Effect of taxifolin on methotrexate-induced oxidative and inflammatory oral mucositis in rats: biochemical and histopathological evaluation

Abstract The role of oxidative stress, as well as inflammation in the pathogenesis of methotrexate (MTX)-induced oral mucositis, is a known fact. The anti-inflammatory, antitumor, antimicrobial, and antioxidant properties of taxifolin—the effect we tested against MTX-induced oral mucosal damage—are well known. Objective Evaluating biochemically and histopathologically the effects of taxifolin on methotrexate-induced oral mucosal damage in rats. Methodology In the taxifolin+MTX (TMTX) group, 50 mg/kg taxifolin was orally administered to rats by gavage. In the MTX and healthy (HG) groups, normal saline was applied to rats as solvent by the same method. One hour after administration of taxifolin and solvent, 5 mg/kg MTX was orally administered to rats in the MTX and TMTX groups. Taxifolin and methotrexate were administered once a day for 30 days. Macroscopic, biochemical, and histopathological evaluations were performed on the inner cheek and tongue tissues of rats. These parts were removed after rats were killed with a high-dose anesthesia. Results Taxifolin with MTX prevented the increase in oxidant and pro-inflammatory parameters, such as malondialdehyde (MDA), tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1β), interleukin 6 (IL-6), on the inner cheek and tongue tissues of rats. Moreover, taxifolin antagonized the decrease in total glutathione (tGSH). Taxifolin decreased MTX-induced histopathological damage. Conclusion These findings suggest that taxifolin may be useful to treat MTX-associated oral mucositis.


Introduction
Methotrexate (MTX) is a folic acid antagonist-an antiproliferative drug, as it is known. 1 Currently, MTX is used in the treatment of pediatric acute lymphoblastic leukemia, choriocarcinoma, and various malignancies.
Moreover, MTX is a gold-standard antirheumatic agent in the treatment of rheumatoid arthritis, psoriasis, systemic scleroderma, and other autoimmune disorders. 2 The use of MTX is preferred in low doses in the treatment of inflammatory diseases while it is preferred in high doses in the treatment of malignant diseases. 1 As well as high-dose MTX is safely applied to most patients during treatment, serious side effects, such as nephrotoxicity, myelosuppression, mucositis, dermatologic toxicity, and hepatotoxicity, can be observed. 3 Moreover, various side effects may occur during the treatment of inflammatory diseases, ranging from mild to severe side effects and even leading to treatment discontinuation. 4 One of the serious side effects of MTX is oral mucositis. 5 Oral toxicities, such as mucositis and stomatitis, are some of the most serious and indispensable side effects related to cancer therapy. 6 Mucositis is a pathological process that causes ulceration and inflammation of the mucosal tissue in the region from the mouth to the anus. 7 Oral mucositis makes feeding difficult and may also increase the risk of infection, limit the dose of the medication, delay the treatment, and extend hospital stay. 8 The role of reactive oxygen species (ROS) and various pro-inflammatory cytokines in the pathogenesis of mucositis have been shown. 9 It has also been showed that MTX increases the levels of malondialdehyde (MDA), an oxidant parameter in the oral mucosa, interleukin 1 beta (IL-1β), and tumor necrosis factor alpha (TNF-α), an indicator of inflammatory response. 10 The literature shows that effective antioxidant, antiinflammatory, antiulcer, and antimicrobial medication may be useful to treat MTX-associated oral mucositis.
Taxifolin (3, 5, 7, 3, 4-pentahydroxy flavanone), which we tested against MTX-induced oral mucosal damage in this study, can be found in plants such as thistle, onion, French maritime, and tamarind. 11 The anti-inflammatory, antitumor, antimicrobial, and antioxidant properties of taxifolin are well known. 12 It has been shown that taxifolin has an antioxidant effect by inhibiting ROS production. 13

Chemicals
The chemicals used were methotrexate, thiopental sodium, and taxifolin and they were obtained from Med-İlaç, Turkey, I.E. Ulagay, Turkey, and Evalar, Russia, respectively.

Experimental groups
The rats used in the experiment were divided into three groups: MTX group , taxifolin+MTX group (TMTX), and healthy group (HG).

Experiment procedure
A total of 50 mg/kg taxifolin 14 was administered orally to the stomach of rats by gavage in the TMTX group (n=6). Normal saline (0.9% NaCl) was applied to rats in MTX (n=6) and healthy (n=6) groups as solvent by the same method. One hour after taxifolin and solvent administration, animals in the MTX and TMTX groups orally received 5 mg/kg MTXo. 15 Taxifolin and methotrexate were administered once a day for 30 days. At the end of this period, all rats were killed with high-dose anesthesia (50 mg/kg thiopental sodium). The inner cheek and tongue tissues were removed and macroscopically evaluated, and then Tissue MDA and tGSH determination Tissue MDA (µmol/g protein) was measured by the method used by Ohkawa, Ohishi and Yagi 16 (1979). On the other hand, tGSH (nmol/g protein) was measured by the method described by Sedlak J and Lindsay RH. 17 Tissue TNF-α, IL-1β, and IL-6 analysis

