Keywords
intestine - mice - morphology - Silymarin - supplementation
Introduction
The investigation of natural compounds for nonpharmacological interventions aimed
at promoting health has gained momentum in the pursuit of an extended lifespan coupled
with an improved quality of life.[1] Notably, numerous studies have demonstrated the potential of nutraceuticals as effective
therapeutic approaches for the prevention and management of various inflammatory and
metabolic disorders, including but not limited to obesity, steatosis, and type-2 diabetes
mellitus.[2]
[3]
[4]
Silymarin (Silybum marianum) exerts hepatoprotective effects through various mechanisms, including antioxidant
activity and stabilization of the hepatocellular membrane.[5] Furthermore, silymarin has demonstrated the ability to decrease plasma cholesterol
and low-density lipoprotein levels in hyperlipidemic animals.[6] Moreover, experimental studies have reported that specific flavonoids present in
silymarin, such as silibinin, exhibit inhibitory effects on renal toxicity induced
by cisplatin.[7]
Silymarin and its flavonoid constituents, particularly silibinin, have demonstrated
emerging potential in the inhibition of tumor growth.[8] A recent study revealed a significant dose-dependent reduction in viability and
migration of gastric cancer cells following silymarin administration.[9] Furthermore, silibinin has exhibited strong inhibitory effects on various epithelium-derived
cancers, including prostate, colorectal, bladder, and lung cancer.[10]
[11]
Understand the effects of phytocompounds on intestinal morphology is of paramount
importance to elaborate supplements for a better quality of life in a variety of health
conditions.[12] Thus, this study aimed to analyze the effects of a nutraceutical supplementation
with silymarin on small intestine morphology.
Materials and Methods
Study Design
All experimental procedures were conducted in strict accordance with the National
Institutes of Health guidelines, and the research protocol received approval from
the Ethics Committee of the University of São Paulo Medical School (FMUSP) under protocol
number 1810/2022. Sixty-day-old adult male C57BL/6 mice were procured from the Central
Vivarium of Mice at FMUSP. The mice were housed in a temperature-controlled room maintained
at (24 ± 2)°C, following a 12-hour light/12-hour dark cycle. The mice were divided
into two groups (n = 5 per group): control and experimental. Both groups received
a standard nonfat diet containing 3.54 kcal/g for a duration of 10 weeks. Following
this initial period, the control group continued to receive the standard nonfat diet,
while the experimental group received the supplementation for an additional 4 weeks
(28 consecutive days). This treatment duration was chosen to assess the long-term
effects of supplementation, as previously reported.[2] At the end of the experimental period, the animals were euthanized with an overdose
of ketamine and xylazine, and fragments of the small intestine were collected for
further analysis.
Supplement Composition
The supplement formulation (Patent number: BR 10 2020 016156 3) utilized in this study
consisted of zinc (Zn), selenium (Se), magnesium (Mg), fructooligosaccharides (FOS),
galactooligosaccharides (GOS), 1.3/1.6-(β-glycosidic bonds) yeast β-glucans (Saccharomyces cerevisiae), and Silybum marianum extract. The mineral percentages were calculated based on dietary reference values,[2]
[3]
[4] and the final product was diluted in mineral water with carboxymethyl cellulose
as the emulsifier.
Histochemical Techniques
Small intestine fragments were fixed in 4% formaldehyde for 24 hours and subsequently
embedded in paraffin for histochemical staining techniques. Tissue sections were subjected
to Masson's trichrome staining to evaluate morphological structures and collagen deposition.[13] In addition, slides were stained using periodic acid-Schiff with Alcian blue to
visualize intestinal glycoproteins (mucins), as previously described.[14] For image capture, approximately five images per animal were obtained using a desktop
microscope (Leica Microsystems DMC 2900, SP, Brazil) equipped with AxioVision software
(Carl Zeiss, White Plains, New York, United States).
Quantitative Analysis
Morphological parameters were assessed using ImageJ software (National Institutes
of Health, United States) to analyze both structure density (%) and numerical quantity.
The color deconvolution tool in ImageJ was employed to unmix the brightfield images
into channels representing the absorbance of individual dyes.[15]
[16] After channel splitting, the images were converted to grayscale to measure the area
fraction of stained structures in contrast to the white background. This quantitative
analysis allowed for the evaluation of the following parameters: acid mucin (Alcian
blue), basic mucin (periodic acid-Schiff), and collagen deposition (Masson's trichrome
staining). Moreover, the numerical quantity of villi per field was determined using
the cell counter tool in ImageJ on sections stained with Masson's trichrome. Morphometric
analysis ([Fig. 1]) was performed on approximately five fields per animal, using AxioVision software
(Carl Zeiss, United States), to measure the following parameters (approximately 5
structures per field for each parameter) on Masson's trichrome staining (µm): crypt
depth, mucosal thickness, villus length, and villus spacing.[17]
Fig. 1 Schematic representation of measurements of intestinal morphology parameters. The
vertical red lines correspond to mucosal thickness, yellow lines indicate villus length,
blue lines indicate crypt depth and horizontal green lines demonstrate how villus
spacing was determined.
