The protective effect of bosentan on methotrexate-induced oral mucositis in rats

Article history: Received October 23, 2022 Accept March 29, 2023 Available online June 10, 2023 This study aimed to evaluate the effectiveness of bosentan as a protective agent on the rat’s cheek mucosa for Methotrexate-induced oral mucositis. Twenty-four Wister-albino rats, about 12-16 weeks and weighing 300-400 g, were used. The animals were divided into three groups. In the first control group, the second group was treated with methotrexate (80 mg/kg), and the third group was treated with methotrexate and bosentan 60 mg/kg. The microscopical parameters were estimated, the pro-inflammatory marker (TNF-α) and intercellular adhesive molecule-1(ICAM-1) measurement in the buccal mucosa. The animals were sacrificed on day 15, and the buccal mucosa was dissected for histological and immunohistochemical analysis. Bosentan decreased the severity of the Methotrexateinduced oral mucositis by a significant decrease in the histological score and a significant decrease in TNF-α and ICAM-1 immune histochemical expression at day fifteen. Bosentan at a concentration of 60 mg/kg/day produced protection against Methotrexate-induced oral mucositis by its anti-inflammatory action and reduction in the histological score and, therefore, can be used as a protective agent against Methotrexate-induced oral mucositis.


Introduction
Oral mucositis is an inherent complication of cancer therapy for many patients and plays a significant role in the physical and psychosocial aspects of patients undergoing cancer therapy. It is a larger problem than is currently recognized and represents a therapeutic challenge frequently encountered in cancer patients because of a significant number of patients affected by this side effect. The drugs used in chemotherapy, like Methotrexate, destroy rapidly dividing cells in the body, like the gastrointestinal epithelium and buccal epithelium (1,2). Methotrexate, the rival of folic acid, is one of the remedies for tumor treatment (3,4). Oral mucositis affects the quality of life and nutritional state of the patient, who may require hospitalization, thus increasing health care costs (5). Mucositis reportedly consists of steps beginning with the formation of reactive oxygen species (ROS), progressing with the release of pro-inflammatory cytokines, such as tumor necrosis factor (TNF-α) and interleukin-1 beta (IL-1 β), resulting in mucosal damage, infection, and cell death (6). Oral mucositis causes either discontinuation or reduced chemotherapy dose (7,8). Therefore, numerous studies have been conducted about protection against mucositis, but there are no specific medications in clinical practice for the treatment of mucositis. Bosentan is a mixed endothelin receptor antagonist recognized as the first orally bioavailable endothelin receptor antagonist approved by the Food and Drug Administration for treating patients with pulmonary arterial hypertension (9,10). Pre-clinical studies have suggested the potential of using bosentan to treat a wide range of inflammatory diseases, including arthritis (11), cancer (12), uveitis (13), and depression (14). Thus, bosentan has been a crucial tool in investigating endotheline-1 roles in disease and the perspective of targeting endotheline-1 to treat diseases.
To date, the anti-inflammatory activities of bosentan against methotrexate-induced oral mucositis have not been investigated. Therefore, this investigation was designed to evaluate the preventive effect of bosentan against methotrexate-induced oral mucositis.

Ethical approve
The study protocol was approved by the institutional animal ethics committee from Al-Mosul university in the college of dentistry (UoM.Dent/A.L.14/22).

Experimental rats
Prior to the experiment, adult albino male rats that are weighing 300-400g were domiciled in four per cages for 7 days. The animals were raised in a laboratory affiliated with the College of Dentistry and were subjected to standard conditions of temperature, humidity, and light, in addition to being them with water and the necessary food throughout the research period.

Drugs
Remedy Methotrexate was provided by Ebewe company at 2.5 mg/ml, and bosentan from Cipla company volume of dose admiration was 2ml/kg

Experimental pattern
This study was conducted on 24 adult male rats. The rats were divided into three groups (n= 8/group). I received distal water and served as a control, while the other group II was exposed to the methotrexate induction at 80 mg/kg intraperitoneally. Group III was administered bosentan 60mg/kg orally, seven days before and after the induction by 80 mg/kg of Methotrexate.

Preparation of drugs
On the day of the experiment, the Methotrexate and bosentan were freshly prepared before administration. Estimated drugs were prepared as suspensions in distilled water. The dose of bosentan 60 mg/kg was determined based on a previous study (15). Methotrexate (80 mg/kg) was used as chemotherapeutic-induced oral mucositis (16).

