| Abstract|| |
Introduction: Mucositis is a common complication in around 40% of patients receiving chemotherapy which profoundly impact the quality of life. Despite the availability of many therapeutic agents that claim to prevent or reduce the severity of oral mucositis, no intervention that is completely successful at preventing oral mucositis exists. The present study aimed to demonstrate the effect of local honey on methotrexate-induced mucositis of the tongue. Materials and Methods: The study was done in albino rats (n = 24), divided into four groups: honey-normal saline group (H/NS), distilled water-NS group (DW/NS), DW-methotrexate group (DW/MTX), and H/MTX group. Local honey was given (2.5 g/kg) twice a daily for 8 days. NS or methotrexate (60 mg/kg) interrupted the experiment procedure on day 4. At day 8, rats were sacrificed and middle third of tongue was removed for preparation of histological slides. Slides were examined for inflammatory cell infiltration (ICI), cell vacuolization (CV), congested blood vessels (CBV), thickness of epithelium (both dorsal and ventral mucosa), and thickness keratin. Results: Statistically significant association was found between the groups for ICI and CV, χ2 (3) = 11.97, P= 0.003 for ICI, and χ2 (3) = 8.307, P= 0.023 for CV. Mean number of CBV per microscopic filed was 0.445, 0.389, 1.28, and 0.5 for H/NS, DW/NS, DW/MTX, and DW/H groups, respectively. Mean value of dorsal epithelial thickness (μm) was 96.78 ± 13.59, 100.50 ± 9.75, 75.53 ± 9.24, and 98.75 ± 10.92 for H/NS, DW/NS, DW/MTX, and DW/H groups, respectively. Mean value of ventral epithelial thickness (μm) was 70.45 ± 13.60, 58.38 ± 14.26, 37.04 ± 5.37, and 52.80 ± 7.97 for H/NS, DW/NS, DW/MTX, and DW/H groups, respectively. The thickness of epithelium of DW/MTX group was significantly different from other groups (P < 0.05). Conclusion: The present study demonstrated the palliative effect of local honey over the mucositis induced by the chemotherapeutic drug (methotrexate).
Keywords: Cell vacuolization, chemotherapy, epithelium, honey, oral mucositis
|How to cite this article:|
Khanal L, Yadav P, Baral P, Shah R, Rauniar GP. Effect of local bee honey on dihydrofolate reductase enzyme inhibitor-induced mucositis: A histological study on albino wistar rats. Indian J Dent Res 2019;30:708-15
|How to cite this URL:|
Khanal L, Yadav P, Baral P, Shah R, Rauniar GP. Effect of local bee honey on dihydrofolate reductase enzyme inhibitor-induced mucositis: A histological study on albino wistar rats. Indian J Dent Res [serial online] 2019 [cited 2021 Jun 19];30:708-15. Available from: https://www.ijdr.in/text.asp?2019/30/5/708/273433
| Introduction|| |
Cancers are one of the major causes of death worldwide. Until the last century, the only treatment available for the patients with the solid tumor was surgery, associated with high morbidity and mortality. Over the past 45 years, chemotherapy resulted in a progressive improvement in the survival rates of patients with such tumors. Chemotherapeutic drugs affect all the cells with rapid replication (high mitotic activity index), especially cells of gastrointestinal tract buccal cavity, and bone marrow; therefore, the patients may suffer from mucositis of those regions. Mucositis is a common complication in around 40% of patients receiving chemotherapy. Uncontrolled mucositis can profoundly impact quality of life by causing immense pain, increases the risk of infection and dysphagia, affecting nutrition and hydration status, leading to weight loss.
The first experimental study of chemotherapy-induced oral mucositis was performed to observe the effect of the antimetabolite 5-fluorouracil on hamsters. Since then, other studies have been conducted in rats and mice, with different inducing agents.,,,,,
Methotrexate has been widely used for the treatment of rheumatoid arthritis. The mechanisms of action of methotrexate are complex; developed as a folic acid analog, methotrexate inhibits purine, and pyrimidine synthesis, which accounts for its efficacy in the therapy of cancer as well as for some of its toxicities. Adverse effects due to its action as a folate antagonist include anemia, neutropenia, stomatitis, and oral ulcers. Recently, many studies have focused on the adenosine-mediated anti-inflammatory effects of methotrexate.
Since oral mucosa has a high level of mitotic activity and high cell turnover, there is a continuous need for cell multiplication to repair the oral mucosa. Such epithelial tissues with high levels of mitotic activity respond rapidly to the drugs and radiation during the cell cycle. Chemoradiotherapy causes various changes in normal tissues, depending on the closely interrelated factors of total dose, fractionation schedule, and volume treated. Histologically, mucositis proceeds in four phases: inflammatory/vascular, epithelial, ulcerative/bacterial, and healing phase. These stages are interdependent and result from cytokine-mediated effects of chemotherapeutic drugs on the epithelium and the composition of the oral microbiota.
