ORIGINAL ARTICLE
Mangiferin ameliorates the intestinal inflammatory response
and the impaired gastrointestinal motility in mouse model
of postoperative ileus
Talita Cavalcante Morais&Bruno Rodrigues Arruda&Hebert de Sousa Magalhães& Maria Teresa Salles Trevisan&Daniel de Araújo Viana&
Vietla Satyanarayana Rao&Flavia Almeida Santos
Received: 12 August 2014 / Accepted: 19 January 2015 / Published online: 5 February 2015
#Springer-Verlag Berlin Heidelberg 2015
Abstract Our previous study has shown that mangiferin
(MGF), a glucosylxanthone fromMangifera indica, exerts
gastrointestinal prokinetic action involving a cholinergic mechanism. Postoperative ileus (POI) is a temporary distur-bance in gastrointestinal motility following surgery, and intes-tinal inflammatory response plays a critical role in the patho-genesis of POI. The present study investigated to know wheth-er MGF having anti-inflammatory and prokinetic actions can ameliorate the intestinal inflammation and impaired gastroin-testinal transit seen in the mouse model of POI. Experimental POI was induced in adult male Swiss mice by standardized small intestinal manipulation (IM). Twenty-four hours later, gastrointestinal transit was assessed by charcoal transport. MGF was administered orally 1 h before the measurement of GIT. To evaluate the inflammatory response, plasma levels
of proinflammatory cytokines TNF-α, IL-1β, IL-6, and
che-mokine MCP-1, and the myeloperoxidase activity, nitrate/ nitrite level, and histological changes of ileum were deter-mined in mice treated or not with MGF. Experimental POI in mice was characterized by decreased gastrointestinal transit and marked intestinal and systemic inflammatory response. MGF treatment led to recovery of the delayed intestinal transit
induced by IM. MGF in ileum significantly inhibited the myeloperoxidase activity, a marker of neutrophil in-filtration, and nitrate/nitrite level and reduced the plasma
levels of TNF-α, IL-1β, IL-6, and MCP-1 as well.
MGF treatment ameliorates the intestinal inflammatory re-sponse and the impaired gastrointestinal motility in the mouse model of POI.
Keywords Postoperative ileus . Mangiferin . Intestinal inflammation . Gastrointestinal transit
Introduction
Postoperative ileus (POI) refers to the time after open abdom-inal surgery (generally >5 days) before coordinated electromotor bowel function resumes, resulting in gut inflam-mation that leads to impaired motility of the entire gastroin-testinal tract or selectively the stomach, small intestine, or
colon (Boeckxstaens and de Jonge2009; Johnson and Walsh
2009). The impaired contractility and motility in POI is
pos-tulated to be multifactorial, with principal mediators being inflammatory cell activation, autonomic dysfunction, activa-tion of gut opioid receptors, modulaactiva-tion of gastrointestinal hormone activity, and electrolyte derangements. POI slows recovery, increases the risk of developing postoperative com-plications, and confers a significant financial burden on
healthcare institutions (Vather et al.2014). Currently used
drugs for the treatment of POI that include neostigmine, a reversible acetylcholinesterase inhibitor, and alvimopan, a
se-lective and peripherally acting μ-opioid receptor antagonist,
although are proved to be effective in shortening POI (Fucuda
et al.2006; de Giorgio and Knowles2009), have undesirable
side effects, and higher cost limits their usefulness (Becker T. C. Morais:B. R. Arruda
:
V. S. Rao:
F. A. Santos (*)Department of Physiology and Pharmacology, Faculty of Medicine, INCT–IBISAB–Brazilian Semi-Arid Institute of Biomedicine, 60430-270 Fortaleza, Ceará, Brazil
e-mail: fasufc@gmail.com
H. de Sousa Magalhães:M. T. S. Trevisan
Department of Organic and Inorganic Chemistry, Federal University of Ceará, 60451-970 Fortaleza, Ceará, Brazil
D. de Araújo Viana
and Blum2009; Johnson and Walsh2009; Yeh et al.2009). Therefore, novel therapeutic strategies that target the POI war-rant further investigation.
Mangiferin (1,3,6,7-tetrahydroxy-2-[3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]xanthen-9-one) (MGF) is a nat-ural polyphenol found in many plant species, among which the mango tree (Mangifera indica) is one of the primary sources. Mangiferin has been reported to exhibit
immuno-modulatory (Rajendran et al.2013), anticytotoxic (Rao et al.
