Topical Administration of an Ointment Prepared From Satureja sahendica Essential Oil Accelerated Infected Full-Thickness Wound Healing by Modulating Inflammatory Response in a Mouse Model

Surgical infections are a major issue among patients, and infection influences mechanisms and physiologic stages involved in the wound healing process.^1,2 Extracellular matrix molecules, soluble mediators, various resident cells, and infiltrating leukocyte subtypes interact in the wound healing process.³ Different factors such as growth factor-related cytokines, mast cells, platelets, and other damaged stromal cells have crucial roles in the wound healing process.⁴ Interleukin 1 beta (IL-1ß) aggregates the neutrophils at the site of infection.^5,6 Interleukin 4 (IL-4) activates connective tissue cells and promotes increased extracellular matrix macromolecules,⁷ but IL-10 has anti-inflammatory properties.^8-10 Transforming growth factor beta 1 (TGF-ß1) is infiltrated by neutrophils and macrophages that finally activate fibroblasts and start the proliferative stage.^4,11 Inflammatory chemokines affect leukocytes recruitment toward the site of inflammation. The CXCL-l signaling calls neutrophils and lymphocytes and manages the first stages of healing.^6,12

Insulin-like growth factor 1 (IGF-1) is an important modulator in wound healing that induces its effects by promoting the cellular granulation process, cell proliferation,^13,14 and myofibroblast differentiation and proliferation.¹⁵ Vascular endothelial growth factor (VEGF) and other related factors stimulate angiogenesis, induce vascular permeability, and facilitate wound healing.^10,16 Fibroblast growth factor 2 (FGF-2) has a clear association with hair bulbs at the wound edge and also with basal keratinocytes of the normal and hyperproliferative wound epidermis.^10,11

Pathogens attack skin scars and induce inflammation and infection.¹⁷Pseudomonas aeruginosa and Staphylococcus aureus are the most common bacteria that induce infection in the wound.^9,10P aeruginosa and S aureus are opportunistic bacteria that cause infection and are observed in the upper and deepest region of the wound bed, respectively. Colonization of S aureus and P aeruginosa at the wound site increases inflammation and delays the wound healing process.⁹ Some wounds, such as chronic wounds, cannot be healed by normal mechanisms and require advanced dressings for healing.¹⁸ A safe antibacterial agent can reduce bacterial count and inflammation compared with synthetic agents. It appears that a natural and safe antibacterial and anti-inflammation agent can have beneficial effects in infected wounds. Medicinal plants are commonly used in wound healing. Satureja sahendica is a perennial plant that belongs to the Lamiaceae family. It is an aromatic plant found in Iran flora whose essential oil components are used in culinary, medicinal, and perfume industries. The main constituents of the S sahendica essential oil (SSO) are the phenols carvacrol and thymol, p-cymene, ß-caryophyllene, linalool, and other terpenoids that have antibacterial properties.¹⁹ The genus Satureja is known to have antioxidant, antibacterial, and anti-inflammatory properties^20,21 to manage diarrhea and wound healing.²² Several studies have shown the positive effects of ointments prepared from medicinal plants to decrease inflammation and promote wound healing in infected wounds.^9,23,24 The bacteria cause inflammation, and SSO may accelerate wound healing because of its anti-inflammation properties. This study was conducted in response to the hypothesis that an ointment prepared from SSO improves infected wounds by decreasing the inflammation and gene expression involved in the proliferative phase in a mouse model.

Plant material

Essential oil prepared from aerial parts and seeds of S sahendica was purchased from Barij Essence Company. A voucher specimen (No. 110) was deposited in the herbarium of the same company. Based on recommendations, carvacrol, thymol, ɣ-terpinene, p-cymene, ß-caryophyllene, linalool, and other terpenoids were the most important compounds.

Experimental animal

Ninety healthy BALB/c mice were used as models and had free access to standard rodent laboratory food and tap water. This experimental study was approved by the Animal Research Committee of Islamic Azad University, Urmia Branch, West Azerbaijan Province, Iran with Ethical No. (IAUUB, No. 10693).

