Role of Sulforaphane in Protection of Gastrointestinal Tract Against H. pylori and NSAID-Induced Oxidative Stress

Abstract

Background:
Sulforaphane (SFN), a phytochemical found in abundance in broccoli sprouts, potently induces a variety of antioxidant enzymes, and thereby protects cells from injury induced by various kinds of oxidative stresses. It has been suggested that both H. pylori infection and intake of non-steroidal anti-inflammatory drugs (NSAIDs) induce chronic oxidative stress in gastrointestinal (GI) mucosa, thereby causing mucosal injury in the GI tract. Therefore, it would be a reasonable assumption that SFN protects GI mucosa against oxidative injury induced by H. pylori or NSAIDs.

Methods:
We examined the effects of SFN on H. pylori viability in vitro, levels of gastritis in H.pylori-infected mice in vivo, and in H.pylori-infected human subjects. We also examined the effects of SFN on NSAID-induced small intestinal injury in mice.

Results:
Our data from the H. pylori infection study clearly demonstrated that SFN inhibited H. pylori viability both in vitro and in vivo, and mitigated H. pylori-induced gastritis in mice and humans. Similarly, our study on NSAID-induced small intestinal injury showed that SFN not only mitigated aspirin-induced injury of small intestinal epithelial cells in vitro, but also ameliorated indomethacin-induced small intestinal injury in mice in vivo.

Conclusions:
These data strongly suggest that SFN contributes to the protection of GI mucosa against oxidative injury induced by H. pylori or NSAIDs.

Full text

Current Pharmaceutical Design
Impact
Factor:
3.45
ISSN: 1381-6128
eISSN: 1873-4286
BENTHAM
SCIENCE
no.
Akinori Yanaka*
Division of Gastroenterology, Hitachi Medical Education and Research Center, Faculty of Medicine, University of Tsukuba, 2-1-1,
Jonan-cho, Hitachi-shi, Ibaraki-ken, 317-0077, Japan
A R T I C L E H I S T O R Y
Received: January 5 , 2017
Accepted: February 2 , 2017
DOI:
10.2174/1381612823666170207103943
Abstract: Background: Sulforaphane (SFN), a phytochemical found in abundance in broccoli sprouts, potently
induces a variety of antioxidant enzymes, and thereby protects cells from injury induced by various kinds of oxi-
dative stresses. It has been suggested that both H. pylori infection and in take of non-steroidal anti-inflammatory
drugs (NSAIDs) induce chronic oxidative stress in gastrointestinal (GI) mucosa, thereby causing mucosal injury
in the GI tract. Therefore, it would be a reasonable assumption that SFN protects GI mucosa against oxidative
injury induced by H. pylori or NSAIDs.
Methods: We examined the effects of SFN on H. pylori viability in vitro, levels of gastritis in H.pylori-infected
mice in vivo, and in H.pylori-infected human subjects. We also examined the effects of SFN on NSAID-induced
small intestinal injury in mice.
Results: Our data from the H. pylori infection study clearly demonstrated that SFN inhibited H. pylori viability
both in vitro and in vivo, and mitigated H. pylori-induced gastritis in mice and human s. Similarly, o ur study on
NSAID-induced small intestinal injury showed that SFN not only mitigated aspirin-induced injury of small intes-
tinal epithelial cells in vitro, but also ameliorated indomethacin-induced small intestinal injury in mice in vivo.
Conclusions: These data strongly suggest that SFN contributes to the protection of GI mucosa against oxidative
injury induced by H. pylori or NSAIDs.
Keywords: Sulforaphane, Helicobacter pylori, stomach, aspirin, indomethacin, small intestine.
1. INTRODUCTION
Sulforaphane (SFN), an abundant antioxidant phytochemical
found in broccoli sprouts (BS), potently induces a variety of anti-
oxidant enzymes, which protect cells and organs against various
kinds of oxidative stresses [1]. Recent studies have shown the anti-
bacterial activity of SFN ag ainst Helicobacter pylori (H. pylori) in
vitro [2]. We have recently shown that SFN induces antioxidant
enzymes in the gastro-intestinal (GI) tract of mice, and protects GI
mucosa against in juries induced by H. pylori and NSAIDs [3-5].
Our studies show that SFN not only enhances the antioxidant activ-
ity of GI mucosa, but also demonstrates antibacterial activity
against H. pylori in gastric mucosa and anaerobic bacteria in the
small intestine of mice [5]. We have conducted clinical trials with
H. pylori-infected human subjects who consume BS, and have
shown that SFN clearly inhibits H. pylori activity and mitigates H.
pylori-induced gastritis [4]. This review introduces our recent data
on the protective effects of SFN, which demonstrate that SFN pre-
vented H. pylori- and NSA ID-induced GI mucosal injury.
2. ROLE OF SFN IN PROTECTION OF THE CELLS
AGAINST OXIDATIVE STRESS
It has been suggested that environmental factors, especially
dietary factors, are more important in the development of GI cancer
than genetic factors [6,7]. Recent studies have clearly associated the
development of gastric cancer not only with H. pylori infection
[8,9], but also with the intake of a high salt diet [10,11]. In contrast,
it has also been reported that daily intake of fruits and vegetables
*Address correspondence to this author at the Division of Gastroenterology,
Hitachi Medical Education and Research Center, Faculty of Medicine, Uni-
versity of Tsukuba, 2-1-1, Jonan-cho, Hitachi-shi, Ibaraki-ken, 317-0077,
Japan; Tel: +81-294-23-1111, Fax: +81-294-23-8767;
E-mail: ynk-aki@md.tsukuba.ac.jp
decreased the risk of developing GI cancers [6,12]. However, the
mechanisms by which intake of fruits and vegetables decreased the
risk of GI cancers were not clarified until recently. It is naturally
important to id entify the protectiv e substances from the various
phytochemicals in fruits and vegetables. Furthermore, it is neces-
sary to clarify the mechanisms by which each substance protects the
GI tract against oxidative injury. Finally, the effects of such sub-
stances on human health should be examined through well-designed
clinical trials.