Current treatments for oral mucositis in the clinical settings are local anesthetics, paliferin, glutamine, caphosol mouth rinse, amifostine, and antimicrobial agents., Despite the availability of many therapeutic agents that claim to prevent or reduce the severity of oral mucositis, no intervention that is completely successful at preventing oral mucositis exists. Many authors have reported the prophylactic use of various bee products, green tea extract, coffee plus honey, topical steroids, chamomile extracts, and olive oil for prevention of oral mucositis induced by chemotherapeutic agent like 5-fluorouracil, methotrexate, and also by combine form of chemoradiotherapy.,,,,,, The present study aimed to evaluate the preventive role of orally given local natural honey in methotrexate-induced mucositis of the tongue.
| Materials and Methods|| |
The study was performed in animal laboratory of the Department of Anatomy (BP Koirala Institute of Health Sciences). Twenty-four adult white albino rats were used for the experimental procedure. The focal animal sampling method was used for the sampling. The sample size was calculated by the resource equation method (with a number of group as 4 and value of the degree of freedom (E) as 20). Ethical clearance was taken from the Institutional Review Committee: (IRC/0742/016).
E = Total number of animals − Total number of groups
In our study, Total number of animals: 24; Total numbers of groups: 4
So, the value of E comes to be 20.
Young Wistar rats (Rattus norvegicus) having 200–240 g body weight were procured for the study. The rats were fed with normal rat diet and normal water ad libitum for 2 weeks before commencement of the project. The cages were kept constantly clean to prevent any disease to the rats. Rats were kept at room temperature on 12 h light/dark cycle. All the possible safety precautions were taken for handling the rats. Animal care was done according to “ethical guidelines for the care and use of animal in health research” published by Nepal Health Research Council.
Induction of mucositis
Mucositis was induced by giving intraperitoneal methotrexate at dose of 60 mg/kg. According to the study done by Fijlstra et al., rats start to show the signs and symptoms of mucositis from the second day until the 5th day; thereafter effect gradually recovered. On the day 4, histological changes and clinical symptoms become most severe; for this reason, rats were sacrificed on the day 4 after the experiment.
Honey information and identification
Nepal is regarded as one of the countries that produce high-quality honey due to its diverse climatic conditions and biodiversity. Sunsari district is one of the major honey production sites in Nepal.
Asian hive bee (Apis cerana) which is the common indigenous species of honey bee in upper tropical and subtropical region of Nepal.
Cultivation and collecting season
A. cerana produces honey two times a year (March–May and November–December). Honey produced in the second phase was used in the study.
Honey was plant specific and multiflora in origin, containing pollens from more than one plant with no dominance of particular species of plant.
Honey was processed by traditional method of extraction by squeezing the honeycomb. By consistency, it was liquid honey that was thicker in consistency without visible crystals.
Preparation of honey
The preparation was done as per the method applied by Al Refai. Natural unprocessed thick, brown and sticky honey was purchased from local market of Dharan. Raw honey was mixed with distilled water in the concentration of 1.25 g honey per milliliter of water. The prepared solution was first filtered by fine cloth and then with filter paper [Figure 1].
|Figure 1: Logistics preparation for experiment procedure. Oral feeding syringe (a), digital weighing scale (b), and use of filter paper for the preparation of honey (c)|
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Rats were divided into control group and experimental group with 12 rats in each group. Both experimental and control groups were again divided into H-treated group (six rats) and DW-treated group (six rats). H-treated group was given honey at a dose of 2.5 g/kg twice a day, and DW-treated group was given the same quantity of water as that of the H-treated group. The treatment was continued for 8 days. At day 4, intraperitoneal methotrexate at a dose of 60 mg/kg interrupted the experimental procedure in experimental group; similarly, physiological normal saline (0.9% Na Cl) at a dose of 60 mg/kg (intraperitoneal route) interrupted the experimental procedure in control group [Figure 2]. Groups were named as honey + normal saline (H/NS) and distilled water + normal saline (DW/NS) in control group and honey + methotrexate (H/MTX) and distilled water + methotrexate (DW/MTX) in the experimental group. The provision of honey and distilled water was done by syringe feeding method [Figure 1] and [Figure 2] as suggested by Atcha et al. The observer who recorded the observation of the experiment was blinded about the provision of the experiment.
