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EXCLI Journal 2021;20:1106-1117 – ISSN 1611-2156
Received: April 14, 2021, accepted: June 15, 2021, published: June 29, 2021
1106
Original article:
MOLECULAR HYDROGEN IS COMPARABLE TO SULFASALAZINE
AS A TREATMENT FOR DSS-INDUCED COLITIS IN MICE
Tyler W. LeBaron†1-3 , Fereshteh Asgharzadeh†4,5 , Majid Khazei4,5* ,
Branislav Kura1, Alex Tarnava6, Jan Slezak1*
1 Centre of Experimental Medicine, Institute for Heart Research, Slovak Academy of
Sciences, Faculty of Natural Sciences of Comenius University, 841 04 Bratislava,
Slovak Republic
2 Molecular Hydrogen Institute, Utah, USA
3 Department of Kinesiology and Outdoor Recreation, Southern Utah University, Cedar
City, 84720, Utah, USA
4 Department of Physiology, Faculty of Medicine, Mashhad University of Medical Sciences,
Mashhad, Iran
5 Metabolic Syndrome Research Center, Mashhad University of Medical Sciences,
Mashhad, Iran
6 Drink HRW and Natural Wellness Now Health Products Inc., Unit C 60, Braid St,
New Westminster, BC, Canada
† TWL and FA contributed equally to the work.
* Corresponding authors: Majid Khazaei, PhD; Department of Physiology, Faculty of
Medicine, Mashhad University of Medical Sciences; Metabolic Syndrome Research
Center, Mashhad University of Medical Sciences; E-mail: KhazaeiM@mums.ac.ir;
Professor Ján Slezák, MD, PhD, DSc, Centre of Experimental Medicine, Institute for Heart
Research, Slovak Academy of Sciences, Dúbravská cesta 9, 841 04 Bratislava, Slovak
Republic. Tel.: +421 903 620 181, E-mail: jan.slezak@savba.sk
http://dx.doi.org/10.17179/excli2021-3762
This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0/).
ABSTRACT
Colitis is an inflammatory condition of the bowels associated with abdominal pain, diarrhea, fatigue, and fever.
Its etiology is multifactorial but related to the overproduction of inflammatory and oxidative mediators. There is
currently no cure for this disease, and drugs used to manage it often have deleterious side effects. H2 is recognized
as having anti-inflammatory and antioxidant effects, which may qualify it as a novel therapeutic for colitis. We
induced an acute model of colitis in mice by administering dextran sulfate sodium (DSS) in drinking water for
seven days. Mice were divided into five groups (n=6); normal, colitis, H2-treated colitis, sulfasalazine-treated co-
litis, and H2 plus sulfasalazine-treated colitis. From days three to ten, mice were given H2, sulfasalazine, or both.
H2 was administered via dissolving a hydrogen-generating tablet in water to make hydrogen-rich water (HRW),
which was ingested ad libitum and via oral gavage (200 μL). The Disease Activity Index (DAI), histological
changes, and markers of inflammation and oxidative stress were assessed. HRW and sulfasalazine significantly
improved bodyweight, DAI, mucosal damage, crypt loss, and spleen weight compared to control. Both treatments
significantly decreased inflammation (high-sensitive C-reactive protein) and restored redox balance (total thiol,
superoxide dismutase, catalase activity). There was a trend for the combination treatment to be more effective than
either HRW or sulfasalazine alone. Furthermore, HRW tended to be as effective as, and often more effective than,
sulfasalazine. HRW may serve as a therapeutic for ameliorating DSS-induced colitis in mice.
Keywords: Molecular hydrogen, colitis, inflammation, sulfasalazine, oxidative stress
EXCLI Journal 2021;20:1106-1117 – ISSN 1611-2156
Received: April 14, 2021, accepted: June 15, 2021, published: June 29, 2021
1107
INTRODUCTION
Idiopathic inflammatory bowel diseases
are associated with a high burden of morbid-
ity and progressive disability (Piovani et al.,
2019). Colitis is one of the principal forms of
inflammatory bowel disease and there is no
effective treatment or cure (Piovani et al.,
2019). The annual costs associated with its
management are estimated to be as high as
nearly $ 15 billion in the USA alone
(Gajendran et al., 2019). The presumed etiol-
ogy of colitis is considered to be multifacto-
rial involving genetics, autoimmunity, the mi-
crobiome, and various environmental factors
(Gajendran et al., 2019; Piovani et al., 2019).