Histopathological analyses
The inner cheek and tongue tissues were fixed in 10% neutral formalin solution. The tissues were subjected to routine alcohol-xylol series after fixation.
Later, 5 µm sections of the sample were placed in paraffin blocks and six random areas were stained with hematoxylin and eosin. Sections were stained with hematoxylin and eosin. The evaluation was made according to

Results of the macroscopic evaluation of the inner cheek and tongue tissues
In the MTX group, the inner cheek tissue was quite swollen and presented severe hemorrhage, hyperemia, and ulcerated areas. In the TMTX group, the inner cheek tissue was less swollen and there was moderate swelling and hyperemia. When the tongue tissue was macroscopically evaluated, hyperemic muscle-due to the loss of the full-thickness epithelial layer on the tip of the tongues of the MTX group-complete loss of papillae, significant swelling of the entire tongue, hemorrhage, and hyperemia were observed. In the tongues of the TMTX group, moderate hyperemia was observed in the body and tip of the tongues, with a decrease in the papillary prominence ( Figure 1).

Results of the MDA analysis in the inner cheek and tongue tissues
The amount of MDA in the inner cheek and tongue tissues of rats treated with MTX increased significantly when compared to the HG, as it can be observed in Figure 2 (p<0.001). The increase in MDA was significantly inhibited by taxifolin (p<0.001). There was a significant difference between the healthy and TMTX groups in terms of MDA values in the inner check tissue (p=0.036), but there was no significant difference between samples in the tongue tissue (p=0.630).

Results of the tGSH analysis in the inner cheek and tongue tissues
As it can be seen in Figure 2, the amount of tGSH in the inner cheek and tongue tissues of rats treated with MTX was lower than that of the HG (p<0.001).
Taxifolin significantly inhibited the reduction of tGSH in the inner cheek and tongue tissues of rats receiving MTX <0.001 ( Figure 2).

Results of the TNF-α analysis in the inner cheek and tongue tissues
As it can be seen in Figure 2, the amount of TNF-α in the inner cheek and tongue tissues of rats treated with MTX was higher than that of the HG (p<0.001).
Taxifolin significantly inhibited the reduction of TNF-α in the inner cheek and tongue tissues of rats receiving MTX (p<0.001).

Results of the IL-1β analysis in the inner cheek and tongue tissues
MTX increased the amount of IL-1β in the inner cheek and tongue tissues of rats (p<0.001). However, taxifolin significantly inhibited the increase in IL-1β in rats receiving MTX (p<0.001) (Figure 2).

Results of the IL-6 analysis in the inner cheek and tongue tissues
The amount of IL-6 in the inner cheek and tongue tissues of rats treated with MTX increased significantly when compared to the HG, as it can be observed in group than in the TMTX group. Statistically significant differences were observed between groups in terms of histopathological findings (Table 3; p<0.05).
The tongue tissue presented a normal histological appearance in rats in the HG ( Figure 3A). Keratinized papillae were slightly shaped in the TMTX group while no keratinized papillae were observed in the tongues of the MTX group. The reduction in epithelial thickness was moderate in the MTX group and mild in the TMTX group. Swelling was moderate in the MTX and TMTX groups ( Figure 3B-E).
The inner cheek tissue presented a normal histological appearance in rats in the HG ( Figure   4A). Interstitial inflammatory cell infiltration and hemorrhage were severe in the MTX group while these histopathological findings were mild in the TMTX group.
Similarly, muscle fiber degeneration was severe in the MTX group and mild in the TMTX group ( Figure 4B-E).

Discussion
The effects of taxifolin on MTX-induced oxidative and inflammatory oral mucosal damage in rats were evaluated macroscopically, biochemically, and histopathologically in this study. Our macroscopic findings showed that mucositis developed in the   Oral mucositis is one of the serious side effects of MTX, as aforementioned. 5 Mucositis is a pathological process characterized by ulceration and inflammation of the mucosal tissue. 18 Another symptom of mucositis is bleeding of the mucosa. 19 In our study, symptoms such as swelling, hyperemia, ulcer, and hemorrhage were observed in the inner cheek and tongue tissues of rats treated with MTX, which is in accordance with the literature.  In our study, the amount of MDA in the TMTX group was close to that of the HG. These findings suggest that taxifolin is supported by studies that show its inhibitory effect on LPO and ROS production. 13,25 Taxifolin has also been reported to have a scavenging effect on other ROS, especially hydroxyl radical (HO). 26 The fact that taxifolin has an inhibitory effect on ROS 27 can be considered as its mechanism to prevent MTX from decreasing tGSH in the oral mucosa. These findings suggest that tGSH was insufficient to neutralize ROS in the MTX group. GSH is a tripeptide consisting of L-glutamate, L-cysteine, and glycine that found in many cells, as it is known. 28 GSH, when catalyzed by selenium-glutathione peroxidase, reacts with hydrogen peroxide (H 2 O 2 ) and chemically detoxifies H 2 O 2 and protects cells from ROS damage. 22,29 Our biochemical findings showed that MDA, TNF-α, IL-1β, and IL-6 increased and tGSH decreased in the MTX group. In a study that support our findings, it was reported that MTX increased the levels of MDA, IL-1β and TNF-α in the oral mucosa. 10

Conflict of interest
The authors declare no conflict of interest in this study.