Investigation of the Effects of Micromeria congesta Essential Oil Extract on Wound Healing in Rabbits and Molecular Genetics Applications

Healing of skin wounds is a complicated series of biological events that involves completion of the intertwined stages of hemostasis and inflammation, proliferation, maturation and remodeling (Gonzalez et al., 2016; Wang and Windbergs, 2017; Preethi et al., 2018). In vivo and in vitro studies have determined that plants increase wound recovery as topical antimicrobial substances and they do this by the fibroblast growth effects of the terpenoids, flavonoids, tannins and saponins in their chemical composition (Budovsky et al., 2015; Ribeiro et al., 2015, Agyare et al., 2016, Slobodníková et al., 2016; Yamani et al., 2016). In veterinary practice, studies related to the contribution of herbal products to wound healing are constantly investigated (Chopda et al., 2015; Mohan et al., 2018).
       
It was reported that Micromeria species belonging to the Lamiaceae family are represented by 14 species and 22 taxa in the flora of Turkey and 12 species among these are endemic (Kirimer et al., 1991; Tabanca et al., 2001; Duru et al., 2004; Herken et al., 2012; Dincer et al., 2017). The plant of M. congesta that naturally grows in the mountainous parts of the provinces of Þanlýurfa and Gaziantep is utilized by locals by boiling its roots and above-ground parts and drinking its water or for antibacterial purposes in respiratory tract diseases and coughing (Kirimer et al., 1991; Tabanca et al., 2001; Herken et al., 2012; Akan et al., 2013).
       
Gene expressions in wound healing have been investigated for a long time. It is known that IL6 and IL8 play an important role in wound healing, the fibroblast proliferation of macrophages, lymphocytes, several tissues and cells, in addition to collagenase, neutrophil chemotaxis. The chemokine receptor 1 (CXCR-1) is a specific receptor for interleukin 8 (IL8), which is a chemoattractant for neutrophils and important in the inflammatory response (Pradhan et al., 2011; Scarel-Caminaga et al., 2011; Ünüvar 2017).
       
This study aimed to investigate the effects of M. congesta extract, which is used as a wound healing agent by the local people, on wound recovery.

Plant material
 
The volatile parts of M. congesta were collected from the Germüþ village in the province of Þanlýurfa (altitude: 550 m) in July-August 2015, which was the plant’s flowering period. The identification of the collected plants was carried out by Dr. Mustafa Aslan, a faculty member of the department of Biology Education at the Education Faculty of Harran University with the access number of Harran University Herbarium no: 4521 HURUB.
 
Preparation of the plant material
 
The M. congesta samples were dried in open air and kept in polyethylene bags until usage. The essential oils of the dried parts were obtained by the method of hydro-distillation for 8 hours by using a Clevenger device. The oil samples were dried over hydrous sodium sulfate and stored at +4°C. By adding M. congesta extract (4 ml) into liquid vaseline (100 ml), a 4% mixture was prepared.
       
The product Madecassol® (1% pomade, Bayer, Istanbul) which contained 10 mg of titrated C. asiatica extract in 1 g of pomade was obtained from a local pharmacy.
 
Sample
In the study, 21 female, 6 months old New Zealand rabbits at weights of 2500±200 g were used. The experiment was conducted in Dollvet R&D Center in 2017. The rabbits were accommodated individually in standard cages. They were fed in a natural light cycle, at a constant temperature of 24±2°C and with a normal diet and ad libitum water. The study was approved by the Animal Experiments Local Ethics Board of Dollvet A.Ş. (DOLLVET-HADYEK) with the approval number 2016/13.
       
The 21 New Zealand rabbits that were used in the study were randomly divided into 3 groups so that there would be 7 rabbits in each group.
Group I: M. congesta essential oil extract.
Group II: C. asiatica extract in pomade form.
Group III: Control.
 
Anesthesia
 
After xylazine hydrochloride (Alfazyne® 2%, Alfasan International B.V. Netherlands) (4 mg/kg, IM) premedication, anesthesia was achieved by ketamine hydrochloride (Alfamine® 10%, Alfasan International B.V. Netherlands) (50 mg/kg, IM) (Satar et al., 2013).
 
Operation procedure
 
After laying each rabbit in a dorsoventral position and shaving its dorsal region, the skin region where the wound would be induced was prepared by using PVP iodine. Wounds with dimensions of 2.8 cm were created to cover full-thickness skin in the region close to the caudal border of the scapula. Hemostasis was achieved by compression with sterile pads.
 