2012), antiviral (Zheng and Lu 1990), antioxidant
(Matkowski et al. 2013), antidiabetic (Sellamuthu et al.
2013), gastroprotective (Carvalho et al. 2007), prokinetic
(Cavalcante Morais et al.2012), radioprotective (Menkovic
et al.2010), hepatoprotective (Das et al.2012), and
anti-inflammatory (Gong et al.2013) properties. Since POI is
char-acterized by impaired gut motility and intestinal inflammation
(de Winter2003; van Bree et al.2014), this study investigated
whether MGF could ameliorate intestinal inflammation and impaired gastrointestinal transit seen in the mouse model of POI.
Materials and methods
Plant material and isolation of mangiferin
M. indicawas collected from the field localized in Beberibe, Ceará, Brazil by Luquesio Petrola de Melo Jorge. The plant was identified and authenticated by Dr. Francisco Edson de Paula, from Herbário Prisco Bezerra, Federal University of Ceará (voucher specimen number 32628). Mango root bark powder (35 kg) was extracted with 95 % ethanol for about 120 h by percolation and the resulting extract obtained was filtered and evaporated to dryness at 40 °C by rotary evapo-ration under reduced pressure. The extract was dissolved in methanol, and after cooling a yellow precipitate (42 g) was obtained. The yellow precipitate was approximately 98 %
pu-rity; mass spectrometric analysis gave a [M-H]−ion atm/z
421 and typical fragmentation behavior of C-glycoside with fragment ions at 301 (M-H-120 Da) and 331 (M-H-90 Da), melting point 271 °C, and after NMR was identified as
mangiferin (MGF; Fig.1) as reported earlier (Barreto et al.
2008).
Animals
Swiss albino male mice (20–25 g) were used in this
study. They were housed in environmentally controlled
conditions (23± 2 °C, 12-h light–dark cycle, and
humid-ity 55± 10 %), with free access to a standard diet and water ad libitum. Mice were fasted for 15 h prior to the experiments, but allowed free access to water. The In-stitutional Animal Care and Use Committee for Federal University of Ceará approved the study protocol (no. 31/11), and all animal experiments were performed ac-cording to the Regulations for the Care and Use of Laboratory Animals in Federal University of Ceará, Fortaleza.
Experimental study of postoperative ileus (POI)
POI was induced in four groups of mice (n=6) as per
proce-dures described earlier (Li et al.2013; Mueller et al.2011). In
brief, under anesthesia [intraperitoneal (i.p.) injection of a mixture of ketamine (Ketalar® 100 mg/kg) and xylazine (Rompun® 10 mg/kg)], animals were laparotomized and followed by sham operation (group 4) or small bowel manipulation (groups 1, 2, and 3). The small bowel was pulled out gently onto moist gauze and systematically manipulated from the ligament of Treitz to the terminal ileum for 5 min with two moist cotton applicators to induce postoperative ileus. The laparotomy was closed with a running suture, and all animals recovered quickly from surgery and generally began to eat and drink with-in several hours after surgery. Surgery was performed under sterile conditions.
Gastrointestinal transit and inflammatory responses of POI were investigated 24 h after surgery. Blood and ileum were collected for analysis that included determination of cytokine and chemokine levels in mouse plasma by ELISA as well as myeloperoxidase activity, nitrate/nitrite level, and histological changes in ileum.
Gastrointestinal transit
POI mice were treated as follows: group 1 received 10 mL/kg vehicle (the diluent of mangiferin, 2 % DMSO in distilled water); groups 2 and 3 were treated orally with mangiferin in doses of 30 or 100 mg/kg, respectively, whereas group 4 remained sham operated and received no treatment. Forty-five minutes following treatments, gastrointestinal transit was measured using the charcoal propulsion test (Capasso et al.
1976). Each mouse was orally given 0.1 mL of charcoal meal
from pylorus to the ileo-cecal junction) and expressed as follows:
Gastrointestinal transit %ð Þ
¼ distance traveled by the charcoal
.
total length of the small intestine 100:
Myeloperoxidase activity
The degree of neutrophil infiltration was quantified by the mea-surement of myeloperoxidase (MPO) activity (Bradley et al.
