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Original Article

Anat Cell Biol 2024; 57(1): 129-142

Published online March 31, 2024

https://doi.org/10.5115/acb.23.230

Copyright © Korean Association of ANATOMISTS.

Bisphosphonate’s effect on the tongue in adult male albino rats and the possible protective role of rutin: light and scanning electron microscopic study

Dalia El-sayed El-ghazouly , Rania Ibrahim Yassien

Department of Histology and Cell Biology, Faculty of Medicine, Menoufia University, Al Menoufia, Egypt

Correspondence to:Dalia El-sayed El-ghazouly
Department of Histology and Cell Biology, Faculty of Medicine, Menoufia University, Shibin Al-kom 32511, Al-Menoufia, Egypt
E-mail: daliaelghazouly@yahoo.com

Received: September 3, 2023; Revised: December 14, 2023; Accepted: December 21, 2023

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Alendronate sodium (ALS) is a nitrogen-containing bisphosphonate used for the treatment of different bone disorders. However, its adverse effect on oral soft tissue has been detected. Rutin (RUT) is natural flavonoid with antioxidant and anti-inflammatory properties. This work aimed to investigate the possible effect of ALS on the tongue of adult male albino rats and to evaluate the possible protective role of RUT. Forty adult male albino rats were equally divided into four groups: group I (control), group II (RUT): Received RUT 50 mg/kg, group III (ALS): Received ALS 1 mg/kg, group IV (ALS+RUT): Received ALS and RUT with the same doses as pervious groups. The drugs were given once daily for 5 weeks. Tongue specimens were taken and processed for light and scanning electron microscopic inspection. ALS treated group revealed structural changes in the tongue in the form of decrease in the height of the filiform papillae with blunt ends, marked atrophy in some papillae with areas of focal loss, loss of some epithelial cells, pyknotic nuclei and cytoplasmic vacuoles in some epithelial cells. The lamina propria showed inflammatory cellular infiltration with congested blood vessels. Statistically, there were highly significant decrease in the number of proliferating cell nuclear antigen immunopositive cells, area percentage of Bcl-2 immunoexpression and highly significant increase in the collagen content compared to control group. Administration of RUT with ALS minimizes these changes. RUT protected the rat tongue against the histological and immunohistochemical changes induced by ALS through its antioxidant and anti-inflammatory properties.

Keywords: Bisphosphonates, Rutin, Tongue, Proliferating cell nuclear antigen, Scanning electron microscopy

Bisphosphonates (BPs) are category of antiresorptive agents widely used for osteoporosis treatment. These drugs raise the bone density and decrease the danger of fracture in osteoporotic individuals. In addition, calcium metabolic diseases as hypercalcemia of malignancy, Paget’s disease, multiple myeloma are treated by these agents [1].

BPs are synthetic analogues of pyrophosphate, highly efficient in binding bone hydroxyapatite. These agents perform anticatabolic role by changing osteoclast mobilization, differentiation and function, and/or by triggering apoptosis, thus leading to a reduction in bone resorption [2, 3].

BPs are divided into two types which differ in their mechanism of action: nitrogen-containing bisphosphonates (N-BPs) and non-nitrogen-containing bisphosphonates (non-N-BPs) [4]. N-BPs possess the most potent antiresorptive impact as alendronate, zoledronic acid, ibandronate, and risedronate [5].

Alendronate and risedronate are the most commonly used agents for osteoporosis. In comparison with other BPs, alendronate is more active and selective because it has an amino acid group in its side chain [6].

The therapeutic uses of BPs have been explored for many years with reports of many side effects [3]. Concerning the gastrointestinal tract, many adverse effects were documented including dyspepsia, abdominal pain, nausea, vomiting, bleeding, diarrhea, constipation, oral ulcers, esophagitis, esophageal ulcers, gastric ulcer, cancers of gastrointestinal tract, and hepatotoxicity [7]. There is evidence in some studies that oral BPs (including alendronate) in high doses may possess adverse effects on the mucosa of oral cavity with the induction of an inflammatory reaction in the tongue and buccal mucosa [7-9]. Moreover, several case reports have described negative impacts of oral BPs on the oral soft tissues of the patient such as great discomfort and pain [3].