Statistical Analysis
We conducted unpaired Student's t-test to examine the difference between the groups and data were expressed as mean ± standard
error. The statistical analyses were performed using GraphPad Prism 5.0 software (GraphPad
Prism, Inc., San Diego, California, United States). The alpha level was set at the
0.05 level, and all tests were two-tailed.
Results
In [Fig. 2], we can see the quantitative analysis, as well as the representative images for
each group. Regarding morphometric analysis, animals submitted to the supplementation
had a significant decrease of crypt depth (71.61 ± 2.09 vs. 64.08 ± 1.03µm), mucosal
thickness (446.5 ± 11.6 vs. 321.2 ± 7.314µm), villus length (330.7 ± 11.56 vs. 210.2 ± 6.78µm),
and villus spacing (109.3 ± 4.78 vs. 22.2 ± 0.73µm). In contrast, we observed a significant
increase of villus per field (10.5 ± 1.25 vs. 20.1 ± 1.19 villus/field). However,
color deconvolution did not show significant difference in collagen deposit or acid
mucins between the groups. On the other hand, basic mucin was decreased in the experimental
group (2.84 ± 0.23 vs. 1.87 ± 0.17%).
Fig. 2 Quantitative analysis between the groups. (A) Acid mucins (%); (B) basic mucins (%); (C) collagen density (%); (D) quantity of villus per field; (E) crypt depth (µm); (F) mucosal thickness (µm); (G) villus length (µm); (H) villus spacing (µm). Representative images are shown above for both Masson's trichrome
and periodic acid-Schiff with Alcian blue (PAS + AB). Scale bar = 50µm.
Discussion
Our study aimed to investigate the effects of a novel nutraceutical supplementation
on the components of the small intestine in mice. We observed an increase in the number
of villi per field, suggesting an enlargement of the absorption area. This result
was accompanied by a reduction in villus spacing, length, crypt depth, and mucosal
thickness.
Previous research has shown that only a fraction of orally administered silymarin
is absorbed from the gastrointestinal tract, as it undergoes extensive enterohepatic
circulation.[18] However, an experimental study in mice demonstrated that flavonoids from silymarin,
in both free and conjugated forms, exhibited good distribution in various examined
tissues.[19] The observed phenomenon in our study can be partially explained by the formulation
of the supplement. Silymarin has low permeability across intestinal epithelial cells,
low aqueous solubility, and is rapidly excreted in bile and urine, resulting in low
bioavailability. Experimental studies often combine its supplementation with compounds
such as phospholipids,[20] liposomes,[21] and β-cyclodextrins[22] to enhance its bioavailability.
Dietary minerals can be absorbed through the epithelial cells lining the gastrointestinal
tract, enabling transcellular mineral transport even at low concentrations in the
intestinal lumen.[23] Therefore, mineral conjugates are utilized to enhance gastrointestinal absorption.
Prebiotics, which promote the growth of beneficial bacteria in the gastrointestinal
tract, have been shown to enhance the absorption capacity when combined with minerals.[24] Furthermore, the addition of β-glucan, a dietary fiber found in various sources
including yeast, has demonstrated antioxidant activity and glucose control due to
its bioavailability.[25]
However, it is important to consider the limitations of our study. We solely employed
histochemical techniques to elucidate our findings, and the bioavailability of silymarin
was not directly measured in the experimental group. Despite the limitation posed
by a modest sample size, our group's prior investigations have demonstrated compelling
outcomes of this nutraceutical in preclinical models of obesity and type 2 diabetes,
utilizing an identical sample size.[2]
[3]
[4] These earlier studies yielded statistically significant results, thus providing
a strong foundation for the current study's rationale and potential implications.
However, it is crucial to acknowledge the necessity for larger sample sizes in future
research to further validate and consolidate these findings, ensuring greater generalizability
and robustness of the conclusion drawn. Notwithstanding these limitations, our study
boasts several noteworthy strengths. We introduced a novel nutraceutical supplementation
that potentially modulates small intestine morphology, thereby increasing absorption
capacity. Future investigations utilizing immunohistochemical and molecular techniques
may further elucidate the underlying pathways involved in villus genesis and the effects
of each individual compound within this nutraceutical on the gastrointestinal system.
In conclusion, the combination of silymarin, prebiotics, yeast β-glucan, and minerals
in a dietary supplementation regimen may play a crucial role in shaping small intestine
morphology and enhancing absorption capacity. Thus, silymarin's role in shaping small
intestine morphology and its combination with other compounds for improved bioavailability
are noteworthy, offering implications for the nutraceutical industry.