1982). Ileum tissue (50 mg) from each animal was minced and
homogenized in 0.5 mL of 50 mmol/L phosphate buffer solution (PBS) (pH 6) that contained 0.5 % HTAB. The homogenate was
subjected to three cycles of freezing (−70 °C) and thawing
(37 °C) and brief periods (15 s) of sonication, after which they
were centrifuged at 12,000×gfor 15 min at 4 °C. The supernatant
(0.1 mL) was mixed with 2.9 mL of 50 mmol/L PBS (pH 6),
which contained 0.167 mg/mLo-dianisidine dihydrochloride
and 0.0005 % hydrogen peroxide. The change in absorbance at 470 nm was then measured for 5 min using a spectrophotometer.
Nitrate/nitrite level
Nitric oxide levels were measured as total nitrate/nitrite levels
with the use of the Griess reagent (Green et al.1982). The ileum
was homogenized in 50 mM potassium phosphate buffer
(pH 7.8) and centrifugated at 11,000×g for 15 min at 4 °C.
One hundred microliters of the supernatant was mixed with
100μL Griess reagent [0.1 %N-(1-naphthyl) ethylenediamide
dihydrochloride, 1 % sulfanilamide in 5 % phosphoric acid] and after 10 min the absorbance was measured at 540 nm. The stan-dard curve was obtained by using sodium nitrite. The results were expressed as micromoles nitrate/nitrite per microgram of protein. The protein concentration of the sample was determined
by the Bradford assay (Bradford1976) and the nitrate/nitrite
levels were expressed as micromolar per microgram of protein.
ELISA assay
Plasma TNF-αand IL-6 (eBioscience, San Diego, CA, USA),
IL-1β, and MCP-1 (R&D Systems, Minneapolis, MN, USA)
were measured by enzyme-linked immunosorbent assay (ELISA). Assays were performed following the
manufac-turer’s instruction. The levels were calculated from the
stan-dard curve and expressed as picograms per milliliter.
Histological analysis
In order to investigate morphological changes of the gut dur-ing POI, histological evaluation was performed on ileum
samples. After washing with normal saline, intestinal samples were fixed in 4 % paraformaldehyde overnight and embedded
in paraffin. Thereafter, tissue slices (5μm) were stained with
hematoxylin and eosin (H&E) and evaluated under light microscopy.
NF-κB and iNOS immunohistochemistry
Immunohistochemical analysis of the expression of NF-κB
and inducible nitric oxide synthase (iNOS) was performed.
Sections of ileum (4 μm) were transferred to a
gelatin-coated slide. Sections were deparaffinized and rehydrated through xylene and graded alcohols and endogenous
peroxi-dase activity was blocked by incubation with 3 % H2O2
(10 min). Nonspecific protein binding was blocked by incu-bating the tissue sections with blocking solution (10 % goat serum, 0.2 % Triton X-100 in PBS) for 45 min. The slides were then incubated overnight with primary rabbit
anti-NF-κB (1:200; Santa Cruz Biotechnology, Santa Cruz, CA,
USA) or rabbit anti-iNOS (1:400; Sigma-Aldrich, St. Louis, MO, USA) diluted with blocking solution. Sections were rinsed three times for 10 min each in Tris-buffered saline and subsequently incubated for 2 h at room temperature, with goat anti-rabbit secondary antibodies conjugated with a fluorophore (Alexa Fluor 594, diluted 1:500 with blocking solution; Invitrogen, Carlsbad, CA, USA). After rinsing three times for 10 min in Tris-buffered saline, the sections were
stained with DAPI (4′,6-diamidino-2-phenylindole; Vector
Laboratories, UK, 1:250 dilution), rinsed, mounted with
Fluoromount (Sigma), and stored at −20 °C until analysis.
Negative-control sections were processed simultaneously as described above but with the first antibody being replaced by blocking solution. Sections were visualized with a laser scan-ning confocal microscope (LSM 510 Meta; Zeiss).
Sham Vehicle MGF 30 MGF 100 mg/kg 0
20 40 60 80 100
POI
a
b
b
G
a
st
ro
in
te
st
in
a
l t
ra
n
si
t (
%
)
Statistical analysis
Results are expressed as the mean±standard error of mean (SEM). Statistical evaluation was done by one-way analysis
of variance (ANOVA) followed by Student–Newman–Keuls
multiple comparisons test. A value ofp<0.05 was considered
statistically significant.
