Open Veterinary Journal, (2026), Vol. 16(4): 2344-2351

Research Article

10.5455/OVJ.2026.v16.i4.34

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Immunomodulatory effects of oregano extract ointment on the expression of IL-10 and VEGF in white rat excision wounds

Gegana Wimaldy Airlangga¹ and Indah Amalia Amri^2*

¹Laboratory of Veterinary Biochemistry, Faculty of Veterinary Medicine, Universitas Brawijaya, Malang, Indonesia

²Laboratory of Veterinary Microbiology and Immunology, Faculty of Veterinary Medicine, Universitas Brawijaya, Malang, Indonesia

*Corresponding Author: Indah Amalia Amri. Laboratory of Veterinary Microbiology and Immunology, Faculty of Veterinary Medicine, Universitas Brawijaya, Malang, Indonesia. Email: indahamaliaamri [at] ub.ac.id

Submitted: 15/09/2025 Revised: XX/XX/XXXX Accepted: 09/03/2026 Published: XX/XX/XXXX

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ABSTRACT

Background: Wound healing involves a sequential transition from inflammation to proliferation, requiring a balanced immune response. The disruption of this balance can impair repair. Origanum vulgare (oregano) has been reported to exert immunomodulatory properties; however, evidence regarding its influence on IL-10 and Vascular Endothelial Growth Factor (VEGF) expression in excisional wound healing remains limited.

Aim: This study aimed to evaluate the effect of topical oregano extract ointment on the expression of IL-10 and VEGF in a rat excisional wound model.

Methods: Twenty-four male white rats were randomly assigned to four groups (n=6 per group): vehicle control and oregano extract ointment at concentrations of 3%, 6%, and 9%. A standardized 5-mm full-thickness excisional wound was created on the dorsal skin. Treatments were applied topically twice daily for 14 days. On day 14, skin tissues were harvested for immunohistochemical analysis of IL-10 and VEGF expression. Immunohistochemical analysis was performed using Immunohistochemistry Profiler, and statistical analysis was conducted using one-way ANOVA followed by Tukey’s post hoc test (p < 0.05).

Results: IL-10 expression showed an upward trend in the oregano-treated groups compared with the vehicle control; however, the differences did not reach statistical significance. In contrast, VEGF expression was significantly increased in the 6% oregano extract group compared with the vehicle control (p < 0.01), while the 3% and 9% groups demonstrated non-significant increases. No significant differences were observed among the oregano-treated groups.

Conclusion: Oregano extract modulated the expression of molecular markers associated with wound healing, with the 6% concentration significantly increasing VEGF expression. IL-10 showed a non-significant upward trend across the treatment groups. These results suggest that oregano extract may support pathways involved in tissue repair, with 6% representing a potentially optimal concentration under the conditions of this study.

Keywords: IL-10, Immunohistochemistry, Origanum vulgare, VEGF, Wound healing.

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Introduction

Wound healing is a dynamic biological process that occurs through four overlapping and well-coordinated stages: hemostasis, inflammation, proliferation, and remodeling. Each stage is regulated by interactions among immune cells, growth factors, and signaling pathways that work together to repair tissue and restore homeostasis (Wang et al., 2022). During the inflammatory phase, innate immune cells release pro-inflammatory cytokines and recruit leukocytes to the wound area. This process is followed by adaptive immune regulation, which influences the proliferative and remodeling phases through cytokine-dependent mechanisms. Disruption at any stage can interfere with proper wound closure, delay tissue regeneration, and increase the likelihood of chronic wound formation (Avishai et al., 2017).

Several factors can disrupt normal wound healing, particularly immune dysregulation and microbial infection. An imbalanced immune response can alter cytokine production and prolong inflammation, leading to ongoing tissue damage (MacLeod and Mansbridge, 2016). In addition, bacterial colonization creates a hypoxic and acidic environment that favors microbial persistence while hindering tissue regeneration (Versey et al., 2021). These findings highlight the importance of therapeutic approaches that regulate immune responses to restore balance within the wound microenvironment. By modulating immune signaling pathways, such strategies may support tissue repair while also reducing complications related to infection (Cao et al., 2024).

