Abstract
Ulcerative colitis is a chronic and intermittent illness. The current treatment failed to cure the disease which requires to investigate other drug with minimal side effects. The goal of the research is to assess the histological outcome, antioxidant and anti-inflammatory effects of cinnarizine in comparison with that of sulfasalazine (salazosulfapyridine) in experimentally induced colitis in rats. Acetic acid 4% (vol/vol) was used rectally to induce experimental colitis in rats. After induction, rats were administered either sulfasalazine 100mg/kg or cinnarizine 20 mg/kg as a therapeutic dose in rats orally for one week. The duration of treatment was depended on previous studies. There were estimation of histopathological and clinical parameters also the expression of cytokines (tumor necrosis factor alpha (TNF-α) and interleukin-4 (IL-4)), oxidative stress markers (malondialdehyde (MDA) and myeloperoxidase (MPO)), and adhesion molecules (intercellular adhesion molecule-1 (ICAM-1) and endothelial (E)-Selectin) in the colonic tissue. Results showed that both cinnarizine and sulfasalazine significantly reduced the clinical and histological injury in colon that induced by acetic acid. In addition to the down regulation of the increased colonic cytokines, MDA, MPO parameters and adhesive molecules. These results concluded that cinnarizine had an effective therapeutic role which is comparable with sulfasalazine on the experimental colitis through anti-inflammatory and antioxidant actions with down regulation the colonic adhesion molecule.
Main Subjects
Highlights
1. Ulcerative colitis is a chronic, intermittent illness and the current treatment failed to cure the disease.
2. The goal of the research is to assess the histological outcome, antioxidant and anti-inflammatory effects of cinnarizine in comparison with that of sulfasalazine in experimentally induced colitis in rats.
3. Cinnarizine and sulfasalazine significantly reduced the clinical, histological and immunohistochemical expression of colonic cytokines, oxidative stress markers and adhesive molecules
4. Cinnarizine has a therapeutic effect which is comparable to that of sulfasalazine in experimentally induced colitis.
Full Text
Comparative treatment of induced ulcerative colitis in male rat
model by using cinnarizine and sulfasalazine
R.K. Attarbashee1 and A. Abu-Raghif2
1Department of Dental Basic Science, College of Dentistry, University of Mosul, Mosul, 2Department of Pharmacology and Therapeutics, College of Medicine, Al- Nahrain University, Baghdad, Iraq
Emails: 1ranaatarbashe@gmail.com, 2ar-armat1967@yahoo.com
(Received October 15, 2019; Accepted December 4, 2019; Available online September 2, 2020)
Abstract
Ulcerative colitis is a chronic and intermittent illness. The current treatment failed to cure the disease which requires to investigate other drug with minimal side effects. The goal of the research is to assess the histological outcome, antioxidant and anti-inflammatory effects of cinnarizine in comparison with that of sulfasalazine (salazosulfapyridine) in experimentally induced colitis in rats. Acetic acid 4% (vol/vol) was used rectally to induce experimental colitis in rats. After induction, rats were administered either sulfasalazine 100mg/kg or cinnarizine 20 mg/kg as a therapeutic dose in rats orally for one week. The duration of treatment was depended on previous studies. There were estimation of histopathological and clinical parameters also the expression of cytokines (tumor necrosis factor alpha (TNF-α) and interleukin-4 (IL-4)), oxidative stress markers (malondialdehyde (MDA) and myeloperoxidase (MPO)), and adhesion molecules (intercellular adhesion molecule-1 (ICAM-1) and endothelial (E)-Selectin) in the colonic tissue. Results showed that both cinnarizine and sulfasalazine significantly reduced the clinical and histological injury in colon that induced by acetic acid. In addition to the down regulation of the increased colonic cytokines, MDA, MPO parameters and adhesive molecules. These results concluded that cinnarizine had an effective therapeutic role which is comparable with sulfasalazine on the experimental colitis through anti-inflammatory and antioxidant actions with down regulation the colonic adhesion molecule.
Keyword: Cinnarizine, Ulcerative colitis, Acetic acid, Oxidative stress, Adhesion molecules
DOI: 10.33899/ijvs.2019.126170.1254, ©2020, College of Veterinary Medicine, University of Mosul.
This is an open access article under the CC BY 4.0 license (http://creativecommons.org/licenses/by/4.0/).
