Royal jelly and propolis therapies reduce inammation and stimulate healing of oral mucositis in rats

Purpose This study aimed to evaluate the effects and mechanisms of action of royal jelly (RJ) and propolis in an animal model of oral mucositis (OM). Methods Seventy-two male Wistar rats were randomly allocated to four groups (n = 18): control (no treatment), PBMT (intraoral laser, 6 J/cm2), RJ, and propolis. On days 0 and 2, animals received injection of 5-uorouracil (5-FU). The buccal mucosa was scratched (days 3 and 4) and the treatments were initiated on day 5. Six animals of each group were euthanized on days 8, 10, and 14. Phytochemical analysis (thin-layer chromatography-TLC), clinical, histopathological, immunohistochemical of pS6, pAKT and NF-κB and oxidative stress markers were also investigated. Results TLC revealed the presence of large amounts of sucrose (Rf 0.34) in RJ and avonoids in propolis. On day 8, lower clinical OM scores (and day 10) and morphological were observed in the PBMT, RJ and propolis groups (p < 0.05). On day 8, immunoexpression of pS6, pAKT and NF-κB was increased when compared to control. On day 14, GSH antioxidant levels were increased in the propolis group compared to control (p < 0.05). Conclusions Our results showed that RJ and propolis, as well as PBMT, are effective in the treatment of OM. The RJ and propolis may explain their excellent wound healing activity and anti-inammatory effects. Considering that not all patients who develop OM have access to PBMT, the present study demonstrated that topical application of RJ and propolis may be an important alternative for the treatment of OM.


Introduction
Oral mucositis (OM) is a common and severe complication of cancer treatment. It is estimated that more than 90% of patients undergoing radiotherapy for head and neck cancers have some degree of OM. This condition is therefore considered one of the most important acute side effects observed in patients with head and neck cancer submitted to radiotherapy and/or chemotherapy [1][2][3][4].
In general, OM clinically manifests as erythematous, erosive and/or ulcerative lesions of variable clinical severity. The ulcers are usually painful and therefore interfere directly with the quality of life of the patient. Additionally, OM increases the risk of secondary infections, which may lead to a delay, interruption or discontinuation of oncological treatment, with a consequent increase in the morbidity and mortality of cancer patients [3,5].
The pathogenesis of OM comprises several stages: initiation, positive regulation and activation that result in the production of in ammatory mediators, signal ampli cation, ulceration, and healing. The initiation phase is characterized by tissue damage due to oxidative stress resulting from the production of reactive oxygen species (ROS), direct DNA damage, and activation of the innate immune response. This oxidative stress causes damage to cell membranes and triggers the activation of macrophages and nuclear factor kappa B (NF-κB). Activated NF-κB induces gene expression and the release of proin ammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, IL-1β, and cyclooxygenase 2 (COX-2). The healing phase occurs after the harmful stimuli are eliminated and is characterized by increased epithelial cell proliferation and differentiation, associated with reorganization of the extracellular matrix [4,[6][7][8][9][10].
Some authors have linked the development of OM and aphthous ulcerations to activation via mammalian target of rapamycin (mTOR) [4,11,12]. The phosphoinositide 3-kinase (PI3K)-mTOR pathway is activated by AKT (serine/threonine kinase). This pathway is part of two complexes: mTORC1 (that leads to the phosphorylation of S6) and mTORC2 (that leads to the phosphorylation of AKT) [4,12,13]. The activation of PIK3-mTOR regulates the synthesis of proteins necessary for cell cycle progression and cell growth and survival, increasing the proliferation and migration of epithelial cells and thus accelerating wound healing [7,[13][14][15][16].
Studies have investigated the use of natural compounds in an attempt to identify alternatives for the prevention and treatment of OM since photobiomodulation therapy (PBMT), the gold standard for the prevention and treatment of OM, is not always available at cancer treatment centers [3]. Additionally, some studies have sought to understand the physiopathology of OM and the effects of treatment with these natural compounds on signaling pathways involved in the development of this condition [2,5,[17][18][19][20]. Among the natural products available for the treatment of different conditions, royal jelly (RJ) and propolis, two important compounds produced by honey bees of the genus Apis, have been used worldwide as traditional nutrients and in pharmacology since ancient times [21][22][23][24][25][26].
Within this context, the present study aimed to investigate the wound healing and anti-in ammatory effects of RJ and propolis in an animal model of OM by clinical and histological analyses. In addition, the mechanisms of action were assessed based on the oxidative stress pattern and analysis of NF-κB and PI3K/mTOR/AKT.

