Tissue and Cell 69 (2021) 101483

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Tissue and Cell

journal homepage: www.elsevier.com/locate/tice

S-methyl cysteine sulfoxide ameliorates duodenal morphological

alterations in streptozotocin-induced diabetic rats
Valéria Milena Dantas de Castro a, Karina Carla de Paula Medeiros a,
Licyanne Ingrid Carvalho de Lemos b, Lucia de Fátima Campos Pedrosa b,
Fernando Vagner Lobo Ladd a, Thaís Gomes de Carvalho c,
Raimundo Fernandes de Araújo Júnior a, Bento João Abreu a, *,
Naisandra Bezerra da Silva Farias a
a
Department of Morphology, Federal University of Rio Grande do Norte, Natal, Brazil
b
Department of Nutrition, Federal University of Rio Grande do Norte, Natal, Brazil
c
Health Sciences Center, Federal University of Rio Grande do Norte, Natal, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Diabetes mellitus (DM) is a metabolic disease associated with several intestinal disorders. S-methyl cysteine
Diabetes mellitus sulfoxide (SMCS) is an amino acid present in Allium cepa L with hypoglycemic effects. However, the effects of
Histomorphometry SMCS on diabetic intestinal changes are unknown. Thus, we aimed to investigate the effects of SMCS on duodenal
Small intestine
morphology and immunomodulatory markers in diabetic rats. Twenty-six rats were divided into three groups:
SMCS
control (C), diabetic (D) and diabetic +200 mg/kg SMCS (DSM). DM was induced by intraperitoneal injection of
streptozotocin (50 mg/kg). After 30 days, duodenum samples were processed to assess histopathological and
stereological alterations in volume, villus length, and immunohistochemical expression of NF-kB, IL-10, BCL-2,
and caspase-3. SMCS reduced hyperglycemia and mitigated the increase in total reference volume of the duo­
denum, the absolute volume of the mucosa, and the length of the intestinal crypts in the DMS group when
compared to D. IL-10 immunostaining was reduced in D when compared to C, while NF-kB was increased in D in
comparison to the other groups. SMCS supplementation could decrease the NF-kB immunostaining observed in
D. Positive staining for BCL-2 and caspase-3 were not statistically different between groups. In summary, SMCS
decreased hyperglycemia and mitigated the morphological changes of the duodenum in diabetic animals, and
these beneficial effects can be partially explained by NF-kB modulation.

1. Introduction enzymatic mechanisms and promotes long-term systemic tissue damage

Diabetes mellitus (DM) is a metabolic disease characterized by
chronic hyperglycemia resulting from the body’s inability to produce or
use insulin properly (Okur et al., 2017). With a global occurrence, it is
estimated that over 640 million people will present this disease by 2040
(IDF, 2019).
Type I DM (DM1) is the second most prevalent type, comprising 5–10
% of all DM cases (Kahanovitz et al., 2017), and results from the
destruction of pancreatic β-cells by autoimmune reaction leading to
deficient insulin production (Almahfoodh et al., 2017). The hypergly­
cemia generated by this process stimulates inflammatory, oxidative, and
(Basha and Sankaranarayanan, 2016; Tormo et al., 2002).
Among the chronic DM complications (Melendez-Ramirez et al.,
2010), gastrointestinal tract disorders may lead to dysphagia, gastro­
esophageal reflux disease, chronic diarrhea, nonalcoholic fatty liver
disease, and gastroparesis (Boland et al., 2013; Krishnan, 2013). These
disorders are associated with a thicker and stiffer tissue (Zhao et al.,
2009) and enhancement of several immunomodulatory markers,
including transforming growth factor-beta 1 (TGF-B1) (Sha et al., 2018),
interleukin-10 (IL-10) (Malik et al., 2018), and imbalance of cell pro­
liferation, cell migration, and apoptosis (Delgado et al., 2016).
Natural compounds have been increasingly used as adjuvant therapy

* Corresponding author at: Department of Morphology, Federal University of Rio Grande do Norte, Av Salgado Filho, Universitary Campus, 59072-970. Natal,
Brazil.
E-mail address: abreubj@gmail.com (B.J. Abreu).