Flavonoids are natural polyphenols present in several fruits, seeds and leaves [10]. Flavonoids affect human health by providing a high level of protection against reactive oxygen species (ROS) [11]. Rutin (quercetin-3-rutinoside, RUT) is a nutritional substance from the flavonoid group formed of quercetin combined with the disaccharide Rutinose. It is recognized as vitamin P [12]. Orange, tomato, sweet potato, carrot, apple peels and black tea are copious sources of RUT [13]. RUT has been found to exhibit antioxidant, vascular prophylactic, anticoagulant, anti-apoptotic, cytoprotective, anti-inflammatory, antiulcerogenic, cardioprotective, neuroprotective, renoprotective, and hepatoprotective, activities as well as gastroprotective efficacy [11]. RUT can attach to the iron ion, prohibiting it from combination with hydrogen peroxide and hence protects the cell from damage by oxygen free radicals [14]. A wide variety of the biological and pharmacological activities of RUT has been studied, which reveals that RUT may be used as a key molecule for an advanced clinical use [11].

From the foregoing, it seems that BPs have adverse side effect on the oral soft tissues including the tongue. Thus, the purpose of the current work is to investigate the possible impact of alendronate sodium (ALS), as one of N-BPs, on the tongue of adult male albino rats and to evaluate the possible protective role of RUT through light and scanning electron microscopic study (using histological and immunohistochemical techniques).

Materials

Drugs

ALS was found in tablet form 70 mg with trade name Fosamax (M.O.H Reg. No. 195/2014; Merck & Co. Inc.). The drug was dissolved in distilled water and administrated orally to rats using gastric tube. To get solution having 0.2 mg/1 ml, we dissolved one tablet in 350 ml of water.

RUT was bought from Sigma-Aldrich chemical company. RUT was available in capsule form 450 mg. The drug was dissolved in distilled water and administrated orally to rats using gastric tube. To get solution having 10 mg/1 ml, we dissolved one capsule powder in 45 ml of water.

Animals

Forty adult male albino rats with a weight range 180–200 g were utilized in this work. Animal care and hygiene were preserved to retain them in good and sanitary atmosphere. They were given balanced food with free access to water. All animal methods were carried out according to approved protocols and in accordance to the recommendations for suitable care and usage of experimental animals (approval number: 2/2023 HIST17).

Experimental design: the animals were divided into 4 groups (10 rats in each group). In each group, the rats received their drugs once daily for 5 weeks.

Group I (control group): animals of this group were subdivided into 2 subgroups of 5 rats each:

· Subgroup Ia: left without treatment throughout the period of experiment.

· Subgroup Ib: received 1 ml distilled water orally by gastric tube.

Group II (RUT group): received RUT 50 mg/kg [11] orally using gastric tube. Each animal received 1 ml of RUT solution.

Group III (ALS group): received ALS 1 mg/kg [6] orally using gastric tube. Each animal received 1 ml of ALS solution.

Group IV (ALS+RUT group): received ALS concomitantly with RUT using the same doses like groups II, III.

Methods

After the last dose by 24 hours, the animals were anaesthetized using pentobarbital (35 mg/kg) [15] given intraperitoneally and then sacrificed. The tongues were excised and washed. The anterior 2/3 of each tongue was divided along the midline into 2 halves. One half was prepared for light microscopic inspection while the other half was prepared for scanning electron microscopic inspection.

Light microscopic study

Specimens were put in formalin 10% and prepared by the usual method to get paraffin blocks. 4 μm thick sections were cut and the following studies were carried out: (1) Histological study: Hematoxylin & eosin (H&E) stain and Masson’s trichrome [16]. (2) Immunohistochemical study: for detection of proliferating cell nuclear antigen (PCNA, cell proliferation marker) [17] and Bcl-2 (an antiapoptotic marker) [18].

It was done on 4-mm thick Paraffin sections by utilizing streptavidin–biotin complex method. The sections were incubated with primary mouse monoclonal anti PCNA antibody (CAT.# MS-106-R7, dilution1:200; Lab Vision Corporation) and mouse monoclonal anti Bcl-2 (Cat.# sc-7382, dilution1:50; Santa Cruz Biotechnology). The positive control of the primary antibodies was rat tonsil for PCNA and mouse colon for Bcl-2. The negative control was done by adding phosphate buffer solution instead of the primary antibody. PCNA appeared as brown nuclear staining. Bcl-2 immunoexpression appeared as brown cytoplasmic staining.

Scanning electron microscopic study

Specimens of the tongues were put in 2.5% glutaraldehyde and prepared for examination by scanning electron microscope (JSM-52500LV; JEOL Ltd.) in Unit of Electron Microscope, Faculty of Medicine, Tanta University, Egypt [19].

Morphometric measurements

1. Height of the filiform lingual papillae in H&E‐stained sections magnified at 400.

2. Thickness of the ventral epithelium in H&E‐stained sections magnified at 400.

3. Area percent of collagen fiber content in Masson’s trichrome-stained sections magnified at 200.

4. Number of PCNA positive cells in the dorsal epithelium was counted in PCNA immuno-stained sections magnified at 400.

5. Area percentage of Bcl-2 immunoexpression in the dorsal epithelium was estimated in Bcl-2 immuno-stained sections magnified at 400.

