COMPARISON OF THE HEALING POTENTIAL OF PROPOLIS AND BEE VENOM ON SURGICALLY INDUCED WOUND IN RAT’S BUCCAL MUCOSA.

Lina Samir Shalaby 1 , Ahmed Helmy Salama 2 , Sahar Mahmoud Shawkat 2 , Amira Elham Fares 2 and Ahmed Gaafar Hegazi 3 . 1. Oral Biology Department, Faculty of Dentistry, Modern Sciences and Arts University (MSA), Egypt. 2. Oral Biology Department, Faculty of Oral and Dental Medicine, Cairo University, Egypt. 3. Oral Biology Department, Faculty of Oral and Dental Medicine, Cairo University, Egypt. 4. Oral Biology Department, Faculty of Oral and Dental Medicine, Cairo University, Egypt. 5. Microbiology and Immunology, National Research Center, Egypt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Bee products include honey as well as propolis, royal jelly and venom which are used in medicine, not only as an immune system stimulant but also as a wound healing promoter. Aim of work: was to assess and compare the healing potential of propolis and bee venom on the healing wounds in rat's buccal mucosa. Materials and Methods: Foutry five adult male Wistar albino rats divided into in 3 groups of 15 each (control, propolis & bee venom). A wound (3.5 mm diameter) was made in the cheek of each rat with a punch biopsy. Samples were prepared and investigated histopathologically and immunohistochemically by proliferating cell nuclear antigen (PCNA) after 1, 3 and 7 days of wounding. Results: Bee venom showed the highest healing potential followed by propolis through stimulated epithelial cells proliferation and reepithelization compared to the control group. Conclusion: Bee venom and Propolis had contributed positively to the wound healing in the surgically induced wounds of rat's buccal mucosa. With an increasing necessity for a natural medication, the current medical treatment modalities directs attention to the natural products and biological methods to be used in the clinical practice due to the high costs and side effects of the traditional treatments (Majtan et al., 2010). The aim of the present study was to assess and compare the healing potential of propolis and bee venom on surgically induced wound on rat's buccal mucosa.

Materials and methods:-
The experimental protocol of the present study follow the recommendations and got the approval of the Ethics Committee on animal's experimentation of the Faculty of Oral and Dental Medicine, Cairo University. Propolis and bee venom were kindly obtained from Apery farm of Professor Ahmed Hegazi (National Research Center, Dokki, Giza, Egypt) during spring season 2015. For intra oral irrigation of the wound lesion, 1 gm of the dried propolis was dissolved in 10 ml of saline to obtain propolis extract in a form of solution, while 0.1 gm of collected dried bee venom was freshly mixed with 10 ml of saline solution. Saline solution only was used as a control.
Animals:-45 adult male Wistar albino rats of average weight 150-200 grams were used from the Animal House (Faculty of Medicine, Cairo University, Egypt). Rats were housed in stainless-steel cages, 5 rats per cage. All rats were maintained on basic diet of regular rat chow and distilled water adlibitum. Rats were divided into 3 groups; each group was further subdivided into 3 subgroups according to sacrifice date as illustrated in the following table All animals were anesthetized with a mixture of 13 mg/kg of xylazine (2%) and 33 mg/kg of ketamine via intramuscular injection. After anesthesia, the cheek was pulled and a wound was induced opposite to the mid buccal premolar area using sterile punch biopsy instrument (3.5 mm diameter) limited to mucosal tissues without involving the muscular layer. Immediately after wound induction, for intra oral irrigation a syringe with an attached sterile metal tube was loaded either with 0.5 ml of propolis, bee venom or saline solution and each wound lesion was irrigated for 30 seconds once daily for 7 days successively. The rat head was held in a horizontal position during irrigation to allow direct contact between the solution and the wound lesion for 30 seconds.
The animals were sacrificed with cervical dislocation after 1, 3 and 7 days of treatment. The buccal mucosa containing the induced buccal wound was dissected. Each wound was excised using a 5.5 mm punch biopsy, maintaining approximately 2 mm of intact mucosa around the marked wound lesion. Then fixed in 10% formalin solution and subjected to routine laboratory studies after sectioning at a thickness of 4 µ. Samples were stained with hematoxylin and eosin and immunohistochemically examined using proliferating cell nuclear antigen (PCNA) followed by histomorphometric analysis.

Statistical analysis:-
The statistical analysis was performed using the arithmetic mean, standard deviation (S.D.) and one way ANOVA with tukeys post hoc test.