https://doi.org/10.1016/j.tice.2020.101483
Received 6 October 2020; Received in revised form 8 December 2020; Accepted 22 December 2020
Available online 31 December 2020
0040-8166/© 2020 Elsevier Ltd. All rights reserved.
V.M.D. Castro et al.
for DM due to their promising effects (Governa et al., 2018). S-methyl
cysteine sulfoxide (SMCS) is a cysteine amino acid derivative present in
allium vegetables, such as garlic (Allium sativum) and onion (Allium cepa
L), and it is associated with reduced blood glucose, circulating lipids,
and oxidative stress in experimental models of DM (Yin et al., 2007). It
has already been demonstrated that SMCS has protective effects that
may delay diabetic deterioration by stimulating insulin secretion and
reducing pro-inflammatory cytokines (Hsu et al., 2004). Moreover, its
antidiabetic action has already been compared with insulin and glen­
benzamine (Li and Perera, 2012).
Despite these findings, the mechanisms responsible for the SMCS
action in DM1 remain elusive and, to the best of our knowledge, no
studies investigated the role of SMCS in the small intestine’s morphology
in DM. Thus, we hypothesized that SMCS supplementation would pre­
vent intestinal alterations in DM1 through factors associated with
inflammation and apoptosis. Here, we investigated the effects of SMCS
administration on the duodenum morphology of streptozotocin-induced
diabetic rats by evaluating histopathological and stereological parame­
ters and the immunohistochemical expression of B-cell lymphoma pro­
tein 2 (BCL-2), caspase-3, IL-10, and nuclear factor kappa beta (NF-kB).
Our results indicate that SMCS was able to mitigate the morphological
changes in the duodenum of diabetic animals, and these beneficial ef­
fects were partially explained by NF-kB modulation.
2. Materials and methods
2.1. Animals and experimental design
The sample size was calculated using the G* power software (version
3.1.6) based on the analysis of variance (ANOVA), considering the gly­
cemic level as the primary outcome, a minimum effect size of 0.6, an
statistical power of 80 %, and an alpha level of 5%. Twenty-six male 90-
day-old Wistar rats (Rattus norvegicus) weighing between 250 and 300 g
were housed in polypropylene cages (up to 4 animals per cage) with
climate-controlled conditions (12 h light/dark cycle, 22–24 ◦ C, and
50–60% relative humidity) and received food and water ad libitum. This
study was approved by the Research Ethics Committee on the Use of
Animals of the Federal University of Rio Grande do Norte (protocol no.
012.018/2017) and was carried out following the Guide for the Care and
Use of Laboratory Animals of the National Institutes of Health.
The animals were randomly distributed into three groups: healthy
control group (C, n = 8), untreated diabetic group (D, n = 9), and dia­
betic group treated with SMCS (DSM, n = 9). DM1 was induced via
single intraperitoneal injection of 50 mg/kg of streptozotocin (STZ,
Sigma-Aldrich, St. Louis, MO, USA) dissolved in sodium citrate solution
(10 mmol/L, pH 4.5). Equal vehicle volumes were injected into the non-
diabetic rats. Blood glucose levels were measured using an Accu-Chek
Advantage glucometer (Roche Diagnostics, Indianapolis, USA) on the
fifth day after the induction. Animals with blood glucose ≥ 250 mg/dL
were considered diabetic (Hernandes Bortolin et al., 2015).
Food and water intake were measured using a weight scale and a
measuring beaker before euthanasia. Polydipsia, polyphagia, and poly­
uria were observed in all diabetic rats, which are compatible with the
DM1 experimental model (Silva et al., 2017). After seven days of DM
induction, SMCS [2-amino-3-(methylmercapto) propionic acid, 99 %
purity, Sigma-Aldrich, St Louis, USA] dissolved in sterile saline was
administered to DSM for 30 consecutive days via oral gavage. The 200
mg/kg dose of SMCS was chosen based on previous studies demon­
strating antidiabetic, antioxidant, and hypolipidemic effects (Kumari
and Augusti, 2007). C and D were treated with equal volumes of saline
solution. Two animals in D died during the experimental period. At the