These contribute to increased inflammation
and excessive production of reactive oxygen
species, both of which further induce and ex-
acerbate colitis (Gajendran et al., 2019;
Piovani et al., 2019).
Molecular hydrogen (H2 gas) has recently
been recognized as a novel medical therapeu-
tic with anti-inflammatory, antioxidant, and
signal modulating effects (LeBaron et al.,
2019a). Molecular hydrogen is a stable dia-
tomic gas and can be administered either via
inhalation or ingestion of water that contains
dissolved H2, hydrogen-rich water (HRW)
(LeBaron et al., 2019a). Clinical studies have
demonstrated its benefits in many different ar-
eas including exercise medicine (LeBaron et
al., 2019b, c), cognitive impairments
(Nishimaki et al., 2018), stroke (Ono et al.,
2017), cancer (Akagi and Baba, 2019), meta-
bolic syndrome (LeBaron et al., 2020), and re-
cently in patients with COVID-19 (Guan et
al., 2020; Russell et al., 2020).
HRW has been previously studied in an
animal model of inflammatory bowel disease
with favorable effects (Kajiya et al., 2009).
However, it is unclear how effective HRW is
compared to standard drug treatment such as
sulfasalazine. Additionally, we used drinking
HRW and oral gavage instead of H2-rich sa-
line, and a higher concentration of HRW. Ac-
cordingly, we induced an acute model of co-
litis induced by dextran sodium sulfate (DSS)
and compared and contrasted the antioxidant
and anti-inflammatory effects of HRW with
the drug sulfasalazine.
MATERIALS AND METHODS
Drugs and chemicals
High-concentration HRW was made via
hydrogen-producing tablets (HRW Natural
Health Products Inc., New Westminster BC,
Canada). For drinking, HRW was prepared
twice daily every 12 h by dissolving one tablet
in a sealed 500-mL soda bottle with no head-
space. The initial concentration of hydrogen
water was > 1.5 mM and remained > 0.1 mM
by end of 12 h as measured by redox titration
(H2Blue; H2Sciences, Las Vegas, Nevada).
Dextran sodium sulfate (DSS‐40kD), Hema-
toxylin and Eosin (H&E), and malondialde-
hyde (MDA), total thiol, superoxide dis-
mutase (SOD), and catalase materials were all
purchased from Sigma (Sigma Chemical Co.,
USA). The colitis drug sulfasalazine was also
obtained from Cayman Co.
Ethics statement
Thirty C57BL/6 male mice (6–8 weeks
old) were provided by the Pasteur Institute of
Iran (Tehran, Iran) and maintained according
to the standard protocol of the Guidelines on
Institutional Animal Care from Mashhad Uni-
versity of Medical Sciences. Mice received
water and food ad libitum and were kept in an
air-conditioned room with a laboratory tem-
perature of (22–25 °C), 12 hr light/dark cycle.
Murine colitis model and experimental
protocol
As illustrated in Table 1 mice were ran-
domly divided into five groups (n = 6 for each
group): i) the control group, which received
drinking water for the full 10 days, ii) the co-
litis group, which received 1 % (w/v) DSS in
drinking water for 7 days (≈ 3.3 mL/mouse/
day) followed by normal drinking water for
the next 3 days, iii) the sulfasalazine-treated
group, which received 1 % (w/v) DSS in
drinking water (≈ 3.3 mL/mouse/day) from
day 1-7, and 100 mg/kg/day of sulfasalazine
from day 3–10 via oral gavage, iv) the HRW-
EXCLI Journal 2021;20:1106-1117 – ISSN 1611-2156
Received: April 14, 2021, accepted: June 15, 2021, published: June 29, 2021
1108
treated group, which received DSS 1 % (w/v)
in drinking water (≈ 3.3 mL/mouse/day) from
day 1–7, and HRW from day 3–10 both from
drinking and 200 μL (> 1.5 mM H2) via daily
oral gavage, and v) the combination-treated
group, which received 1 % (w/v) DSS in
drinking water (≈ 3.3 mL/mouse/day) from
day 3-7, and 100 mg/kg/day sulfasalazine and
HRW via drinking (ad libitum) and via oral
gavage (200 μL) from day 3–10.