SFN has been studied extensively in the past two decades, and
several studies have suggested the possibility that SFN may con-
tribute to cancer chemoprotection [13]. SFN, a member of the Iso-
thiocyanate (ITC) family, is abundant in cruciferous vegetables, in
particular Broccoli Sprouts (BS) [13,14]. Previous studies have
shown that the unique molecular functional group -N=C=S is com-
mon to ITCs, which accounts for the pungency of these vegetables
[15] (Fig. 1). Different ITCs have been reported in a variety of
foods. For example, Wasabi and mustard oils are rich in allyl ITCs
[15]. Phenethyl ITC and 4-(methylthio)-3-butenyl ITC are found
abundantly in Daikon and Cresson, respectively [16]. As previously
stated, BS are rich in SFN. SFN potently induces v arious antioxi-
dant (or phase 2) enzymes, such as glutathione S-transferase (GST),
heme oxygenase-1 (HO-1), and NAD(P)H: quinone oxidoreductase
1 (NQO1), via Nf-E2 related factor 2-Kelch-like ECH-associated
protein 1 (Nrf2-Keap1)-dependent pathways (17), thereby enhanc-
ing the antioxidant activity of the cells in th e GI tract [4,5] (Fig. 2).
In raw BS, SFN exists in the biologically inactive form of sul-
foraphane glucosinolates (SGS) [14]. The transformation of SGS to
SFN occurs during the chewing process, where SGS is subjected to
the action of myrosinase, also a component of BS [14]. SGS is also
transformed to SFN in the in testinal lum en, by myrosinase of the
intestinal microflora [14]. As previously stated, SFN upregulates
1873-4286/17 $58.00+.00 © 2017 Bentham Science Publishers
Send Ord ers for Reprints to reprin ts@bentham science.ae
4066
Current Pharmaceutical Design, 2017, 23, 4066-4075
REVIEW ARTICLE
Role of Sulforaphane in Protection of Gastrointestinal Tract Against H. pylori
and NSAID-Induced Oxidative Stress

Role of Sulforaphane in Protection of Gastrointestinal Tract Against H. pylori Current Pharmaceutical Design, 2017, Vol. 23, No. 27 4067
Fig. (1). Broccoli Sprouts Contai n High Concentration of Sulforaphane
(SFN)
SFN, a member of the isothiocyanate (ITC) family, is abundant in crucifer-
ous vegetables, especially broccoli sprouts (BS). (A) Fifty grams of raw BS
contain 128 mg of sulforaphane glucosinolate, a precursor of SFN. (B) SFN
possesses -N=C=S, which is a common chemical structure found in the ITC
family. This molecular structure accounts for the pungency of the crucifer-
ous vegetables.
phase 2 enzymes via N rf2-Keap1-depend ent mechanisms [17].
Under basal conditions, Nrf2 proteins are located in the cytoplasm
and are biologically inactive as Nrf2 is bound to the Keap1 protein.
Following exposure of the cells to oxidative stresses or SFN, Nrf2
proteins become dissociated from Keap1 and translocate into the
nucleus. After entering the nucleus, Nrf2 protein binds to the anti-
oxidant response element, and upregulates expression of a variety
of xenobiotic and antioxidant enzymes [18-20] (Fig. 2). It has been
shown that induction of the phase 2 enzymes by SFN lasts almost
for 72 h [17]. In addition to activation of the Nrf2-Keap1 pathway
within cells, SFN has also been shown to inhibit H. pylori viability
in vitro; these effects were demonstrated in both the clarithromycin-
sensitive and resistant strains [2]. Furthermore, other studies have
shown that SFN decreases colonization in mice stomach ex vivo
[21], and in a small number of human cases [22].
Fig. (2). Sulforaphane (SFN) Enhances Antioxidant Activity via Nrf2-
Keap1 System.
SFN potently induces various antioxidant (or phase 2) enzymes, such as
glutathione S-transferase (GST), heme oxygenase-1 (HO-1), and NAD(P)H:
quinone oxidoreductase 1 (NQO1), via Nf-E2 related factor 2 (Nrf2) -
Kelch-like ECH-associated protein 1 (Keap1) dependent pathways. These
are responsible for the enhanced antioxidant activity of the cells.
Based on these results, we aimed to determine if oral intake of
SFN contributed to the protection of GI mucosa against GI diseases
induced by oxidative stresses, such as those induced by H. pylori
and NSAIDs.
3. ROLES OF SFN IN PROTECTION AGAINST H. PYLORI-
INDUCED GASTRIC MUCOSAL INFLAMMATION
H. pylori infection induces chronic oxidative stress in gastric
mucosa, which eventually induces gastric cancer. However, SFN
has been shown to mitigate various kinds of oxidative stresses. In
this study, we aimed to determine if SFN inhibited H. pylori activ-
ity in vitro, and if SFN mitigated H. pylori-induced oxidative injury
in gastric mucosa in mice and humans in vivo.
3.1. Effect of SFN on Urease Activity and H. pylori Viability In
Vitro
In this series of experim ents, w e examined whether SFN
showed direct antibacterial activity against H. pylori in vitro [23].
The H. pylori strain Sydney Strain-1 (SS-1) was used in this study.