The measurements were picked by computerized image analyzer (Leica Q500 MC Program; Leica Microsystems Ltd.) and carried out into ten sections of 5 animals for every group.

Statistical analysis

SPSS program, version 17 (IBM Co.) was used for statistical analysis of morphometric results. The mean±standard error of mean (SEM) was used to demonstrate data. ANOVA followed by “Tuckey” post-hoc test was utilized to compare between the groups. Comparisons were considered highly significant in case of P-value <0.001, significant in case of P-value <0.05 and non-significant if P-value >0.05 [20].

In the present work, no mortality was recorded throughout the experimental period. All subgroups of the control group didn’t show any difference in the histological, immunohistochemical or morphometric and statistical results. Therefore, subgroups (Ia & Ib) were referred to as the control group. Regarding group II (RUT group), it also showed no difference in all result parameters compared with group I (control group).

Light microscopic results

Histological study

H&E staining: H&E-stained sections of control group (I) exhibited the normal well-known histological structure of the rat tongue. The dorsal surface was covered by a mucous membrane that revealed numerous filiform papillae. Filiform papillae had a conical shape with characteristic pointed ends. The papillae had regular distribution and orientation. Each papilla was composed of connective tissue (CT) core having small blood vessels and covered with stratified squamous keratinized epithelium sitting on basement membrane with numerous epithelial ridges (Fig. 1A, B). The four layers of stratified epithelium are basal cell layer, spinous cell layer, granulosum cell layer and superficial corneum layer (Fig. 1C). The CT cores were continuous with the lamina propria (Fig. 1B). Among filiform papillae, there were few fungiform papillae scattered in-between them. They had distinctive mushroom shape and were formed of a CT core containing small blood vessels and an epithelial cover (Fig. 1D). A smooth mucous membrane was found to cover the ventral surface of the tongue and was formed of stratified squamous keratinized epithelium with underlying lamina propria (Fig. 1E). Underneath the lamina propria, the skeletal muscle fibers were arranged in different directions (Fig. 1A, B, E). On the other hand, ALS group (III) revealed severe alterations in the tongue. The dorsal surface showed apparent reduction in filiform papillae height with blunt ends and separated parts of keratin layer (Fig. 2A, C). Disorganization of the epithelial cells was observed with focal loss of some cells (Fig. 2A–D). There were deeply stained pyknotic nuclei and cytoplasmic vacuoles in some epithelial cells of the filiform and fungiform papillae (Fig. 2B, D, E). Also, some epithelial cells revealed mitotic activity and other superficial cells showed many basophilic granules (Fig. 2D). The underlying CT cores and lamina propria exhibited inflammatory cell infiltration and dilated congested blood vessels. Areas of CT core and lamina propria were lost (Fig. 2A, C–E). The ventral surface revealed areas of thinned and thickened stratified epithelium with separated keratin layer. Dilated congested blood vessels with inflammatory cell infiltration were seen in the underlying lamina propria (Fig. 2F, G). Some muscles fibers appeared disrupted with wide spaces in between. Hypereosinophilic muscles fibers were noticed (Fig. 2C, F, H). Interestingly, sections from group IV (ALS+RUT) exhibited much preservation of the histological features of the tongue. Most lingual papillae revealed almost normal structure with normal epithelium and keratin layer. However, few disfigured papillae and some pyknotic nuclei were noticed in few areas of the dorsal surface. The CT core and lamina propria appeared normal except for some dilated congested blood vessels (Fig. 3A–C). The ventral surface had a normal appearance with a thin keratin layer over the stratified epithelium, some pyknotic nuclei were noticed (Fig. 3D). Most of the muscles fibers had a normal appearance (Fig. 3A, D).

