Keywords
dyslipidemia - cholesterol - salivary glands - fibrosis - image analysis - simvastatin
Introduction
The global rise in obesity and metabolic syndrome has been closely linked to increased
consumption of high-fat diet (HFD), which is known to induce systemic metabolic disturbances,
including dyslipidemia. These systemic alterations have profound effects on various
organs such as the liver, heart, brain, periodontium, etc.[1]
[2]
[3] Moreover, the consumption of HFD also affects the salivary glands, which play a
crucial role in oral health by maintaining mucosal integrity, initiating digestion,
and providing antimicrobial action.[4]
[5]
[6]
Recent studies have found that HFD can induce significant cellular alterations, which
could lead to salivary gland dysfunction.[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13] For example, HFD disrupts intracellular calcium regulation and enhances reactive
oxygen species (ROS) production, leading to mitochondrial dysfunction and apoptosis.[9]
[12] Some studies investigated the alterations in morphology and functional proteins.
Generally, the changes in acini and ductal systems were reported.[6]
[7]
[8]
[10]
[13] However, a consensus on these changes is lacking in previous studies, and their
association with cellular changes remains uncertain.
Statins, such as simvastatin, are widely prescribed lipid-lowering agents. In addition
to cholesterol reduction, simvastatin has demonstrated anti-inflammatory and antioxidant
properties.[14]
[15]
[16]
[17] One study has reported the protective effects of simvastatin against salivary gland
inflammation in arthritis.[18] Another study reported that simvastatin treatment in hyperlipidemic rats resulted
in a reduction in lipocyte accumulation in the parotid gland; however, chronic inflammation
persisted.[8] The protective effect of simvastatin on the alterations in the submandibular and
sublingual glands induced by HFD remains unrevealed.
As the submandibular and sublingual glands play a critical role in maintaining oral
health, it is essential to understand the impact of HFD on these glands as well as
the potential protective effects of simvastatin. However, current evidence remains
inconclusive regarding the effects of HFD, and the protective role of simvastatin
remains unrevealed. Therefore, our study aimed to investigate the effect of HFD and
the protective effect of simvastatin on histological morphology and mucin production
in the submandibular and sublingual glands of rats.
Materials and Methods
Animal Ethics
The protocol of animal experiment was approved by the Naresuan University Animal Care
and Use Committee under animal ethics no. 6702004. All animals were housed at the
Center for Animal Research, Naresuan University, which has been certified by the Association
for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International.
Animal Experiment
The experiment was performed on 7-week-old male Wistar rats (Rattus norvegicus) obtained from Nomura Siam International Co., Ltd., Bangkok, Thailand. Constant environmental
conditions, i.e., 12-hour light/dark cycle, stable temperature of 22 ± 1°C, and relative
humidity of 55 ± 10%, were maintained throughout the entire study. After 1 week of
an acclimation period, the rats were randomly divided into three equally numbered
groups of six animals: (1) control (C) group, (2) HFD (H) group, and (3) HFD with
simvastatin (S) group.
Each group received a distinct diet for 12 weeks. The C group was fed a standard diet
(SmartHeart Hamster Food Complete & Balance, Perfect Companion Group Co., Ltd., Formula
Code 082), consisting of 4.5% fat, 24.2% protein, and 44.8% carbohydrates. Rats in
the H and S groups were fed with HFD (27.42% fat, 11.85% protein, 41.94% carbohydrates).
The S group additionally received 40 mg/kg/day of simvastatin, administered alongside
the HFD. Food consumption and body weight were monitored weekly.
After the 12-week experimental period, all rats underwent a 12-hour fasting period.
Anesthesia was induced via intraperitoneal injection of sodium thiopental (50 mg/kg
body weight). Blood samples were subsequently collected from the inferior vena cava.
Both submandibular and sublingual salivary glands were then harvested and immediately
immersed in 4% paraformaldehyde prepared in phosphate-buffered saline at 4°C for 2
days.
Blood Cholesterol Analysis
Serum samples were analyzed for total cholesterol (TC), low-density lipoprotein cholesterol
(LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglyceride (TG) levels
using an automated analyzer (Cobas c 311 analyzer, Roche).