end of the 4-week SMCS administration, the animals were weighed,
fasted for 12 h, and euthanized using a lethal dose of xylazine (12
mg/kg) and ketamine hydrochloride (> 90 mg/kg). Blood sampling was
collected within a few minutes after anesthesia to determine the final
glycemia.
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Tissue and Cell 69 (2021) 101483
2.2. Tissue histomorphometry
The intestinal segments were removed and immersed in 4 % para­
formaldehyde. When sectioning the intestinal tubes, the stereological
uniform and random sample (SURS) principle was applied, and rings
resulted from the serial section were allocated to the cassettes. (Brown,
2017). Histological processing followed the standard laboratory proto­
col. Briefly, tissues were dehydrated in increasing alcohol concentra­
tions from 70 to 100 %, clarified with xylene, and infiltrated with liquid
paraffin at 60 ◦ C. Thick sections of 5μ and 4μ were stained with hema­
toxylin and eosin (HE) and immunohistochemistry, respectively.
Regarding the morphometric analysis, the images were acquired
using an OLYMPUS CX21 optical binocular microscope (OLYMPUS,
Center Valley, USA) with a 10x objective lens and coded for blinded
analysis. Quantification was performed using the ImageJ software (NIH,
Bethesda, MD, USA). The length of five villi and five crypts was
measured according to the method used by (Atiq et al., 2019). The villi
were measured from the apex to the villus-crypt junction, while the
crypt was measured from the base to the crypto-villous junction.
2.3. Stereological procedures
To estimate the duodenum volume, we adopted the following
methods that provide absolute and relative values: the reference volume
(Vref), following the Cavalieri principle; the volume density (Vv),
following the Delesse principle; and the absolute volume, which is the
product of Vv (mucosa, submucosa, and muscle) and Vref wall (Garcia
et al., 2007). The entire organ was sectioned in a series of parallel planes
spaced at a distance t. To avoid bias, the first section was considered
uniform and randomized in an interval of 0 - t. The areas of the super­
ficial sections were estimated using a grid of points with a known area
associated with each a/p point (Marcos et al., 2012). The numbers of
points that “touched” the areas of interest were converted into reference
volumes (i.e., lumen, wall, and total Vref) using the following formula:
∑
Vref = pxa/pxtxk,
∑
Thus, p was considered the sum of all points in the test system
touching the region of interest (wall or lumen), a/p was the area asso­
ciated with each point in the grid test system, t was the distance between
the cross-sections, and k was the inverse of the ½ fraction used in the
SURS sampling of duodenal rings (Gundersen et al., 2013). After this, the
total volume (Vtot) was obtained from the sum of the wall Vref and
lumen Vref as follows:
Vtot = Wall Reference Volume + Lumen Reference Volume
2.4. Immunohistochemistry procedures
Three random fragments of duodenal microsections from each ani­
mal (three animals per group) were deparaffinized in xylene and washed
in a series of ethanol and phosphate buffer (PBS) concentrations, and the
endogenous peroxidase was blocked with 3% hydrogen peroxide. Tissue
sections were incubated overnight at 4 ◦ C in primary antibodies (anti-B-
cell lymphoma protein 2 [BCL-2] - from Abcam [San Francisco, CA,
USA]), anti-caspase-3 acquired from the Invitrogen™ (Carlsbad, CA,
EUA), anti-interleukin [IL-10] (Santa Cruz Biotechnology, Santa Cruz,
CA, USA), and anti-NF-kB (Santa Cruz Biotechnology, Santa Cruz, CA,
USA) at a dilution of 1:400, as recommended by the manufacturer. Slices
were washed with PBS and incubated with a streptavidin/HRP-
conjugated secondary antibody (Biocare Medical, Concord, CA, USA)
for 30 min. Immunoreactivity to NF-kB, IL-10, BLC-2, and caspase-3 was
visualized with a colorimetric-based detection kit (TrekAvidin-HRP
Label + Kit from Biocare Medical, Dako, USA). Sections were counter-
stained with hematoxylin. Known positive and negative controls were
V.M.D. Castro et al. Tissue and Cell 69 (2021) 101483