Figure 1. A photomicrograph of H&E-stained sections of the anterior 2/3 of rat’s tongue from control group (I). (A) The dorsal surface showing regular orientation of numerous filiform papillae with tapering ends (arrows). Skeletal muscle fibers appear in different directions; some of them appear longitudinal (L), others appear polygonal (P). (B) A higher magnification of (panel A) showing each papilla (arrows) formed of a connective tissue (CT) core (*) with small blood vessels (Bv, blue arrow) and is covered by a keratinized stratified squamous epithelium (E) resting on a basement membrane with many epithelial ridges (arrowhead). The CT cores are continuous with the lamina propria (LP). Muscle fibers appear polygonal (P) with acidophilic cytoplasm and peripherally positioned nuclei. (C) A higher magnification of (panel B) showing the keratinized stratified squamous epithelium (E) with its basal cell layer (B), spinous cell layer (S), granulosum cell layer (G) and superficial corneum layer formed of keratin (K). (D) The dorsal surface showing fungiform papillae (arrowheads) in between the filiform ones (arrows). It has CT core (*) with small Bv and an E cover. (E) The ventral surface showing a smooth surface without papillae. It shows a thin keratin layer (arrows) over the stratified squamous E cover and the underlying LP (*). H&E stained, (A) ×100, (B, E) ×200, (C, D) ×400.
Figure 2. A photomicrograph of H&E-stained sections of the anterior 2/3 of rat’s tongue from alendronate sodium group (III). (A) The dorsal surface showing an apparent decrease in the height of the filiform papillae with blunt ends (arrows). Focal loss of epithelial cells, connective tissue (CT) core and lamina propria is observed (*). Disorganized epithelial cells (arrowhead) are observed. Separated parts of keratin layer (K). (B) A higher magnification of (panel A) showing disorganized epithelial cells (arrowheads). Focal loss of epithelial cells is observed (*). Some epithelial cells appear with deeply stained pyknotic nuclei (arrows). (C) The dorsal surface showing an apparent decrease in the height of some filiform papillae with blunt ends (black arrow). Focal loss of epithelial cells is observed (*). The underlying CT core and lamina propria show inflammatory cellular infiltration (I, blue arrows). Disrupted muscle fibers (arrowheads). (D) The dorsal surface showing focal loss of epithelial cells (*), pyknotic nuclei and vacuoles of some epithelial cells (arrowheads). Mitotic activity is obvious in some epithelial cells (black arrow). Many basophilic granules were seen in the superficial cells (blue arrows). Congested blood vessel (Bv) in CT core. (E) The dorsal surface showing a fungiform papilla with pyknotic nuclei and vacuoles of some of its epithelial cell cover (arrows). The taste bud shows pyknotic nuclei in some cells (arrowhead). The CT core shows dilated congested Bv. (F) The ventral surface showing separation of the K layer from the underlying epithelium (E, arrow). The lamina propria shows dilated congested Bv. Disrupted muscles fibers (arrowheads). (G) The ventral surface showing areas of thinned and thickened stratified E. Vacuolated epithelial cells with pyknotic nuclei are observed (arrows). The lamina propria shows inflammatory cell (I) with elongated dilated congested Bv. (H) Showing disruption in the muscle fibers (arrows). Some muscle fibers appear hypereosinophilic (*). Wide spaces (S) between the muscles. H&E stained, (F) ×100, (A, C, G, H) ×200, (B, D, E) ×400.
Figure 3. A photomicrograph of H&E-stained sections of the anterior 2/3 of rat’s tongue from alendronate sodium+rutin group (IV). (A) The dorsal surface showing regular orientation of the lingual papillae. Most of filiform papillae are long with pointed tips and covered by stratified squamous keratinized epithelium (arrows), few papillae appear disfigured (arrowheads). Dilated congested blood vessels (Bv) are seen in the connective tissue (CT) core and lamina propria. Normal appearance of muscles (M) that run in different directions. (B) A higher magnification of (panel A) showing the keratinized stratified squamous epithelium with most of the cells appearing normal with vesicular nuclei (black arrows), few ones appear with pyknotic nuclei (orange arrows). One papilla appears disfigured (arrowhead). Dilated congested Bv in the CT core and lamina propria. (C) The dorsal surface showing normal appearance of the fungiform papilla (arrow). Some pyknotic nuclei are seen (arrowhead). (D) The ventral surface showing a thin keratin layer (arrow) over the stratified squamous epithelial cover (E). Some pyknotic nuclei are seen (arrowhead). Most of the muscle (M) fibers show normal appearance. H&E stained, (A, D) ×200, (B, C) ×400.

Masson’s trichrome staining: The control group (I) exhibited normal distribution of regularly arranged collagen fibers in the CT core, lamina propria and thin rims in between the muscle fibers (Fig. 4A). In contrast, ALS group (III) revealed markedly increased collagen fibers that appear disorganized and wavy in CT core, lamina propria and in between the muscle fibers (Fig. 4B). Sections from group IV (ALS+RUT) exhibited moderately increased collagen fibers in CT core, lamina propria with normal distribution of collagen fibers appearing as thin rims in between the muscle fibers (Fig. 4C).

Figure 4. A photomicrograph of Masson’s trichrome-stained sections of the dorsal surface of the anterior 2/3 of the rat’s tongue from control and treated groups. (A) Control group (I) showing normal distribution of regularly arranged collagen fibers (green color) in the connective tissue (CT) core, lamina propria (*) and thin rims in between the muscle fibers (arrows). (B) Alendronate sodium (ALS) group (III) showing marked increase in the amount of the collagen fibers which appear disorganized and wavy in the CT core, lamina propria (*) and in between the muscle fibers (arrows). (C) ALS+rutin group (IV) showing moderate increase in the amount of the collagen fibers in the CT core, lamina propria (*) with normal distribution of collagen fibers appearing as thin rims in between the muscle fibers (arrows). Masson’s trichrome, (A–C) ×100.