Experimental Procedure
The study was approved by the Ethics Committee on Animal Use (CEUA, Approval number 2018 − 0298) of the University Hospital of Porto Alegre (HCPA). Seventy-two male Wistar rats (Rattus norvegicus albinus), 8 to 12 weeks old and weighing 275 ± 25 g, were used. The animals were housed as described by Curra et al. (2015) [27] and were divided randomly into four groups: All animals were submitted to the protocol of OM induction proposed by Sonis et al. (1990) and modi ed by Leitão et al. (2007) (moderate severity) [28,29]. Brie y, 5-uorouracil was applied intraperitoneally on days 0 (60mg/kg) and 2 (40mg/kg), in a volume of up to 5 mL/kg. On days 3 and 4, anesthesia was induced in the animals with iso urane (4-5%) and maintained (1-2%) with vaporized iso urane in 0.5 L/min of 100% oxygen. Both sides of the buccal mucosa were scratched twice with the tip of a sterile needle by the same operator. The treatments were initiated on day 5.

Parameters of Photobiomodulation Therapy
The animals of the PBMT group received one daily intraoral laser application with an aluminum gallium Finally, a solvent system consisting of toluene: ethyl acetate (TLC) and formic acid (5:5:0.5, v/v/v) (co-TLC) was used for the analysis of 10% propolis gel, and 1-isopropanol: ethyl acetate: distilled water: formic acid (4:0.5:0.5:0.5, v/v/v/v) was used for the analysis of fresh RJ. The results were compared to standards by observing the chromatographic spot color and retention factor (Rf).
After elution of the mobile phase, the plates were developed using two regents: sulfuric vanillin as universal developer and 0.2% Natural Reagent (diphenylboryloxyethylamine) speci c for avonoids. The plates were observed under ultraviolet (UV) light at 365 nm. Sucrose was used as standard for fresh RJ because of the presence of sugars. Propolis gel was compared to the avonoid standards luteolin, quercetin, and kaempferol (Sigma-Aldrich®). Euthanasia On day 8, 10 and 14, six animals of each group were euthanized with an overdose of the anesthetic iso urane (severity: no recovery). Iso urane was vaporized in 100% oxygen (oxygen ow of 0.5 L/min) at a concentration ≥ 5% and administered by inhalation (chamber, nose cone, or face mask). Before biopsy, the right buccal mucosa was photographed for clinical examination and then xed in 10% buffered formalin for histopathological and immunohistochemical analysis. The left buccal mucosa was stored in liquid nitrogen in a freezer at -80 o C Clinical and Histopathological Analyses The clinical and histopathological analyses were carried out by a previously calibrated and blinded professional according to the parameters proposed by Lima et al. (2005) [30].
For analysis of immunostaining, the slides were photographed (Pannoramic MIDI, 1.15 SPI, 3D HISTECH®, Budapest, Hungary) selecting three different elds in the oral mucosal epithelium using the borders of the wound or the area with evidence of previous tissue damage as a reference. The slides were analyzed by a single examiner using the Image J program (National Institute of Mental Health, Bethesda, Maryland, USA) in a blind analysis. A modi ed semiquantitative immunoreactivity score (IRS) (REMMELE; STEGNER 1987) ( Table 1) was calculated for pS6 (positive cytoplasmic staining) andpAkt 473 [31]. Immunoexpression of NF-κB (positive nuclear staining) was analyzed quantitatively by calculation of the labeling index (LI) as follows (SEVERO et al., 2018): LI (%) = (number of immunopositive cells)/(total number of counted cells) × 100 [32]. Table 1 Immunohistochemical classi cation based on immunoreactivity score (IRS) Reduced glutathione (GSH) exerts antioxidant activity by deactivating free radicals. The method described by Costa et al. (2006) was used to measure GSH levels in OM. Brie y, the buccal mucosa samples (n = 18 per group) were homogenized in 0.25 mL of 5% EDTA and 320 µL distilled water and 80 µL of 50% trichloroacetic acid (TCA) were added [33]. The samples were centrifuged at 3000 ×g for 15 min at 4 o C. The supernatant (400 µL) was combined with 800 µl of0.4 M Tris buffer (pH 8.9) and 20 µL of 0.01 M dithiobis-2-nitrobenzoic acid. Absorbance was read at 420 nm and the results are reported as GSH unit/mg tissue.

