Protective effect of nano vitamin D against fatty degeneration in submandibular and sublingual salivary glands: A histological and ultrastructural study

Abstract

Background: Poor nutritional habits and a low level of physical activity are associated with obesity, leading to increased caloric and fat intakes. A high-fat diet can significantly impact oral health through the accumulation of lipids in the salivary glands, which ultimately affect salivary gland function. Recently, an increasing number of supplement nano-formulations, such as nano vitamin D, have become available. However, only few studies have explored the effects of nano vitamin D on the maintenance of oral health.

Objective: This study aimed to compare the histological effects of nano vitamin D to those of regular vitamin D on fatty degeneration in submandibular and sublingual salivary glands using a rat model.

Methods: Twenty-four adult male albino Sprague-Dawley rats were divided into the following groups: untreated group, high-fat diet group, high-fat diet and regular vitamin D group, and high-fat diet and nano vitamin group.Thereafter, samples of the submandibular and sublingual salivary glands were dissected for histological and electron microscopic studies. Morphometric digital image analysis was used to quantitatively measure the changes in the size and number of acini and secretory granules.

Results: Regular vitamin D had a partial protective effect. However, vitamin D could fully restore cellular structures to their normal state, thereby protecting against fatty degeneration of the salivary tissue and immune cell infiltration, particularly in the submandibular serous tissue. Nano vitamin D was more efficacious than regular vitamin D at restoring the number and size of submandibular serous secretory granules.

Conclusion: Employing nano vitamin D as a supplement to high-fat diets could protect against high-fat diet-induced salivary gland damage in rats.

Keywords: Degeneration; High-fat diet; Histology; Nano vitamin D; Rat; Salivary gland; Sublingual; Submandibular.

Figure 1

Histological image of the submandibular salivary gland serous acini stained with H&E. The untreated group (Panel A, A1) has serous acini (S.A.) with spherical basal nuclei. The connective tissue stroma (C.T.) has striated ducts (S.D.) separated by thin inter-acinar connective tissue. Granular convoluted tubules (G.C.T.) appear with granular cytoplasmic content. The high-fat diet group (Panel B, B1) shows a decrease in acinar cells (S.A.) with a loss of the regular round outlines (black arrow), pyknotic darkly stained nuclei (arrowhead), multiple vacuolizations within the acinar cells (V), and cellular remnants scattered throughout the gland (white arrow). An increase in the number of hypertrophied irregular outlines of granular convoluted tubules (G.C.T.) with signs of degeneration and cytoplasmic vacuolization (V) and inflammatory cellular infiltration (asterisks) is observed. The high-fat diet and vitamin D group (Panel C, C1) shows restored acinar architecture with deeply stained nuclei (S.A.) despite variable degrees of minor cytoplasmic vacuolization (V). Well-formed granular convoluted tubules (G.C.T.) are observed. The high-fat diet and nano vitamin D group (Panel D, D1) shows acinar cells (S.A.) with homogenous cytoplasm, few vacuoles (V), and intact multiple granular convoluted tubules with an increase in its granular content (G.C.T.). Note: The excretory ducts have a healthy cell lining and clear lumen (E.D.).

Figure 2

Histological images of the sublingual salivary gland mucous acini stained with H&E. The untreated group (Panel A, A1) has mucous acini (M.A.) with pale basophilic spongy cytoplasm, flattened basal nuclei, and myoepithelial cells (black arrow) grasping the acini. The connective tissue stroma (C.T.) has an intercalated duct (I.D.), striated ducts (S.D.), and an execratory duct (E.D.). The high-fat diet group (Panel B, B1) has densely packed mucous acinar cells with irregularly shaped giant acini (white arrows). Thickened and fibrotic connective tissue septa (C.T.) with cellular infiltration (asterisks) and diminished and/or absent interlobular spaces are observed. The high-fat diet and vitamin D group (Panel C, C1) has packed mucous acinar cells with some giant acini (white arrow); a decrease in the fibrotic connective tissue septa (C.T.) with no cellular infiltration and the interlobular spaces has started appearing (dotted arrow). The high-fat diet and nano vitamin D group (Panel D, D1) shows the typical general architecture of the mucous acini and duct system. The wide area between the acini is clear, with no fibrotic connective tissue or inflammatory cells observed. Note: Few giant acini are observed (white arrow).