مقارنة علاج قرحة القولون المحدثة فی ذکور الجرذان باستعمال السیناریزین والسلفاسالازین
رنا خیری عطار باشی1و احمد ابو رغیف2
1فرع علوم طب الأسنان الأساسیة، کلیه طب الأسنان، جامعه الموصل، الموصل،
2فرع الأدویة والعلاجات، کلیه الطب، جامعه النهرین، بغداد، العراق
الخلاصة
ان مرض التهاب القولون التقرحی ذو طبیعة مزمنة ومتقطعة. وان فشل العلاج بعد فترة وجیزة کان دافعا کبیرا لفحص تأثیرات علاج أخر وبأقل التأثیرات الجانبیة. إن هدف البحث هو تقییم النتیجة النسیجیة، التأثیرات المضادة للأکسدة والالتهاب لعلاج السیناریزین بالمقارنة مع علاج السلفاسالازین فی قرحة القولون المحدثة فی الجرذان. تم استعمال حامض الخلیک المخفف بترکیز 4% عن طریق المخرج لتحدیث قرحة القولون فی الجرذان. بعد إعطاء الحامض للجرذان تمت معاملتها بعقار فموی وهی20ملغ /کغم من علاج السیناریزین وبجرعة 100 ملغم/کغم من علاج السلفاسالازین من وزن الجسم على التوالی لفترة 7 أیام. وان الفترة العلاجیة کانت بالاعتماد على مصادر سابقة. لقد تم احتساب بعض الأعراض السریریة بالإضافة الى الفحص النسیجی. کذلک تم قیاس کل من السایتوکینات (معامل الورم التنخری الفا والانترلوکین الرابع)، معاییر الإجهاد التأکسدی (المالوندایالدیهاید والمایلوبیروکسیدیز) ومستقبلات التصاق الجزیئات. لقد أظهرت النتائج أن علاجات السیناریزین والسلفاسالازین تسببت فی تقلیل ظهور الأعراض السریریة والنتیجة النسیجیة فی عینة القولون المحدثة بواسطة حامض الخلیک. هذا بالإضافة الى التقلیل فی مستوى السایتوکینات ومعاییر الإجهاد التأکسدی والجزیئات اللاصقة. یستنتج من هذا للسیناریزین دور فعال فی معالجة القرحة المحدثة للقولون من خلال التأثیرات المضادة للالتهابات والأکسدة مع تثبیط إنتاج الجزیئات اللاصقة فی انسجه القولون.
Introduction
Ulcerative colitis is one of the chronic, recurrent and inflammatory intestinal illness. Although its etiology is unknown, but several studies indicated that altered immunity, genetic and environmental factors are intercepted with the pathogenesis of colitis. In the past years despite the high awareness for that disease, the utilization of remedies is still insufficient (1,2). The induction of colitis by acetic acid is widely used as experimental pattern (3). It is effective for the investigation the pathogenesis of ulcerative colitis and novel options for treatment (4). Aminosalicylates, immunomodulators, glucocorticoids, and monoclonal antibodies are pharmacological treatment for ulcerative colitis. Regardless, the increased proportion of undesirable effects at the same time with insufficient therapy get by necessary to inspect novel drug with high effectiveness (5).
Cinnarizine is a drug, it belongs to piperazine derivative. The proposed therapeutic mechanism is through blocking effect of histamine (H1) and calcium channel receptors (6,7). Histamine (8) and calcium influx (9) have great effect concerning the inflammatory role in gastrointestinal tract. Also cinnarizine has antiserotonergic effect (10) and serotonin play an integral role in pathogenesis of ulcerative colitis (11). Cinnarizine was suggested to inhibit the inflammatory response through the oedema reduction caused by indomethacin in rats (12). The current study assesses the protective effect of cinnarizine, a potent antagonist activity on histamine H1 and calcium channel receptors, versus sulfasalazine in case of experimentally induced ulcerative colitis.
Materials and methods
Forty-eight Wister albino adult male rats 200-220 g were used throughout this study (supplied from animal house of the national center for drug control and researches). Prior to the experiment, animal was placed five per cage that was supplied with a large wire -mesh floor for 7 days and were allowed to administer water and laboratory chow pellet. Study protocol were approved by institutional animal ethics committee from the Al-Nahrain university in the college of medicine.