The viability of H. pylori was determined by evaluating the number
of Colony Forming Units (CFU) after incubation of the H. pylori in
the absence or presence of various concentrations of SFN for 3 h.
The urease activity of H. pylori was assessed by measurement of
the concentration of ammonia released into the medium during
incubation with 5 mM urea for 1 h. The effects of SFN on urease
activity and viability of H. pylori were examined at ambient pH 7.4
in vitro. At doses from 1-100 μg/mL, SFN dose-dependently de-
creased urease activity and viability of H. pylori (Fig. 3). These
results suggest that SFN shows antibacterial activity against H.
pylori in vitro.
Fig. (3). Sulforaphane (SFN) Markedly Inhibits Urease Activity and
Viability of H. pylori
The H. pylori strain Sydney Strain-1 (SS-1) was used in this study. The
urease activity of H. pylori was assessed by measurement of the concentra-
tion of ammonia released into the medium during a 1-h incubation period
with 5 mM urea. The viability of H. pylori was determined by the number of
colony forming units (CFU) after incubation of the H. pylori in the absence
or presence of various concentrations of SFN for 3 h. The effects of SFN on
H. pylori viability and urease activity were examined at ambient pH 7.4 in
vitro. At doses of 1-100 μg/mL, SFN dose-dependently decreased the viabil-
ity of H. pylori. The representative trace in left panel shows that SFN dose-
dependently inhibits H. pylori urease activity. The right panel shows that
SFN dose-dependently decreased H. pylori viability. n: number of experi-
ments. *P < 0.05; significant difference from the corresponding values in
the absence of SFN.
3.2. Effect of SFN on H. pylori Colonization and Gastric Mu-
cosal Inflammation in H. pylori-Infected Mice In Vivo
Based on the in vitro experimental data, we conducted the next
series of studies to determine if SFN inhibited colonization of H.
pylori and mitigated inflammation in H. pylori-infected gastric
mucosa in mice in vivo.
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4068 Current Pharmaceutical Design, 2017, Vol. 23, No. 27 Akinori Yanak a
Gastric mucosal infections with H. pylori Sydney Strain-1 were
established in 6-week-old female C57BL/6 mice of both wild-type
(Nrf2+/+) and Nrf2 knockout (Nrf2-/-) strains by inoculation with 5
× 107 CFU of H. pylori [24]. The H. pylori-infected mice were fed a
high-salt diet (7.5% NaCl) for 2 months in order to exacerbate
inflammation in gastric corpus mucosa [11]. The wild-type and
Nrf2-/- mice were fed the homogenized BS (+BS), or no BS (−BS).
Approximately 3 μmol/mouse/day of SGS was administered to the
+BS group. Eight weeks later, all mice were sacrificed. Gastric
mucosal preparations were fixed with formalin, stained with hema-
toxylin and eosin, and examined using light microscopy. The de-
gree of gastric mucosal inflammation was measured using the up-
dated Sydney system [25]. Activities of the gastric mucosal phase 2
detoxification enzymes, NQO1 and GST, were measured by
ELISA. H. pylori colonization of mice gastric mucosa was assessed
as described in our previous report [4].
3.2.1. Effect of BS Treatment on Gastric Mucosal Inflammation
Morphological examination by light microscopy of the gastric
mucosae in H. pylori-infected Nrf2+/+ mice showed massive infil-
tration of inflammatory cells in the mice not receiving BS, while
the mice fed BS showed less inflammation (Fig. 4).
Fig. (4). Broccoli Sprouts Markedly Attenuate Corpus Gastritis in H.
pylori- infected Mice Fed High Salt Diet
Gastric mucosal infections with H. pylori Sydney strain-1 were established
in 6-week-old female C57BL/6 mice of both the wild-type (Nrf2+/+) and
knockout (Nrf-/-) mice by inoculation of 5 × 107 CFU of H. pylori [24]. The
H. pylori-infected mice were fed for 2 months with a high-salt diet (7.5%
NaCl) in order to exacerbate inflammation in gastric corpus mucosa. The
mice were fed with the homogenized Broccoli Sprouts (+BS), or without BS
(−BS). Approximately 3 μmol/mouse/day of SGS were administered into
the +BS group. All the mice were sacrificed at 8 weeks later. Gastric mu-
cosal preparations, fixed with formalin and stained with hematoxylin and
eosin, were examined by light microscopy.
A representative histology of gastric mucosa of the H. pylori-infected mice
without BS treatment shows massive infiltration of inflammatory cells (up-
per panel), while the histology of the mice fed with BS shows less inflam-
mation (lower panel).
Scale bar: 200 μm.
Following the administration of BS, activation of the antioxi-
dant enzymes NQO1 and GST increased significantly in Nrf2+/+,
but not Nrf2-/- mice, as expected [26]. In agreement with th ese
findings, inflammation of the gastric corpus mucosa in H. pylori-
infected mice was substantially attenuated by treatment with BS in
Nrf2+/+, but not in Nrf2-/- mice (Fig. 4, Fig. 5).
3.2.2. Effect of BS Treatment on H. pylori Colonization
BS treatment induced an almo st 2-log reduction in H. pylori
colonization in wild-type mice but not in Nrf2-/- mice (Fig. 5), thus
confirming the integral role of the SFN-induced Nrf2 activation in
protection against H. pylori-induced gastric inflammation.
