The effect of hippophae rhamnoides extract on oral mucositis induced in rats with methotrexate

ABSTRACT Objective: To investigate the effect of HRE (Hippophae rhamnoides extract) on oral mucositis induced in rats with MTX. Material and Methods: Experimental animals were divided into groups as healthy (HG), HRE+MTX (HMTX), and control group, which received MTX (MTXC). HMTX group received 50 mg/kg HRE while MTXC and HG groups received equivolume distilled water with gavage once a day. After one hour of HRE and distilled water administration, HMTX and MTXC groups received a single dose of oral MTX 5 mg/ kg. This procedure was repeated for one month. Results: The levels of MDA, IL-1β, and TNF-α were found to be significantly higher in the cheek, lower lip, and tongue tissue of the animals receiving MTX, compared with HG and HMTX groups; however, these parameters were lower in the cheek and low lip tissue, and a milder damage ocurred in these tissues, compared with the tongue tissue in MTXC group. No histopathologic damage was observed in the cheek, lower lip, and tongue tissues of the rats treated with HRE. Conclusion: This findings indicate that HRE as a natural product is an important advantage compared with synthetic drugs for prophylaxis of oral mucositis developed due to MTX.


INTRODUCTION
Methotrexate (MTX) is an antiproliferative folic acid antagonist used in the treatment of various and high doses are used to treat malignancies 2 . However, high-dose MTX cause serious adverse effects 29 . MTX and some antineoplastic drugs destruct the proliferating cells of the mucosal layer, creating damage in the oral tissues 14 . This side effect, known as oral mucositis, occurs in 40% of patients receiving chemotherapy 12 . Mucositis reportedly consists of steps beginning with the formation of reactive oxygen species (ROS), mucosal damage, infection, and cell death 21 . Oral mucositis causes either discontinuation or reduced chemotherapy dose 19,28 . Therefore, numerous studies have been conducted about protection against mucositis, but there are no specific medications in clinical practice for the treatment of mucositis. MTX has been reported to decrease levels of myeloperoxidase (MPO), malondialdehyde necrosis factor (TNF-), which are indicators of inflammatory response in the gastrointestinal system 1,16 . This information from the literature indicates that MTX may also damage the oral antiulcer, and antimicrobial effects, and inhibiting the production of proinflammatory cytokines. Hippophae rhamnoides fruit extract (HRE), tested in this study against MTX oral mucositis, showed antioxidant, antiulcerogenic, antiinflammatory, antimicrobial, and proinflammatory cytokine antagonist properties 18,27,31 . Hippophae rhamnoides L. plant, member of the Eleagnaceae family, contains carotenoids ( C, tocopherol, tocotrienol, folic acid, tannin, and fatty acids 18,31 . No studies on the protective effect of HRE against oral mucositis induced by MTX were found in the literature screening. Therefore, the objective of this study is to investigate and evaluate the effect of HRE on oral mucositis induced in rats with MTX through biochemical, gene expression, and histopathologic examinations.

Animals
Experimental animals were obtained from Atatürk University Medical Experimental Application and Research Center. A total of 21 rats weighing 224-232 g were used. Before the experiment, animals were doused and fed as groups (n-7) in the pharmacology laboratory at normal room temperature (22°C

Chemical agents
The chemical agents used in the experiment Thiopental sodium from I.E. Ulagay-Turkey, and Hippophea rhamnoides extract from Karen Bilim-Turkey.

The experiment
Experimental animals were divided into groups as healthy (HG), HRE+MTX (HMTX), and control group, which received MTX (MTXC). HMTX group of rats (n-7) was given 50 mg/kg HRE while MTXC (n-7) and HG (n-7) groups were given equivolume distilled water with gavage once a day. After one hour of HRE and distilled water administration, HMTX and MTXC groups received a single dose of oral MTX 5 mg/kg. This procedure was repeated for one month. At the high-dose anesthesia. Then the amounts of MDA and tGSH were determined in the removed cheek, TNF-gene expressions were measured, and all the tissues were histopathologically studied.

Histopathologic examination
The cheek mucosa, lower lip, and tongue 10% formalin solution for 24 h. Following routine μm thick sections and stained with hematoxylin and eosin (H&E). All the sections were evaluated by a pathologist who was blinded to the treatment protocols under optic microscope (BX-52; Olympus, Tokyo, Japan).

Statistical analysis
Experimental results were expressed as the difference between the groups was determined using one-way ANOVA test followed by LSD test. All statistical analyses were performed using the SPSS Statistics Version 22 statistical software, and  (Figure 3a). Whereas, the amount of tGSH were found as 1.78±0.33, 6.81±0.37, and 7.38±0.46 nmol/gr protein in the tongue tissues of these groups (Figure 3b).