Drug-induced oral mucositis model
All the experimental rats were kept in the animal house for 7 days without any treatment to adapt to the environmental conditions. Oral mucositis was induced by the Intraperitoneal administration of the chemotherapeutic agent methotrexate at 80 mg/kg (16). The third group was given bosentan seven days before methotrexate treatment and was continued for 7 days after induction (totaling 15 days of treatment) with a dose of 60 mg/kg (17). After the experiment ended, the animals were anesthetized with ketamine 50 mg/kg and xylazine 5 mg/kg in the different syringes, and their buccal mucosa was removed for histological and immunohistochemical studies.

Histological evaluations
Formalin 10% was used to fix the mucosa samples, then the sample was dehydrated, embedded in paraffin, deparaffinized with xylene, cut into 4 µm sections, and stained by hematoxylin and eosin (H&E). Slides were examined and scored to evaluate the histopathological changes. During the evaluation, the slides were coded to prevent observer bias (18)(19)(20)(21). All tissue sections were examined in a blinded fashion by an experienced histopathologist. The classification was made following previous studies (22) as follows: 1 = epithelial and connective tissue without vasodilatation, absence or low cellular infiltration, absence of edema, ulceration, and abscess; 2 = scattered vasodilatation, areas of reepithelization, diffuse cell infiltration with multiple mononuclear leukocytes, and absence of bleeding, edema, ulcers, and abscesses; 3 = moderate vasodilatation, hydropic epithelial degeneration (vacuolization), moderate cell infiltration dominated by polymorph nuclear leukocytes, the presence of hemorrhagic areas, edema and rarely small ulcers but the absence of abscesses; 4 = marked vasodilation, cell infiltration with multiple polymorph nuclear leukocytes, presence of hemorrhagic sites, presence of edema and ulceration, and absence of abscess; 5 = severe vasodilatation and inflammatory infiltration, characterized by neutrophils, abscesses, and diffuse ulcers.

Immunohistochemistry of TNF-α and ICAM-1
This technique is based on the detection of the product of gene expression (protein) in the cells of normal, mucositis and treated groups using specific polyclonal and monoclonal antibodies. The bound primary antibody is then detected by secondary antibody (usually mouse anti rat), which contains specific label (peroxidase labeled polymer conjugated to goat anti rat immunoglobulin). The substrate is 3,3diaminobenzidine in chromogen Solution. Positive reaction will result in a brown colored precipitate at the antigen site in tested tissue (23). Immunohistochemistry (IHC) directly demonstrated the affected tissue's cells (24). The development of specific antibodies for immunohistochemical reactions, simultaneously with the evaluation of the production of several biochemical markers in intestinal samples that were paraffin-embedded to measure the pro-inflammatory cytokine TNF-α and adhesive molecule ICAM-1. Quantification of IHC was performed in accordance to the following semiquantitative scores (25), which were based on the percentage of positively stained cells as follows :0, no staining; 1, ≤ 25%; 2, 26-50%; 3, 51-75 %; and 4, 76-10%.

Statistical analysis
Two software programs: SPSS (statistical package for social science) version 23 and Microsoft Office Excel 2013, were used to summarize, analyze and present the data. Quantitative (numeric) variables were expressed as mean and standard error. A paired t-test was utilized to determine the differences in the mean of dependent variables between the two groups. One-way ANOVA was used to study the difference in mean of quantitative variables among more than two groups, followed by a post hoc least significant difference (LSD) test to evaluate the mean difference between any two groups. The level of significance was considered at p ≤ 0.05 (26).

Results
This current study exhibits the histological changes in oral mucosal tissue in methotrexate treated group, primarily with focal infiltration of inflammatory cells in submucosa, necrosis, and atrophy of skeletal muscles with hyaline degeneration and edema between it, as displayed in figures 1 and 2.  In addition, the bosentan-treated group showed a significant reduction in the microscopical score as judged by intact mucosa and submucosa with slight edema between muscle fibers as displayed in table 1 and figures 3-5. Table 1 and figures 6-9 showed there was a highly significant difference of TNF-α and ICAM-1 level in the methotrexate treated group in comparison with bosentan and methotrexate treated group. However, there were no statistical differences between the control group and Methotrexate in bosentan treated group.