Immunohistochemical study

Proliferating cell nuclear antigen immunostaining: The control group (I) revealed strong positive brown nuclear immunoexpression for PCNA in numerous epithelial cells in the basal and suprabasal layers of dorsal epithelium (Fig. 5A). In contrast, ALS group (III) showed weak PCNA immunoexpression in few epithelial cells mainly in the basal layer (Fig. 5B). As regards group IV (ALS+RUT), it exhibited strong PCNA immunoexpression in many epithelial cells of basal and suprabasal layers of the dorsal epithelium (Fig. 5C).

Figure 5. A photomicrograph of proliferating cell nuclear antigen (PCNA)-stained sections of the dorsal surface of the anterior 2/3 of the rat’s tongue from control and treated groups. (A) Control group (I) showing strong positive brown nuclear immunoreaction for PCNA in numerous epithelial cells in the basal and suprabasal layers of the epithelium (arrows). (B) Alendronate sodium (ALS) group (III) showing weak positive nuclear immunoreaction for PCNA in few epithelial cells mainly in the basal layer of the epithelium (arrows). (C) ALS+rutin group (IV) showing strong positive nuclear immunoreaction for PCNA in many epithelial cells in the basal and suprabasal layers of the epithelium (arrows). PCNA stained, (A–C) ×400.

Bcl-2 immunostaining: The control group (I) revealed strong positive cytoplasmic immunoexpression to Bcl-2 in numerous epithelial cells (Fig. 6A). On the other hand, ALS group (III) exhibited negative cytoplasmic reaction to Bcl-2 in almost all epithelial cells (Fig. 6B). Sections from group IV (ALS+RUT) showed moderate positive cytoplasmic immunoexpression to Bcl-2 in many epithelial cells (Fig. 6C).

Figure 6. A photomicrograph of Bcl-2-stained sections of the dorsal surface of the anterior 2/3 of the rat’s tongue from control and treated groups. (A) Control group (I) showing strong positive cytoplasmic immunoexpression to Bcl-2 in numerous epithelial cells (arrows). (B) Alendronate sodium (ALS) group (III) showing negative cytoplasmic immunoexpression to Bcl-2 in the epithelial cells. (C) ALS+rutin group (IV) showing moderate positive cytoplasmic immunoexpression to Bcl-2 in many epithelial cells (arrows). Bcl2 stained, (A–C) ×400.

Scanning electron microscopic results

The scanning electron microscopic results of control group (I) revealed abundant, orderly arranged, long filiform papillae appearing conical in shape with tapering ends which had the same direction. Few fungiform papillae were scattered in-between filiform ones. The fungiform papillae appeared broad and dome shaped with well-defined centrally located taste pore and circular keratin packs (Fig. 7). In contrast, ALS treated group (III) showed marked changes in the filiform papillae which oriented in different directions. The filiform papillae appeared thin and atrophied, while others appeared short and destructed. In addition, disfigured filiform papillae were observed which appeared short with irregular surface and blunt ends. Marked atrophy was observed in some papillae which appeared widely separated with areas of focal loss. Also, fissures were noticed on the dorsal mucosal surface in between the atrophied papillae. Less prominent and distorted fungiform papillae were seen in-between filiform papillae (Fig. 8). As regards group IV (ALS+RUT), it showed almost preservation of lingual papillae. Most filiform papillae appeared long and conical in shape with tapering ends which had the same direction. Few papillae appeared short with blunt ends. The fungiform papilla appeared normal with centrally located well-defined taste pore (Fig. 9).