Superoxide dismutase
The enzyme superoxide dismutase (SOD) catalyzes the dismutation of superoxide to oxygen and hydrogen peroxide. SOD exerts an important antioxidant activity in cells exposed to oxygen. The activity of SOD was estimated following the method of Beauchamp and Fridovich (1971) [34]. The sites labeled test, standard and control were exposed to a 15-W uorescent lamp for 10 min in a chamber covered with aluminum foil. Immediately after light exposure, the optical density of all reactions was read at 560 nm. The units of enzyme present in the samples were calculated using the following formula: SOD activity × 100/total protein (mL/100µL).

Malondialdehyde
Malondialdehyde (MDA) is an end product of membrane lipid peroxidation. In this study, MDA levels were quanti ed in the samples analyzed as described by Siddique et al. (2012) and Bradley et al. (1982) [35,36]. The buccal mucosa samples were resuspended in Tris-HCl (1:5,w/v) and cut into small pieces with scissors for 15 seconds on an ice-cold plate. The resulting suspension was homogenized for 2 min in an automated Potter homogenizer and centrifuged at 2,500 × gfor 10 min at 4 o C. The MDA content was determined in the supernatants by reading absorbance at 586 nm. The results were expressed as nanomole MDA per gram tissue.

Statistical Analysis
The clinical, histopathological, pS6 and pAkt 473 immunoexpression, and oxidative stress data were evaluated by one-way analysis of variance (ANOVA), followed by Tukey's multiple comparisons test. The Mann-Whitney test was used for the analysis of transcription factor NF-κB. Statistical analysis was performed with the GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA), adopting a level of signi cance of 5% (p < 0.05).

Results
The thin-layer chromatography (TLC) analysis of RJ revealed a dark brown color spot (Rf 0.42), suggesting the presence of sugars due the characteristic color. RJ showed a spot with the same Rf and color of the standard sucrose (Rf 0.34) (Fig. 1A).
The chromatograms of propolis developed with sulfuric vanillin revealed a predominance of compounds with a terpene-like structure (red-brown, yellow-brown or dark green, blue and blue-purple bands), avonoides (yellow and green bands) and saponins, (blue, blue-purple and sometimes red or yellowbrown bands) (Fig. 1B).
Co-TLC of propolis eluted and developed with 0.2% Natural Reagent A (speci c for the detection of avonoids) followed by UV detection at 365 nm showed the presence of yellow and green uorescence, indicating the presence of aglycone avonoids such as quercetin (yellow-orange uorescence and Rf 0.41), luteolin (yellow uorescence and Rf 0.40), and kaempferol (green color andRf 0.58), using the color characteristics and Rf as reference ( Fig. 1C and 1D).
All animals developed OM on day 5. Figure 2 shows the clinical features of OM in all groups over the experimental period (8, 10, and 14 days). In general, animals of the treated groups (PBMT, RJ and propolis) exhibited lower clinical OM scores on days 8 and 10 compared to the control group (p < 0.05) ( Fig. 2A and 2B). No signi cant differences were observed between the groups studied on day 14 (p > 0.05).
Regarding the histopathological ndings (Fig. 3), accelerated healing of OM was observed on day 8 in the PBMT, RJ and propolis groups compared to control, with the difference in scores being statistically signi cant(p < 0.05) ( Fig. 3A and 3B). The main ndings in these groups were reepithelization, mild or absent hyperemia, a mild to moderate chronic in ammatory in ltrate, and the absence of ulceration and abscesses (score 0 or 1). Particularly interesting ndings were the increased number of new broblasts amidst the developing extracellular matrix and the presence of immature skeletal muscle cells (Fig. 3A).
No signi cant differences were observed between the groups studied on day 10 or 14 (p > 0.05).
On day 8, the PBMT, RJ and propolis groups exhibited an increase in the immunoexpression of proteins pS6 and pAKT (p < 0.05) when compared to the control group (Fig. 4A, 4B and 4C). However, on day 10, only the expression of pS6 appeared to be increased in the RJ group compared to control (p < 0.05) (Fig. 4B). No signi cant differences in the immunoexpression of these proteins were observed between groups on day 14 (p > 0.05) (Fig. 4B and 4C).
Immunoexpression of NF-κB was increased in the treated groups compared to control throughout the experimental period (p < 0.05) (Fig. 4A and 4D). Comparison between the treated groups (PBMT, RJ and propolis) showed no signi cant difference in these markers (p > 0.05), regardless of the day of analysis (Fig. 4). Figure 5 illustrates the results of the oxidative stress assays (GSH, SOD and MDA). As can be seen, only on day 14 did the propolis group produce higher GSH levels when compared to the control group (p < 0.05) (Fig. 5A). No signi cant difference in SOD or MDA levels was observed when the control group was compared to the PBMT, RJ and propolis group on the days analyzed (p > 0.05) (Fig. 5B and 5C).