Wound infection and ointment

A ketamine/xylazine cocktail (ketamine 50 mg/kg, xylazine 10 mg/kg) was intraperitoneally administered to induce anesthesia. Following common surgical procedures, 2 skin wounds were made on the dorsal surface of each animal using a 7-mm punch. Wounds were inoculated with 50 µL of suspension containing 5 × 10⁷ colony-forming units (CFUs) (50 µL) of 2 bacteria (P aeruginosa and S aureus, strains ATCC 25923 and ATCC 27853, respectively). The mice were divided into 5 groups (n = 18) and received topical administration of soft yellow paraffin (control group), mupirocin 2% ointment (mupirocin group), and 1, 2, and 4 g SSO mixed in 100 g of soft yellow paraffin (1%, 2%, and 4% SSO), respectively. Soft yellow paraffin and mupirocin were prepared by Pars Daruo. The ointments were applied once-daily, 24 hours after wound and bacterial colonization. To conduct sampling and minimize experimental errors, the animals per group were divided into 3 subgroups (n = 6). The samples were collected from 6 animals per group on days 3, 7, and 14 after induction of the wound.^6,9,14

Wound healing rate

The rate of wound closure was calculated based on previous studies.^6,14 The wound area was immediately measured by tracing, using a transparent paper placed over the wound. A graph sheet calculated the area of impression on days 3, 7, and 14 after induction of the wound.

Bacteriologic examination of granulated tissue

To evaluate the bacterial count, the granulated tissues were aseptically excised, and 0.1 g of the prepared samples were then crushed and homogenized in a sterile mortar containing 10 mL of sterile saline. The homogenized samples were serially diluted in tubes containing 9 mL of sterile saline. The diluted samples were superficially cultured on plate count agar (Merck KGaA) and duplicated. The cultured plates were incubated at 37ºC for 24 to 48 hours. Following incubation, all colonies were counted, and the results were reported based on CFU/g of granulation tissue.^6,14

Histologic analysis

A special carbon dioxide device was used to euthanize the animals per subgroup (n = 6). Sample tissues with 1 to 2 mm from the surrounding normal skin with an appropriate depth (approximately 2 mm) were excised at days 3, 7, and 14 after induction of the wound. The samples were then fixed in neutral buffered formalin (10%). Tissue samples were routinely processed, embedded in paraffin wax, sectioned at 5 µm, stained with Masson’s trichrome, and assessed by light microscopy (Olympus CX31RBSF attached camera; Olympus Corporation). Three parallel sections were obtained per specimen. Immune cells and fibroblasts per 1 mm² of tissue, edema, and collagen deposition were analyzed. The deposition of collagen was calculated by Image-Pro Insight software (background intensity section; Media Cybernetics, Inc). All parameters were analyzed in 5 per high-power fields. The epithelial thickness was assessed using a morphometric lens (Olympus) and reported in micrometers.^{6, 14}

