Curcumin mitigates the histopathological and molecular changes in sublingual glands in diabetic male rats

Abstract

Objective

demonstrate the possible palliative role of curcumin in the prospective molecular and histological alterations in the sublingual glands of diabetic male rats.

Methods

Twenty-one male adult rats were used and randomly assigned into three groups (n = 7). The control group consisted of rats administered a single IP injection of saline. The diabetic group included rats receiving one dose of alloxan (140 mg/kg). The nanocurcumin-treated rat group (NC group) contained diabetic rats administered 200 mg/kg nanocurcumin. After 42 days, the salivary glands were dissected and assessed for Masson trichrome, Hematoxylin and eosin (H&E) staining, expression of c-kit and β-catenin, and histomorphometric analysis.

Results

The sublingual glands of diabetic rats revealed altered histology. In addition, the salivary tissues depicted an apparent change in β-catenin, and c-kit expression. The NC group resumed the normal sublingual gland architecture and c-kit and β-catenin expression.

Conclusion

Curcumin treatment rescued the histological and molecular changes in the sublingual glands of the diabetic rats. The ameliorating effect of curcumin on sublingual tissues of diabetic rats may be due to resuming of expression of β-catenin suggesting restoration of Wnt/β-catenin signaling in the glandular tissues which can maintain the c-kit + glandular stem/progenitor cells.

Graphical abstract

Highlights

• •Sublingual salivary glands (SLG) of diabetic rats revealed deteriorated structure.
• •Diabetic rats' SLG depicted altered c-kit and β-catenin expression.
• •Curcumin restored normal architecture of SLG of diabetic rats.
• •Curcumin resumed regular expression of c-kit and β-catenin in diabetic rats' SLG.

1. Introduction

The sublingual salivary gland is one of the major glands which produces a mixed type of saliva rich in mucins. The parenchymal part of the sublingual glands includes the secretory acini and the duct system. The acinar cells are the secretory cells which drain their secretion into the duct system. The interlobular excretory ducts are the largest in the duct system and are encircled by collagen fibers. They collect the salivary secretion from the intralobular striated ducts. The smallest duct in the salivary glands is the intralobular intercalated duct and it is poorly developed in the sublingual glands.¹ In addition, in the salivary tissues reside the stem cells/progenitors that are responsible for self-renewal upon exposure to stress or injury. These cells express several markers including c-kit.²

Canonical Wnt signaling, also known as Wnt/β-catenin signaling, is pivotal for cellular proliferation, differentiation, adult tissue homeostasis, adult stem cell maintenance, tissue repair and regeneration.³ In the salivary glands, active Wnt/β-catenin cascade is crucial for normal functioning salivary glands.⁴ Canonical Wnt signaling is activated via binding of Wnt ligands to frizzled receptors and the co-receptors low density lipoprotein receptor-related protein 5/6. This binding leads to inactivation of the destruction complex and cytoplasmic β-catenin accumulation. Thereafter, β-catenin is translocated into the nucleus to share in the formation of the active transcriptional complex.³ In adult salivary gland, canonical Wnt signaling is maintained in the ductal epithelium and is upregulated during glandular regeneration.⁵

Diabetes is a chronic condition with characteristic high blood sugar levels due to altered insulin secretion or function.⁶ Chronic hyperglycemia leads to complications in the body organs as a result of oxidative stress.⁶ Diabetic patients develop hyposalivation which may result from changes in the salivary gland structure.⁷ In addition, in diabetic rats, oxidative damage leads to structural damage in the salivary gland tissues.^{8, 9, 10, 11, 12, 13, 14} Therefore, antioxidants have high potential as antidiabetic therapeutic agents.⁶

Curcumin is derived from the turmeric flowering plant, and it has an antioxidant, anti-inflammatory, antibacterial, and anticancer effect. Curcumin can alleviate diabetic complications in various conditions.⁶ It plays a crucial role in improving tissue damage in the hippocampus,¹⁵ spinal cord,¹⁶ and periodontal ligament.¹⁷ Curcumin could rescue degenerative alterations in submandibular gland of diabetic animals¹⁸ and reverse nephrotoxicity¹⁹ and submandibular gland damage induced by methotrexate in animal models.²⁰

In comparison to other major glands^18,20, there is a lack of data on the possible curative effect of curcumin on sublingual salivary gland in diabetic rats. To address this, alloxan-induced diabetic rat model was utilized to assess the prospective palliative effect of nanocurcumin on the possible histological and molecular sublingual gland abnormalities in diabetic male rats.