Histological Section and Staining
The submandibular and sublingual glands were subsequently processed and embedded in
paraffin after fixation, then sectioned at a thickness of 4 µm, and stained with hematoxylin
and eosin (H&E) to assess general tissue architecture. Masson's trichrome staining
was performed to detect collagen deposition (fibrosis), periodic acid–Schiff (PAS)
staining was used to visualize neutral mucins, and alcian blue staining was applied
to identify acidic mucins.
Histological Analysis
Stained tissue sections were scanned using a slide scanner (ZEISS Axio Scan.Z1) equipped
with an LED light source (ZEISS Colibri 7) and image acquisition software (ZEN Blue
2.3). Quantitative histological analysis was conducted using ImageJ software.
Both submandibular and sublingual glands were analyzed for acinar cell size, striated
duct size, and the percentage area occupied by collagen. The process of image analysis
was blinded by assigning number codes for the images. The investigator performing
the analysis was unaware of the experimental groups. At the end of the experiment,
the number codes were revealed to collect the data and perform statistical analysis.
For acinar cell size, five nonoverlapping images at 200× magnification were selected
per sample. These images were taken from five distinct regions: two from the outer
edges of the gland, two from the central area, and one from the region adjacent to
the neighboring gland. In each image, the total acinar area was measured in μm2 and divided by the number of visible nuclei to calculate the average acinar cell
size. The values from all five images were then averaged to obtain a representative
value for each animal, which was subsequently used to calculate group means.
For striated ducts, 10 nonoverlapping areas containing striated ducts at 200× magnification
were selected. The duct area was measured in μm2 and based on clearly defined and traceable duct boundaries. Approximately 20 to 30
ducts were yielded from each rat.
The percentage area of collagen was calculated from Masson's trichrome-stained sections
by selecting blue-stained regions and dividing by the total gland area, then multiplying
by 100. The data were shown as a percentage of the total gland area.
Statistical Analysis
The serum lipid parameters, the average acinar cell size of the submandibular gland,
and the mean percentage areas of collagen are presented as means ± standard deviation
(SD). These data were analyzed using one-way analysis of variance (ANOVA) followed
by Tukey's post-hoc test. The average acinar cell size of the sublingual gland was
nonparametric data, presented as median and quartiles 1 and 3. These data were analyzed
using the Kruskal–Wallis test followed by Dunn's test. The size of striated ducts
is presented as median, minimum, and maximum. All statistical analyses were performed
using JASP software (Version 0.17.2; JASP Team, 2024),[19] and a p-value of < 0.05 was considered statistically significant.
Results
Blood Lipid Profiles
LDL-C levels differed significantly across all groups (p = 0.006), whereas TC, HDL-C, and TG levels showed no statistically significant differences
(p = 0.129, 0.065, and 0.157, respectively). HFD consumption resulted in increased levels
of all lipid parameters in the H group compared with the C group, but only the increase
in LDL-C was statistically significant (p = 0.004). Simvastatin treatment (S group) reduced all serum lipid levels compared
with the H group; however, these reductions were not statistically significant. Additionally,
TC, HDL-C, and TG levels in the S group were lower than those in the C group, while
the LDL-C level was higher. Nevertheless, none of these differences were statistically
significant ([Table 1]; [Supplementary Tables S1] and [S2], available in the online version only).
Table 1
Blood lipid profiles of rats in the standard diet (C), high-fat diet (H), and high-fat
diet with simvastatin (S) groups. Values are presented as mean ± standard deviation
(SD) for total cholesterol (TC, mg/dL), high-density lipoprotein cholesterol (HDL-C,
mg/dL), low-density lipoprotein cholesterol (LDL-C, mg/dL), and triglyceride (TG,
mg/dL).
|
Blood profile
|
Control
|
High-fat diet
|
High-fat diet with simvastatin
|
|
N
|
6
|
6
|
6
|
|
TC (mg/dL)
|
57.8 ± 16.9
|
86.2 ± 30.2
|
55.5 ± 8.67
|
|
HDL-C (mg/dL)
|
35.7 ± 11.1
|
39.3 ± 14.2
|
23.7 ± 6.44
|
|
LDL-C (mg/dL)
|
9.50 ± 3.78a
|
37.0 ± 19.8b
|
20.2 ± 6.24a,b
|
|
TG (mg/dL)
|
28.8 ± 15.3
|
33.0 ± 12.7
|
19.3 ± 5.09
|
Note: Groups sharing the same letter (a, b) are statistically not different, while those with different letters are significantly different.