RNA extraction and quantitative real-time polymerase chain reaction

The samples (3–5 g) also were prepared to evaluate the gene expression profile on days 3, 7, and 14. The samples were transferred into tubes containing RNase solution (Qiagen) and sent for laboratory analysis. Following homogenization, RNA was extracted using Trizol reagent (Roche Life Science) per manufacturer instructions. The complementary DNA was prepared from cell RNA using the Exiqon complementary DNA synthesis kit based on manufacturer instructions. The samples were incubated at 25ºC for 5 minutes and then 42ºC for 60 minutes. The reaction was finally terminated by heating at 70ºC for 5 minutes. A LightCycler 96 instrument (Roche Life Science) was used to evaluate the messenger RNA expressions of the target genes, such as IL-1ß, FGF-2, VEGF, IL-4, IGF-1, and IL-10. The primers sequences were IL-1ß, forward (5'-AAC AAA CCC TGC AGT GGT TCG-3') and reversed (5'-AGCTGCTTCAGACACTTGCAC-3'); FGF-2, forward (5'-GGA ACC CGGCGGGACACGGAC-3') and reverse (5'-CCGCTGTGGCAGCTCTTG GGG-3'); VEGF, forward (5'-GCTCCGTAGTAGCCGTGGTCT-3') and reverse (5'-GGAACCCGGCGGGAC ACGGAC-3'); IL-10, forward (5'-CCATCATGCCTGGCTCAGCAC-3') and reverse (5'-TGTACTGGCCCCTGCTGATCC-3'); IGF-1, forward (5'-TAGGTGGTTGATGAATGGT-3') and reverse (5'- GAAAGGGCAGGGCTAAT-3'); CXCL-1, forward (5'-CAGACTCCAGCCACACTCCAA-3') and reverse (5'-CAGCGCAGCTCATTGGCGATA-3'); and IL-4, forward (5'-CAGACTCCAGCCACACTCCAA-3') and reverse (5'-CAGCGCAGCTCATTGGCGATA-3').^6,9

Statistical analyses

The results were reported as mean ± standard deviation. All analyses were carried out by GraphPad Prism, version 18.0 (GraphPad Software Inc). The model assumptions were evaluated by examination of the residual plot. One-way analysis of variance was used for the analysis of the results, and Dunnett test compared the means.

Wound area

A decrease in the rate of wound area was observed in all treated groups compared with the negative control group on all days (Figure 1). Wound area was significantly reduced (P <.05) in the animals treated with 4% SSO in comparison with those in the mupirocin-treated group at all days after induction of the wound (Figure 2A). The best responses were observed in 2% and 4% SSO groups.

Topical application of 1% SSO decreased wound area in comparison with that in the control group, but administration of mupirocin had better efficiency compared with that of 1% SSO (Figure 2A). On day 3, the values were 37.32 ± 2.90 mm², 32.51 ± 3.33 mm², 33.21 ± 1.20 mm², 31.21 ± 1.50 mm², and 31.12 ± 1.12 mm² in the control, mupirocin, 1% SSO, 2% SSO, and 4% SSO groups, respectively. The results did not show a significant difference between treated animals on day 3 (P >.05).

On day 7, the values were 28.89 ± 3.00 mm², 22.10 ± 2.30 mm², 24.10 ± 1.30 mm², 20.61 ± 2.00 mm², and 14.12 ± 1.90 mm² in the control, mupirocin, 1% SSO, 2% SSO, and 4% SSO groups, respectively. This means the administration of 4% SSO decreased the wound area by more than 56% compared with mupirocin on day 7.

The values were 7.23 ± 1.80 mm², 2.23 ± 0.74 mm², 4.21 ± 0.74 mm², 0.87 ± 0.52 mm², and 0.20 ± 0.10 mm² in the control, mupirocin, 1% SSO, 2% SSO, and 4% SSO groups, respectively, on day 14. This means the administration of 4% SSO decreased the wound area by more than 89% compared with mupirocin on day 14.

Granulation tissue total bacterial count

Topical administration of SSO and mupirocin significantly diminished the rate of total bacterial count (P <.05) in all the days after induction of wound compared with the control group (Figure 2B). The best responses were observed in the animals treated with SSO at the highest levels.