Our null hypothesis was that nanocurcumin will not affect the possible glandular alterations in the sublingual gland of diabetic male rats.

2. Materials and methods

2.1. Ethical statement

All the animal work was accepted by the Research Ethics Committee of Faculty of Dentistry at Minia University (Ethical Code: RHDIRB2017122001, Approval No. 913) and adhered to the guidelines of the national laws on animal protection and of the ARRIVE, and the NIH's Guide for the Care and Use of Laboratory Animals.^21,22

2.2. Animals and experimental design

Twenty-one adult Spargue Dawley male rats (weight 150–200 g) were used and divided equally into 3 groups (n = 7). The animals were housed in separate cages for each group under controlled conditions of humidity and temperature (22–25 °C) with food and water ad libitum. The control group was administered an IP injection of saline, followed by daily administration of distilled water by oral gavage for 42 days. The diabetic group contained rats receiving an IP administration of alloxan (140 mg/kg), followed by daily intake of distilled water by oral gavage. The nanocurcumin treated rat group (NC group), contained diabetic rats which received daily administration of nanocurcumin 200 mg/kg, by oral gavage for 42 days. The sample size and study design were based on previous work.¹⁸

2.3. Induction of diabetes mellitus

Rats were fasted for 12 h prior to IP administration of alloxan 140 mg/kg of the body weight (S D fine chem limited, Mumbai, India). Alloxan was freshly dissolved in 4 % saline. Three days of post-injection, blood samples were obtained from the tail vein, and the level of blood glucose was measured utilizing a Glucometer (RIGHTEST, Wiz Plus, BIONIME CORP., Taiwan). Animals with levels of blood glucose greater than 300 mg/dL were considered diabetic²³.

Seven days post-injection of alloxan, the control and diabetic groups received distilled water by oral gavage, while NC group received nanocurcumin at a dose of 200 mg/kg/day (NanoTech, Cairo, Egypt) in distilled water via oral gavage.²⁴

2.4. Dissection of sublingual glands

After 42 days of administration of nanocurcumin, the rats were euthanized by IP injection of phenobarbital 60 mg/kg.²⁵ The salivary glands were bilaterally dissected, washed in PBS and fixed in 10 % formaldehyde for 24 h.

2.5. Sample preparation for histological and histochemical evaluation

Sublingual salivary glands were washed in PBS, dehydrated in 3 changes of each alcohol concentration (70 %, 95 %, 99.5 %), 30 min per change. Then, the specimens were bathed in (4–5 changes) of xylene till clearing, 10 min per change. Finally, the samples were embedded in paraffin wax after^4,5 wax changes and 20 min per change. Five-micron-thick tissue sections were obtained utilizing microtome (CUT 5062, SLEE medical GmbH, Germany). Afterwards, the tissue sections were deparaffinized in 3 baths of xylene (5 min per change), rehydrated in descending alcohol concentrations (99.5 %, 95 %, 70 %), 5 min for each bath and rinsed in PBS. For general examination, the rehydrated tissues were stained with H&E (Life chemicals group, Alexandria, Egypt). Thereafter, the sections were washed in tap water, followed by dehydration in ascending changes of ethanol and soaked in xylene. After mounting (DPX EXTRA PURE, Alpha Chemicka, India), the tissues were cover-slipped (COVER GLASS, SAIL BRAND, China). To study collagen distribution, the tissue sections were stained with Masson's trichrome utilizing the staining kit (Atom Scientific, Machester, UK), according to the manufacturer instructions. The collagen fibers stained green in color with Masson trichrome stain ²⁶.