Wound diameter measurement
 
Wound diameters were measured on the postoperative 7^th, 14^th, 21^st and 28^th days in the vertical and horizontal directions by using a compass and average values were recorded. Biopsy specimens were collected on the postoperative 7^th, 14^th, 21^st and 28^th days in a clockwise direction.
 
Histopathological analysis
 
Tissue specimens were taken from the wound area on the 7^th, 14^th, 21^st and 28^th days of the study and fixated in 10% buffered formaldehyde. In the study, histopathological analysis of the specimens was done using the method reported by Luna (1968).
 
Immunohistochemical analysis
 
For the purpose of showing epithelization and increased connective tissue among the tissues, the Avidin Biotin Peroxidase Complex (ABC) technique was carried out with a commercial kit (Zymed, Histostain Plus Kit, California, USA) based on the standard procedure. As the primary antibodies, active Vimentin (dilution ratio: 1/25) and Cytokeratin (Novocastra) (dilution ratio: 1/25) were used, while AEC was utilized as the chromogen. PBS (pH 7.4) was applied on the tissues as the negative control, while primary antibodies were applied on the control tissues recommended by primary antibody manufacturers as the positive control.
 
Genetic analysis
 
The full-thickness wound specimens taken from the backs of the rabbits were kept at -80°C until isolation. After the specimens were homogenized at 6500 rpm for 40 s and by 30-35 mg, RNA isolation was carried out with a high pure FFPET RNA isolation kit (Roche®). The cDNA synthesis stage was performed by a transcriptor first strantc DNA synthesis kit (Roche^®). The realtime PCR mix was prepared to include each primer by 1 µl (10 pmol) (Table 1) and LC480 real time sybrgreen I (Roche^®) kit at 10 µL and 3 µl of PCR grade water. For each specimen, 5 μl of cDNA was added onto the protocol above and with the final volume of 20 μl, the specimen was studied with a Lightcycler 480 Realtime PCR system. For denaturation, 5 min at 95°C, 45 cycles for 5 s at 95°C, 10 s at 60°C and 5 c at 72°C, 1 single reading was made. For melting analysis, 5 s at 95°C, 60 s at 50°C, continuous at 50°C-95°C and 60 s at 40°C reading was made.


       
Before calculating the results of the specimens, their amplification curves and whether or not the crossing point (Cp) values of each sample were within desired levels were checked. Afterwards, with the concentration values determined based on the Cp results of the specimens, the results were obtained as 2^-ΔΔCT. In the real-time PCR procedure, the expression levels of the target genes were measured by using the housekeeping gene Beta actin (ACTB) as a reference gene for the samples.
 
Statistical analysis
 
After obtaining histopathological data, the differences between days and groups were determined by using Kruskal Wallis analysis and “all pairwise” multiple comparisons tests. The expression data were calculated with median (min-max) values. The significant differences in gene expression among all samples were determined by two-way ANOVA. SPSS version 23 for Windows was used for the statistical analyses and p<0.05 was accepted as statistically significant.

Wound measurement findings
 
The mean unhealed wound area (mm2) values based on days for the M. congesta essential oil extract (group I), C. asiatica extract in pomade form (group II) and control (group III) groups are shown in Table 2. In all groups, it was observed that wound healing increased significantly through the 7^th, 14^th, 21^st and 28^th days (p<0.001) (Fig 1). The unhealedwound areas in group I and group II were significantly smaller than those in group III on the 7^th, 14^th, 21^st and 28^th days (p<0.001) (Table 2).
 

 

 
Histopathological and Immunohistochemical findings
 
In the histopathological analyses, it was observed that the findings belonging to the week following the implementation did not differ among the groups. On the other hand, in tissues belonging to the 2^nd week, the histopathological findings of inflammation, increased connective tissue and vascularization were the highest in group II, while these were significantly higher in group I than group III (p<0.05). Likewise, in the analysis of the cross-sections for the 3^rd and 4^th weeks, in addition to other findings, the increase in epithelization and increased connective tissue were significantly higher in Group II in comparison to the others (p<0.05). Additionally, findings on inflammation were not seen in group II in the 3^rd week, while these were milder in group I than group III (p<0.05) (Fig 2).
 