In recent years, natural compounds have gained attention as complementary approaches in wound management. Many natural compounds have been reported to modulate cytokine production and enhance angiogenesis in injured tissues (Kaban et al., 2024; Khazaei et al., 2024a,b; Arafa et al., 2025). These findings indicate that phytochemicals may offer safe and cost-effective options to support wound healing (Mustapha and J, 2024). Among these plants, Origanum vulgare (oregano) has received increasing interest. Oregano contains various bioactive compounds with reported immunomodulatory properties, which may contribute to the regulation of inflammatory responses during tissue repair (Trinh et al., 2022).

Experimental studies show that oregano can reduce the production of major pro-inflammatory cytokines such as Tumor necrosis factor-alpha (TNF-α), IInterleukin-1 beta (IL-1β), and Interleukin-6 by regulating the TLR4/NF-κB signaling pathway. This includes lowering Nuclear Factor kappa B (NF-κB) p65 expression, inhibiting IκB Kinase activity, and preventing IκB phosphorylation (Liu et al., 2019). In vivo findings also demonstrated that oregano treatment attenuated paw edema and reduced IL-1β and PGE2 levels, further supporting its modulatory effects on inflammatory responses in experimental models (Lima et al., 2013). Overall, these findings suggest that oregano extract may help regulate excessive inflammatory activity during tissue injury (Las Heras et al., 2024).

Despite these promising findings, there is limited direct evidence linking oregano extract to the regulation of IL-10 and Vascular Endothelial Growth Factor (VEGF) in excisional wound healing. Clarifying this aspect is important to better understand the therapeutic potential of oregano in wound management. Therefore, this study examined the effect of oregano extract ointment on IL-10 and VEGF expression in rat excisional wounds. We hypothesize that oregano extract at appropriate concentrations may support wound healing by enhancing IL-10-mediated resolution of inflammation and VEGF-associated angiogenic signaling, thereby contributing to improved tissue repair.

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Materials and Methods

Research site

All experimental work was conducted at the Faculty of Veterinary Medicine, Universitas Brawijaya, Malang, Indonesia: Laboratory of Veterinary Microbiology and Immunology, Laboratory of Experimental Animals, and Laboratory of Anatomic Pathology.

Experimental animals

Twenty-four male white rats (Rattus norvegicus), aged 3 months and weighing 250 g, were obtained from the Laboratory Animal Unit, Faculty of Veterinary Medicine, Universitas Brawijaya. Animals were acclimatized for 7 days under standard laboratory conditions (room temperature, humidity, 12:12 hours light/dark cycle) with ad libitum access to standard food and water. Rats were randomly assigned to four groups (n=6 per group): a vehicle control group (K–) consisting of rats with excisional wounds treated with Vaseline, and three treatment groups (P1, P2, and P3) with excisional wounds treated with oregano extract ointment at concentrations of 3%, 6%, and 9%, respectively. The selected concentrations were based on our previous study, which demonstrated differential biological responses to oregano extract in excisional wound tissue.

Collection and extraction of plant material

Fresh oregano leaves (Origanum vulgare) were collected from wild populations in Malang, East Java, Indonesia. The leaves were washed, shade-dried, and ground to a powder. A representative 500 g of dried powder was macerated in 1,000 ml of 96% ethanol in a sealed glass container for 24 hours at room temperature and agitated on a shaker at 50 rpm. The mixture was filtered through cloth and filter paper; the filtrate was remacerated for an additional 24 hours, and the combined filtrates were concentrated using a rotary evaporator (~2 hours). The crude extract was further dried in an oven at 70°C to remove residual solvent.