2.6. Immunohistochemistry

As described previously in El-Badawy et al. the immunohistochemistry was performed.²⁷ The sublingual salivary gland tissues were immunostained with β-Catenin (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA, USA) and c-kit (Ventana medical system, Arizona, USA). The dewaxed glandular tissue sections underwent citrate buffer antigen retrieval in a microwave, for 10 min. After 30 min cooling, the sections were rinsed in 3 baths of distilled water and rinsed in phosphate buffered saline, for 5 min. Afterwards, they were immersed in 3 % H2O2 to block the endogenous peroxidases with subsequent rinsing. The primary antibody was applied to the tissues for 35 min, followed by washing in PBS. The tissues were incubated with the secondary antibody (EnVision FLEX/HRP, DAKO, Denmark) for 20 min. After rinsing, the antigenic sites were visualized by using Diaminobenzidine chromogen (DAB + Chromogen, EnVision FLEX, DACO, Denmark) for 15 min.²⁷ The tissues were rinsed in distilled water, counterstained with hematoxylin for 45 s, rinsed in distilled water, dehydrated in ethanol, immersed in xylene and mounted.

2.7. Histomorphometric analysis

The c-kit immunostaining area percent was performed utilizing image J software (version 1. 48, NIH, United States of America). The area percent of c-kit immunolabelling of the acini was acquired by using standard measuring frame for analyzing 5 non-overlapping fields for each specimen (n = 7) at ×400 magnification. For this, image J was calibrated, and the pixel measurement unit was converted to micrometer unit. Following grey calibration, to determine the c-kit immunolabelling's brownish sites, the grey-marked sites were covered by red binary color. Finally, to measure the area percent, the red binary color was calculated relative to the standard measuring frame. Thereafter, the mean value for each sample was analyzed.²⁸

2.8. Statistical analysis

Analysis of the level of glucose in the blood of control and diabetic rats was performed utilizing Mann Whitney test. In addition, the c-kit immunolabelling area percent of the three studied groups was analyzed by Kruskal Wallis test followed by Dunn's post hoc test using International Business Machines Corporation, Statistical Package for the Social Sciences software package, version 20.0. (Armonk, NY: IBM Corporation). p ≤ 0.05 was set as significant.

3. Results

3.1. Higher blood glucose level in diabetic animals

Analysis of the blood glucose level in diabetic animals (389.3 ± 34.21) showed significant increase in comparison to the control animals (111.9 ± 9.48) (p < 0.001) (Fig. 1a) (Table 1).

Fig. 1.

Abnormal sublingual gland histology in diabetic rats and regained normal histology in the nanocurcumin-treated diabetic rats. (a) is a graph depicting higher levels of glucose in the blood of diabetic rats in comparison to their controls (p < 0.001). A photomicrograph of the control group (b, e, h), the diabetic group (c, f, i) and the nanocurcumin-treated group (NC group, d, g, j). (b) is a photomicrograph of the sublingual gland of control group showing tubular mucous acini with basal nuclei (black arrowhead) and capped with serous acinar cells (white arrow), the striated ducts with central nuclei and basal striations (black arrow). (c) is an image of the sublingual gland of diabetic rats depicting vacuolization in the serous acinar cells (double black arrow), distorted striated duct with loss of basal striation (black arrow) and cytoplasmic vacuolization (black arrowhead). (d) is an image of the NC group showing restoration of the regular acinar (double black arrow) and striated duct structure (black arrow). (e) is an image showing the excretory ducts with pseudostratified columnar epithelial lining (black arrow). (f) Shows the abnormal excretory duct with discontinuity in the epithelial lining (black arrow), vacuolization (black arrowhead), and hyperchromatic nuclei (white arrow). (g) Shows restoration of the regular excretory duct structure (black arrow). (h) is an image depicting an apparent thin layer of collagen fibers surrounding the interlobular excretory duct in the control sublingual gland (black arrow). (i) Shows an abnormal thick detached layer of collagen fibers in the sublingual gland of diabetic rats (black arrow). (j) is an apparent thin layer of collagen fibers around the interlobular excretory duct of the NC group (black arrow, c). (H and E, magnification ×400 (b–g); Masson trichrome, magnification ×400 (h–j)).

Table 1.

Comparison between the control and diabetic rats according to blood glucose level.