On day 3, the values were 6.10 ± 0.51 CFU/g, 4.00 ± 0.42 CFU/g, 5.30 ± 0.42 CFU/g, 3.70 ± 0.30 CFU/g, and 2.50 ± 0.42 CFU/g in the control, mupirocin, 1% SSO, 2% SSO, and 4% SSO groups, respectively. The total bacterial count decreased by more than 62.5% with administration of 4% SSO compared with mupirocin on day 3. The values were 4.20 ± 0.40 CFU/g, 2.50 ± 0.30 CFU/g, 3.20 ± 0.30 CFU/g, 2.00 ± 0.30 CFU/g, and 1.00 ± 0.20 CFU/g in the control, mupirocin, 1% SSO, 2% SSO, and 4% SSO groups, respectively, on day 7. The total bacterial count decreased by more than 40% with administration of 4% SSO compared with mupirocin on day 7. The values were 1.20 ± 0.12 CFU/g, 0.70 ± 0.15 CFU/g, 1.00 ± 0.15 CFU/g, 0.87 ± 0.22 CFU/g, and 0.20 ± 0.10 CFU/g in the control, mupirocin, 1% SSO, 2% SSO, and 4% SSO groups, respectively, on day 14. The total bacterial count decreased by more than 285% with administration of 4% SSO compared with mupirocin on day 14.

Histopathologic analysis

Histopathologic studies of the wound area revealed that the animals treated with SSO showed a significant enhancement in the rate of immune cells infiltration compared with the mupirocin-treated and control groups on day 3. Cross-sections from the SSO-treated groups revealed a marked reduction in the proliferation of immune cells (P <.05) on days 7 to 14 (Table). The animals treated with 4% SSO demonstrated decreased edema after 7 days. The light microscopic analyses of the wound area showed that SSO was able to induce fibroblast proliferation in all days following induction of the wound (P <.05). The animals treated with 2% SSO indicated increased collagen fibers on day 7 compared with the control group, whereas the same results were observed in the SSO-treated animals on day 14 (Table; Figure 3). The scab was completely removed after day 14 in the SSO-treated animals. Some sections from the negative control group showed the scab remained even after 14 days. Migration of epidermal cells was started in the control group on day 7, whereas it was observed on day 3 in the treated animals (Table; Figure 3). Neovascularization was seen in SSO-treated animals on day 3, but it was significantly developed in the 4% SSO group. At day 14, vascularization was significantly enhanced in all SSO-treated groups (Table).