Preparation of the topical ointment

The ointments were made using Vaseline album as the hydrocarbon base. For each 10 g batch, oregano crude extract (100%) was accurately weighed and incorporated into molten Vaseline to produce final concentrations of 3% (0.3 g extract + 9.7 g Vaseline), 6% (0.6 g + 9.4 g), and 9% (0.9 g + 9.1 g). Vaseline was liquefied in a water bath, after which the extract was gradually added with continuous stirring until a uniform preparation was obtained. The resulting formulations were then transferred into airtight containers and stored at room temperature until further use.

Excisional wound model

After acclimatization, animals were anesthetized with ketamine (80 mg/kg BW) and xylazine (8 mg/kg BW) administered intramuscularly. The dorsal area was shaved and cleaned with 70% ethanol, and a standardized full-thickness circular excision wound was created using a sterile 5-mm diameter biopsy punch on the dorsal midline (caudal to the ear and lateral to the vertebral column), extending down to the panniculus carnosus. This punch wound model produces reproducible acute excisional wounds suitable for evaluating topical therapies. Wounds were left open (no suturing), and animals were housed individually on underpads to prevent wound contamination and interference from cage mates.

Treatment regimen and clinical monitoring

After a 7-day acclimatization period, full-thickness excisional wounds were created under anesthesia. Topical treatments were initiated 1 day after wound induction and were applied twice daily for 14 consecutive days. Prior to each application, the wound surfaces were gently cleaned with sterile cotton. The vehicle control group (K–) received Vaseline, whereas the treatment groups (P1–P3) received oregano extract ointment at concentrations of 3%, 6%, and 9%, respectively.

Sample collection

On day 14 after wound induction, animals were euthanized by cervical dislocation. Full-thickness skin samples, including the wound area and approximately 1 cm of surrounding tissue, were excised, trimmed to ~0.5 cm thickness, and fixed in 10% neutral buffered formalin for 24 hours at room temperature. Tissues were then processed for immunohistochemical analysis of IL-10 and VEGF expression.

Immunohistochemistry for IL-10 and VEGF

The immunohistochemical staining procedure was performed in several sequential steps: deparaffinization, rehydration, antigen retrieval, blocking, application of primary and secondary antibodies, enzyme labeling, chromogen development, counterstaining, and mounting. For deparaffinization, the tissue sections were immersed in xylol three times, each for 5–10 minutes, and subsequently rehydrated through a graded ethanol series (absolute, 90%, 80%, and 70%), with each step lasting 15 minutes. The slides were then rinsed in phosphate-buffered saline (PBS) for 9 minute (Khalaf et al., 2019). Antigen retrieval was performed using a microwave oven with buffer adjusted to the appropriate antibody pH, followed by PBS washing.

To block endogenous activity, hydrogen peroxide was applied as an endogenous peroxidase inhibitor and fetal bovine serum as a protein blocker, each for 10 minutes, with PBS rinsing in between. For antibody staining, tissue sections were incubated with mouse monoclonal primary antibodies against IL-10 (clone E-10, Cat. No. sc-365858; Santa Cruz Biotechnology, USA) and VEGF (clone VG-1, Cat. No. sc-53462; Santa Cruz Biotechnology, USA) at a dilution of 1:100 for 60 minutes at room temperature, followed by washing with PBS. Sections were then incubated with an Horseradish Peroxidase-conjugated anti-mouse Immunoglobulin G secondary antibody for 60 minutes and rinsed with PBS (Khan et al., 2020). Immunoreactivity was visualized using diaminobenzidine substrate for 5–10 minutes. Sections were counterstained with Mayer’s hematoxylin, rinsed with PBS, dehydrated, mounted with Entellan, and covered with glass slips. The slides were allowed to dry for 24 hours prior to microscopic examination (Wang et al., 2018).