Histopathological Results
After 1 day, control group showed complete loss of epithelium and tissue fragmentation lateral to the wound lesion with heavy inflammatory cell infiltrates in the underlying connective tissue. While less inflammatory cell infiltrates in propolis group and few in bee venom group were detected. After 3 days, control group showed incomplete epithelial rim covering the wound lesion. The sub epithelial connective tissue showed numerous scattered inflammatory cell infiltrations and dilated blood vessels engorged with blood. While in propolis the newly formed epithelial lining appeared in a form of thin continuous layer of basal and parabasal cells (with mitotic figures) that cover all the wound lesion. A thicker epithelial layer in bee venom group with moderate inflammatory cell infiltrates in the underlying connective tissue in both groups was observed. (Fig. 2

)
After 7 days, the newly formed covering epithelium in the control group showed hyperkeratosis and acanthosis. The subepithelial connective tissue revealed some inflammatory cells. In propolis group, the wound was completely covered with hyperkeratotic acanthotic epithelium (but less than that of the control group) while nearly normal epithelial thickness in bee venom group was observed. The subepithelial connective tissue in both groups showed fewer inflammatory cells compared to the control group. (Fig. 3) PCNA histomorphometric results:-On the basis of one-way ANOVA test the image analysis of PCNA positive epithelial cells of the buccal mucosa sections after 1, 3 and 7 days demonstrated the following: After 1 Day:-PCNA mean percentage among the control and experimental groups showed a statistically non-significant (p>0.05) difference among the different groups (Fig.4A).

After 3 Days:-
PCNA mean percentage among the control and experimental groups showed a statistically significant difference between the control group and P group (p<0.05), a statistically significant difference between the control group and BV group (p<0.05) while a statistically non-significant difference between the P group and BV group (p>0.05) (Fig.4B).
After 7 Days:morphometric analysis showed that the BV group had the highest mean values of PCNA immunoreaction followed by P group and then the control group which had the lowest PCNA mean values. PCNA mean percentage among the control and experimental groups showed a statistically significant difference between the control group and P group (p<0.05), a statistically significant difference between the control group and BV group (p<0.05) and a statistically significant difference between the P group and BV group (p<0.05) (Fig.4C).

Discussion:-
Wound healing is well orchestrated process which is divided into three or four phases, which are hemostasis, inflammation, proliferation, and remodeling. These distinct, complex phases overlap in order to ensure tissue repair (Abreu et al., 2012). The aim of this study was to evaluate and compare the healing potential of Propolis and bee venom as a local treatment modality on surgically induced wound on Wistar rat's buccal mucosa. In the current study which involves the oral mucosa, rats are commonly used in relation to other animals due to the low cost, the easy manipulation and the similarity between the oral mucosa of rats and humans, the oral epithelium of rats is basically made up of epithelial tissues on the surface and subjacent connective tissue (Cavalcante et al., 2011).
Propolis is believed to be a useful product for wound healing and is already used in alternative medicine. It contains a wide-range of chemical compounds that have many biological activities such as, antioxidant, anti-inflammatory and immunomodulatory activities (Kosalec et al., 2004; Savickas et al., 2005). Bee venom comprises a very complex mixture of active peptides, enzymes and amines such as melittin, phospholipase A2, apamin and Adolapin. These substances demonstrated significant antinociceptive, anti-inflammatory, antibacterial effects and immunity boosting action. The therapeutic application of bee venom has been used in the traditional medicine to treat different diseases (Hegazi et al., 2014c).
The healing process was evaluated at three time intervals; one, three and seven days after wound induction. According to literature reviews these time slots were the most commonly used to monitor tissue changes occurs during wound healing process In the current study, histological examination of the wound lesion treated with propolis (P) solution, after 1 day, showed less inflammatory cells in the connective tissue beneath the wound. As propolis displays anti-inflammatory properties in both acute and chronic inflammatory processes, and this is principally due to its large content of polyphenol compounds this is supported with Martinotti and Ranzato (2015b).These findings were These results could be due to BV content of mast cell degranulating peptide (MCD) that lyses mast cells, releasing histamine, serotonine and heparine that causing increased capillary permeability and anti-inflammatory action. Also histamine is one of the major components which dilates blood vessels, increasing the permeability of blood capillaries and increases blood circulation (Moreno and Giralt, 2015).
After 7 days from wound induction, the healing of the control group was obtained by regeneration of the epithelium, wound was decreased in size and was covered with acanthotic and hyperkeratotic layer of epithelium. 7 days after treatment of the lesion with bee venom, the buccal mucosa sections revealed increased number of PCNA positive cells in the newly formed epithelium. We suggested that the higher proliferative expression of PCNA in BV group could be attributed to that the BV could enhance epithelial cells proliferation.

Conclusion:-
Both bee venom and propolis could enhance wound healing through stimulation of epithelial cells proliferation thus increasing the reepithelization, improve wound closure and decreasing inflammation (as prolonged inflammation and inflammatory cytokines release might cause damage to the tissues).Based on the limitation of the present study. Bee venom was proved to have the highest healing potential followed by propolis then saline (control).  In the control group is thin and incomplete rim of basal and parabasal cells (black arrows), numerous scattered inflammatory cells, dilated blood vessels engorged with blood. While (B) In the propolis group it is thick and continuous epithelium (black arrows). And (C) In the bee venom group it is continuous but thicker than that in the propolis group (black arrows). With moderate inflammatory cell infiltrates were observed in both groups (B & C).