|Figure 2: Feeding of honey by oral feeding syringe (a), provision of methotrexate by insulin syringe through intraperitoneal route (b), and process of giving anesthesia in close chamber by the use of ether before cervical dislocation for scarification (c)|
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Light microscopic examination
At day 8, all animals were sacrificed by cervical dislocation after anesthetizing with ether vapor [Figure 2]. Middle third of the tongue was removed and was fixed in 10% formalin (pH 7.4) for 48 h. The tissue specimens were dehydrated in ethyl alcohol followed by clearing by two changes of xylene. The specimens were impregnated and embedded in paraffin wax. Sections of 5 μm thick were cut, dewaxed, hydrated, and stained with routine Harris' hematoxylin for 3 min, then with eosin for 1 min, washed in tap water and dehydrated in ethanol. The compound light microscope was used for examining the slides.
For microscopic analysis, three microscopic fields from each section were analyzed, and pictures were captured. The thickness of epithelium between external surface and epithelial crista and thickness of keratin were measured for every three photographic fields. The presence or absence of inflammatory cells infiltration (ICI) and cell vacuolization (CV) was recorded. Numbers of congested blood vessels (CBV) were also counted for each field. Structures with endothelial lining containing red blood cells in their lumen were counted as CBV. At every 10 measurements, one was repeated to assess intra-examiner reproducibility. All the measurements were done at a total of 400X magnification except for blood vessels counting which was done at a total of 40X. For the purpose of measurement of the thickness of the epithelium, free and valid image analysis software ImageJ (National Institute of Health (NIH), Bethesda, Maryland, USA) was used (source: https://imagej.nih.gov/ij/). To set the scale for the measurement of thickness, stage micrometer was used.
Recorded data and measurements were first entered into the Excel Sheet; then transferred to the statistical package for social science version 11.5 (IBM corp, New York, USA) for further statistical analysis. Paired t-test was used to compare the weight of the animals before the intervention and just before sacrifice of the animals. Other statistical tests used were Fisher's exact test, Kruskal–Wallis test, and analysis of variance with post hoc analysis.
| Results|| |
The present study was carried out to assess the effect of local honey on the animal model of mucositis of the tongue, induced by intraperitoneal methotrexate injection.
The weight of the rat was recorded before the intervention and just before sacrificing the animal [Table 1]. The mean weight of the animals was found to be reduced significantly (P = 0.034) only in DW/MTX group.
|Table 1: Body weight before and after intervention among different groups. (Paired sample t-test)|
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After sacrificing the animal, histological slides were prepared to assess the qualitative and quantitative changes indicating the inflammation of mucosal epithelium of the tongue. To assess the qualitative changes, presence or absence of ICI and CV were recorded from three microscopic fields of each slide. If the findings were found to be positive in any of two fields, it was recorded as a positive finding for that animal. Only one of the rats of H/NS group was found to have the presence of ICI; while in DW/MTX group, five rats were found to have the presence of ICI. ICI was not found in DW/NS and H/MTX groups [Figure 3]. The presence of CV was most frequent in DW/MTX group, followed by H/MTX group. CV was not found in control groups (H/NS and DW/NS) [Figure 4].
|Figure 3: The presence or absence of inflammatory cell infiltration among four groups|
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|Figure 4: The presence or absence of cell vacuolization among four groups|
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Chi-square test of association between treatment groups for ICI and CV showed statistically significant association, χ2 (3) = 11.97, P = 0.003 for ICI and χ2 (3) = 8.307, P = 0.023 for CV. Similarly, the strength of association was found to be significant according to the value of Phi and Cramer's V [Table 2].
|Table 2: Fisher's Exact test for the comparison of inflammation and cell vacuolization among different groups and significance of their difference|
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The quantitative parameter measured for assessing the inflammation was number of CBV per microscopic field under 40X magnification. Three microscopic fields from each slide were assessed, and total count was divided by three to calculate the average number of blood vessels per microscopic field. Mean number of CBV in control group was 0.445 and 0.389 for H/NS group and DW/NS group, respectively. In the experimental group, DW/MTX group showed the maximum number of CBV with mean value of 1.28 per microscopic field. While in H/MTX group, it was reduced to 0.5 per microscopic field. When tested for statistical significance by applying Kruskal–Wallis test, this difference in number of CBV was found to be statistically significant: χ2 (3) = 8.334, P = 0.040 [Table 3].