Chemicals and drugs
Acetic acid and diethyl ether (BDH Chemical Ltd., England), immunohistochemistry kits (Abcam/UK), sulfasalazine 2 ml/kg orally, and cinnarizine 4 ml/kg orally (Sigma -Aldrich) were purchased.
Experimental design
Rats were classified to four groups (n=12 in each group). The Group I received no treatment and served as normal control while the other groups, colitis (group II) was induced by rectal administration of 4% acetic acid (v/v). The group II was administered orally normal saline; while group III was administered cinnarizine 20 mg/kg (4 ml/kg) orally and lastly group IV treated orally with sulfasalazine (Salazosulfapyridine) 100 mg/kg (2 ml/kg), sixty minutes after the induction of colitis for 7 days. The duration of treatment was depended on previous studies of experimental colitis (13,14).
Induction of ulcerative colitis
Before the colitis induction, rats were fasted for at least 24hrs to get proper induction of colitis by evacuation the colon from feces but where be permitted to tap water. Experimental colonic ulceration was accomplished after interruption of water (2 hours) in accordance to that procedure suggested by Mousavizadeh et al. (15) with modification. Briefly, rats were received single intrarectal infusion of 4% acetic acid in a dose 5 ml/kg solution for 30s (8cm into the colon) under light ether anesthesia by flexible plastic tube (2 mm extrinsic diameter). Rats were positioned in horizontal direction for 2 min to prevent the discharge of acetic acid. Rats in group 1 go through the identical procedure by using the same amount of normal saline as an alternative to acetic acid.
Preparation of drugs
The sulfasalazine and cinnarizine were freshly prepared before administration. Estimated drugs were prepared as suspensions in the distilled water. Cinnarizine was used at a dose 20 mg/kg as a therapeutic dose in rats according to the study reporting that the highest inhibitory effect of cinnarizine on edema formation and inflammatory modulation (12). Sulfasalazine 100 mg/kg was served as standard therapy (16,17).
Assessment of colitis
At the end of the experimental period, rats inhaled an excessive dose of diethyl ether in order to sacrifice them. The colon was removed rapidly after dissection of the abdomen. The specimen of colon was opened longitudinally and gently cleaned with normal saline. Then, the assessment of clinical features were achieved by normal observation. Finally, the samples were assessed for histopathologic and immunohistochemical changes.
Disease Activity Index (DAI)
The Disease activity index defined by Niu et al. (18) was used to assess the disease clinically which involve body weight reduction (0=weight gain or no reduction, 1=1-5 % reduction, 2=6- 10% reduction, 3=11-15 % reduction, 4=more than 15% reduction); the consistence of faeces (0=normal, 2=loose faeces, 4=diarrhea) and bleeding of rectum (0= normal, 2= mild bleeding, 4=severe bleeding). The calculation was made by combination of the total scores of DAI.
Colon edema
It was utilized as an indicator of edematous tissue and the intensity of colitis. After the incision was done along the mesenteric margin of each colonic specimen and washed gently, the colon edema was determined by measuring the colon weight by using sensitive balance (19).
Macroscopic colonic score
The colonic samples were examined visually. The macroscopic score based on the clinical features of the colon according to scoring system ranging from 0-6 as follows: 0= absence of inflammation; 1= redness or swelling; 2= swelling and redness; 3= one or two ulcers; 4= one large ulcer or more than two ulcers; 5= initial necrosis; 6, severe necrosis (20).
Histopathological evaluations
Formalin 10% was used to fix the colonic samples. Dehydration, paraffin embodiment and deparaffinization were done on the samples. Colonic samples were cut into sections (4µm) and dyed with Hematoxylin and eosin (H&E).
The histopathological changes were assessed by examining and scoring the slides. Experienced histopathologist tested the tissue sections in a blinded manner and results evaluated according to scoring system ranging from 0-3 (0=normal; 1=focal; 2=zonal; 3=sever) which assessed the extent of: destruction of glands and epithelium, glandular crypts dilation, depletion of goblet cells, infiltration of inflammatory cells, edema, dysplasia, mucosal hemorrhage and crypt abscesses (21).