Table 1 included in each sample set (de Araújo et al., 2014). The rapid score
Glycemia, weight, water and food intake at the end of the 30-day experimental method (Chand et al., 2018) was used to quantify the number of marked
period. cells during the light microscopy analysis of the intestinal fragments.
C D DSM

Final glycemia (dl/mg) 120.6 ± 6.4 700.4*± 37.0 524.5*# ± 28.0

2.5. Statistical analysis
Final body weight (g) 283.1 ± 28.9 223.1*± 24.4 192.8* ± 2.6
Final water intake (ml) 31.8 ± 4.0 220.5* ± 11.4 193.2* ± 8.5 Data were expressed as mean ± standard error of the mean. All
Final feed intake (g) 19.3 ± 0.9 42.8* ± 1.4 37.4* ± 1.8 variables were submitted to normality test and, as all data presented
Data are expressed as mean ± S.E.M. Comparisons between groups were per­ normal distribution, the one-way ANOVA followed by Tukey post hoc
formed using the one-way ANOVA and Tukey post hoc test. *P ≤ 0.05 vs. C; #P ≤ test was performed. A P-value of ≤0.05 was considered statistically
0.001 vs. D. significant.

Fig. 1. Histological examination of the duodenal tissue. C, control group; D, untreated diabetic group; and DSM, diabetic group treated with S-methyl cysteine
sulfoxide. Representative images of the intestinal mucosa in 4x lower magnification and 10x larger magnification highlight the crypts (black double arrow) and the
intestinal villi (yellow double arrow). Bar scales = 500 μm and 100 μm, respectively.

3
V.M.D. Castro et al.
Fig. 2. Quantitative morphometry and stereological data of the duodenum. C, control group; D, untreated diabetic group; and DSM, diabetic group treated with
S-methyl cysteine sulfoxide. A) Reference volume; B) volume density; C) absolute volume and D) crypt length and villus. *P ≤ 0.05; **P ≤ 0.001.
3. Results
3.1. Glycemia, water consumption, and food intake
Glycemia, water intake, and body weight results are shown in
Table 1. Glycemic levels in D and DSM remained higher than C from
disease onset until the end of the study (Table 1). The treatment with
SMCS significantly reduced blood glucose in DSM compared with D (P =
0.001). As expected, a noticeable weight loss was observed in the dia­
betic groups during the experimental period, as well as increased water
and food intake (P = 0.001). The SMCS administration could not prevent
weight loss or change water and food intake (Table 1).
3.2. Stereology and morphometry
Histological findings showed an increased duodenal volume in both
D and DSM groups compared with C (Figs. 1 and 2 ). The wall Vref and
Vtot were higher in D compared with C (Fig. 2A; P = 0.001). The SMCS
supplementation reduced significantly these alterations in DSM (Fig. 2A;
P = 0.001).
When evaluating the proportion occupied by each duodenum layer
(i.e., mucosa, submucosa, and muscle) (Fig. 2B), the mucosa of the
diabetic animals was significantly thicker (81.0 %) compared with C
(71.0 %) (P = 0.001). In the submucosal layer, the proportion decreased
from 10.4 % in group C to 6.4 % and 6.3 % in groups D and DSM,
respectively (P = 0.006). Considering the muscle layer, it decreased
from 18.3 % in group C to 12.6 % in both D and DSM (Fig 2B; P = 0.001).
As an increased wall Vref was observed in D due to greater partici­
pation of the mucosa layer (Fig. 2C), the crypt and villus parameters
were also evaluated to identify which structure was responsible for this
change. No significant differences were observed in crypt length data
between groups (Fig. 2D; P = 0.168), while villus length was signifi­
cantly increased in D and DSM compared with C (Fig. 2D; P = 0.009).
4
Tissue and Cell 69 (2021) 101483
3.3. Immunohistochemistry
Immunohistochemical analyzes showed changes in the expression of
proteins associated with inflammation and apoptosis. Fig. 3A presents
the cross-sections counter-stained with light yellow to brown hema­
toxylin (400-fold magnification). The score indicated a significant IL-10
positive staining reduction in D compared with C (Fig. 3B; P = 0.04). NF-
kB immunostaining was significantly increased in D (Fig. 3B; P = 0.001)
compared with C; however, SMCS supplementation reduced NF-kB
staining in DSM compared with D (Fig. 3B; P = 0.001). Immunostain­
ing for BCL-2 and caspase-3 were not statistically different between
groups (Fig. 3B; P > 0.05).
4. Discussion
The main finding of this study was that SMCS presented protective
effects on the morphological changes of the duodenum in diabetic ani­
mals, and NF-kB may participate in this process.
Diabetic animals present several morphological alterations in the
duodenum layers (Da Rosa et al., 2015), probably by increased food
intake (Hvid et al., 2017). SMSC significantly reduced glycemia in these
animals, which agrees with other studies using SMCS to treat
insulin-resistant rats (Lee et al., 2013; Thomas et al., 2015). However, in
contrast with another study (Yin et al., 2007), SCMS administration was
not able to mitigate weight loss, polydipsia, and polyphagia.
A significant increase in wall Vref and Vtot were observed in the
untreated diabetic group, as previously shown in a type II DM (DM2)
experimental model (Hansen et al., 2013). This increase is referred to as
mucosal thickening caused by increased intestinal villi length, and
probably related to adaptive and inflammatory mechanisms in the in­
testinal mucosa of DM1 (Hansen et al., 2013; Macdonald, 1992; Mayhew
and Carson, 1989). Interestingly, the SMCS administration prevented
wall Vref and Vtot increase. This effect may be partially explained by the
positive action of this cysteine derivative in reducing the hyperglycemia
associated with abnormal duodenum morphology (Zhao et al., 2003).
These alterations have been associated with hyperglycemia since
V.M.D. Castro et al. Tissue and Cell 69 (2021) 101483