Figure 7. A scanning electron micrograph of the dorsal surface of the anterior 2/3 of the rat’s tongue from control group (I). (A) Showing numerous filiform papillae (arrows) with scattered fungiform ones in between (*). (B) Showing numerous regularly arranged conical shaped filiform papillae with tapering tips that pointed into the same direction (arrows). (C) Showing a broad dome shaped fungiform papilla (*) with circular keratin packs (arrows) and centrally located well-defined taste pore (arrowhead). (A, B) ×150, (C) ×750.
Figure 8. A scanning electron micrograph of the dorsal surface of the anterior 2/3 of the rat’s tongue from alendronate sodium group (III). (A) Showing filiform papillae oriented in different directions (arrowheads). Some of papillae appear thin and atrophied (orange arrows). Others appear short and destructed (blue arrows). Less prominent and distorted fungiform papilla (*) in between filiform papillae. (B) Showing markedly destructed filiform papillae (arrows). (C) Showing disfigured filiform papillae which appear short with blunt ends (arrows). The surface of papillae appears irregular. Filiform papillae are widely separated with areas of focal loss (*). (D) Showing markedly atrophied filiform papillae (arrows) which appear widely separated with areas of focal loss (*). Fissures (arrowheads) on the dorsal mucosal surface in between the atrophied papillae. (E) Showing distorted fungiform papilla (*) with ill-defined taste pore. (A) ×150, (B, C) ×350, (D) ×200, (E) ×750.
Figure 9. A scanning electron micrograph of the dorsal surface of the anterior 2/3 of the rat’s tongue from alendronate sodium+rutin group (IV). (A) Showing conical shaped filiform papillae with tapering tips (arrows). Few of them appear short with blunt ends (arrowheads). Fungiform papilla (*) interposed in between filiform papillae. (B) Showing long conical shaped filiform papillae with tapering tips that pointed into the same direction (arrows). Fungiform papilla (*) interposed in between filiform papillae. (C) Showing normal fungiform papilla (*) with centrally located well-defined taste pore (arrowhead). (A, B) ×150, (C) ×750.

Morphometric and statistical results

Height of filiform papillae: ALS treated group (III) revealed highly significant reduction in mean height of filiform papillae compared to control group. Group IV (ALS+RUT) demonstrated non-significant decrease in the same parameter compared to the control group. When compared group IV to group III, there was highly significant rise in group IV (Table 1, Fig. 10A).

Table 1 . The morphometric and statistical results in the control and experimental groups

Group IGroup IIGroup IIIGroup IVP-value
Height of filiform papillae (μm)318.88±14.73320.94±15.88214.68±11.11307.36±13.83P1>0.05*
P2<0.001***
P3>0.05*
P4<0.001***
Thickness of ventral epithelium (μm)62.84±2.8763.56±2.9548.17±6.8160.09±4.24P1>0.05*
P2<0.001***
P3>0.05*
P4<0.001***
Area percentage of collagen fibers8.83±1.209.05±0.8852.67±7.3118.59±4.32P1>0.05*
P2<0.001***
P3<0.05**
P4<0.001***
Number of PCNA positive cells132.83±8.04134.67±8.5739.33±3.72121.17±5.64P1>0.05*
P2<0.001***
P3<0.05**
P4<0.001***
Area percentage of Bcl-2 immunoexpression38.52±4.9339.47±5.091.28±1.8021.31±3.80P1>0.05*
P2<0.001***
P3<0.001***
P4<0.001***

P1: group I vs. group II, P2: group I vs. group III, P3: group I vs. group IV, P4: group III vs. group IV. PCNA, proliferating cell nuclear antigen. Non-significant *P>0.05, significant **P<0.05, highly significant ***P<0.001.


Figure 10. Morphometric and statistical results in the control and experimental groups showing. (A) The mean height of filiform papillae (μm). (B) The mean thickness of ventral epithelium (μm). (C) The mean area percentage of collagen fibers. (D) The mean number of proliferating cell nuclear antigen positive cells. (E) The mean area percentage of Bcl-2 immunoexpression.

Thickness of ventral epithelium: ALS group exhibited highly significant decrease in the mean thickness of the epithelium of the ventral surface compared to control group. Group IV (ALS+RUT) demonstrated non-significant decrease in the same parameter compared to the control group. There was highly significant rise in group IV when compared to group III (Table 1, Fig. 10B).

Area percentage of collagen fibers: Highly significant increase in mean area percentage of collagen fibers was detected in ALS treated group (III) in comparison to control group. While group IV (ALS+RUT) revealed significant increase compared to control group. There was highly significant decrease in group IV when compared to group III (Table 1, Fig. 10C).

Number of PCNA positive cells in the dorsal epithelium: ALS treated group (III) showed highly significant decrease in the mean number of PCNA positive cells compared to control group. Group IV (ALS+RUT) revealed significant reduction in the same parameter compared to the control group. There was highly significant rise in group IV compared to group III (Table 1, Fig. 10D).

Area percentage of Bcl-2 immunoexpression: Highly significant reduction in the mean area percentage of Bcl-2 immunoexpression was detected in group III (ALS) and group IV (ALS+RUT) compared to control group. There was highly significant rise in group IV when compared to group III (Table 1, Fig. 10E).

BPs are utilized for the management of several bone diseases as osteoporosis because they can selectively inhibit osteoclast mediated bone resorption.Despite their effectiveness, their undesirable influences have been documented in previous case reports and researches. Oral ulcerations are reported among these adverse effects [7]. This has motivated us to search for new protective agent against these side effects.