Results
POI in mice was characterized by decreased gastrointestinal transit accompanied by a marked intestinal and systemic in-flammatory response. POI caused a significant (p<0.05) de-crease (46.46±4.56 %) of gastrointestinal transit when com-pared to sham control (75.09±1.88 %). The oral treatment with
MGF (30 and 100 mg/kg) led to significant (p<0.05) recovery
of delayed intestinal transit induced by POI (Fig.2). In
vehicle-treated POI mice, there was a significant increase in MPO ac-tivity (75.04±8.84 U/g) in ileum tissue as compared to
sham-operated controls (Fig.3a) that was effectively (p<0.05)
re-duced in animals treated with MGF 30 mg/kg (36.07 ± 8.08 U/g) and 100 mg/kg (30.68±8.51 U/g). Nitrate/nitrite levels were significantly lower in POI animals treated with
MGF 30 mg/kg (2.29±0.44 μM/μg protein) and 100 mg/kg
(1.74±0.45 μM/μg protein) (Fig. 3b). Figure 4 depicts the
levels of inflammatory mediators TNF-α, IL-1β, and IL-6 in
mice on POI treated or not with MGF. MGF (30 and 100 mg/kg)
significantly (p<0.05) reduced the plasma levels of TNF-α
(14.27±5.08 and 16.45±6.15 pg/mL), IL-1β (49.07±1.86
0 20 40 60 80
a
b
b
Sham MGF 30mg/kg MGF 100mg/kgA
Vehicle (p g/m L ) 0 20 40 60 80 a b bB
Sham MGF 30mg/kg MGF 100mg/kg Vehicle IL -1 (p g /ml ) 0 100 200 300 400a
b
b
C
Sham MGF 30mg/kg MGF 100mg/kg Vehicle IL -6 (p g /ml ) 0 20 40 60 80a
b
b
Sham MGF 30mg/kg MGF 100mg/kgD
Vehicle M C P -1 (p g/ m l)Fig. 4 Effect of mangiferin (MGF) on serum TNF-α(a), IL-1β(b), IL-6 (c), and MCP-1 (d) in mice on POI. Results are represented as mean±SEM.n=6. a
p<0.05 compared with sham group,bp<0.05 compared with vehicle group (ANOVA and Student–Newman–Keuls test)
Sham Vehicle MGF 30 MGF 100 mg/kg 0 20 40 60 80 100 POI
A
a
b
b
M P O a cti v it y (U / g we t t is su e)Sham Vehicle MGF 30 MGF 100 mg/kg 0 1 2 3 4 5
a
b
b
POIB
Ni tr a te /n itr it e l ev e l ( M/m g p r ot e in )Fig. 6 Effect of mangiferin on activation of NF-κB in postoperative ileum in mice. iNOS positive immunoreactivity was revealed by Alexa Fluor 594 (red staining), the blue
fluorescence represents DAPI nuclear staining of double-stranded DNA, and merged images of the
immunofluorescence of iNOS and DAPI. Cells viewed at ×400 magnification
A
C
B
D
Fig. 5 Histomorphologicaland 47.45±5.65 pg/mL), and IL-6 (150.80±55.00 and 127.60 ±32.59 pg/mL) compared to respective vehicle control (54.44±
7.71, 71.41±5.61, and 302.20±46.57 pg/mL) (Fig.4a–c). The
levels of chemokine MCP-1 was also greatly reduced in
MGF-treated animals at both doses (Fig.4d).
Histological examination of the ileum from sham-operated control showed normal architecture and absence of
inflamma-tory cells (Fig.5a). In contrast, ileum sections from the POI
group revealed mild inflammatory response infiltrated with neutrophil and macrophage cells in the muscular layer
(Fig.5b). Treatment with mangiferin (30 and 100 mg/kg,
p.o.) reduced the inflammation and protected the ileus from
histological damage induced by POI (Fig.5c, d).
To provide further insight into the effects of mangiferin on the
NF-κB and iNOS inhibition, we performed an
immunohisto-chemical analysis on paraffin-embedded ileum tissue using
NF-κB and iNOS antibodies. Mangiferin treatment reduced the
NF-κB and iNOS immunoreactivity in the ileum (Figs.6and7).
Discussion
Postoperative ileus is a temporary disturbance in gastric and bowel motility following surgery that increases the risk of
postoperative complications and morbidity (Stoffels et al.