Assessment of colitis
During the study, weight change, stool
characteristics, rectal bleeding and rectal pro-
lapse were reported daily. The disease activity
index (DAI) data was given as an average
body weight change score, stool consistency,
and rectal bleeding and prolapse as previously
described (see Table 2) (Cooper et al., 1993).
Histopathological evaluation of colons
At the end of the experiments, mice were
euthanized, and the colon was removed,
washed, and its weight and length were meas-
ured. The formalin-fixed colon tissues were
washed, paraffin-embedded, sectioned, stained
with hematoxylin-eosin (H&E) and Masson's
trichrome, examined by light microscopy, and
graded according to the standard histopatho-
logical criteria provided in Table 3.
Tissue preparation to measure oxidative
stress markers
The colon tissues were weighed and ho-
mogenized in ice with PBS. Then, at 4 ° C, the
homogenate was centrifuged for 20 min at
10000 rpm. The supernatant was collected
and stored at – 70 °C for evaluation of oxida-
tive/antioxidative markers including malon-
dialdehyde (MDA), total thiol, and superox-
ide dismutase (SOD) and catalase activity
(Chassaing et al., 2014).
Table 1: Protocol for inducing and treating experimental colitis in mice
Group
(n=6
mice/group)
Untreated
water
(day 1-10)
1 % DSS H2O
≈ 3.3 mL/
mouse/day
(day 1-7)
Molecular hydrogen
drinking (ad libitum) &
oral gavage (200 μL)
(day 3-10)
Sulfasalazine via
oral gavage
(100 mg/kg/day)
day 3-10
Control
✓
-
-
-
Colitis
-
✓
-
-
Sulfasalazine
-
✓
-
✓
H2 group
-
✓
✓
-
Combination
-
✓
✓
✓
Table 2: Disease Activity Index (DAI) score system for colitis mice
Rectal bleeding
Stool consistency
Rectal prolapse
Weight loss
0
None
Normal
None
< 5 %
1
Red
Soft
Sign of prolapse
5-10 %
2
Dark red
Very soft
Clear prolapse
10-15 %
3
Gross bleeding
Diarrhea
Extensive prolapse
> 15 %
Table 3: Colorectal tissue damage assessment system based on histopathological criteria
Score
0
1
2
3
4
Inflammation
None
Mild
Moderate
Severe
Mucosal
damage
None
Mucus layer
Submucosa
Muscular and
serosa
Crypt loss
None
1/3
2/3
100 % + intact
epithelium
100 % loss of
epithelium
Pathological
change range
None
1-25 %
26-50 %
51-75 %
76-100 %
EXCLI Journal 2021;20:1106-1117 – ISSN 1611-2156
Received: April 14, 2021, accepted: June 15, 2021, published: June 29, 2021
1109
MDA measurement
Malondialdehyde (MDA) was measured
by mixing 1 mL of 10 percent homogenate
with 2 mL of a solution containing thiobarbi-
turic acid, trichloroacetic acid, and HCL in
boiling water for 45 minutes and centrifuged
for 10 minutes. The absorbance was read at
535 nm and the MDA levels were calculated
using the formula C(M) = (A/1.65 = 105).
Total thiol group measurement
Total thiol concentration was calculated
using the di-thio nitrobenzoic acid (DTNB)
reagent. 1 mL of Tris-EDTA buffer (pH=8.6)
was added to the colon homogenate. Test ab-
sorbance was read at 412 nm against the Tris-
EDTA buffer alone (A1). This solution was
applied to 20 μL of DTNB reagents and kept
at room temperature for 15 minutes. Next, the
sample absorbance was reported again (A2).