Fig. (5). Broccoli Sprouts Attenuate Corpus Gastritis and Inhibits H.
pylori Colonization in Nrf2+/+, but not in Nrf2-/- mice
The wild-type (Nrf2+/+) and the Nrf2 knockout (Nrf2-/-) mice infected with
H. pylori were fed with broccoli sprouts (BS) (+BS; ■), or without BS (-BS;
□) (as described in Fig. 4 legend). The degree of inflammation in gastric
corpus mucosa was expressed as the inflammation score as defined in the
updated Sydney system [25]. H. pylori colonization was expressed as the
number of H. pylori Colony Forming Units (CFU) after incubation of the
mucosal homogenates in the specific medium for H. pylori culture, de-
scribed in our previous report [4]. The left panel shows that feeding with BS
significantly mitigated both the inflammation score (left panel) and the
colonization (right panel) in Nrf2+/+, but not in Nrf2-/- mice (*P < 0.05;
significant difference from the corresponding values in the absence of BS;
n=number of animals).
3.2.3. Effect of Daily Intake of SFN-Rich BS on H. pylori
Infection in Human Subjects
Based on the in vivo experimental data from H. pylori-infected
mice, we conducted the next experiments to determine if daily in-
take of SFN-rich BS mitig ated H. pylori-induced gastritis in human
subjects.
Fifty H. pylori-positive volunteers, whose endoscopy showed
no abnormalities other than gastritis, were randomized to either the
BS group (n=25) or the alfalfa sprouts (AS) group (n=25). In this
study, AS were used as the placebo, since they do not contain SGS
or other isothiocyanates (Fig. 6). Subjects were instructed to con-
sume 70 g/day of SGS-rich 3-day-old BS (Broccoli Super Sprout®,
Murakami Pharm Ltd, Japan) for 8 weeks; these sprouts were vali-
dated to have an SGS content of approximately 6 μmol/g [27, 28].
Subjects in the AS (placebo) group were instructed to consume an
equivalent amount of AS for 8 weeks. All participants were re-
quired to attend the hospital for collection of blood and stool sam-
ples at 0, 4, 8, and 16 weeks (eight weeks after the completion of
the intervention); the dates corresponded to study days 0, 28, 56,
and 112, respectively. Stool samples were analyzed for H. pylori
stool antigen (HpSA) using an HpSA-ELISA kit from Meridian
Bioscience, Inc., as previously described [29]. Serum pepsinogens I
and II (PGI and PGII) were measured in blood samples collected
from volunteers at these same time points, and the PGI/PGII ratio
was computed [30, 31]. All test results were compared using a Stu-
dent's t-test. Error bars on all figur es represent ±1 SD from th e
mean.
The study protocol (outlined in Fig. 6) randomized 50 subjects
to daily consumption of either 70 g of BS or a placebo (AS). The
mean age of subjects at randomization was 54.5 years. There were
more female (n=28) than male (n=19) subjects, but there was no
difference in pre-intervention H. pylori infection and inflammation
status for the two groups (Table 1). Two subjects in the AS group
dropped out during the intervention period owing to acute viral
infection. Thus, the data obtained from 25 subjects in the BS group
and 23 in the AS group were analyzed.
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Role of Sulforaphane in Protection of Gastrointestinal Tract Against H. pylori Current Pharmaceutical Design, 2017, Vol. 23, No. 27 4069
Fig. (6). Protocol for the Broccoli Sprouts Clinical Trial on H. pylori
Infection
Fifty H. pylori–positive volunteers were randomized to either the Broccoli
Sprouts (BS) group (n=25), or the Alfalfa Sprouts (AS) group (n=25). In
this study, AS was used as the placebo, since AS do not contain any sul-
foraphane glucosinolates (SGS), the precursor of SFN. Subjects were in-
structed to consume 70 g/day of either the SGS-rich 3-day-old BS that con-
tains SGS of approximately 6 μmol/g dose [27, 28], or an equivalent amount
of AS for 8 weeks. All participants were instructed to attend the hospital for
collection of blood and stool samples at 0, 4, 8, and 16 weeks. Levels of
serum pepsinogens I and II (PGI and PGII) were measured by a commer-
cially available ELISA kit, and PGI/PGII ratios were calculated [30, 31].
Stool samples were analyzed for H. pylori stool antigen (HpSA) using a
HpSA-ELISA kit, as previously described (29) All tests results were com-
pared using a Student's t test for paired comparison. Error bars on all figures
represent ±1 SD from the mean.
1) Effects of BS/AS Treatment on Serum PGI and PGII
During the intervention period, significant reductions in both
PGI and PGII (P < 0.05) compared with baseline levels were only
observed in the BS group, and these returned to baseline values 2
months after th e intervention. The ratio of PGI to PGII, used as a
more robust indicator of changes in gastric inflammation (31), in-
creased significantly (P < 0.05) during the intervention in the BS
group (Fig. 7).
2) Effects of BS/AS T reatment on HpSA
The HpSA levels measured in th e BS interven tion arm w ere
significantly lower (P < 0.05) during the intervention than at base-
line, and return ed to baseline levels (P < 0.05) at 16 weeks
Fig. (7). Effect of Broccoli Sprouts (BS) and/or Alfalfa Sprouts (AS) on
Serum Pepsinogen I/II Ratio
There were significant reductions in both PGI and PGII compared with
baseline levels during the 8-week intervention period in the BS group, and
there was a return to baseline values at eight weeks after the intervention.
Since the magnitude of the reduction in PGII was greater than PGI, the ratio
of PGI to PGII (PGI/PGII) rose significantly during the intervention in the
BS group, suggesting that BS treatment mitigates gastric inflammation. In
contrast, there were no changes in PGI/PGII throughout the entire study
period in AS group. Rectangular overlays represent means for each time
period. n=number of participants.
P < 0.05; Significant difference from the corresponding values at
week 0.
P <0.05; Significant difference from the corresponding values at
week 8.