Immunohistochemistry
Immunohistochemistry (IHC) directly demonstrated the cells in the affected tissue (22). The immunohistochemical reactions were produced by presence of specific antibodies, simultaneously the evaluation of the production a number of biochemical markers in intestinal samples that were paraffin- embedded in order to measure the colonic cytokines (tumor necrosis factor-alpha and interleukin-4), adhesion molecules (intercellular adhesion molecules-1 (ICAM-1) and endothelial selectin (E-selectin) and oxidative stress parameters (Malondialdehyde (MDA), Myeloperoxidase (MPO)).
Quantification of IHC was performed in accordance to the following semiquantitative scores (23), which was based on the percentage of positively stained cells as following :0, no staining; 1, ≤ 25%; 2, 26-50%; 3, 51- 75 %; and 4, 76-100%.
Statistical Analysis
Statistical package for social science version 23 software program was used to summarize, analyze and present the data. Quantitative (numeric) variables were expressed as mean and standard deviation.
One-way ANOVA was used to study difference in mean of quantitative variables among groups; then followed by post hoc least significant difference (LSD) test to evaluate mean difference within groups. The significant level was considered at P ≤ 0.05 (24).
Results
The influence of cinnarizine on clinical parameters
The colonic mucosa was extensively ulcerated and showed necrotic tissue in colitis group in contrast to control normal group. Even so, after the induction of colitis, the rats that received cinnarizine or sulfasalazine elicit orally significant reduction to the disease activity index and colon weight. Additionally, two drugs significantly reduce the macroscopical score. However, cinnarizine produced a higher significant reduction in the macroscopical score in comparison to the sulfasalazine group as displayed in table 1.
The influence of cinnarizine on histopathological score
The current study exhibits the histological changes in colitis group, primarily loss of architecture in the intestinal crypt, extensive mucosal colonic damage, ulcerations and necrosis compared to normal control group.
Furthermore, both sulfasalazine and cinnarizine administered groups elicit significant reduction in the microscopical score as specified by colonic mucosa epithelization, reduction of neutrophil infiltration and edema compared to acetic acid induced colitis group. Even so, cinnarizine exhibited higher significant reduction in the scoring of microscopical parameter compared to sulfasalazine that displayed in table 2 and figures 1, 2, 3.
The influence of cinnarizine on cytokines (IL-4 and TNF-α)
Oral treatment of cinnarizine or sulfasalazine to the colitis group was significant. It was diminished by IL-4 and TNF-α in comparison with colitis. However, cinnarizine exhibited better significant reduction in the IL-4 and TNF-α level compared with sulfasalazine group as displayed in table 2 and figures 4,5.
The influence of cinnarizine on oxidative stress markers (MDA and MPO)
After sulfasalazine and cinnarizine treatment, the high colonic MDA level in the induced group was found to be significantly diminished. Even so, cinnarizine elicit a better significant reduction in the level of MDA in comparison to sulfasalazine group. Also, both drugs produce significant declination in MPO in comparison with induced group as displayed in table 3 and figures 6,7.
The influence of cinnarizine on adhesion molecules (ICAM-1 and E-selectin)
As displayed in table 3 and figures 8,9. There are elevated level of ICAM-1 and E-selectin after the induction by acetic acid in comparison with that of normal group. While these findings elicit significant declination in the cinnarizine and sulfasalazine treatment. However, cinnarizine produced a better significantly declination in the adhesive molecules (ICAM-1) in comparison to sulfasalazine administered group.
Table 1: Macroscopic parameters in study and healthy control groups
Clinical parameters |
Groups (n=12/group) mean ± Standard Deviation (SD) |
|||
Control |
Colitis group |
Sulfasalazine treated group |
Cinnarizine treaterd group |
|
Disease activity index |
0.0±0.0 a |
11.17±1.27 b |
2.33±1.56 c |
1.67±0.78 c |
Colonic weight (gram) |
1.5±0.22 a |
3.54±0.4 b |
1.67±0.39 c |
1.58±0.35 c |
Macroscopic score |
0.0±0.0 a |
5.75±0.36 b |
2.0±0.6 c |
1.5± 0.52 d |
Comparison expressed by letters; dissimilar letters denotes significant difference.
Table 2: Cytokines immunohistochemical score and histopathological parameters
Cytokines parameters |
Groups (n=12/group) mean ±Standard deviation (SD) |
|||
Control |
Colitis |
Sulfasalazine treated group |
Cinnarizine treaterd group |
|
Histopathology |
0.0 ±0.0 a |
3.0 ±0.0 b |
1.67±0.49 c |
0.67±0.49 d |
Interleukin-4 |
1.0 ±0.0 a |
4.0±0.0 b |
2.0 ±0.0 c |
1.33±0.49 a |
Tumor necrosis factor-α |
1.0 ±0.0 a |
4.0±0.0 b |
2.67±0.49 c |
1.33±0.49 a |
Comparison expressed by letters; dissimilar letters denotes significant difference.
