42,43 In our case, the Western blot study of colon tissues from the
DSS group showed a relevant increase in the expression of COXꢀ2 (Figure 7a) and
iNOS (Figure 7b), with apocynin significantly downꢀregulating the expression of both
proteins by 29% and 27%, respectively. Oral treatment with diapocynin also decreases
COXꢀ2 and iNOS colonic protein expression by 24% and 39%, respectively; however,
only the reduction of iNOS reached statistical significance. Nevertheless, after Western
blot analysis of tissue homogenates in other in vivo models of inflammation44 (data not
shown), we have determined that diapocynin reduces inflammation through the
suppression of COXꢀ2 expression.
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The biochemical analyses of biopsies from the colon of mice treated with apocynin
and its dimer were very similar. However, the inhibitory effects of apocynin on proꢀ
inflammatory mediators from LPSꢀactivated macrophages were much lesser than that of
diapocynin. Studies on the pharmacokinetic of apocynin with the aid of LCꢀMS/MS45
demonstrated that apocynin does not generally convert to its dimeric form diapocynin.
Wang et al.25, after intraperitoneal administration of apocynin, observed rapid
glycosylation of the compound in vivo and its transport to the blood and other organs
without conversion to diapocynin. These findings reinforce the evidence that the
beneficial properties of apocynin are not related to its antiꢀoxidant activity. On the other
hand, recently Potje et al.23 have studied the antioxidant effect and the efficacy on blood
pressure reduction of diapocynin and compared to apocynin. The authors found that,
despite in vitro diapocynin antiꢀoxidant effect was higher than apocynin, the efficacy on
blood pressure reduction or its potency as vasodilator in vivo were reduced when
compared to apocynin.
Different studies have used apocynin9ꢀ14 and diapocynin17,18,22 as treatment of
experimental inflammation models to evaluate their molecular mechanism. In our study,
we have tried: 1) To elucidate the activity of diapocynin on inflammatory stimulated
cells, 2) To evaluate its antiꢀinflammatory effect on acute colitis induced by DSS in
mice and 3) To contribute to the knowledge that apocynin is not a prodrug of
diapocynin.
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plasma protein binding evaluation of NADPHꢀoxidase inhibitor apocynin using
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Apocynin
Diapocynin
Figure 1. Chemical structures of apocynin and diapocynin
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Figure 2. Effect of apocynin and/or diapocynin on LPSꢀinduced inflammatory
parameters in RAW 264.7 cells. (a) iNOS and (b) COXꢀ2 expressions and (c) NO
and (d) PGE2 production. The corresponding densitometry analysis is shown
above each Western blot. Each bar is the mean ± S.E.M. of four different
#
experiments in triplicate. *p<0.05, ***p<0.001 relative to LPS group. p<0.05
relative to LPS + apocynin 400 µM group (Bonferroni postꢀtest to compare
replicate means by row).
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Figure 3. Effect of apocynin and diapocynin on NFꢀκB signaling in LPSꢀstimulated
RAW 264.7 cells. Representative western blots and bar diagrams showing densitometric
analyses of (a) NFꢀκB (p65) and (b) phosphoꢀIκBα. Protein expression levels were
measured 30 min after LPS stimulation. PARPꢀ1 and GADPH were used as loading
control for nuclear and whole extracts, respectively. Results are shown as the mean ±
S.E.M. (n=4). *p<0.05 vs LPS group (Dunnett’s tꢀtest).
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40
20
0
,#
*
,#
*
LPS (1ꢀg/mL)
Apocynin
-
-
+
-
+
+
400
-
+
+
100
-
-
-
(ꢀM)
(ꢀM)
Diapocynin
-
-
100
400
Figure 4. Effect of apocynin and diapocynin on LPSꢀinduced intracellular ROS
production by RAW 264.7 cells. Each bar represents the mean ± S.E.M. (n = 8).
#
*p<0.05 relative to LPS group. p<0.05 relative to LPS + apocynin 400 µM group
(Bonferroni postꢀtest to compare replicate means by row).
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Figure 5. Effects of apocynin and diapocynin on DSSꢀinduced (a) histological
manifestations, (b) colonic shortening and (c) MPO activity in colon tissue. Four
representative hematoxylin/eosin sections from blank (A), control (B), DSS +
apocynin (C), and DSS + diapocynin (D) groups. Original magnification: 100×.
Data are expressed as mean ± S.E.M. (n=3ꢀ6). *p<0.05, **p<0.01 relative to DSS
group; calculated with a oneꢀway ANOVA followed by Dunnett’s t test
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Figure 6. Effects of apocynin and diapocynin on DSSꢀinduced (a) ILꢀ6 production and
(b) cytosolic STAT3 phosphorylation in colon tissue. Each bar represents the mean ±
SEM (n = 3ꢀ6). **p<0.01 relative to DSS group; calculated with a oneꢀway ANOVA
followed by Dunnett’s t test.
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Figure 7. Effects of apocynin and diapocynin on the (a) COXꢀ2 and (b) iNOS
expressions in DSSꢀinduced acute colitis in mice. The corresponding densitometry
analyses is shown above each Western blot. Each bar represents the mean ± SEM
(n = 3ꢀ6).*p<0.05, **p<0.01, significantly different from the DSS group;
calculated with a oneꢀway ANOVA followed by Dunnett’s t test.
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