Molecular analysis

Topical administration of SSO significantly increased levels of IL-1ß on day 3 (P <.05) and decreased its expression level on day 7 compared with the control group (Figure 4). The values were 1.00 ± 0.06, 1.70 ± 0.06, 1.75 ± 0.07, 1.30 ± 0.05, and 1.21 ± 0.07 in the control, mupirocin, 1% SSO, 2% SSO, and 4% SSO groups, respectively, on day 3. The administration of SSO at 4% concentration decreased the expression of IL-1ß more than 71% compared with mupirocin on day 3. The values were 1.00 ± 0.07, 1.10 ± 0.05, 1.20 ± 0.08, 0.75 ± 0.08, and 0.70 ± 0.07 in the control, mupirocin, 1% SSO, 2% SSO, and 4% SSO groups, respectively, on day 7. The administration of SSO at 4% concentration decreased the expression of IL-1ß more than 63% compared with mupirocin on day 7. Significant differences between treated groups on day 14 for the expression of IL-1ß were not observed. Topical application of SSO significantly (P <.05) increased the levels of TGF-ß1 and CXCL-1 on days 3 and 7 after induction of the wound compared with the control group, but it was reduced at day 14 (Figure 4). The values were 1.00 ± 0.05, 1.25 ± 0.06, 1.15 ± 0.05, 1.60 ± 0.06, and 1.66 ± 0.07 in the control, mupirocin, 1% SSO, 2% SSO and 4% SSO groups, respectively, on day 3 for the expression of TGF-ß1, whereas the values were 1.00 ± 0.06, 1.60 ± 0.07, 1.40 ± 0.05, 1.86 ± 0.07, and 1.95 ± 0.10, respectively, in the same groups on day 7. The administration of SSO 4% increased the expression of TGF-ß1 75% and 82% compared with mupirocin on days 3 and 7, respectively. However, the values were 1.00 ± 0.05, 1.60 ± 0.06, 1.40 ± 0.07, 1.60 ± 0.09, and 1.65 ± 0.07 in the control, mupirocin, 1% SSO, 2% SSO, and 4% SSO groups, respectively, on day 3 for the expression of CXCL-1, whereas the values were 1.00 ± 0.08, 2.00 ± 0.07, 1.90 ± 0.06, 2.35 ± 0.08, and 2.50 ± 0.15, respectively, in the same groups on day 7. The administration of SSO 4% increased the expression of CXCL-1 71% compared with mupirocin on day 7. The expression of IL-10 was significantly higher (P <.05) in the treated animals compared with the control group (Figure 4). The values in the mupirocin and 4% SSO groups were 1.25 ± 0.04 and 1.40 ± 0.06 on day 3, 1.55 ± 0.06 and 1.95 ± 0.05 on day 7, and 1.10 ± 0.03 and 1.40 ± 0.04 on day 14, respectively. The expression of IL-4 was significantly higher on day 3 in the treated animals, but on days 7 and 14 it was decreased compared with the control group. For the expression of IL-4 on day 3, the values were 1.00 ± 0.07, 1.20 ± 0.06, 1.00 ± 0.05, 1.40 ± 0.06, and 1.45 ± 0.05 in the control, mupirocin, 1% SSO, 2% SSO, and 4% SSO groups, respectively. On day 7, topical administration of SSO significantly increased the messenger RNA levels of IGF-1, FGF-2, and VEGF (P <.05), but it did not have a significant effect on their expression on day 3 (P >.05) (Figure 4). The values for the expression of IGF-1 were 1.00 ± 0.07, 1.20 ± 0.06, 1.00 ± 0.06, 1.50 ± 0.04, and 1.60 ± 0.05 in the control, mupirocin, 1% SSO, 2% SSO, and 4% SSO groups, respectively, on day 7. However, on day 7, the values for the expression of FGF-2 were 1.00 ± 0.06, 1.05 ± 0.05, 1.00 ± 0.04, 1.25 ± 0.05, and 1.30 ± 0.05 in the control, mupirocin, 1% SSO, 2% SSO, and 4% SSO groups, respectively. On day 7, the results for VEGF values showed the data were 1.00 ± 0.07, 1.35 ± 0.06, 1.30 ± 0.05, 1.70 ± 0.06, and 1.95 ± 0.05 in the control, mupirocin, 1% SSO, 2% SSO, and 4% SSO groups, respectively.

Some wounds are prone to infection by gram-positive and gram-negative bacteria, including P aeruginosa, Staphylococcus spp, Streptococcus spp, Escherichia coli, and Corynebacterium pyogenes. If the wound is infected, the healing process is delayed. In the aforementioned process, the inflammatory phase is the most important stage in decreasing cellular debris from tissue and also shows broad responses against microbial infection.^1,2,10

Previous studies have shown that SSO has antibacterial²⁰ and anti-inflammatory²¹ properties. The antibacterial activity of the essential oil is mainly attributed to phenolic compounds.^20,25 The decreased rate of tissue bacteria colonization, edema, and immune cells may be attributed to phenolic compounds. Inflammation is a defense mechanism against bacteria, but prolonged inflammation delays the wound healing process. The histopathology and molecular results also show the application of SSO decreases tissue inflammation during the first days (day 3) and then decreases it by modulating the expression of IL-1ß. Bacterial endotoxins increase the expression of proinflammatory cytokines, for example, IL-1ß that decreases the production of growth factors.²⁶ Proinflammatory cytokines such as IL-1ß are extensively secreted against infection^6,9 and are mainly synthesized by macrophages/monocytes and participate in the initial stage of inflammation. These have major roles in the inflammatory response.⁵ To summarize, it can be stated that SSO decreased the inflammatory phase during the first days of fighting against infection. However, the findings of this study also showed that SSO increased the expressions of IL-4, TGF-ß1, and CXCL-1 in treated animals. IL-4 activates connective tissue cells and promotes increased extracellular matrix macromolecules.⁷ In addition, TGF-ß1 promotes the fibroblasts and starts the proliferative stage.¹¹ TGF-ß also participates in angiogenesis and promotes granulation tissue formation.¹¹ TGF-ß1 stimulates fibroblasts for differentiation into myofibroblast that expresses smooth muscle actin and contracts the wound.²⁷ CXCL-1 gene encodes a surface protein in the cells as a receptor.²⁸ Increased CXCL-1 expression increases CXCR receptors on the surface due to the release of growth factors and inflammatory preventers, increased proliferation of epithelial cells, and improved stability and integrity of the extracellular matrix in the affected area.^6,12 On day 3, the results also showed the expression of IL-10 was significantly higher; IL-10 has anti-inflammatory properties.⁸ Thus, SSO, especially in higher levels, promotes the transition from inflammation to proliferation.   