(2 months after intervention). The placebo group receiving AS
showed no significant change in HpSA (Fig. 8). Of the 25 subjects
in the BS treatment group, eight subjects had HpSA values below
the cutoff (0.100) at the end of the 8-week BS treatment period. In
six of these subjects, the HpSA values b ecame positive again at 8
weeks after cessation of BS consumption, and the HpSA values of
the remaining two subjects b ecame positiv e again, 6 months after
intervention, thus indicating that BS treatment reduced H. pylori
colonization but did not result in complete eradication.
4. DISCUSSION FOR H. PYLORI STUDY
This study was designed to determine whether regular dietary
consumption of BS, which is rich in the SFN precursor SGS, inhib-
ited H. pylori colonization and attenuated inflammation in the gas-
tric mucosa of mice and humans.
Table 1. Baseline data for H.pylori-infected subjects (n=50) after randomization to broccoli sprout and alfalfa sprout supplemented
diets.
Broccoli sprout (n=25) Alfalfa sprout (n=23)
Mean ± SD Range Mean ± SD Range
Age (y) 53.4±11.6 25 - 70 55.7±12.9 23 - 73
UBT (‰) 32.7±14.4 10.0 - 56.8 35.7±25.3 6.7 – 106.1
PG I (ng/mL) 70.3±21.4 33.3 - 118 69.4±26.7 31.5 - 135
PG II (ng/mL) 30.5±14.8 14.7 - 43.7 29.4±8.63 18.3 – 52.5
PGI/PGII (ratio) 2.41±1.00 0.68 - 4.10 2.38±0.70 1.03 – 3.93
NOTE: Note that there were two dropouts, both in the alfalfa group, one due to proton pump inhibitor use and one due to antibiotic use after enrollment, and these subjects are not
included in the table or the data presented in Figs. 7 and 8.
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4070 Current Pharmaceutical Design, 2017, Vol. 23, No. 27 Akinori Yanak a
Fig. (8). Effects of Broccoli Sprouts (BS) and/or Alfalfa Sprouts (AS) on
H. pylori-Specific Stool Antigen (HPSA)
There was a significant reduction in of H. pylori-Specific Stool Antigen
(HpSA) compared with baseline levels during the 8-week in tervention pe-
riod in the BS group, and there was a return to baseline values at eight
weeks after the intervention, suggesting that BS treatment reduces H. pylori
colonization, but does not eradicate H. pylori completely. In contrast, there
were no changes in the HpSA levels in the AS group, throughout the whole
study period. Rectangular overlays represent means for each time period.
n=number of participants.
P < 0.05; Significant difference from the corresponding values at
week 0.
P <0.05; Significant difference from the corresponding values at
week 8.
In this study, we first established that oral intake of BS de-
creased gastric mucosal inflammation in vivo in a well-estab lished
H. pylori-infected animal model. Oral dosing of animals in this
model (approximately 3 μmol/mouse/day) was consistent with the
SFN dosages in a variety of other mouse experiments investigating
carcinogenesis [21,32-35].
Second, the findings strongly suggest that mitigation of gastritis
by SFN was at least partially due to the induction of antioxidant
enzymes via the Nrf2 signaling pathway. SFN is a well-known
activator of antioxidant enzymes and we have shown that these
enzymes are upregulated in BS-treated animals.
Third, we d etermined that H. pylori colonization decreased in
SFN-treated wild-type mice but not in Nrf2-/- mice. This in vivo
finding suggests that SFN may have a direct antibiotic effect on the
level of H. pylori colonization. The primary effect may occur via
the upregulation of the host's systemic protection against oxidative
stress and inflammation, which results in reduced H. pylori coloni-
zation. The mechanisms for the detoxification effects of SFN have
been studied extensively [1, 17, 36-38]. SFN induces cytoprotec-
tive, antioxidant, and anti-inflammatory enzym es via the transcrip-
tion factor Nrf2, which activates the genes that control these en-
dogenous protective responses [20]. H. pylori infection generates a
variety of reactive oxygen species within the mucosa that enhance
gastric mucosal injury and inflammation. Our data show that H.
pylori-induced gastritis in human subjects is mitigated by BS treat-
ment. The data suggest that this effect is induced either by the inhi-
bition of H. pylori colonization (in vitro, SFN is a potent antibiotic
against H. pylori), the upregulation of Nrf2-dependent antioxidant
enzyme activity (a number of in vitro, animal, and clinical studies
have reported this), or by a combination of these two factors. None-
theless, the SFN-induced enhancement of Nrf2-dependent antioxi-
dant enzyme activity in gastric mucosal cells reduced reactive oxy-
gen species from gastric mucosa, which resulted in the m itigation of
H. pylori infection-induced gastritis.
Fourth, daily intake of BS (70 g) for 2 months decreased serum
levels of PGI and PGII and increased the PGI/PGII ratio during the
2-month intervention period, which was consistent with clinical
observations correlating to increased PGI/PGII ratio with reduced
inflammation of gastric mucosa [30, 31]. We also measured reduc-
tions in HpSA (an indicator of recent infection) after the intak e of
BS. All biomarkers returned to baseline levels after the interv ention
was discontinued.
In summary, our data on H. pylori-infected mice and humans
clearly suggested that SFN may have a direct antibacterial effect on
H. pylori, leading to reduced gastritis, and a systemic effect by
increasing the antioxidant response. It is not possible to determine
the relative contributions of these two mechanisms from this study;
however, in light of other evidence which suggests a strong anti-H.
pylori effect of SFN in vitro [2], the findings in this study suggest
that SFN h as promise both as an antibacterial agent directed against
H. pylori and a dietary preven tive agent against the development of
human gastric cancer.