Table 3: Immunohistochemical score for oxidative stress and adhesive molecules
Oxidative stress and Adhesive molecules |
Groups (n=12/group) mean ± Standard Deviation (SD) |
|||
Control |
Colitis |
Sulfasalazine treated group |
Cinnarizine treaterd group |
|
Myeloperoxidase |
1.00 ±0.0 a |
4.0 ±0.00 b |
1.83±0.38 c |
1.67±0.49 c |
Malondialdehyde |
1.0±0.0 a |
4.0 ±0.0 b |
2.58±0.51 c |
1.25±0.452 a |
ICAM-1 |
1.0±0.0 a |
4.0 ±0.0 b |
2.5±0.522 c |
1.5±0.52 d |
E-selectin |
1.0±0.0 a |
4.0 ±0.0 b |
1.92±0.51 c |
1.75±0.4 c |
Comparison expressed by letters; dissimilar letters denotes significant difference.
Discussion
This study demonstrated the effect of cinnarizine on experimentally colitis. It is concluded that cinnarizine significantly reduced the mean DAI. The weight of the inflamed colonic tissue is considered as a reliable and sensitive indicator for the severity and extent of intestinal inflammation (25). However, the current study showed that cinnarizine has decreased the weight of colonic tissue in colitis group which was comparable to the suggestion of Abdel Salam (12) who showed that cinnarizine produced an inhibitory effect on edema formation and gastric mucosal lesion caused by indomethacin in rats. Moreover, cinnarizine in the present study reduced macroscopic and the scoring of histopathological changes in colitis. The proposed protective mechanism of cinnarizine is through blocking effect of histamine and calcium channel receptors (6,7). The increased histamine has an important role in the inflammation of the gastrointestinal tract (8). It showed that calcium is responsible for the stimulation of nuclear factor (NFkB) activity (7).
Figure 1: Histological section throughout the wall of colon reveals A, the normal mucosa and submucosa without any inflammatory signal and conservation of goblet cells in colon; B, extensive ulceration; necrosis with no gland; and mononuclear inflammatory infiltrate (yellow arrow) in experimentally colitis in rat, hematoxylin and eosin stain; 40 X.
Figure 2: Histological section throughout the colonic wall reveals A, extensive ulceration; necrosis with no gland; B, the effects of cinnarizine after 7 days and exhibit (1) the regeneration of mucosa and formation of glands; (2) least severe inflammation and (3) regeneration of goblet cells; B, hematoxylin and eosin stain; 40 X.
Figure 3: Histological section throughout the colonic wall reveals the effects of A, cinnarizine ; B, sulfasalazine after 7 days and exhibit (1) the regeneration of mucosa and formation of glands; (2) least severe inflammation and (3) regeneration of goblet cells; hematoxylin and eosin stain; 40 X.
Figure 4: Immunohistochemical expression of interleukin-4 reveals secretory pattern (brown color in the stromal cells). A, score-4; B, cinnarizine treated group (score-1); C, sulfasalazine treated group (score-2); hematoxylin, 20X.
Figure 5: Immunohistochemical expression of tumor necrosis factor-α (TNF-α) reveals membranous and secretory pattern (brown color in the stromal cells); A, colitis group (score-4); B, cinnarizine treated group (score-1); C, sulfasalazine treated group (score-2); hematoxylin; 20X.
Figure 6: Immunohistochemical expression of myeloperoxidase (MPO) reveals cytoplasmic pattern (brown color in the stromal cells); A, colitis group (score-4); B, cinnarizine treated group (score-1); C, sulfasalazine treated group (score-2); hematoxylin; 20X.
Figure 7: Immunohistochemical expression of malondialdehyde (MDA) reveals cytoplasmic pattern (brown color in the stromal cells); A, colitis group (score-4); B, cinnarizine treated group (score-1); C, sulfasalazine treated group (score-2); hematoxylin; 20X.