Microscopy and image analysis

The Immunohistochemistry (IHC) slides were examined under a Nikon trinocular microscope (model, supplier, country) equipped with a Nikon DS-Fi3 digital camera at 400× magnification. Images were captured under consistent exposure settings. Semi-quantitative analysis of IL-10 and VEGF expression was performed using the IHC Profiler software. The percentage of positive area was calculated by summing the proportion of high-positive and positive staining in each field of view. Expression levels were represented as the percentage of stained area, indicated by brown coloration in the cytoplasm of cells. At least five randomly selected high-power fields per section were analyzed by an observer blinded to the treatment groups. The ImageJ IHC Profiler plugin was used for the objective quantification of staining intensity, providing semi-automated interpretation of immunohistochemical images.

Statistical analysis

Data normality was assessed using the Shapiro–Wilk test, and Levene’s test was used to evaluate the homogeneity of variances. For datasets that met both assumptions (normal distribution and equal variances), one-way ANOVA was conducted, followed by Tukey’s HSD test for post hoc multiple comparisons. All statistical analyses were performed in R Studio (version 2025.05.1+513), with a significance threshold set at p < 0.05.

Ethical approval

This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Universitas Brawijaya (No. 126-KEP-UB-2022). All procedures were performed in accordance with institutional guidelines to minimize animal suffering and ensure humane handling throughout the study.

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Results

Data for IL-10 were first tested for normality using the Shapiro–Wilk test and for homogeneity of variance using Levene’s test, confirming that the dataset met the assumptions for parametric analysis. A one-way ANOVA was then performed to compare the groups. Analysis of IL-10 expression in skin wound tissue showed that the vehicle control group and the 3% oregano extract group had similar IL-10 levels, with no statistically significant difference. The 6% and 9% oregano extract groups exhibited higher mean IL-10 levels compared to the vehicle and 3% groups, although these differences did not reach statistical significance (Fig. 1).

Fig. 1. IL-10 expression levels in wound tissue across the treatment groups. Data are presented as mean ± standard deviation. Data normality was confirmed using the Shapiro–Wilk test, and homogeneity of variances was assessed using Levene’s test. A one-way ANOVA was then performed to compare the groups. No significant differences were observed between the groups.

The data for VEGF were first tested for normality using the Shapiro–Wilk test and for homogeneity of variance using Levene’s test, confirming that the dataset met the assumptions for parametric analysis. One-way ANOVA followed by Tukey’s post hoc test was applied to compare groups. Analysis of VEGF expression in skin wound tissue showed that the vehicle control group had markedly lower VEGF levels compared to the 6% oregano extract group, with a highly significant difference (p < 0.01, Fig. 2). The 3% and 9% oregano extract groups also showed higher VEGF levels than the vehicle group, but these differences did not reach statistical significance. Comparisons among the oregano extract groups revealed no significant differences, indicating that the 6% concentration achieved a substantial enhancement of VEGF expression.

Fig. 2. VEGF expression levels in wound tissue across treatment groups. Data are presented as mean ± standard deviation. Data normality was confirmed using the Shapiro–Wilk test, and homogeneity of variances was assessed using Levene’s test. Group comparisons were performed using one-way analysis of variance followed by Tukey’s post hoc test. Compared with the vehicle control group, VEGF expression was significantly increased in the 6% oregano extract group (**p < 0.01). No statistically significant differences were observed between the oregano extract treatment groups themselves.

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Discussion

Wound healing is a complex and tightly coordinated process that progresses through overlapping stages: hemostasis, inflammation, proliferation, and remodeling. Each stage is controlled by interactions among immune cells, cytokines, and growth factors, which together determine how effectively tissue repair is performed (Rodrigues et al., 2019). Hemostasis initially stops blood loss, followed by an inflammatory phase in which innate immune cells remove pathogens and damaged tissue (Almadani et al., 2021). After inflammation resolves, the proliferative phase begins, characterized by the formation of granulation tissue and re-epithelialization. The final remodeling phase strengthens the extracellular matrix, restoring the structure and function of the tissue. Disruptions at any stage can slow wound closure and increase the risk of developing chronic wounds (Cañedo-Dorantes and Cañedo-Ayala, 2019).