	Control rats (n = 7)	Diabetic rats (n = 14)	U	p
Blood glucose level
Min. – Max.	101.0–126.0	327.0–439.0	0.00	<0.001∗
Mean ± SD.	111.9 ± 9.48	389.3 ± 34.21
Median (IQR)	108.0 (105.0–119.0)	392.5 (367.0–413.0)

IQR: Inter quartile range SD: Standard deviation U: Mann Whitney test.

p: p value for comparing between the three studied groups.

∗: Statistically significant at p ≤ 0.05.

3.2. Aberration in sublingual salivary gland histology in diabetic rats and rescued sublingual salivary gland structure after nanocurcumin administration

To determine the histological alteration in the sublingual glands of diabetic rats, the sublingual gland tissue sections were assessed after H & E, and Masson trichrome staining. The sublingual salivary gland tissue of the control group revealed tubular mucous acini. The mucous cells possessed eosinophilic foamy cytoplasm and flat basal nuclei. Few serous cells capped the mucous acini and had central nuclei. The epithelial lining of the striated ducts consisted of columnar cells with central nuclei and basally located striations. The excretory ducts had empty lumen, lined by pseudo stratified columnar epithelium and surrounded by dense connective tissue (Fig. 1b, e, 1h).

The glandular tissue of the diabetic group depicted irregularly shaped large mucous acini with ill-defined acinar cell boundaries. The striated ducts revealed distorted outline, loss of basal striation, vacuolization and disorganized hyperchromatic nuclei. In addition. The excretory duct showed discontinuity in the epithelial lining and cytoplasmic vacuolization. The lumen showed stagnation of the salivary secretion. Furthermore, some nuclei were abnormally hyperchromatic or pyknotic and an apparent increase in the collagen surrounding the excretory duct was detected (Fig. 1c, f, 1i). The salivary tissue of the NC group revealed regularly shaped tubular acini capped with serous cells, and the acinar cell depicted well-defined outlines. The striated ducts depicted columnar cells with basally situated striations and central nuclei. The excretory ducts displayed normal histology and an apparent regular connective tissue thickness (Fig. 1d, g, 1j)

3.3. Alteration in β-catenin expression in sublingual salivary gland tissues of diabetic rats and its restoration in nanocurcumin-treated rats

To show the localization of β-catenin in the sublingual glands of control rats and to assess the possible alterations in β-catenin expression in diabetic animals and the role of nanocurcumin treatment in diabetic rats, β-catenin immunohistochemistry was carried out. The sublingual salivary gland mucous acini of the control group revealed moderate cytoplasmic immunoreaction for β-catenin and weak membranous immunostaining. The striated ducts revealed strong cytoplasmic and membranous β-catenin immunostaining and moderate nuclear immunoreaction for β-catenin. The excretory duct displayed robust nuclear, cytoplasmic and membranous β-catenin immunoreaction. The salivary gland acini of the diabetic rats displayed weak cytoplasmic immunoreaction for β-catenin with very weak immunoreaction in the acinar wall. The striated ducts of the diabetic rats revealed apparently moderate immunoreaction in the cytoplasm and membranes with very weak immunostaining nuclei. The excretory ducts of the diabetic group revealed areas of very weak β-catenin immunolabelling in the nuclei and cytoplasm and membranes of the duct lining cells, alternate with areas of moderate immunoreaction. The mucous acini, excretory ducts and striated ducts of the NC group resumed the normal β-catenin immunostaining (Fig. 2a–f).

Fig. 2.

Alteration in β-catenin and c-kit immunostaining in the sublingual glands of the diabetic group and their restoration in the nanocurcumin-treated animals. (a, d, g) are photomicrographs of the control group, (b, e, h) of the diabetic group and (c, f, i) of the nanocurcumin-treated group (NC group). An image shows the moderate nuclear β-catenin expression in the striated duct of the control group (black arrow, a), very weak immunostaining in the diabetic rats (black arrow, b), and moderate in the NC group (black arrow, c). Another image shows strong nuclear β-catenin staining in the excretory duct of the control group (black arrow, d), areas of apparent loss of the β-catenin nuclear immunoreaction in the excretory ducts of the diabetic rats (black arrow, e), and restoration of the strong nuclear β-catenin immunolabelling in the NC group (black arrow, f). One of the images shows c-kit⁺ progenitor cells in the sublingual gland of control group (black arrow, g), diabetic group (black arrow, h), and NC group (black arrow, i). (β-catenin immunohistochemistry, magnification ×400 (a–f); c- Kit immunohistochemistry, magnification ×400 (g–i)). (j) is a graph depicting low c-kit immunostaining area percent in the diabetic group in comparison to their controls (p < 0.001), and higher c-kit immunolabelling area percent in the NC group compared to the diabetic rats (p = 0.007).