DTNB reagent absorbance was reported alone
as a blank (B). The total concentration of thiol
(mM) was determined using the following
formula: (mM) = (A2 − A1 − B) × (1.07/0.05
× 13.6).
Determination of SOD activity
SOD activity was measured using a color-
imetric assay described by Madesh and Bal-
asurbamanian (1997). The method is based on
the synthesis of superoxide dismutase by py-
rogallol auto-oxidation and inhibition of su-
peroxide-dependent reduction of 3-(4,5-di-
methyl-thiazol-2-yl) 2,5-diphenyl tetrazolium
bromide (MTT) to its formazan. The reaction
ends with the addition of dimethyl sulfoxide
(DMSO), stabilizing the color. In brief, the
homogenized colon was poured into the wells
and incubated for 5 min at room temperature.
The reaction was stopped by DMSO and then
read as a reference wavelength with a micro-
plate reader at a wavelength between 570 nm
and 630 nm. One unit of SOD is known as the
amount of protein required to inhibit a 50 %
reduction in MTT (Madesh and Balasubra-
manian, 1997).
Determination of CAT activity
Catalase activity was determined by
measuring the rate of H2O2 hydrolysis at
240 nm in sodium phosphate buffer. The ve-
locity of the enzyme response can be deter-
mined by converting H2O2 to H2O and O2
within 1 minute of the normal condition
(Madesh and Balasubramanian, 1997).
Determination of hsCRP
The inflammatory marker, high-sensitive
C-reactive protein (hsCRP) was determined
using the assay based on latex-enhanced tur-
bidimetric immunoassay. The agglutination
of the anti-CRP antibody is detected as an ab-
sorbance change (500 nm). The magnitude of
the change is proportional to the quantity of
CRP in the sample, and concentration is inter-
polated from the prepared standard calibra-
tion curve.
Statistical analysis
Results are presented as mean values ±
standard error of the mean and analyzed fol-
lowing Tukey's multiple comparison tests by
Student's t-test or ANOVA. Software anal-
yses were conducted using SPSS v.20 statisti-
cal software (IBM, Chicago).
RESULTS
H2-rich water improved clinical symptoms
of colitis
Animal weight was monitored daily. The
body weight of mice in the control group con-
tinued to increase, while the body weight of
the mice treated with DSS decreased during
the experiment. In comparison, mice body
weight in the H2, sulfasalazine, and combina-
tion groups significantly improved after treat-
ment (Figure 1A). The DAI scores for the
DSS-treated mice were significantly higher
compared to the control group (P < 0.001).
However, the DAI scores for the sulfasala-
zine, H2, and combination groups were all sig-
nificantly lower compared to the colitis group
(P < 0.001) (Figure 1C).
H2 ameliorated colon tissue damage in
colitis model
In colitis mice, sulfasalazine (P < 0.01)
and sulfasalazine with H2 (P < 0.001) signifi-
EXCLI Journal 2021;20:1106-1117 – ISSN 1611-2156
Received: April 14, 2021, accepted: June 15, 2021, published: June 29, 2021
1110
cantly suppressed DSS-induced colon short-
ening (Figure 2A, B). Similarly, sulfasalazine
with and without H2 attenuated the decreased
colon weight induced by DSS (P < 0.05).
Treatment with H2 had a non-statistical ten-
dency to decrease the colon weight to length
ratio, reflecting a decrease in inflammation
and tissue edema (Figure 2D).
Figure 2: The effects of H2 and sulfasalazine on DSS-induced colon aberrations. Colon length (A, B),
colon weight (C), colon weight / length ratio (D). Results are expressed as means ± SEM (n = 6). ***P
< 0.001 , *P < 0.05 compared to control and ### P < 0.001, ## P < 0.01 and # P < 0.05 compared to
colitis group.