Fig. 3. Immunostaining examination of the duodenal tissue. C, control group; D, untreated diabetic group; and DSM, diabetic group treated with S-methyl
cysteine sulfoxide. A) Photomicrographs with duodenal cross sections counter-stained with hematoxylin showing immunoreactivity for IL-10, NF-kB, BCL-2 and
caspase-3 in yellow to brown. B) Graph representing the quantification of marked cells. 40x magnification. *P ≤ 0.05; **P ≤ 0.001.

morphological alterations were reduced in diabetic rats using insulin
compared with controls (Hansen et al., 2013).
Persistent hyperglycemia triggers the signaling of several pathways
that lead to diabetic complications, including oxidative stress, and the
intestinal mucosa is a vulnerable environment to this process (Bhor
et al., 2004; Volpe et al., 2018). It is known that the binding between
NF-kB and DNA regulates the expression of genes involved in several
pathophysiological processes, such as immune responses, cell prolifer­
ation, and apoptosis (Oguntibeju, 2019; Vandenabeele et al., 2010), and
the NF-kB signaling pathway activation products downstream oxidative
and inflammatory factors related to metabolic diseases (Baker et al.,
2011). In the present study, NF-kB was significantly increased in D
compared with C, whereas SMCS mitigated its increase in DSM. These
results corroborate with previous studies showing that SMCS reduced