3.4. The c-kit immunohistochemical change in sublingual salivary glands of diabetic rats and recovery after nanocurcumin administration

To determine the possible alterations in the c-kit⁺ progenitors in the sublingual glands of diabetic animals and the effect of nanocurcumin treatment on c-kit distribution, c-kit immunohistochemistry was used. The sublingual gland of the control animals revealed numerous c-kit immunopositive cells, however the diabetic group revealed apparent decrease in the c-kit immunolabelled cells. The NC group depicted an apparent increase in the c-kit + progenitors (Fig. 2g–i). The c-kit immunoreaction area percent in the sublingual glands of diabetic rats (0.17 ± 0.04) was decreased in comparison to their controls (0.46 ± 0.13) (p < 0.001). The c-kit immunolabelling area percent in the NC group (0.37 ± 0.06) was insignificantly different from that of their controls (p = 0.366) and was significantly higher than the c-kit immunolabelling area percent of the diabetic group (p = 0.007) (Fig. 2j)(Table 2).

Table 2.

Comparison between the control, diabetic and nanocurcumin treated groups according to the c-Kit immunostaining area percent.

	Control (n = 7)	Diabetic (n = 7)	Curcumin treated (n = 7)	H	p
C-Kit
Min. – Max.	0.36–0.67	0.10–0.24	0.27–0.44	14.182∗	0.001∗
Mean ± SD.	0.46 ± 0.13	0.17 ± 0.04	0.37 ± 0.06
Median (IQR)	0.38 (0.37–0.54)	0.17 (0.15–0.20)	0.37 (0.34–0.42)
Significance between groups	p1 < 0.001∗,p2 = 0.366,p3 = 0.007∗

IQR: Inter quartile range SD: Standard deviation.

H: H for Kruskal Wallis test, Pairwise comparison bet. each 2 groups was done using Post Hoc Test (Dunn's for multiple comparisons test).

p: p value for comparing between the three studied groups.

p₁: p value for comparing between Control and Diabetic.

p₂: p value for comparing between Control and Curcumin treated.

p₃: p value for comparing between Diabetic and Curcumin treated.

∗: Statistically significant at p ≤ 0.05.

4. Discussion

Sublingual salivary gland is one of the major salivary glands that are predominately mucous in nature. In the current work, the role of curcumin in alleviating the possible structural and molecular alterations in the sublingual glands of diabetic rats was evaluated.

In the present work, the sublingual salivary gland of the diabetic animals depicted a deteriorated histological structure of the serous acini, and duct system. Our results are consistent with the findings of the earlier studies on the sublingual glands of diabetic animals.^{8, 9, 10} These degenerative alterations can be induced by persistent hyperglycemia in the diabetic rats that lead to oxidative stress as a result of reactive oxygen species accumulation. The increase in the reactive oxygen species leads to damage in the cellular structures.²⁹ By contrast, Huang et al. reported the absence of histological changes in the sublingual gland. This difference in the findings may be due to species differences.¹¹

In this work, and in contrast to Huang et al.¹¹ who did not detect obvious structural alterations in the sublingual glands of diabetic animals, the serous acinar cells, striated ducts and excretory ducts of the sublingual gland of diabetic rats depicted vacuolization. Our results are in line with the findings in earlier studies on the sublingual glands of diabetic animals.^8,9 The authors explained that these vacuoles could be lipid droplets that formed as a result of the insulin decrease and the consequent decrease in the protein formation and secretory granule production. This is associated with the decrease in the lipid utilized in the synthesis in the secretory granule limiting membrane and the excess of lipids stored as droplets^8,9.