       
In the immunohistochemical analyses that were carried out to determine epithelization and increased connective tissue, similar findings were encountered against the cytokeratin and vimentin antibodies. Both antibodies showed a diffuse positive reaction by the 1^st week especially in group II, while the tissues in group I were more noticeable positive in comparison to group III. These findings were similar to those of the 2^nd and 3^rd weeks. Especially in the 4^th week after the implementation, it was seen that the cytokeratin and vimentin antibodies were severely immunopositive in group I and group II, while there were milder positive reactions in group III (Fig 3 and 4).
 

 

 
Genetic findings
 
According to the gene analysis results, the expression levels of all target genes (IL6, IL8 and CXCR1) in the rabbit tissues in group I and group II were significantly higher than the group III on the 7^th day (p<0.05). Especially the IL6 and CXCR1 genes had higher expression in group I than group II, but this difference was not statistically significant (p>0.05). On the following days, all target gene expressions were observed to decrease (Fig 5a,b,c).
 

       
Several researchers have reported that Micromeria species’ extracts and essential oils have antimicrobial, antioxidant and anti-inflammatory properties (Ali-Shtayeh et al., 1997; Tabanca et al., 2001; Duru et al., 2004; Mishra et al., 2010; Herken et al., 2012; Sonboli A. 2015; Alwan et al., 2016; Alizadeh and Ranjbaran 2017). In this study, it was aimed to investigate the wound healing activity of M. congesta extract by daily observations, histopathological, immunohistochemical and genetic methods in rabbits on which full-thickness skin wounds were inflicted.
       
Many studies have been conducted to investigate the antibacterial and antioxidant activity of different endemically growing Micromeria species. Herken et al., (2012) determined that M. congesta shows a high antibacterial activity against 12 bacterium species that cause infections in humans. Alwan et al., (2016) reported that Micromeria barbata extract is useful against especially multi-drug-resistant pathogens in human infections. Duru et al., (2004) stated that Micromeria cilicica is a strong antimicrobial agent against most microorganisms, especially Candida albicans. Tabanca et al., (2001) showed that Micromeriaristata has a biological activity against human pathogenic microorganisms. Alizadeh and Ranjbaran (2016) stated that Micromeria hedgei shows good antimicrobial activity against 5 significant pathogens. The finding in our study that no infected wounds were observed in the M. congesta group in the recovery period was considered to have been caused by the antibacterial effects of Micromeria species as reported by the authors mentioned above (Tabanca et al., 2001; Duru et al., 2004; Mishra et al., 2010; Herken et al., 2012; Alwan et al., 2016; Alizadeh and Ranjbaran 2017).
       
Studies on wound healing have proven that this process increases the secretion of proinflammatory cytokines suchas interleukins. Additionally, it is known activate several chemokine receptors by multiple ligands and especially IL8 can bind both CXCR1 and CXCR2. The protein that is coded by the CXCR1 gene is a member of the G-protein-coupled receptor family. This protein is a receptor for IL8. It is bonded with IL8 with high affinity and transmits its signal with a G-protein-activated secondary messaging system. Considering gene expressions especially in wounds, Pradhan et al., (2011) found the expressions of the IL6, IL8 and CXCR1 genes in wounds to be high, as in our study.
       
Especially Lin et al., (2003), in similarity to this study, showed that IL6 has significant roles in wound healing possibly by regulating leukocyte infiltration, angiogenesis and collagen accumulation (Lin et al., 2003; Pradhan et al., 2011; Pekmezci and Mutlu, 2019). In this study, the expressions of the same target genes in the first week were found to be substantially higher than the control group by the method of realtime PCR and it was seen that this is important in wound healing. The values decreased on the 21^st and 28^th days, but the recovery in group II was observed to be always better than that in group I.

Consequently, in our study, it was concluded that usage of the plant M. congesta, which is known as a wound healer in traditional folk medicine, in open wounds shortened the mean time of recovery in comparison to the control group, but its recovery time was longer than that of the group treated with C. asiatica. Accordingly, we believe there is a need for more studies to reveal the utilization of the plant M. congesta in open wounds and its therapeutic effects.

Authors declare that there are no conflicts of interest to report.

This study was supported by Harran University, Scientific Research Projects Board with project no: HÜBAK-16120.
This article was presented as an oral presentation at the 4^th International Congress on Veterinary and Animal Sciences held on July 12-15, 2018 in Nevsehir/Turkey.