Histopathologic results
Examinations under optic microscope revealed a normal histopathologic appearance of the cheek, lower lip, and tongue tissues of the healthy group (Figure 4a, 4b, and 4c).
In Figure 4d, dilated congested blood vessels (arrow) and edema (star) were distinguished in the cheek mucosal tissue of MTXC group, which received MTX, while there were mild edema (star) and dilated congested blood vessels (arrow) observed also in the lower lip mucosa of this group (Figure 4e). However, besides dilated congested blood vessels (Figure 4f

DISCUSSION
In this study, the effect of HRE on oral mucositis induced in rats with MTX was investigated and evaluated through biochemical, gene expression, and histopathologic examinations. Experimental results showed that the amount of MDA was increased and the amount of tGSH was decreased in the cheek, lower lip, and tongue tissues of the in the tissues in which MDA was significantly increased. MDA is known to be an oxidant and GSH an antioxidant parameter 17 . Therefore, increased MDA and decreased tGSH amounts indicate the development of oxidative stress. MTX has been experimentally demonstrated to increase the tissue levels of MDA, which is a marker of lipid peroxidation to decrease the level of tGSH 15,20 . HRE, which is used against the toxicity of MTX, was found decrease of tGSH in the cheek, lower lip, and tongue tissues. This demonstrates that HRE protects the tissues against oxidative stress. Fruits of Hippophae rhamnoides contain numerous phytochemicals, such as vitamins A, E, C, carotens, fatty acids, and 10,18,31 . These antioxidant phytochemicals are used to treat cancer, ulcer, liver, and skin diseases 32 . Many researchers have reported that lipid peroxidation products are increased, and the plasma level of vitamin E is decreased in the tissues of cancer patients due to chemotherapy 30 . Lipid peroxidation products increased with chemotherapy have been reported to decrease free radical scavengers antioxidant vitamins, such as vitamins A, E, and C 4,26 . In another in vivo and in vitro study, the use of vitamins E, A, and C against oxidative stress caused by chemotherapy has been found to enhance the therapeutic effect and also protect normal cells against apoptosis 5 . It has been reported in a cell culture and animal model that the combinations of vitamins A, B6, B12, C, D, and E of chemotherapy prolonged survival time and increased response to treatment 26 . Furthermore, it has been argued in a study evaluating riskand multivitamin combinations that vitamins had  (HEx40); e: Mild edema (star) and dilated congested blood vessels (arrow) are monitored in the lip mucosa of MTXC group given MTX (HEx40); f: Dilated congested blood vessels are seen in the tongue mucosa of MTXC group given MTX (HE&100); g: Fibroblastic proliferation (star) and fat cells replaced muscle layer are monitored in the tongue mucosal tissue of MTXC group given MTX (arrow) (HE&40); h: Dilated, congested, proliferated blood vessels (arrow) are seen in the tongue mucosa of MTXC group given MTX (HE&100); i: Edema (star) and dilated congested blood vessels (arrow) are monitored in the tongue mucosal tissue of MTXC group given MTX (HE&100); j: Appearance of the cheek tissue of HMTX group treated with Hippophae rhamnoides extract (HRE) resembling normal mucosa (HEx10); k: Appearance of the lip tissue of HMTX group treated with HRE resembling normal mucosa (HEx20); l: Appearance of the tongue tissue of HMTX group treated with HRE resembling normal mucosa (HEx100)  (2015) reported that MTX gene expressions in the kidney tissue. In our gene expressions. Vitamin C, alpha-tocopherol, and beta-carotens, also found in HRE, are known to have ihibitor 23 . Again, palmitic, oleic, and linoleic fatty acids appear to and TNF- 13 .
Macroscopically, no mucosal ulcerations were found in the cheek, lower lip, and tongue tissues of the animals given MTX. In addition, we found histopathological signs including mild edema and dilated congested blood vessels in the cheek and lower lip tissues, which have lower levels of than the tongue tissue. However, marked congested blood vessels, layer, proliferated blood vessels, and edema were observed in the tongue mucosal tissue.
cheek, lower lip, and tongue tissues of the healthy and HMTX groups, suggesting that histopathologic results were consistent with biochemical and gene expression results. Vascular congestion, 11 . Nothing was found in the literature about excessive fat accumulation in the muscle layer due to MTX. However, several studies associate the accumulation this is a pathologic event 9 . Edema caused by MTX in the cheek, lower lip, and tongue tissue might be a result of vascular changes. In the literature, edema vascular changes 24 . In conclusion, in this study we demonstrated with biochemical, gene expression, damage in the cheek and lower lip tissues and more severe damage in the tongue tissue of rats.
HRE was found to protect the cheek, lower lip, and tongue tissue against the toxic effect of MTX. the prophylaxis of oral damage due to MTX.