Figure 8: Expression of ICAM-1 by Immunohistochemistry reveals membranous pattern (brown color in the stromal cells); A, colitis group (score-4); B, cinnarizine treated group (score-1); C, sulfasalazine treated group (score-2 ); hematoxylin; 20X.
Figure 9: Immunohistochemical expression of E-selectin reveals membranous pattern (brown color in the stromal cells); A, colitis group (score-4); B, cinnarizine treated group (score-1); C, sulfasalazine treated group (score-2 ); hematoxylin; 20X.
The effect of calcium ions on Ieukotriene synthesis with the enzyme 5-lipooxygenase from arachidonic acid has been suggested and the enzyme 5-lipooxygenase is calcium dependent and requires adenosine triphosphate (26). Also cinnarizine has antiserotonogeric effects (10). Serotonin (5-Hydroxytryptamine) receptors are highly expressed in intestinal tissues and play an integral role in pathogenesis of ulcerative colitis (11).
There was a close relation between ulcerative colitis and inflammation (27). Tumor necrosis factor-α is a crucial proinflammatory cytokine liberated from the lymphocytes and macrophages in the initial step of inflammation, also, it has been noted that TNF-α has great participation in the pathogenesis of colonic ulceration (28, 29). Interleukin -4 is a key immunoregulatory cytokine (T-helper-2) and it contribute in down regulation the release of pro inflammatory cytokines (30). The dysregulation of IL-4 may assist in inflammation and pathogenesis of intestinal ulceration by reducing resistance in intestinal epithelial cells (31), goblet cell hyperplasia (32) and exotoxin expression in colonic mucosa (33). Kasaian et al. showed the involvement of IL-4 in experimental colitis that induced by oxazolone, a model of ulcerative colitis in mice (34). The present study explained the administration of cinnarizine causes significant reduction in the expression of immunohistochemistry for colonic cytokines (interleukin-4 and tumor necrosis factor-α) in experimentally induced colitis in rats. Previous study showed that Cinnarizine has anti-inflammatory through itʼs suppressant effect on pro inflammatory cytokines TNF-α, IL-1, and IL-6 release in experimentally-induced bronchial asthma in rats (35).
Malondialdehyde produced from the peroxidation process of lipid (36, 37) which causing destruction of colonic tissue in ulcerative colitis (38) and cell necrosis (39). Myloperoxidase is a hemoprotein enzyme abdudantly released from the neutrophils granules by inflammatory stimuli that catalyzes the formation of a number of reactive species (40). Studies have indicated that oxidative stress results from the shift of equilibrium between the pro-oxidant and anti-oxidant systems in favor of the pro-oxidant system which result of excessive production of free oxygen radicals and neutrophil infiltration (41, 42). The study has also shown that administration of cinnarizine significantly reduce the expression of immunohistochemistry for markers of oxidative stress (myeloperoxidase and malondialdehyde) in colonic mucosa of experimentally induced colitis in rats. Studies have suggested that cinnarizine reduces the immune response through inhibition of endothelial oxidative stress and nuclear factor (NF-κB) action (the transcription factor through signaling pathways of phospholipase C and the phosphatidylinositol) (35).
Inter Cellular adhesive molecule (ICAM-1) engage the leukocytes association to endothelial cells, that simplify their penetration to the inflammatory site. Endothelial selectin is particularly interesting because it is only found in the activated endothelium, in contrast with other adhesion molecules, which have a more widespread tissue distribution. The demonstration of E-selectin can thus be considered conclusive evidence of endothelial activation (43). Endothelial selectin is biologically active, facilitating the early phase of polymorphonuclear adhesion to the endothelial cell, constituting an early marker of the inflammatory response (44). The present study showed that cinnarizine significantly reduced immune histochemical expression of adhesive molecules in rat colonic mucosa in contrast to colitis group and this evidence is corresponding with Haress (45). that showed cinnarizine can inhibit the cell adhesion molecules, and chemotactic factors.
Conclusions
Cinnarizine has a therapeutic effect through the anti-oxidant and anti-inflammatory effects which is comparable to that of sulfasalazine in experimentally induced colitis.
Acknowledgements
I would like to express my thanks for pathologist Dr. Ban Jumaa Qasim /College of medicine /Al-Nahrain University for support in evaluation of research. Also, I am very grateful to the University of Mosul/College of Dentistry for their provided facilities, which helped to improve the quality of this work.
Conflict of interest
There is no conflicts of interest.