RUT is a natural flavonoid with antioxidant, anti-inflammatory, cytoprotective, and gastroprotective activities and has been reported to have protective effect against intestinalmucositis [21].

Therefore, the target of our work was to explore the possible influence of ALS, as one of N-BPs, on the rat tongue and to estimate the probable protective role of RUT.

In our study, ALS was found to cause severe structural alterations in the tongue as observed in the light and scanning electron microscopic results of ALS group (group III). There were reduction in the filiform papillae height with blunt ends, marked atrophy in some papillae with areas of focal loss, fissures on the dorsal mucosal surface in between the atrophied papillae and distorted fungiform papillae. Also, disorganized epithelial cells with focal loss of some cells, pyknotic nuclei and cytoplasmic vacuoles in some epithelial cells were detected. Moreover, there were inflammatory cell infiltration, dilated congested blood vessels in lamina propria and disrupted skeletal muscles fibers with wide spaces in between. These results were consistent with several case reports describing adverse impacts of BP on the oral soft tissues including mucositis and severe oral ulceration most frequently affecting the tongue [3]. Moreover, our findings were in line with other experimental studies [7, 9, 22] on the effect of alendronate in oral mucosa and the researchers detected epithelial degeneration or ulcers, perivascular and/or subepithelial cellular infiltration, reduction of the epithelial cell layers with basement membrane rupture, pyknotic nuclei and increase in the intercellular spaces between the epithelial cells with destroyed desmosomes.

In the current study, the histological changes induced by ALS could be explained by the occurrence of oxidative damage as a result of infiltration by neutrophil. Alendronate causes the activation and the migration of neutrophils which are responsible for the production of oxygen metabolites that can trigger mucosal damage. These toxic metabolites involved in alendronate-induced mucosal damage are mainly OH+, peroxynitrite, H2O2, hypochlorite and lipid peroxyl radicals that can cause severe disruption of the cell membrane resulting in DNA damage and cell death of mucosa. In addition, alendronate induces depletion of glutathione (an important intracellular antioxidant) in the mucosa as a result of mitochondrial damage. Glutathione depletion and lipid peroxidation triggered by reactive oxygen species are considered substantial causes of mucosal damage in gastrointestinal tract [23, 24].

Moreover, Papamitsou et al. [7] attributed the effect of alendronate on tongue to the disturbance in the saliva secretion as a result of adverse effect of alendronate on the structure and the secretory activity of the main salivary glands, saliva has protective effect on the tongue.

In our study, the atrophy of the papillae and the decreased epithelial thickness observed in group III treated with ALS were supported by the statistical results which exhibited highly significant reduction in the papillae height and the ventral epithelial thickness. This could be interpreted by decreased proliferation of the epithelial cells on both the dorsal and ventral surfaces. Such explanation was confirmed by our immunohistochemical and the statistical results for PCNA which exhibited a decrease in PCNA immunoexpression with highly significant decrease in PCNA positive cells number in comparison with control group. PCNA is located into the cell nucleus and used as a marker for cell proliferation. It is essential for DNA replication. PCNA reaction increases in cells undergoing division at G1and S phases of cell cycle [25]. The decreased epithelial proliferation observed in our study was in harmony with previous study in which the researchers took biopsy from the oral mucosa of patients under treatment with oral alendronate and they detected cutback in epithelial proliferation with nuclear abnormalities [8]. Pourgonabadi et al. [26] attributed the reduction in the epithelial cell proliferation to negative impact of alendronate on the stem cells which play an essential role in the development and repair of tissues. They added that the reduction in cell proliferation was associated with an increase in sub-G1 phase indicating apoptosis into cell cycle.

The results of the current work demonstrated that ALS could trigger apoptosis in the epithelial cells as manifested in H&E-stained sections in which pyknotic nuclei were observed in the epithelial cells and this was interpreted as apoptosis by some researchers [9]. Such finding was supported by our immunohistochemical and the statistical results for Bcl-2 which exhibited highly significant reduction in area percentage of Bcl-2 immunoexpression in comparison with control group. Bcl2 is anti-apoptotic marker which is considered a member of Bcl-2 family. Increased Bcl-2 expression promotes the viability and differentiation of cells and decreases apoptosis. Significant reduction of Bcl2 immunoexpression occurs in cells that expose to stimuli triggering apoptosis [24]. The decreased Bcl-2 immunoexpression in our study was in line with the findings of prior studies which stated that alendronate induces cytotoxicity with the involvement of apoptosis through decreased expression of Bcl-2 [24, 26]. Moreover, Theodora et al. [9] reported that BPs reduce cell survival, migration capability, and raise the rate of apoptosis in the oral mucosa.