Our results in the NC group showed that curcumin alleviated the sublingual salivary glands abnormalities in the diabetic rats. Our data are consistent with the earlier studies showing the role of curcumin in treating the damaged tissue including the submandibular gland of diabetic rats,¹⁸ submandibular gland of methotrexate-treated rats,²⁰ periodontitis in diabetic patients,³⁰ and the abnormalities in the hippocampus induced by intermittent hypoxia.¹⁵ This could be due to the antioxidant and anti-inflammatory effect of curcumin. Moreover, it has been demonstrated that the regular intake of curcumin can improve the metabolic condition including blood glucose and cholesterol.³¹ In addition, curcumin can upregulate canonical Wnt signaling which plays a role in tissue regeneration.^{15, 16, 17}

When Wnt ligand binds to their receptors and coreceptors, a cascade of interactions takes place and cytoplasmic accumulation of β -catenin takes place, followed by its translocation into the nucleus and canonical Wnt cascade activation. Active Wnt/β-catenin signaling has a crucial role in many cellular processes, in addition to tissue regeneration.³ Curcumin regulates canonical Wnt signaling in several tissues and enhances their regeneration in various diseases.^{15, 16, 17} In this work, nuclear β-catenin was detected in the striated and excretory ducts of the sublingual gland tissue and was apparently deceased in these glandular tissues of diabetic rats. In addition, the expression was apparently restored in the ducts of the nanocurcumin-treated rats. Our data are consistent with the findings of previous work on the spinal cord¹⁶ and hippocampus,¹⁵ showing the regenerative role of curcumin by activating Wnt/β-catenin in these injured tissues; spinal cord¹⁶ and hippocampus. In the hippocampus, the authors demonstrated that curcumin increased the proliferation of the nerve stem cells and enhanced neonatal neurons' development.¹⁵ In line with our findings, active canonical Wnt signaling was detected in the submandibular gland ducts of adult mice⁵ and was upregulated after main duct ligation during the duct regeneration process.⁵ Moreover, the osteogenesis induced by the periodontal ligament stem cell extracellular vesicles was improved after utilizing curcumin and was associated with Wnt/β-catenin upregulation.¹⁷

The c-kit is one of the markers of the salivary gland stem cells/progenitors. These cells are capable of expansion and differentiated into salivary acinar and ducal cells.² In the current study, c-kit immunoreaction was localized in the tissues of the control sublingual glands. The c-kit immunostaining area percent in the glandular tissues of the diabetic rats was diminished, however, was restored in the NC-treated animals. Our results are in line with the previous studies in the submandibular salivary gland, in which glandular regeneration is linked to upregulation of canonical Wnt signaling,⁵ and expansion of c-kit + stem cells/progenitors.² In addition, in the hippocampus, curcumin could increase the proliferation level of the nerve stem cells, may be due to the capacity of curcumin to upregulate canonical Wnt signaling cascade.¹⁵

The limitation of the current work was the absence of functional investigations and the inability to determine the mechanism behind β-catenin dysregulation in diabetic rats. Therefore, further studies are needed to investigate the functional restoration of the sublingual glands in diabetic rats after curcumin treatment. In addition, future comparative studies with other glands affected by diabetes such as pancreas and biochemical anti-inflammatory and antioxidant analyses are also recommended to broaden our knowledge about the possible ameliorative effect of curcumin on the different tissues. Furthermore, future molecular studies are also needed to understand more about the nature of the molecular alterations in the gland of diabetic cases for proper therapeutic manipulation of the accompanied hyposalivation.

5. Conclusion

The sublingual salivary glands of diabetic male rats showed disfigured structure and altered β-catenin and c-kit expression. Nanocurcumin treatment rescued the glandular histology, c-kit and β-catenin immunostaining in the diabetic rats. The present data suggest an ameliorating role of nanocurcumin in treating the damaged sublingual tissues of diabetic rats. This ameliorating role may have resulted from the restoration of the Wnt/β-catenin transduction cascade in the salivary tissues which may maintain the stemness of the c-kit⁺ salivary gland cells.

CRediT authorship contribution statement

Conceptualization, Data curation, Formal analysis, Funding acquisition; investigation, methodology, Resources, Validation, Visualization, Writing - original draft, writing-review and editing.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors.

Declaration of competing interest

None. We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Data availability

All data are available from the corresponding author upon a reasonable request.