Histological Analysis
Submandibular and sublingual glands were observed as follows:
Acinar Units
In the submandibular gland, all groups displayed similar morphology. Normal acinar
cells with purple-red cytoplasm and basally located nuclei were observed ([Fig. 1A–C]). The acinar units in the C group appeared larger in size and densely packed ([Fig. 1A]) compared with acinar units in H and S groups, which exhibit smaller acinar units
with visible spaces between some units ([Fig. 1B, C], red arrow). In addition, the H group represented a higher number of clear round
structures inside the cell ([Fig. 1A–C], yellow arrows) compared with the C and S groups. These structures resembled vacuoles
or lipid droplets. However, oil red O staining, which indicates the presence of lipid
droplets, showed similar staining patterns among the groups (data not shown), suggesting
an increase in non-lipid vacuoles in theses group. Next, the average size of acinar
cells was measured (shown in [Fig. 1G]; [Supplementary Tables S3] and [S4], available in the online version only) and found to be significantly smaller in
both the H and S groups compared with the C group (p = 0.002 and p < 0.001, respectively). No significant difference in acinar size was observed between
the H and S groups (p = 0.274).
Fig. 1 High-fat diet consumption reduced acinar cell size in both submandibular and sublingual
glands. Hematoxylin and eosin staining of the submandibular (A–C) and sublingual (D–F) glands. Intercellular spaces (red arrow) were evident in the high-fat diet (B, E)
and the high-fat diet with simvastatin (C, F) groups compared with control (A, D),
suggesting reduced size of acinar cells in both glands. In addition, numerous cytoplasmic
vacuoles (yellow arrow) were observed in the acinar cells of all groups, but prominently
seen in the high-fat diet group (A–C). Data of the submandibular gland shown as mean ± SD
(G). Data of the sublingual gland shown as median and quartile (H). Scale bars: 50 μm. **p < 0.01, ***p < 0.001. AC, acini; GD, granular duct; SD, striated duct; SD, standard deviation.
Similarly, acinar units in the sublingual glands of all groups exhibited comparable
morphology ([Fig. 1D–F]). Smaller acinar units with visible spaces between some of the units in the H and
S groups ([Fig. 1E, F], red arrow) were observed. In contrast, more densely packed acinar units were revealed
in the C group ([Fig. 1D]). However, vacuoles in the acinar cells were not detected. Consistent with the submandibular
gland, a significantly smaller acinar cell size was found in both the H and S groups
compared with the C group (p = 0.017, p = 0.005, respectively; [Fig. 1H], [Supplementary Tables S3] and [S4], available in the online version only). However, no significant difference was exhibited
between the H and S groups (p = 0.665).
Ducts
Both the submandibular and sublingual glands contained excretory ducts, striated ducts,
and intercalated ducts, each characterized by distinct epithelial morphologies. In
the submandibular gland, prominent convoluted (granular) ducts with red-stained cytoplasm
were also observed. All ductal cells exhibited well-defined cell borders, and no obvious
morphological changes were noticed across the groups.
Striated ducts were lined by cuboidal to columnar cells with centrally located dark
nuclei and red-stained cytoplasm (SD; [Fig. 1A–F]). The size of the striated ducts in both glands varied across individual rats; the
sizes were found to be similar between the groups. The median, minimum, and maximum
size of striated ducts in both glands are presented in [Supplementary Fig. S1], available in the online version only. No distinct morphological changes were observed
across the groups.
Collagen Deposition
Masson's trichrome staining revealed collagen as blue-stained areas, as shown in [Fig. 2(A–F)]. In both salivary glands, collagen accumulation was predominantly observed in the
periductal area, particularly around large ducts. Light blue staining was observed
in the C group ([Fig. 2A, D]), particularly in the submandibular gland, while more intense blue coloration was
evident in the H and S groups ([Fig. 2B, E] and [2C, F], respectively). Quantitative analysis ([Fig. 2G, H]) demonstrated a statistically significant increase in collagen deposition in the
submandibular gland among the three groups (p = 0.003). In the submandibular gland ([Fig. 2G]), statistically significant differences were found in both the H and S groups compared
with the C group (p = 0.005 and p = 0.011, respectively), with no significant difference between the H and S groups
(p = 0.914). A similar pattern of collagen deposition was observed in the sublingual
gland ([Fig. 2H]); however, the differences among groups were not statistically significant (p = 0.699).