The proliferative phase is crucial because it marks the transition from inflammation to tissue regeneration (Landén et al., 2016). Fibroblasts produce extracellular matrix to support the wound structure, keratinocytes move to cover the wound surface, and new blood vessels develop to deliver oxygen and nutrients (Wilkinson and Hardman, 2020). Effective immune control is crucial during this phase, since persistent inflammation can impair fibroblast function and interfere with the formation of new blood vessels. Anti-inflammatory cytokines and pro-angiogenic growth factors work together to support repair (Eming et al., 2014). Among these, IL-10 helps control excessive inflammation, while VEGF promotes the growth of new blood vessels. Both are essential for creating the immune and vascular conditions necessary for successful wound regeneration (Martin and Nunan, 2015).

IL-10 is widely recognized as a central anti-inflammatory cytokine in wound healing (Saraiva and O’Garra, 2010). In this study, the topical application of oregano extract tended to increase IL-10 expression in excisional wounds (Figs. 1 and 3), with the 6% concentration showing the highest mean levels. Although these differences did not reach statistical significance, this upward trend may suggest a potential role for oregano extract in modulating the inflammatory phase (Ip et al., 2017 ; Saraiva et al., 2020).

The absence of statistical significance in IL-10 expression may be attributed to several biological and methodological factors. IL-10 is tightly regulated and exhibits a temporally dynamic pattern during wound healing, with peak expression typically occurring within a defined inflammatory phase window (Lima et al., 2013). Consequently, assessment at a single time point may fail to capture its maximal or transient upregulation. Furthermore, IL-10 production is orchestrated through complex interactions among macrophages, regulatory T cells (Treg), and the surrounding cytokine microenvironment (Abbas AH Lichtman and Pillai, 2022). Therefore, the lack of statistical significance does not definitively indicate the absence of biological modulation, but may instead reflect the inherent regulatory complexity of IL-10 expression.

VEGF plays a central role in the proliferative phase of wound healing by promoting angiogenesis, which ensures adequate oxygen and nutrient delivery to regenerating tissue (Apte et al., 2019). In the present study, VEGF expression increased significantly only in the 6% oregano extract group, whereas the 3% and 9% concentrations showed non-significant changes despite higher mean values (Figs. 2 and 4). This pattern suggests a concentration-dependent response, indicating an optimal therapeutic window. At 3%, the level of bioactive compounds, such as carvacrol and thymol, may have been insufficient to consistently stimulate VEGF expression across all samples, resulting in a sub-threshold biological effect

Fig. 3. Immunohistochemical staining of IL-10 in skin wound tissue: (A) Vehicle control, (B) Oregano extract ointment 3%, (C) Oregano extract ointment 6%, and (D) Oregano extract ointment 9%. Magnification ×400.

Fig. 4. Immunohistochemical staining of VEGF in skin wound tissue: (A) Vehicle control, (B) Oregano extract ointment 3%, (C) Oregano extract ointment 6%, and (D) Oregano extract ointment 9%. Magnification ×400.

In contrast, the absence of significance at 9% may reflect a shift in biological activity at higher concentrations. Oregano and its major components have been reported to exert antiproliferative and, under certain conditions, anti-angiogenic effects. At elevated concentrations, these properties may partially counterbalance pro-angiogenic signaling, thereby attenuating the overall increase in VEGF expression. This biphasic response is consistent with the concept of hormesis, in which moderate doses stimulate regenerative pathways while higher doses activate inhibitory or regulatory mechanisms (Han and Parker, 2017).