Figure 3

Electron micrograph image of the submandibular salivary gland serous acini. The untreated group (Panel A, A1) shows regular-shaped serous acinar cells with a vesicular nucleus (N) and secretory granules with different electron densities (S.G.). The high-fat diet group (Panel B, B1) has acinar cells with marked cell organelle degeneration (asterisk), leaving few strands of rough endoplasmic reticulum (R.E.R.), small darkly stained degenerated mitochondria (M), lipid droplet deposition (F), and deformed dense nuclei (N). Note: The number of secretory granules decreased, and the granules appeared to fuse with each other (white arrows). The high-fat diet and vitamin D group (Panel C, C1) has serous acinar cells clustered with irregular-shaped electron-dense nucleus (N), immature light, dense serous granules (S.G.), few intracytoplasmic vacuolizations (V), and decreased lipid deposition (F). The high-fat diet and nano vitamin D group (Panel D, D1) shows active secreting acinar cells with a vesicular nucleus (N) surrounded by parallel arrays of the rough endoplasmic reticulum (R.E.R.). Numerous round, mature, and immature serous secretory granules (S.G.) in the process of releasing their content into the lumen (curved arrow) are observed. Note: The absence of cytoplasmic vacuolization and one to two lipid droplets are observed (F).

Figure 4

Electron micrograph image of the sublingual salivary gland mucous acini. The untreated group (Panel A, A1) has mucous acinar cells with an open-faced nucleus (N) surrounded by parallel arrays of the rough endoplasmic reticulum (R.E.R.), numerous lightly stained, lucent secretory granules, and mitochondria (M) with parallel patterns of crests. The high-fat diet group (Panel B, B1) shows acinar cells with malformed nuclei (N) and various alterations in the cytoplasmic organelles, including decreased secretory granules (S.G.), cell organelle degeneration (white arrows), dilated Golgi apparatus (G), intracytoplasmic vacuolization (V), and lipid droplet deposition (F). The high-fat diet and vitamin D group (Panel C, C1) shows an intact nucleus (N) and standard light mucous secretory granules, but a decreased number compared to the untreated group. Some cytoplasmic vacuolization (V) and lipid droplet deposition (F) in between is observed. Note: Wide intercellular canaliculi (asterisks). The high-fat diet and nano vitamin D group (Panel D, D1) has healthy appearance of acinar cells encircling the lumen (L) with intact nucleus (N) and clusters of electron-lucent mucous secretory granules (S.G.). Note: Few cytoplasmic vacuolizations (V) and one to two lipid droplets are observed (F).

Figure 5

Graph of the average number and area of acini in the submandibular and sublingual salivary glands. Digital morphometric analysis was performed on serous and mucous acini H&E images. (A) Average number of submandibular serous acini. (B) Average number of sublingual mucous acini. (C) Average size of the submandibular serous acini (μm²). (D) Average size of the sublingual mucous acini (μm²). Data are presented as mean ± SD. Differences between groups were identified using one-way ANOVA, followed by Tukey's multiple comparison post-test, indicated above the bars. a: comparison between the untreated and high-fat diet group; b: comparison between the untreated and vitamin D group; c: comparison between the untreated and nano vitamin D group; d: comparison between the high-fat diet and vitamin D group; e: comparison between the high-fat diet and nano vitamin D groups; and f: comparison between the vitamin D and nano vitamin D group. Subscript numerals indicate the p-value: ⁴p˂0.001, ³p˂0.005, ²p˂0.01, ¹p˂0.05, ⁰no significant difference.

Figure 6

Graph of the average number and area of secretory granules per acini in the submandibular and sublingual salivary glands. Digital morphometric analysis was performed on the serous and mucous TEM images. (A) Average number of submandibular serous secretory granules. (B) Average number of sublingual mucous secretory granules. (C) Average size of the submandibular serous secretory granules (nm²). (D) Average size of the sublingual mucous secretory granules (nm²). Data are presented as mean ± SD. Differences between groups were identified using ANOVA, followed by Tukey's multiple comparison post-test, indicated above the bars. a: comparison between the untreated and high-fat diet group; b: comparison between the untreated and vitamin D group; c: comparison between the untreated and nano vitamin D group; d: comparison between the high-fat diet and vitamin D group; e: comparison between the high-fat diet and nano vitamin D group; and f: comparison between the vitamin D and nano vitamin D group. Subscript numerals indicate the p-value: ⁴p˂0.001, ³p˂0.005, ²p˂0.01, ¹p˂0.05, ⁰no significant difference.