5. ROLES OF SFN IN PROTECTION OF THE SMALL IN-
TESTINE AGAINST ASPIRIN/NSAID-INDUCED MUCOSAL
DAMAGE
The current prevalence of the prescription of aspirin and/or
other NSAIDs to prevent cardiovascular events and relieve pain
from osteoarthritis [39-41] is rising. It is well-established that aspi-
rin and/or NSAIDs frequently cause peptic ulcer disease and bleed-
ing from the upper GI tract. In order to prevent aspirin and/or
NSAID-induced GI mucosal injuries, potent acid inhibitors that
significantly ameliorate these injuries, such as Proton Pump Inhibi-
tors (PPI), have been prescribed [42]. However, recent advances in
capsule video endoscopy have revealed that the ulcers caused by
aspirin and/or NSAIDs were not only localized to the upper GI
tract, but also the small intestine [43-45]. Studies in human volun-
teers using capsule endoscopy have shown PPIs do not offer effec-
tive protection of the small intestin al mucosa against injury [46]. A
recent study in rat has shown that PPI exacerbates NSAID-induced
ulcers in the small intestine [47]. Although misoprostol has some
beneficial effects on NSAID-induced gastrointestinal ulcers
[48,49], it causes several adverse reactions such as abdominal pain,
diarrhea, and abortion [50]. A recent study in rats has shown that
geranylgeranylacetone (GGA), a mucosal protective agent known
to induce HSP70 in gastrointestinal mucosa, prevents small intesti-
nal mucosa from injury induced by loxoprofen [51]. A few clinical
trials using a small number of human volunteers have shown that
aspirin/NSAID -induced small intestinal injuries detected by capsule
video endoscopy are ameliorated by GGA [52] or rebamipide
[53,54], both of which have been used as gastric mucosal protective
agents. Currently, however, there is no direct evidence that such
agents are clinically effective for protection against NSAID -
induced small intestinal ulcers. In the present study, we investigated
the phytochemical sulforaphane (SFN) as a possible candidate drug
for prevention of NSAID-induced small intestinal injuries. Since
activation of antioxidant enzymes by SFN persists for 48-72 h, the
protective effects of SFN against oxidative stress may be more
potent than those of other antioxidant substances are. As NSAIDs
induce oxidative stress in GI mucosa, it is reasonable to assume that
SFN may be useful for protection of the small intestinal mucosa
against oxidative stress induced by NSAIDs. Recent studies have
shown that NSAID exaggerates small intestinal injuries, at least in
part by causing overgrowth of anaerobic bacteria in the intestinal
lumen [55,56]. Therefo re, it seems reasonable to assum e that SFN
may inhibit colonization of anaerobic bacteria in the intestinal lu-
men, thereby preventing invasion of the bacteria into the mucosa
and consequently mitig ating small intestinal injuries. Therefore, we
decided to determine if SFN could protect small intestinal mu cosa
from NSA ID-induced small intestinal injuries.
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Role of Sulforaphane in Protection of Gastrointestinal Tract Against H. pylori Current Pharmaceutical Design, 2017, Vol. 23, No. 27 4071
5.1. Role of SFN in Protection of IEC6 cells against Aspirin-
induced Injury in vitro
IEC6 cells, derived from rat small intestinal mucosa, were in-
cubated with or without various doses of SFN. After incubation for
12 h, the cells were treated with various dosages of aspirin. In some
experiments, the effect of zinc protoporphyrin-IX (ZnPP), an in-
hibitor of HO-1, was also examined. The viability of the IEC6 cells
was assessed by the measurement of 3-[4,5-dim ethylthiazol-2-yl]-
2,5- diphenyltetrazolium bromide (MTT) incorporation into the
cells. Expression of HO-1 protein by IEC6 cells were evaluated by
western blot analysis, using a polyclonal antibody to HO-1 (Hsp 32)
as the primary antibody.
5.1.1. Effects of Aspirin on Viability of IEC-6 Cells
Aspirin, at doses between 10 to 40 mM, dose-dependently de-
creased viability of IEC-6 cells after incubation for 12 h (data not
shown). Therefore, in the next series of experiments, we decided to
use 20 mM aspirin, which is a submaximal dose to cause injury in
IEC-6 cells.
5.1.2. Effect of Pretreatment with SFN on Aspirin-Induced Injury
in IEC6 Cells
We confirmed that incubation of the cells with 5 μM SFN for 6
h did not cause injury to IEC-6 cells, but did induce HO-1 expres-
sion, as discussed later. Therefore, we decided to use 5 μM SFN in
the following experiments. In this series of experiments, the cells
were initially exposed to 5 μM SFN for 6 h, followed by incubation
without SFN for a further 12 h. Then, the cells were exposed to
various doses of aspirin. Pretreatment of the cells with SFN attenu-
ated the 20 mM-aspirin-induced death of IEC6 cells (Fig. 9).
Fig. (9). Sulforaphane (SFN) attenuates Aspirin-Induced Injury in
IEC6 Cells
The cells were initially exposed to 5 μM SFN for 6 h, followed by incuba-
tion without SFN for a further 12 h. The cells were subsequently exposed to
various doses of aspirin. Pretreatment with 5 μM SFN attenuated aspirin (20
mM)-induced decrease in the viability of IEC6 cells. The data are expressed
as Mean ± SEM. n= number of experiments; * P < 0.05; statistically signifi-
cant difference from the corresponding value in the absence of SFN.
5.1.3. Effect of Pretreatment with SFN on HO-1 Expression in
IEC-6 Cells
The cells were exposed to 5 μM SFN for 6 h, followed by incu-
bation without SFN for a further 12 h. Western blot analysis
showed that HO-1 expression in IEC-6 cells increased significantly
after incubation with SFN for 6 h, and the effects persisted at 12 h
after removal of SFN from the medium (Fig. 10). These results
indicated that pretreatment with SFN upregulated HO-1 expression,
and that the effect persisted during the subsequent exposure to aspi-
rin.