The mechanism by which oregano extract modulates IL-10 and VEGF may involve the regulation of T helper cell differentiation pathways. Experimental evidence indicates suppression of pro-inflammatory cytokines such as IL-17 and IFN-γ, accompanied by downregulation of lineage-specific transcription factors including RORγt. This inhibition of Th1 and Th17 pathways reduces sustained inflammatory signaling within the wound microenvironment. Concurrently, oregano enhances IL-4 expression and promotes activation of GATA3, the master transcription factor driving Th2 differentiation. The shift toward a Th2 phenotype favors anti-inflammatory immune polarization and supports tissue-protective responses. In parallel, increased expression of FoxP3 suggests expansion or stabilization of Treg, which further contribute to immune suppression and resolution of inflammation (Vujicic et al., 2015).

The dominance of Th2 and Treg responses has important downstream effects on innate immune cells, particularly macrophages. IL-4 derived from Th2 cells promotes alternative macrophage activation (M2 polarization), while inhibition of Th1/Th17-derived cytokines prevents classical (M1) pro-inflammatory activation. M2 macrophages are key producers of IL-10 and growth factors involved in tissue repair (Abbas AH Lichtman and Pillai, 2022). Through this coordinated immune shift, IL-10 levels may increase as part of a regulatory feedback mechanism that dampens TNF-α and IL-1β signaling and limits NF-κB activation (Liu et al., 2019). Moreover, M2 macrophages contribute to angiogenesis by secreting VEGF, thereby linking adaptive immune modulation to vascular remodeling during the proliferative phase of wound healing (Murray and Wynn, 2011). These integrated immune cells provide a mechanistic framework explaining how oregano may simultaneously regulate inflammation and promote angiogenic repair.

Several limitations should be acknowledged in this study. First, a comprehensive phytochemical characterization of the oregano extract was not performed. Although oregano is known to contain bioactive compounds, such as carvacrol and thymol, the absence of quantitative phytochemical profiling limits the precise attribution of the observed immunomodulatory and angiogenic effects to specific constituents. Future studies incorporating chromatographic analysis would strengthen mechanistic interpretation and improve reproducibility. Second, the assessment of wound healing relied primarily on molecular markers, particularly IL-10 and VEGF expression, without integrating additional objective healing indicators, such as wound closure rate, histological scoring, collagen deposition, re-epithelialization thickness, or microvessel density. The inclusion of morphometric and histopathological parameters would provide a comprehensive evaluation of tissue regeneration. A longitudinal design would better clarify expression kinetics and help define the optimal therapeutic window.

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Conclusion

Oregano extract demonstrated immunomodulatory activity in an excisional wound model. A statistically significant increase in VEGF expression was observed at the 6% concentration, while IL-10 showed a non-significant upward trend across treatment groups. These findings suggest that oregano may modulate molecular mediators involved in the transition from inflammation to proliferation during wound healing. However, interpretation of these results should consider several limitations, including the absence of detailed phytochemical characterization of the extract, reliance primarily on molecular markers without comprehensive histological and functional wound assessment, and evaluation at a single time point that may not fully capture the dynamic kinetics of cytokine and growth factor expression. Further studies incorporating standardized phytochemical profiling, longitudinal analysis, and direct assessment of tissue remodeling and vascular parameters are warranted to clarify the biological significance and enhance translational relevance of the observed molecular changes.

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Acknowledgments

The authors wish to thank the Laboratory of Veterinary Microbiology and Immunology, Faculty of Veterinary Medicine Universitas Brawijaya, for their assistance throughout this study.

Conflict of interest

The author declares no conflict of interest.

Funding

This study was funded by the Universitas Brawijaya through the 2022 HPP research grant scheme No. 974.28/UN10.C10/PN/2022.

Authors’ contribution

Conceptualization: I.A.A.; data collection: I.A.A.; data analysis and interpretation: G.W.A., I.A.A.; manuscript drafting: G.W.A., I.A.A.; critical review/revision: G.W.A., I.A.A. All authors have read, reviewed, and approved the final version of the manuscript.

Data availability

The data have already been included as part of the submitted manuscript.

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