Discussion
Oral mucositis is a common and clinically signi cant side effect in patients undergoing antineoplastic treatments [3,37]. These treatments frequently result in severe damage to the oral mucosa, with the patients developing ulcerative lesions that cause great discomfort and intense pain. If OM is not prevented or treated adequately, the discomfort generated can reduce the patient's ability to tolerate treatment [4,20]. Within this context, considering that not all patients who develop OM have access to PBMT, the present study demonstrated that topical application of natural compounds derived from bees may be an important alternative for the treatment of OM. Our results showed that both RJ and propolis promoted clinical improvement of OM, increasing reepithelization and reducing in ammation.
One of the compounds studied was RJ, which has shown high healing, antibacterial, anti-in ammatory, vasodilating, disinfectant, and antioxidant activities, among others [24,26,38]. Fresh RJ is composed chemically of proteins, sugars, lipids, vitamins, and free amino acids [39]. Sugars make RJ an important nutritional source, while the other components confer pharmacological properties such as healing, antiin ammatory, antimicrobial and antioxidant activity [40,41]. The present result showed that the chromatographic band that could be visualized in the mobile phase used was dark colored, suggesting the presence of sugars, which prevented the visualization of other bands that could suggest more compounds.
The phytochemical properties of RJ and propolis apparently had an impact on the clinical response and histochemical assays in the present study, promoting anti-in ammatory and healing effects in OM. We also reported a signi cant decrease in lesion size and the absence or reduction of ulcers and local in ammation [1,20,21,23,25,27,44]. We therefore consider that the use of fresh RJ and 10% propolis was as effective as PBMT by accelerating healing and reducing in ammation in cases of chemically induced OM.
We also evaluated the mechanisms of action of RJ and propolis. The results showed that the acceleration of healing by the two compounds is associated with activation of the PIK3-mTOR pathway.
Studies report that activation of this pathway regulates the synthesis of different proteins involved in cell cycle progression and cell growth and survival, increasing the proliferation and migration of epithelial cells and thus accelerating wound healing [13][14][15][16].This study demonstrated increased immunoexpression of pS6 and pAKT on day 8, a critical period for tissue repair and wound closure, in animals treated with PBMT, RJ and propolis compared to the control group. Immunoexpression of pS6 continued to be high until day 10. In experimental studies on rats, Squarize et al. (2010) and Castilho et al. (2013) found that activation of mTOR drastically increases the proliferation and migration of epithelial cells and wound healing [13,14]. Curra et al. (2015) observed clinical improvement of OM (day 10) and higher NF-κB activation levels (days 10 and 15) during the development and healing of OM in animals submitted to PBMT when compared to the control group [27]. The present results suggest that the accelerated clinical and microscopic improvement of OM in the PBMT, RJ and propolis groups was due to the activation of the PIK3-mTOR pathway in the early stages of tissue repair and to the high expression of NF-κB in the nal stages. High levels of NF-κB were observed throughout the experimental period until day 14, stimulating proliferation, apoptosis, morphogenesis and cell differentiation until the end of the healing process [12].
Furthermore, the ability of propolis to reduce the redox potential in tissues is widely reported in the literature. This antioxidant activity is related to the action of avonoids. Flavonoids act as scavengers of free radicals that are produced in tissues and can cause extensive damage to macromolecules such as DNA. In addition, avonoids can catalyze the nal products of lipid peroxidation resulting from cell membrane damage [2,43,45]. In the present study, increased GSH levels were observed in animals of the propolis group in the nal stages of healing. However, this nding did not negatively in uence tissue repair because, although late, it suggests that GSH acted by deactivating ROS in the wound, which favored the healing process [41]. The presence of avonoids explains the reduction of tissue oxidative stress in the propolis group.
In conclusion, this is the rst study to investigate the PI3K-Akt-mTOR pathway in the healing process of chemically induced OM treated with RJ and propolis. The study reported promising results for the treatment of OM with RJ and propolis. These products exhibited biological properties, including high healing and anti-in ammatory activity demonstrated by the activation of NF-κB in more advanced stages of healing and of the PIK3-mTOR cell proliferation pathway. The ndings were similar to the clinical, histopathological and molecular effects found in the group submitted to photobiomodulation.  Tables   Table 1 Immunohistochemical