Discussion
Methotrexate, a folic acid antagonist, is widely preferred as a cytotoxic chemotherapeutic agent, but its efficacy is limited due to its side effects (16). Despite the availability of many therapeutic agents that claim to prevent or reduce the severity of oral mucositis, no intervention that is entirely successful at preventing oral mucositis exists. In the present study, the combined treatment of Methotrexate and bosentan ameliorated the buccal mucositis induced by Methotrexate alone.  In the initial inflammatory/vascular phase of mucositis, the epithelial, endothelial, and connective tissue cells in the buccal mucosa release free radicals, modified proteins, and pro-inflammatory cytokines, including interleukin-1B, prostaglandins, and Tumor Necrosis Factor (TNF). These inflammatory mediators cause further damage either directly or indirectly by increasing vascular permeability and angiogenesis, thereby enhancing cytotoxic drug uptake into the oral mucosa (1). Tumor necrosis factor-α is a crucial proinflammatory cytokine liberated from the lymphocytes and macrophages in the initial step of inflammation (27).
In this study, we also found that immunohistochemical expression of TNF-α in the cheek tissues was significantly increased in the rats' given Methotrexate, compared with healthy and bosentan groups. Chang et al. noted that the levels of pro-inflammatory cytokines, such as IL-1β and TNF-α, were elevated in the oral tissue with mucositis developed due to chemotherapy (28). Another study shows that the levels of IL-1β and TNF-α were increased, and inflammatory ulcers were developed in the intestinal tissue with Methotrexate (29). Endothelial adhesion molecules, including intercellular adhesive molecule-1 (ICAM-1) and leukocyte extravasation during inflammation, are mainly regulated by the pro-inflammatory nuclear factor kappa-b (NF-Κb) pathway, which controls the transcription of adhesion molecules and mediators of inflammatory processes (30,31). Inter Cellular adhesive molecule (ICAM-1) engages the leukocytes association with endothelial cells, simplifying their penetration to the inflammatory site (32).
Chemotherapy-induced mucositis is characterized by damage to mucous membranes (33). Cell adhesion molecules interact with extracellular matrix components to regulate cell behavior, including apoptosis, proliferation, migration, and differentiation. Differential expression of Cell adhesion molecules has been demonstrated at the injury and healing phases of mucositis (34). Recently, the subepithelial extracellular matrix changes have been recognized as a hallmark of mucositis development. The cellular receptors that bind to the extracellular matrix are called cell adhesion molecules. Cell adhesion molecules are well known for their role in the regulation of cell kinetics through extracellular matrix regulation. Furthermore, they play a central role in inflammation, which is a key process in the pathogenesis of mucositis (34).
In this study, TNF-α expressions were significantly increased in the tissues in which ICAM-1 was significantly increased. Mafra et al. (35) reported the induction of the immunohistochemical expression of TNF-α and ICAM-1 in the 5 fluorouracil (5-FU) Induced Oral Mucositis. Histopathology also exhibited the histological changes in Methotrexate treated group, primarily with focal infiltration of inflammatory cells in submucosa, necrosis, and atrophy of skeletal muscles with hyaline degeneration and edema between it. In contrast, vacuolar degeneration of some epithelial cells, flattening or shortening of the rete ridge, and an increase in the number of congested blood vessels in the connective tissue are among the histopathologic findings of oral mucositis reported by Sultan (16) in an attempt to examine the protective effect of green tea extract.
Bosentan is a low molecular weight, potent, competitive, dual endothelin A and endothelin B receptor antagonist that is selective for the endothelin system (36). Bosentan antagonizes the detrimental effects of endothelin in various experimental models and exhibits vasodilating, antiproliferative, anti-inflammatory, and anti-fibrotic effects. Bosentan also improves endothelial function. Those effects may be responsible for bosentan's long-term efficacy in treating pulmonary arterial hypertension, which slows disease progression and increases survival (37). In our experiment, bosentan, which significantly prevented the increase of ICAM-1, also significantly prevented the increase of TNF-α expressions. Thus, bosentan probably inhibits inflammation by blocking various inflammatory network agents. It has been shown that treating animals with bosentan resulted in a substantial decrease in several proinflammatory mediators, including TNF-α, endothelin-1 (38), and ICAM-1 (39). This observation provides insight into the mechanism of bosentan as an inhibitor of the inflammatory reaction. In a murine model of inflammatory bowel disease, the anti-inflammatory effect of bosentan was investigated, and it was determined that bosentan reduced inflammation in this animal model (40). Another study showed that prophylactic oral administration of bosentan reduced clinical inflammation in trinitrobenzene sulphonateinduced colitis in rats (41).

Conclusion
Bosentan has a protective effect through the antiinflammatory action and reduction in histological score.