The revascularization of the injured area bed and redevelopment of the extracellular matrix are obtained by cell proliferation and the production of granulation tissue. Increased wound contraction in the treated group may be related to increased fibroblast activity and elimination of bacteria by SSO. The slow rate of wound closure in the control group might be attributed to the presence of microorganisms and their metabolites.²⁹

IGF-1 increases the rate of fibroblast proliferation in the wound site^30-32 and also stimulates glucose transport in the short term over the healing process.³⁰ Data from this study revealed that topical administration of SSO improves cellularity by increasing the expression of IGF-1.

Angiogenesis supports the cellularity and incorporates nutrients delivery during inflammation and cell proliferation at different stages of the healing process. VEGF stimulates angiogenesis by reducing tissue hypoxia and metabolic deficiencies and induces vascular permeability and facilitates wound healing.¹⁶ The results also showed that topical administration of SSO increased VEGF expression and subsequent angiogenesis in the wound site. Further, FGFs are responsible for the proliferation and regulation of various cells of mesodermal, ectodermal, and endodermal origins.¹⁶ The FGFs have cytoprotective and supportive effects on cell survival under stress conditions.³¹ FGF-2 and VEGF assist stimulation of angiogenesis by supporting the cellular nutrient, oxygen, and energy supplements.³³ SSO improves cellularity, cellular migration, and angiogenesis by upregulating FGF-2 expression. In summary, infected wounds cause inflammation, and the use of an anti-inflammatory agent of SSO decreased inflammation and promoted wound healing. This study was conducted in a mouse model, and the results are limited to animals. Skin physiology in animals and humans is different, and the wounds in mice healed rapidly in comparison with those in humans. A human study is required for the investigation of the effects of ointments prepared from SSO.

This study was conducted in a murine animal model with an acute wound, in which no disease was present; substantially more research is needed prior to applying these findings to clinical wounds in humans.

Using SSO on an infected wound reduces the risk of infection and improves the healing process by promoting the transition from inflammation to proliferation and increasing expression of IGF-1, FGF-2, VEGF, TGF-ß, and CXCL-1. It can be recommended to use the SSO at higher levels for the treatment of infected wounds. Essential oils are low cost and can be used as safe compounds in the structure of ointments when in lower concentrations. Future studies should explore the application of SSO on chronic wounds, such as diabetic foot ulcers and pressure ulcers.

Authors: Khaled Omarizadeh, DVM¹; Mohammad Reza Farahpour, DVM, PhD¹; and Mahshid Alipour, MS²

Affiliations: ¹Department of Clinical Sciences, Faculty of Veterinary Medicine, Urmia Branch, Islamic Azad University, Urmia, Iran; ²Department of Microbiology, Faculty of Veterinary Medicine, Urmia Branch, Islamic Azad University, Urmia, Iran

Correspondence: Mohammad Reza Farahpour, DVM, DVSc, Department of Clinical Sciences, Faculty of Veterinary Medicine, Urmia Branch, Islamic Azad University, Urmia, 57159-44867, Iran; mrf78s@gmail.com

ORCID ID: orcid.org/0000-0001-8631-071X

Disclosure: The authors disclose no financial or other conflicts of interest.