2014). This study investigated the beneficial effects of
mangiferin (MGF), a glucosylxanthone in murine model of postoperative ileus (POI). In this animal model, we observed inflammatory responses characterized by leukocyte infiltra-tion in the intestinal tissue, an elevated level of inflammatory mediators in plasma, and a significant delay in gastrointestinal transit 24 h after intestinal manipulation, consistent with
ear-lier reports (Kalff et al.1998,1999). Oxidative stress plays a
key role in the pathogenesis of POI and mangiferin is a known anti-inflammatory and antioxidant agent. The xanthonoid structure of mangiferin with C-glycosyl linkage and polyhy-droxy components contributes to its free radical-scavenging ability, leading to a potent antioxidant effect as well as multi-ple biological activities including anti-inflammatory and
prokinetic actions (Matkowski et al.2013; Cavalcante Morais
et al.2012).
The recovery of gastrointestinal transit is considered the hallmark of clinical outcome measure in POI (van Bree et al.
2014). Therefore, we focused on studying the effect of MGF
on gastrointestinal transit. In the present study, MGF by virtue
of its antioxidant (Matkowsky et al.2013), anti-inflammatory
(Gong et al.2013), and prokinetic actions (Cavalcante Morais
et al. 2012) might have caused effective suppression of
Fig. 7 Effect of mangiferin on activation of iNOS in postoperative ileum in mice. iNOS positive immunoreactivity was revealed by Alexa Fluor 594 (red staining), the blue
fluorescence represents DAPI nuclear staining of double-stranded DNA, and merged images of the
intestinal inflammatory response and reversal of delayed gas-trointestinal transit. Past studies reveal that Daikenchuto, a
traditional herbal medicine (Tokita et al.2007), Jatrorrhizine,
a protoberberine alkaloid isolated from the medicinal plants Berberis aristataandCoptis chinensis (Zhang et al.2012), and neostigmine, a reversible inhibitor of acetylcholinesterase
(Luckey et al.2003), could overcome the delayed
gastrointes-tinal transit in rat postoperative ileus. The use of several prokinetics may overcome the delayed gastrointestinal transit during POI, but routine administration of prokinetics for pre-vention of postoperative ileus is not recommended as their effectiveness is compromised by the inflammatory process
(Traut et al.2008). It is well accepted that intestinal
inflam-mation caused by surgical stress plays an important role in the
development and progression of POI (Stoffels et al.2014; van
Bree et al.2013; Li et al.2013). To understand MGF
mecha-nism of action on gastrointestinal transit, this study analyzed the inflammatory response by studying MPO activity and the levels of nitrite nitrate in ileal tissue as well as the plasma
levels of TNF-α, IL-6, IL-1β, and MCP-1. Consistent with
earlier findings, a significant increase in the levels of these proinflammatory cytokines was observed in serum of POI
animals (Stoffels et al.2014; Endo et al.2013; Wehner et al.
2005). These inflammatory mediators may contribute to the
decreased gastrointestinal motility through various mecha-nisms such as direct cytotoxic effects and induction of nitric
oxide (NO) and prostanoids (Kalff et al.2000; Wehner et al.
2012). MPO is an enzyme abundantly stored in azurophilic
granules of neutrophils and is released into extracellular fluid in the inflammatory process. Therefore, MPO activity is a prognostic biomarker for inflammatory response in a variety of acute and chronic inflammatory conditions (Nussbaum
et al.2013). Thus, an intestinal inflammatory response may
be responsible for delayed gastrointestinal transit seen in POI after intestinal manipulation in mice. Agents that suppress the development of intestinal inflammation are likely to be effec-tive in the treatment of POI. In this regard, this study evi-denced that MGF administered orally could effectively pre-vent the inflammatory response induced in POI mice through suppression of inflammatory mediators, MPO activity, and nitrate/nitrite levels, and thereby the impaired gastrointestinal motility. Histological studies on ileum tissues further con-firmed the anti-inflammatory nature of MGF.
In conclusion, our results suggest that intestinal inflamma-tion and impaired gastrointestinal transit account for intestinal manipulation-induced POI. MGF administered orally could prevent this inflammatory response and ameliorate gastroin-testinal motility during POI, suggesting its likely clinical util-ity to shorten POI.
Acknowledgments This research was supported by the grants and fel-lowships from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico
e Tecnológico (CNPq), and Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP). We are grateful to Aguinéa Rocha de Morais for their excellent technical assistance. We are also grateful to Prof. Geanne Matos de Andrade, Kelly Rose Tavares Neves, and Francisco Arnaldo Viana Lima for immunohistochemical analysis.
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