|Table 3: Comparison of numbers of congested blood vessels per microscopic field among 4 groups (Test: Kruskal Wallis test)|
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The thickness of epithelium of dorsum and ventral surface of the tongue and the thickness of keratin layer on dorsum of tongue were measured [Table 4]. All measured values were presented in micrometer (μm). Mean values of the thickness of keratin were 62.86 ± 13.17 for H/NS group, 67.97 ± 13.91 for DW/NS group, 62.27 ± 12.36 for DW/MTX group, and 65.73 ± 6.97 for H/MTX group. The difference between the groups was not significant for the thickness of keratin (F = 0.296, P = 0.828). The thickness of epithelium on dorsum of the tongue was the thinnest in DW/MTX group (75.53 ± 9.24), followed by DW/NS group (100.50 ± 9.75), H/MTX group (98.75 ± 10.92) and H/NS group (96.78 ± 13.59). Mean difference of dorsal epithelial thickness among the groups was statistically significant (F = 6.750, P = 0.003). Multiple comparisons were done to find out the difference between the specific groups applying post hoc analysis. Significant difference was found between H/NS group and DW/MTX group (P = 0.003), DW/NS group and DW/MTX group (P = 0.001), DW/MTX group and H/MTX group (P = 0.002) [Table 5]. The thickness of epithelium on ventral surface of the tongue was lowest for DW/MTX group (37.04 ± 5.37) followed by H/MTX group (52.80 ± 7.97), DW/NS group (58.38 ± 14.26) and H/NS group (70.45 ± 13.60). The difference of mean values of the thickness of epithelium on ventral surface of tongue among the groups was statistically significant (F = 9.640, P < 0.001) [Table 4]. Multiple comparison showed significant difference between H/NS group and DW/MTX group (P = <0.001), H/NS group and H/MTX group (P = 0.011), DW/NS group and DW/MTX group (P = 0.003), and between DW/MTX group and H/MTX group (P = 0.022) [Table 5].
|Table 4: Descriptive statistics of histological parameters and significance of their differences between the groups. (Test: ANOVA)|
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|Table 5: Multiple comparison of the histological dimensional parameters between the groups. (Test: post hoc analysis)|
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Other characteristics of histological section
The control group (honey-normal saline group and distilled water-NS group)
Dorsum of tongue exhibited filiform papilla as sharp conical projections with pointed tips. These papillae were in regular distribution covered with keratinized epithelium. The connective tissue of the papillae was normal without ICI. Fibers of skeletal muscle were seen as running in different directions beneath the connective tissue. Ventral surface of tongue exhibited thin stratified squamous epithelium covered with very thin layer of keratin [Figure 5].
|Figure 5: Histological pictures of dorsal (A1 and B1) and ventral (A2 and B2) surface of the tongue from the animal of group H/NS (A1 and A2) and group DW/NS (B1 and B2) at the total of ×400|
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Distilled water-methotrexate group
The dorsal surface of tongue revealed filiform papilla with loss of the normal appearance and some of them with flattened tips. Vacuolizations of some epithelial cells were also seen. The shape of fungiform papillae appears similar to normal but with some cellular vacuolization. Underlying connective tissue exhibited the presence of ICI and presence of CBV. The mucosa of ventral epithelium showed a reduction in its thickness as compared to control group with some CV and ICI [Figure 6].
|Figure 6: Histological pictures of dorsal (C1 and D1) and ventral (C2 and D2) surface of the tongue from animal of group DW/MTX (C1 and C2) and group H/MTX (D1 and D2) at a total of × 400. Footnote: Thinning of the epithelial thickness can be appreciated in both C1 and C2. There was the presence of inflammatory cell infiltration in lamina propria (arrow in C1) and also presence of engorged blood vessels (arrows in C2)|
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Dorsum of tongue showed restoration of normal histology of the epithelium and underlying connective tissue. Nearly normal histology of filiform papilla and fungiform papilla was seen. Very few CV and ICI were seen. Ventral epithelium also showed the restoration of normal thickness of epithelium with the absence of engorged blood vessels and ICI [Figure 6].
| Discussion|| |
The tongue consists largely of skeletal muscle, partly invested by thin and smooth ventral or lingual mucosa and thick dorsal mucosa. In rats, the mucosa consists of an outermost keratinized stratified squamous epithelium beneath which is a dense network of connective tissue called the lamina propria containing numerous blood capillaries. The dorsal mucosa, unlike the ventral is thrown into papillae of different shapes and types [Figure 5]A1. Within each papilla is a projection of tunica propria into the overlying epithelium forming secondary or corial papillae. Since the ventral mucosa does not present a thick keratinized layer, it is more easily affected by chemotherapy and was thus chosen in this experimental study for assessing the effects of methotrexate.