Fig. 2 Collagen deposition surrounding salivary ducts increased in response to high-fat
diet consumption. Masson's trichrome staining of the submandibular (A–C) and sublingual (D–F) glands presented collagen accumulation around salivary ducts. Collagen appeared
as light blue in the control group (A, D) and showed increased intensity in the high-fat
diet (B, E) and high-fat diet with simvastatin (C, F) groups. Data shown as mean ± SD
in submandibular (G) and sublingual (H) glands. Scale bars: 50 μm. *p < 0.05, **p < 0.01. AC, acini; GD, granular duct; SD, striated duct; SD, standard deviation.
Mucin
The magenta coloration in PAS staining ([Fig. 3A–F]), indicating the presence of neutral mucin, was prominently observed in the acini
of the submandibular gland, and increased intensity of the staining was observed in
the H and S groups ([Fig. 3A–C]). The staining was also found in the periacinar area of the sublingual gland, particularly
H and S groups ([Fig. 3D–F]).
Fig. 3 Periodic acid–Schiff (PAS) staining revealed neutral mucin in the submandibular and
sublingual glands. Magenta staining indicates the presence of neutral mucin within
acinar cells (A–F). The prominent staining was observed in serous acini of the submandibular gland
(A–C). Sublingual glands show increased intensity of the staining in the surrounding
areas of H and S groups (D–F). Scale bars: 50 μm. AC, acini; GD, granular duct; SD,
striated duct.
Alcian blue staining ([Fig. 4A–F]), indicating the presence of acidic mucin, was prominently observed in the acinar
cells of the sublingual gland ([Fig. 4D–F]). Additionally, thicker pale pink structures with dark-staining nuclei surrounding
the mucous acini were observed in the H and S groups ([Fig. 4E, F], red arrow).
Fig. 4 Alcian blue staining revealed acidic mucin in submandibular and sublingual glands.
Blue staining denotes acidic mucin within acinar cells of both glands (A–F). The staining was prominently observed in the sublingual gland (D–F). Additionally,
thicker pale pink structures surrounding acini (red arrow) were observed in the sublingual
gland of H and S groups. Scale bars: 50 μm (A–F). AC, acini; GD, granular duct; SD,
striated duct.
Discussion
HFD consumption is known to elevate serum lipid levels—including TC, LDL-C, and TGs,
which contribute to systemic metabolic disturbances that affect multiple organ systems.
Secretory glands, such as the liver, pancreas, and lacrimal gland, are particularly
vulnerable to the changes, as they play vital roles in maintaining homeostasis.[20]
[21]
[22] Among these, the salivary glands are essential for preserving oral health and homeostasis;
the impact of HFD on their structure and function remains inconclusive—particularly
in relation to the submandibular and sublingual glands.
Our findings demonstrate that HFD consumption induces significant morphological alterations
in both glands, including a marked reduction in acinar cell size and increased collagen
deposition, and minimal mucin alteration. These structural changes were associated
with elevated LDL-C levels. Simvastatin treatment did not significantly prevent these
alterations.
Animal models, particularly rats, are widely used for organ changes associated with
metabolic diseases.[23] HFD feeding in rats is a well-established method for inducing dyslipidemia and other
metabolic disturbances.[23]
[24] A previous study has reported that rat diets containing 20 to 60% fat can trigger
various metabolic abnormalities.[23] However, the specific formulation of the diet plays a crucial role, as different
fat compositions have been shown to produce varying blood lipid profiles.[13]
[24]
[25]
[26] In our study, we used a diet containing 24.2% fat, which is on the lower end compared
with other experimental HFD models.[12]
[24]
[26]
[27] Nevertheless, our diet formula was sufficient to increase LDL-C and TC levels in
the H group, indicating that metabolic changes were successfully induced in our rat
model. Administration of simvastatin at a moderate dose (40 mg/kg) resulted in a reduction
of LDL-C levels, consistent with previous findings showing that simvastatin can lower
LDL-C by approximately 30 to 49% through inhibition of HMG-CoA reductase activity.[28] These results confirm the effectiveness of simvastatin treatment in our study.