5.1.4. Effects of HO-1 Inhibitor on the Protective Effects of SFN
Against Aspirin-Induced Cell Injury
The cells were exposed for 6 h to 5 μM SFN in combination
with 0.1 μM ZnPP, an HO-1 inhibitor, followed by incubation
without SFN and ZnPP for a further 12 h. After this, the cells were
exposed to 20 mM aspirin. Co-administration of ZnPP with SFN
tended to attenuate the protective effects of SFN on the aspirin-
induced IEC6 cell injury (Fig. 11), which indicated that HO-1 may
contribute to the protective effect of SFN against aspirin-induced
cell injury. Furth er experiments are required to confirm this as-
sumption.
Fig. (10). Sulforaphane (SFN) Up-Regulates Heme Oxygenase (HO)-1
Expression in IEC6 Cells
The cells were exposed to 5 μM SFN for 6 h, followed by incubation with-
out SFN for a further 12 h. Western blot analysis shows that HO-1 expres-
sion increased significantly at 6 h after incubation with 5 μM SFN, and that
this effect still persists a t 12 h after removal of SFN from the medium. The
data are expressed as Mean ± SEM. n=number of experiments; * P < 0.05;
significant difference from the corresponding value in the absence of SFN.
Fig. (11). ZnPP, an Heme Oxygenase (HO)-1 inhibitor, Tends to At-
tenuate Protective Effects of Sulforaphane (SFN) against Aspirin-
Induced Injury of IEC6 Cells
The cells were initially exposed for 6 h to 5 μM SFN in combination with
0.1 mM ZnPP, an HO-1 inhibitor, followed by incubation without either
agent for a further 12 h. The cells were then exposed to 20 mM aspirin. Co-
administration of ZnPP with SFN attenuated the protective effects of SFN
on aspirin-induced IEC6 cell injury. The data are expressed as Mean ±
SEM. n=number of experiments; a, P < 0.05; significant difference from the
corresponding value in the absence of SFN; b, P = 0.09; difference from the
corresponding value in the absence of ZnPP.
5.2. Role of SFN in Protection of the Small Intestinal Mucosa
against Indomethacin (IND)-Induced Injury in vivo
Male ddY mice aged 7 weeks were used in this study. Small
intestinal injuries were induced by two subcutaneous injections of
IND (20 mg/kg), as described elsewhere [57]. The time interval
between the first and second IND injections was 24 h. The mice
were sacrificed 6 h after the second IND injection. The small intes-
tinal mucosa was carefully examined for lesions under a dissecting
microscope.
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4072 Current Pharmaceutical Design, 2017, Vol. 23, No. 27 Akinori Yanak a
MPO activity was measured by a previously described method
[58]. Vascular permeability was assessed by measuring the amount
of leakage of Evans b lue into the small intestinal tissue, after intra-
venous injection of the dye at 1 h before sacrifice [59]. Enterobacte-
ria within the small intestinal mucosa were quantified by a previ-
ously described method [60]. In brief, the homogenates of the small
intestinal mucosa were placed on Gifu anaerobic medium agar and
incubated for 24 h under anaerobic conditions. The number of en-
terobacteria was expressed as log CFU/g of tissue. SGS was orally
administered at 0.5 h before and 6 h after the administration of
IND.
5.2.1. Effects of IND on Small Intestinal Mucosae
Treatment of the mice with IND induced small bowel shorten-
ing, increases in the lesion score (Fig. 12), vascular permeability
(Fig. 13), and MPO activity (Fig. 14); this suggested that treatment
with IND cau sed small intestinal mucosal injury in mice.
Fig. (12). Oral Intake of Sulforaphane Glucosinolates (SGS) Amelio-
rates Indomethacin (IND)-Induced Injury of Small Intestine
Oral administration of 17 mg/kg SGS did not affect the lesion score in the
IND-untreated mice. In contrast, pretreatment with SGS inhibited the in-
crease in the lesion score in the IND-treated mice. The data are expressed as
mean ± SEM. n=number of experiments; a, P<0.05; significant difference
from the corresponding value without IND; b, P<0.05; significant difference
from the corresponding value without SGS.
Fig. (13). Oral Intake of Sulforaphane Glucosinolates (SGS) Amelio-
rates Indomethacin (IND)-Induced Increase in Mucosal Permeability of
Small Intestine in Mice
Oral administration of 17 mg/kg SDS did not influence the mucosal content
of Evans blue in the IND-untreated negative control mice. In contrast, pre-
treatment of with SGS inhibited the increase in the mucosal content of
Evans blue in the IND-treated mice. The data are expressed as mean ± SEM.
n=number of experiments; a, P < 0.05; significant difference from the corre-
sponding value without IND; b, P < 0.05; significant difference from the
corresponding value without SGS.
5.2.2. Effect of SGS on the IND-induced Injuries in Small Intes-
tinal Mucosa
Oral administration of SGS did not affect the lesion score, vas-
cular permeability, or mucosal MPO activity in the IND-untreated
negative control mice. In contrast, pretreatment of the mice with
orally administrated SGS inhibited the increases in lesion score,
vascular permeability, and MPO activity in the IND-treated mice
(Fig. 12, 13, 14), suggesting that SGS attenuates IND-induced small
intestinal injuries in mice.