In group III treated with ALS, some superficial epithelial cells showed many basophilic granules, most probably keratohyline granules. This could be explained by a disturbance in keratinization process. Such explanation was supported by the results of Donetti et al. [8] who detected excessive keratin condensation in the upper layers of the epithelium with several aggregates of tonofilaments suggesting abnormalities in the terminal differentiation process in the group treated with alendronate.

In the current study, ALS treated group showed inflammatory cell infiltration associated with dilated congested blood vessels which are considered inflammatory signs. This could be interpreted by the production of reactive oxygen metabolites and the formation of various proinflammatory cytokines which increase the vascular permeability [27].

In the present work, disrupted skeletal muscles fibers were noticed with wide spaces in between in group III treated with ALS. Such findings matched with the results of Farag and Mehanny [4] who detected degeneration in the muscle fibers of the tongue following zoledronic acid (N-BP) treatment. The authors attributed such degeneration to the activation nuclear factor kappa-B (NF‑κB) signaling pathways due to oxidative stress and proinflammatory cytokines and this activation was confirmed in their results by high immunoreactivity for NF‑κB in the lingual muscle fibers. NF‑κB has been known to be one of the most significant signaling pathways associated with skeletal muscle loss. NF‑κB activation in the skeletal muscle results in degradation of specified proteins in muscles, induction of inflammation and fibrosis, and prevention of muscle regeneration following damage [28].

Masson’s trichrome-stained sections of group III treated with ALS revealed marked rise in collagen fibers amount which seemed disorganized and wavy in the CT core, lamina propria and in between the muscle fibers and statistical results revealed that this increase was highly significant compared to the control. Similar finding were detected by Farag and Mehanny [4] who reported disorganized curly collagen fibers with an increase in the collagen area percentage. The researchers explained the increase in the collagen fibers by a response of fibroblasts that form collagen fibers in order to substitute the degenerated muscle fibers. They added that the persistent inflammation changes the environment around the cells and induces the release of different inflammatory cytokines that have a contribution in fibrosis.

In our study, the administration of RUT with ALS in group IV protected the tongue against histological and immunohistochemical changes induced by ALS. There was much preservation of the normal tongue structure except for few disfigured papillae, some pyknotic nuclei and dilated congested blood vessels. This may reflect the protective effect of RUT against ALS-induced changes. Such finding matched with a previous study [21] in which the investigators reported that RUT reversed the histopathological and morphometric changes of intestinal mucositis triggered by 5-fluorouracil.

The protective impact of RUT could be interpreted by its antioxidant and anti-inflammatory characteristics. The antioxidant effect of RUT was proved by some authors [10] who reported that RUT could increase the antioxidant enzyme activities (GSH-Px, SOD) which inhibit lipid peroxidation and hence protect cells from oxidative damage. Fideles et al. [21] detected that RUT exerts its antioxidant effect by decreasing MDA (lipid peroxidation product) level and increasing GSH (glutathione) level.

Also, other researchers [12] stated that RUT could protect cell against damage caused by oxidative stress through inhibiting the mitochondrial dysfunction and activation of anti-oxidant enzymes which are highly efficient in scavenging free oxygen species.

The anti-inflammatory effect of RUT could be interpreted by its capability to reduce the generation of proinflammatory mediators (interleukin 6, tumor necrosis factor-α) and to reduce the expression levels of COX-2 and NF-κB [29].

The immunohistochemical and the statistical results for Bcl-2 in group IV exhibited highly significant rise in area percentage of Bcl-2 immunoexpression compared to group III. Such finding was in harmony with prior study [30] on retina in which the RUT treatment significantly increased the expression of Bcl-2. The authors stated that the increased Bcl-2 expression has contributed to the viability of the retinal cells.

Concerning the collagen content of group IV (ALS+RUT), there was highly significant decrease compared to group III reflecting the anti-fibrotic effect of RUT. Such finding was in harmony with a prior study [31] on the effect of RUT on lung fibrosis and the investigators detected reduced expressions of fibrosis-related biomarkers and prevention of collagen deposition by RUT.

In conclusion, based on the present work, ALS has been proven to trigger histological and immunohistochemical alterations in the rat tongue. The administration of RUT with ALS protected the rat tongue against these changes through its antioxidant and anti-inflammatory effects. So, RUT is recommended for people who are treated with ALS for a long time to reduce its adverse effects.

Conceptualization: RIY. Data acquisition: DEE, RIY. Data analysis or interpretation: DEE. Drafting of the manuscript: DEE. Critical revision of the manuscript: RIY. Approval of the final version of the manuscript: all authors.

No potential conflict of interest relevant to this article was reported.

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