Mucositis is an inflammatory, painful, and debilitating condition that could occur with anticancer therapy, particularly in patients undergoing radiotherapy and/or chemotherapy for head and neck cancer. Sequelae include severe pain, impairment of oral and pharyngeal function, bleeding episodes, and increased risk for local and systemic infections., Since oral mucosa comprises cells with high mitotic index (rapid epithelial turnover and maturation rates), this renders the mucosa vulnerable to the adverse effects of chemotherapy and radiotherapy.,
A complex mechanism is involved in the pathophysiology of mucositis induced by chemotherapy and radiotherapy; both generate reactive oxygen species which are deleterious to the DNA of epithelial cells. Chemotherapy and radiation also activate the apoptotic pathway, leading to mucosal disintegration. This exposes the nerve ends causing severe pain and bacterial infections., Methotrexate, which is a drug widely used in bone marrow transplantation was used in this study to induce mucositis in rats. As indicated by Al Refai (2014), the total dose of the drug was administered during four consecutive days. Although it increases the researcher's involvement, this schedule reduces the toxicity of drug resulting in lower mortality of the animals.
Bee honey has been used as a medicament since ancient times. It has been used for infected wounds, burns, ophthalmic mucosa, and even for extraction sockets. Chiba et al. stated that the anti-inflammatory action and stimulating effect of honey on tissue repair could relieve oral discomfort arising from radiotherapy. The composition of honey is difficult to define since it is not a generic product. The ingredients and their relative amounts depend on the flora of the geographical area from which honey bees collect pollen. Honey typically contains <20% water, a high concentration of sugars, proteins, vitamins, and enzymes.
Although the exact mechanism of action of “honey” is not known, osmolality, acidity, and the production of hydrogen peroxide have been proposed to be the main factors. By reducing prostaglandin synthesis at the site of application, honey lowers plasma prostaglandin concentrations. It also has antioxidant and anti-inflammatory properties and increases nitric oxide in the lesions. It is proposed that sweet substances per se stimulate the salivation reflex due to their hyperosmolarity.
A randomized control trial (RCT) by Khanal et al. among patients with oral carcinoma scheduled for radiotherapy showed that honey applied topically to the oral mucosa of patients undergoing radiation therapy provided a distinct benefit by limiting the severity of mucositis.
Statistically significant change (P < 0.05) was seen in severity of oral ulcers with topical application of honey in a patient diagnosed with cancer and planned for concomitant chemotherapy and radiotherapy. In an another RCT study, among patients with grade 2 and 3 mucositis, topical application of honey showed a significant reduction (P < 0.05) in recovery time of mucositis as compared to control group. These study results support the finding of the present study result stating that honey is an effective agent to ameliorate the effect of chemotherapy-induced oral mucositis by decreasing the inflammatory parameters as compared to control group [Table 3] and [Table 4].
A study by Alrefai on Wistar rats had shown statistically significant difference (P < 0.01) in the thickness of epithelium between MTX/water group (19.22 ± 3.56) and the other three groups. In the present study, the thickness of the ventral mucosa in MTX/water group was 37.04 ± 5.37 which was also significantly different from other groups (P < 0.001) [Table 4] and [Table 5]. A similar finding was observed for a number of CBV in different groups. According to Al-Refai, there was a statistically significant difference (P < 0.01) present between MTX/water group (5.30 ± 0.22) and the other three groups regarding the number of CBV. The present study also demonstrated the maximum numbers of CBV in DW/MTX group (1.28 ± 0.65) followed by other groups with nearly equal numbers (0.445 ± 0.502 for H/NS group, 0.389 ± 0.390 for DW/NS group and 0.50 ± 0.35 for H/MTX group) [Table 3]. The lesser count of present study result for CBV in each group as compared to study by Al-Refai was due to methodological difference of expressing the value. The present study had expressed the count of CBV per microscopic field (by making an average of three microscopic fields) while a study by Al-Refai had presented the value by adding the number of all the three microscopic fields.
| Conclusion|| |
The present study result showed that feeding of local honey could decrease the ICI in lamina propria, number of CBV in lamina propria, and CV of epithelial cell produced by the provision of chemotherapeutic drug (methotrexate) among experimental animals. Similarly, local honey was found to be effective in restoring the thickness of mucosa of the tongue which was thinned by the administration of methotrexate. The loss of weight produced by the administration of methotrexate was also restored by the use of local honey. The present study concluded that the use of honey was more effective in alleviating oral mucositis in comparison to placebo.
Financial support and sponsorship
BP Koirala Institute of Health Sciences.
Conflicts of interest
There was no conflict of interest.
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Dr. Laxman Khanal
Associate Professor, Department of Human Anatomy, BP Koirala Institute of Health Sciences, Dharan
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]