To date, only a few studies have examined the morphological effects of HFD on major
salivary glands, and the alteration remains inconclusive.[6]
[7]
[8]
[10]
[11]
[13] The enlarged acinar cells were reported in the parotid and submandibular glands,
while shrinkage in acini was reported in the sublingual gland.[8]
[10]
[13] However, the cytoplasmic vacuolization was commonly found in all major salivary
glands.[6]
[7]
[10] In addition, ductal alterations, for example, dilated ducts, a decreased number
of ducts, and changes in epithelial lining in the parotid and submandibular glands
were observed in some studies.[6]
[7]
[8] In our study, we found acinar shrinkage, but ductal changes were inconspicuous.
The discrepancies among the previous studies and our findings gave us two observations.
First, the duration of HFD feeding might affect the characteristics of the damage.
In the submandibular gland, Kandeel and Elwan[10] reported the enlargement of acinar cells in rats fed a HFD for 6 weeks. In contrast,
our 12-week HFD feeding model showed acinar cell shrinkage. When a similar feeding
duration (12 weeks) was applied in our model and Hamada et al,[13] acinar cell shrinkage was reported in the sublingual gland. Second, different types
of salivary glands may respond differently to HFD. In a 12-week HFD feeding model,
the parotid gland was enlarged,[8] whereas the submandibular and sublingual glands were reduced in size, as shown in
our study. Most previous studies focused on alteration in a single gland, suggesting
that future research should investigate multiple glands to obtain more consistent
results.
Increased collagen deposition—indicative of fibrosis—is a common finding and is consistently
reported across previous studies, and our results align with this observation.[6]
[8] Additionally, our findings from PAS and alcian blue staining revealed a thicker
periacinar area in the sublingual glands of the H and S groups, suggesting potential
alterations in the surrounding components, such as the basement membrane.[29] The accumulation of extracellular matrix components, including collagen, is considered
a hallmark of tissue damage and a precursor to fibrosis.[30]
[31]
Our study observed a slightly increased PAS staining intensity in the acinar cells
of the H group, a finding consistent with a previous report that also noted increased
alcian blue intensity in the sublingual gland.[13] Only a few studies have reported on mucin in the salivary gland under HFD conditions.
However, a report of increased mucin in the gallbladder suggests a link to inflammation
and fibrosis.[32] Thus, the mucin alteration in our study possibly relates to inflammation and the
protective mechanism of the organ. Nevertheless, no recent evidence has reported these
changes.
Taken together, HFD consumption leads to alterations in glandular and surrounding
structures. The alterations provide evidence of early tissue injury, which may progress
toward apoptotic activity and, ultimately, fibrosis.[30]
[31] These collective cellular alterations may impair salivary gland function, consequently,
a decrease in salivary flow, which is a clinical manifestation of xerostomia.[5]
[33]
Simvastatin, widely used for cholesterol lowering, has been reported to have anti-inflammatory
and antifibrotic properties in the liver and parotid gland.[18]
[34] Daskala and Tesseromatis reported that simvastatin significantly reduced blood lipid
parameters, but insignificantly mitigated ductal and acinar cell alterations in the
parotid glands of HFD-fed rats.[8] Similarly, simvastatin reduced serum cholesterol levels, but did not significantly
protect against alterations in glandular and gland-surrounding structures in our study.
We suggest that simvastatin is ineffective in glandular protection from HFD consumption
in our model.
Our study is limited by the use of histological analysis, which represents morphology
and structures. However, morphology might not disclose a distinct alteration in the
early stage, but the functions of the organ have already changed.[35] To gain a more comprehensive understanding of the cellular and molecular changes
induced by HFD consumption, future studies should incorporate molecular techniques—such
as immunohistochemistry, gene expression profiling, or protein assays—to validate
and extend our histological findings. Combining morphological and molecular approaches
would provide stronger evidence for the mechanisms underlying salivary gland alterations
in metabolic disorders.
Conclusion
HFD consumption led to elevated LDL-C levels and induced acinar cell shrinkage and
increased collagen deposition. The alteration in mucin requires further molecular
studies to assess glandular function. Simvastatin did not prevent structural changes
and fibrosis in the glands. These findings highlight the vulnerability of salivary
glands to HFD, raising clinical concern for oral health risks and the imperative protective
strategies.