Fig. (14). Oral Intake of Sulforaphane Glucosinolates (SGS) Blocks
Indomethacin (IND)-Induced Increase in Myeloperoxidase (MPO)
Activity of Small Intestinal Mucosa in Mice
Oral administration of 17 mg/kg SDS did not affect the mucosal MPO activ-
ity in the IND-untreated mice. In contrast, pretreatment with SGS inhibited
the increase in the mucosal MPO activity in the IND-treated mice. The data
are expressed as Mean ± SEM. n=number of experiments; a, P < 0.05; sig-
nificant difference from the corresponding value without IND; b, P < 0.05;
significant difference from the corresponding value without SGS.
5.2.3. Effect of SGS on Anaerobic Enterobacterial Count in Small
Intestinal Mucosa of IND-Treated Mice
Oral administration of SGS significantly inhibited the over-
growth of anaerobic enterobacteria in IND-untreated mice. Fur-
thermore, pretreatment of the mice with SGS completely abolished
the increase in the overgrowth of anaerobic enterobacteria the IND-
treated mice (Fig. 15).
Fig. (15). Oral Intake of Sulforaphane Glucosinolates (SGS) Inhibits
Indomethacin (IND)-Induced Increase in Anaerobic Bacteria of Small
Intestinal M ucosa in Mice
Oral administration of 17 mg/kg SDS significantly inhibited amount of
mucosal anaerobic enterobacteria in the IND-untreated mice. Pretreatment
of the mice with SGS totally abolished the increase in the mucosal amount
of anaerobic enterobacteria the IND-treated mice. The data are expressed as
Mean ± SEM. n=number of experiments; a, P < 0.05; significant difference
from the corresponding value without IND; b, P < 0.05; significant differ-
ence from the corresponding value without SGS.
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Role of Sulforaphane in Protection of Gastrointestinal Tract Against H. pylori Current Pharmaceutical Design, 2017, Vol. 23, No. 27 4073
6. DISCUSSION FOR NSAID STUDY
The data from in vitro experiments show adaptive cytoprotec-
tion by SFN against aspirin in IEC6 cells. We have confirmed that
SFN enhanced HO-1 expression in small intestinal mucosa in this
experimental system. Furthermore, the present finding that the pro-
tective effects of SFN are mitigated by an HO-1 inhibitor, ZnPP,
strongly support our hypothesis that SFN affords adaptive cytopro-
tection against aspirin via Nrf2-dependent pathways. It has been
reported that aspirin causes oxidative stress in cells by damaging
mitochondrial respiration [61]. Thus, we assume that pretreatment
with SFN enhances the antioxidant system in the cells, thereby
eliminating oxidative stress induced by aspirin. Our data from in
vivo studies also showed th at pretreatment with orally administrated
SGS protects small intestinal mucosa against IND-induced injury,
suggesting that SFN also provides adaptive cytoprotection to the
small intestinal mucosa against IND-induced injury in vivo. The
present study showed that SFN enhanced HO-1 expression in vitro.
We also confirmed that SGS treatment enhanced HO-1 expression
in small intestinal mucosa in mice in vivo (unpublished observa-
tions). In addition, the present study also indicated that SGS inhib-
ited invasion of anaerobic enterobacteria into the mucosa in vivo.
Thus, it seems reasonable to assume that SFN and/or SGS have
twofold beneficial effects on small intestinal mu cosa ag ainst IND-
induced small intestinal injury.
It has been reported that mild irritants protect gastric mucosa
from the ensuing severe stress by induction of endogenous prosta-
glandins, a phenomenon named “adaptive cytoprotection” by Andre
Robert [62]. However, a number of other studies have shown that
“adaptive cytoprotection” cannot be explained by endogenous pros-
taglandins. More recent studies have suggested possible candidates
for adaptive cytoprotection. For example, it has b een shown that
mild stress induces heat shock proteins in gastric mucosa, thereby
protecting the mucosa from severe stress, which also accounts for
“adaptive cytoprotection” [63]. Alternatively, Yamamoto et al.
have recently demonstrated that mild oxidative stress induces anti-
oxidant enzymes via the Nrf2-Keap1 system, which strengthen
antioxidant activity of the cells against more severe oxidative stress
[64]. Since NSAIDs cause oxidative stress in cells [62,65], it is
reasonable to assume that SFN protects GI mucosa from NSAID-
induced oxidative stress by strengthening Nrf2-Keap1 mediated
antioxidant systems. Our findings strongly support this hypothesis
[5]. In contrast, recent studies have suggested a role of anaerobic
enterobacteria in the pathogenesis of NSAID-induced intestinal
mucosa [47,55,56], which indicated that anaerobic bacteria gener-
ally exacerbate NSAID-induced injury in the small intestine, al-
though the exact mechanisms remain to be elucidated. As SFN
shows antibacterial activity against H. pylori [1,4], it may be possi-
ble that SFN also demonstrates antibacterial activity ag ainst an-
aerobic enterob acteria in the small intestine, thereby ameliorating
NSAID-induced small intestinal injury. In conclusion, we propose
that SFN is a strong candidate for the prevention or treatment of
aspirin/NSAID -induced small intestinal ulcers in clinical practice.
Clinical trials of the effects of SFN on NSAID-induced injury in the
small intestine of humans should be conducted imminently.
In summary, our data suggest that the activation of Nrf2-Keap1
dependent antioxidant enzyme activity by SFN contributed to pro-
tection of small intestinal mucosa against NSAID-induced oxid ative
stress. Our data also indicated that SFN ameliorates NSAID-
induced small intestinal injuries by suppression of the invasion of
anaerobic bacteria into th e mucosa.
CONCLUSION
The present study strongly suggest that SFN contributes to the
protection of GI mucosa against oxidative injury induced by
H.pylori or NSAIDs.
CONFLICT OF INTEREST
The authors confirm that this article content has no conflict of
interest.
ACKNOWLEDGEMENTS
Declared none.
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