Figure 1: Clinical symptoms of DSS-induced
colitis in different groups. (A) The % initial
weight change was shown as the average
daily weight of days 1 to 10. (B) scores of the
Disease Activity Index (DAI) daily. (C) Highest
DAI during the experiment. Results are ex-
pressed as means ± SEM (n = 6). ***P < 0.001
compared to control and ### P < 0.001 com-
pared to the colitis group.
EXCLI Journal 2021;20:1106-1117 – ISSN 1611-2156
Received: April 14, 2021, accepted: June 15, 2021, published: June 29, 2021
1111
Histopathological evaluation
We compared colonic tissue histological
damage (Figure 3A) between H2 treated with
and without sulfasalazine to the colitis model
and control mice.
DSS administration resulted in pathologi-
cal alterations such as inflammation of the
mucosa and cell infiltration (Figure 3B), de-
struction of the epithelium cell layer (Figure
3C), and crypt loss (Figure 3D) resulting in
significantly higher histological scores (Fig-
ure 3E) compared to control group. On the
other hand, both H2 and sulfasalazine, espe-
cially in combination, significantly reduced
these histological aberrations in comparison
with colitis mice (Figure 3B-E). H2 was
slightly more effective (P < 0.001) than sul-
fasalazine (P < 0.01) in preventing mucosal
damage compared to the colitis group. Fur-
thermore, as expected the spleen weight and
spleen weight to body weight ratio were in-
creased by DSS-administration, which is gen-
erally associated with the extent of inflamma-
tion. However, H2 administration both with
and without sulfasalazine was able to improve
these manifestations in DSS-induced colitis
mice (P < 0.001; Figure 3F, G).
H2 and sulfasalazine improves
inflammation in DSS-induced colitis
The effects of hydrogen treatment alone
or in combination with sulfasalazine on high-
sensitive C reactive protein (hs-CRP) levels
were also evaluated in DSS-induced colitis.
Figure 3: H2 has mitigated deteriorating colonial histopathological changes and inflammation in DSS-
induced colitis mice. Representative H&E staining images from various treatments of the colonic sec-
tions (A), inflammation score (B), mucosal damage score (C), crypt loss (D), histological scores (E),
spleen weight (F), and spleen weight to body weight ratio (G) were measured in different groups. Results
are expressed as means ± SEM (n = 6). ***P < 0.001 , **P < 0.01 and *P < 0.05 compared to control
and ### P < 0.001 and ## P < 0.01 compared to colitis group.
EXCLI Journal 2021;20:1106-1117 – ISSN 1611-2156
Received: April 14, 2021, accepted: June 15, 2021, published: June 29, 2021
1112
As illustrated in Figure 4, DSS treatment
significantly elevated hsCRP levels above
control in all groups. However, compared to
the colitis group, hsCRP was significantly re-
duced in the hydrogen (P < 0.01), sulfasala-
zine (P < 0.05), and the combination (P <
0.001) groups.
H2 and sulfasalazine decreases colon fibro-
sis in colitis
The colitis group showed more fibrosis in
collagen colon tissues of colitis mice as visu-
alized through Masson's trichrome staining
(see Figure 5).
H2 and sulfasalazine significantly reduced
the collagen deposition in the colon induced
by DSS (P < 0.001). Compared to sulfasala-
zine, H2 appeared to be more effective statis-
tically (P < 0.05), with maximum effect pro-
vided by the H2 sulfasalazine combination (P
< 0.001) (Figure 5A, B).
Figure 4: Masson's trichrome staining of colon tis-
sues of treated and untreated colitis mice com-
pared to control (Figure 5A). Digital image analy-
sis of percent collagen content. Results are ex-
pressed as means ± SEM (n = 6).***P < 0.001, **P
< 0.01 and *P < 0.05 compared to control. ### P
< 0.001 and ## P < 0.01 compared to colitis group.
+++ P < 0.001, ++ P < 0.01, + P < 0.05 compared
to sulfasalazine group.