5
V.M.D. Castro et al.
the expression of NF-kB p65 pathway (Chou et al., 2019) and suppressed
the protein expression of NF-kB p50 and NF-kB p65 (Liu et al., 2014) in
nerve growth factor differentiated PC12 cells under kainic acid-induced
injury and hypoxia, respectively.
The significant NF-kB increase in D may be due to the lack of insulin,
a hormone with an anti-inflammatory action that suppresses this tran­
scription factor (Dandona et al., 2005). Various stimuli can induce the
NF-kB complex, and its action is influenced by both the circumstance
and cell type (Perkins, 2007). This complex can control the susceptibility
of cells to apoptosis-inducing agents in which, depending on the context,
NF-kB can be both pro-apoptotic and anti-apoptosis (Barkett and Gil­
more, 1999). Therefore, the NF-kB reduction caused by SCMS may have
improved insulin sensitivity and suppressed NF-kB (Thomas et al.,
2015).
On the other hand, IL-10 is an interleukin with an anti-inflammatory
function that inhibits the release of other inflammatory cytokines
(Couper et al., 2008) and may participate with other cytokines in the
intestinal barrier integrity (Winer et al., 2017), promoting intestinal
mucus production (Hasnain et al., 2013). We found that IL-10 was
reduced in D compared with C. Similar results were found in studies
analyzing inflammatory markers (Uluisik and Keskin, 2017), oxidative
stress, inflammation, apoptosis (Nna et al., 2019), and diabetic ne­
phropathy (Lu et al., 2014).
Programmed cell death is an important process to maintain homeo­
stasis between cell proliferation and apoptosis in the intestinal mucosa,
and diabetic animals present an imbalance in the expression of the
molecules involved in these mechanisms (Wang et al., 2016). Conse­
quently, the intestinal mucosa of these animals may become thicker
(Wismann et al., 2018; Zhao et al., 2017). Although a tendency towards
the suppression of apoptosis was observed in group D, no significant
intestinal mucosal cell proliferation changes were found in the immu­
nostaining of BCL-2 and caspase-3 between groups. Interestingly, the
NF-kB transcriptional activity has been related to augmented apoptosis
in hyperglycemic conditions (Guan et al., 2014), and the activation of
Toll-like receptor 4 (TLR4)/NF-kB p65 signaling pathway produced
aberrant cell proliferation and apoptosis in the offspring tooth germ of a
maternal diabetes model (Chen et al., 2017). Thus, we cannot neglect
that the reduction in NF-kB immunoexpression elicited by SMCS sup­
plementation may have interfered with the imbalance of cell prolifera­
tion and apoptosis in hyperglycemic conditions and contributed to
mitigate the duodenal changes.
The submucosa is a layer comprised mainly of connective tissue
enriched with collagen proteins (Sadowska-Bartosz and Bartosz, 2015),
and the reduction of the intestinal submucosa in DM1 may be attributed
to protein glycation (Yin et al., 2007), which, in turn, makes collagen
more challenging to be broken down (Brings et al., 2015). Although
hyperglycemia compromises the collagen synthesis (de Lourdes Pessole
Biondo-Simões et al., 2009), it is unlikely that the treatment with SMCS
exerts positive effects at this stage.
Considering both the muscular and the submucosal layers, Vv
decreased in the diabetic groups, as observed on the muscular layer
surface of diabetic rats (Chen et al., 2012). Regarding the absolute
volume, both layers were not significantly different between groups, as
observed in the thickness analysis of these same layers (Zhao, 2013;
Zhao et al., 2002)
This study has some limitations. Although the streptozotocin-
induced DM1 model is widely used and quite useful to assess the ef­
fects of hyperglycemia, it may not accurately mimic the DM1 alter­
ations. Moreover, a thirty-day period may not be enough to generate an
imbalance between the targets evaluated in this model. Therefore,
future studies must assess the long-term effects of SMCS on DM.
Overall, the present study showed that changes in duodenum volume
and villus length of diabetic rats might be associated with NF-kB
expression. In addition, SMCS had positive effects on hyperglycemia
and mitigated morphological changes caused by DM1. Thus, the SMCS
administration can be a promising alternative therapy for intestinal
6
Tissue and Cell 69 (2021) 101483
changes caused by DM. However, further studies are needed to fully
understand the underlying molecular mechanisms involved in diabetic
intestinal disorders.
Financial support
This research was supported by the Research Support Foundation of
the Federal University of Rio Grande do Norte and the Coordination for
the Improvement of Higher Education Personnel (grant 087/2015 and
Finance Code 001).
CRediT authorship contribution statement
Valéria Milena Dantas de Castro: Investigation, Formal analysis,
Writing - original draft. Karina Carla de Paula Medeiros: Conceptu­
alization, Investigation, Visualization. Licyanne Ingrid Carvalho de
Lemos: Conceptualization, Investigation. Lucia de Fátima Campos
Pedrosa: Resources, Supervision, Conceptualization. Fernando Vagner
Lobo Ladd: Writing - review & editing, Writing - original draft. Thaís
Gomes de Carvalho: Investigation, Data curation. Raimundo Fer­
nandes de Araújo Júnior: Investigation, Data curation. Bento João
Abreu: Writing - review & editing. Naisandra Bezerra da Silva Farias:
Conceptualization, Writing - review & editing.
Declaration of Competing Interest
None.
Acknowledgements
The authors would like to thank the technicians of the histotechnical
laboratory (Melyna Souto, Sara Queiroz, and Rhudson Cruz) for their
assistance with the histological procedures.
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