Figure 5: Effects of H2 and sulfasalazine on oxidative and antioxidant markers in DSS-induced colitis.
Results are expressed as means ± SEM (n = 6). ***P < 0.001 , **P < 0.01 and *P < 0.05 compared to
control. ### P < 0.001 and ## P < 0.01 compared to colitis group. +++ P < 0.001, ++ P < 0.01, + P <
0.05 compared to sulfasalazine group.
A
B
EXCLI Journal 2021;20:1106-1117 – ISSN 1611-2156
Received: April 14, 2021, accepted: June 15, 2021, published: June 29, 2021
1113
H2 and sulfasalazine improves redox status
in DSS-induced colitis
DSS significantly increased oxidative
stress and reduced antioxidant status com-
pared to control. However, H2 and sulfasala-
zine either alone or in combination were pro-
tective against DSS-induced colitis as illus-
trated in Figure 6.
Although sulfasalazine significantly re-
duced MDA levels compared to the colitis
model (P < 0.001), H2 with and without sul-
fasalazine was significantly more effective
compared to sulfasalazine treatment (P <
0.001), and essentially prevented the DSS-in-
duced MDA increase. Similarly, although all
treatments significantly improved superoxide
dismutase (SOD), total thiol, and catalase
(CAT) levels compared to colitis (P < 0.001),
H2 with and without sulfasalazine was signif-
icantly more effective (P < 0.001). Addition-
ally, H2 with and without sulfasalazine essen-
tially prevented the DSS-induced decline in
these antioxidant levels. The combination
treatment had a general trend of being more
effective, but it was only statistically greater
than H2 at improving catalase activity levels
compared to sulfasalazine (P < 0.01 vs. P <
0.001 for H2 and sulfasalazine, respectively).
Figure 6: Effects of H2 and sulfasalazine on oxidative and antioxidant markers in DSS-induced colitis.
Results are expressed as means ± SEM (n = 6). ***P <0.001, **P <0.01 and *P < 0.05 compared to
control. ### P <0.001 and ## P < 0.01 compared to colitis group. +++ P<0.001, ++ P<0.01, + P<0.05
compared to sulfasalazine group.
EXCLI Journal 2021;20:1106-1117 – ISSN 1611-2156
Received: April 14, 2021, accepted: June 15, 2021, published: June 29, 2021
1114
DISCUSSION
In the present study we induced an acute
mouse model of colitis using 1 % DSS in
drinking water, as has been described previ-
ously (Binabaj et al., 2019). Consistent with
previous data, we found that DSS led to
symptoms of colitis, and that treatment with
molecular hydrogen and or sulfasalazine ex-
erted protective effects. From a phenotypic
perspective molecular hydrogen was either as
effective as, and more effective than, sulfasal-
azine in improving body weight, attenuating
the Disease Activity Index, and in maintain-
ing colon length and weight. Furthermore,
this comparative/superior effect was also ob-
served in the histopathological evaluation
looking at inflammation, mucosal damage,
crypt loss, and spleen weight as well as patho-
logical colon fibrosis. However, the combina-
tional therapy of molecular hydrogen with
sulfasalazine was often more effective than
when either was administered alone.
Similar benefits were observed in an ace-
tic acid-induced colitis rat model where H2-
rich saline was administered once every other
day for two weeks (He et al., 2013). They re-
ported reduced weight loss and diarrhea, and
less mucosal damage (He et al., 2013). In an-
other report, DSS was used to induce irritable
bowel syndrome, which H2 treatment also sig-
nificantly attenuated (Shen et al., 2017).
On a molecular level, hydrogen has been
demonstrated to exert anti-inflammatory and
antioxidant effects in animal and human stud-
ies (LeBaron et al., 2019a). DSS induces ex-
cessive inflammation and depletes endoge-
nous antioxidants leading to oxidative stress
(Kajiya et al., 2009). Our results show that
molecular hydrogen can attenuate inflamma-
tion as noted by the reduction of DSS-induced
increase in high-sensitivity C-reactive protein
(hsCRP). In this case, HRW tended to be
slightly more effective than sulfasalazine, but
not as effective as the combination. Similarly,
HRW prevented the DSS-induced increase in
the marker of lipid peroxidation malondialde-
hyde. This may be due to hydrogen’s ability
to prevent the DSS-induced decline in the an-
tioxidants, superoxide dismutase and cata-
lase, as well as in the total thiol concentration,
as we observed in this study.
These antioxidant effects of hydrogen are
in line with previous research, including in
humans. For example, ingestion of high-con-
centration hydrogen water for six months in
subjects with metabolic syndrome (n=60) re-
sulted in decreased markers of inflammation
and improved antioxidant status in addition to
improving metabolic and clinical parameters
(LeBaron et al., 2020). One of the mecha-
nisms by which H2 exerts its antioxidant ef-
fect is via induction of the Nrf2/Keap1 anti-
oxidant system (Kura et al., 2018). The Nrf2
transcription factor regulates the production
of over 200 cytoprotective proteins involved
in antioxidation and detoxification (Ma,
2013). In an earlier study, the protective ef-
fects of hydrogen on DSS-induced colitis
were dependent on the induction of heme-ox-
ygenase-1 expression (Shen et al., 2017),
which is a downstream target of Nrf2. How-
ever, the exact mechanism responsible for H2-
induced Nrf2 activation remains elusive
(LeBaron et al., 2019a).
Interestingly, there is a strong positive re-
lationship between the inflammatory condi-
tions of colitis and rheumatoid arthritis
(Attalla et al., 2019). Accordingly, the drug
sulfasalazine is a standard drug for both of
these inflammatory conditions (Attalla et al.,
2019), for which hydrogen has also been
shown to be effective. For example, in small
clinical studies, treatment with molecular hy-
drogen has shown pronounced therapeutic ef-
fects in patients with rheumatoid arthritis
(Ishibashi et al., 2012, 2014). Additionally,
the drug acarbose, an α-glucosidase inhibitor,
has been suggested to be used as a treatment
for colitis due its ability to increase hydrogen
production from intestinal bacteria (Zhang et
al., 2012).
Dysregulation of the microbiome repre-
sents an important factor for numerous pa-
thologies and conditions including colitis
(Shen et al., 2018). Although not investigated
in our study, several studies suggest that
HRW favorably modulates and improves the
EXCLI Journal 2021;20:1106-1117 – ISSN 1611-2156
Received: April 14, 2021, accepted: June 15, 2021, published: June 29, 2021
1115
microbiome in mice (Higashimura et al.,
2018; Xiao et al., 2018), goats (Wang et al.,
2017), piglets (Zheng et al., 2018), and hu-
mans (Sha et al., 2018). Accordingly, HRW
may have promoted improved microbiome
homeostasis in the DSS-induced colitis mice,
contributing to the favorable effects observed
in our study.
In conclusion, our study provides addi-
tional evidence that HRW may serve as a po-
tential therapy for patients suffering from co-
litis, and as a corollary, strongly warrants
clinical investigation. Additionally, research
into the exact molecular mechanisms of hy-
drogen, and its influence on the gut microbi-
ome, will help further the understanding and
development of this safe and simple therapeu-
tic.
Acknowledgments
We thank Mr. Alex Tarnava, CEO of
HRW Natural Health Products Inc. for kindly
donating Drink HRW tablets for this study
and thank Mashhad University of Medical
Sciences for their support.
Conflict of interest
TWL has received travel reimbursement,
honoraria, and speaking and consultancy fees
from various academic and commercial enti-
ties regarding molecular hydrogen. AT is the
CEO of HRW Natural Health Products Inc.,
whose company provided product and addi-
tional funds for biomarker measurements. All
other authors report no conflict of interest.
Funding
This study was partially supported by Slo-
vak Research and Development Agency
(APVV)-0241-11, APVV-15-0376; ITMS
26230120009; Scientific grant agency of the
Ministry of Education of the Slovak Republic
(VEGA) 2/0063/18, and by HRW Natural
Health Products Inc.
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