Therapeutic Potential of Linalool in Endometriosis: Modulation of Oxidative Stress, Inflammation, and Apoptosis in a Rat Model

ABSTRACT

Endometriosis (EMS) is a chronic inflammatory disease affecting 10%–15% of women of reproductive age and is a major cause of infertility. This study explores the therapeutic effects of linalool, a natural compound with antioxidant and anti‐inflammatory properties, on EMS in a rat model. Twenty‐four female rats were divided into four groups: sham, EMS control, and two treatment groups receiving linalool at 50 mg/kg and 100 mg/kg. EMS was induced by implanting uterine tissue, and linalool was administered for 28 days. Oxidative stress markers (MDA, SOD, GPx, CAT), inflammation indicators (TNF‐α, IL‐1β, NF‐κB), apoptosis regulators (Bax, Bcl‐2, caspase‐3), autophagy marker (Beclin1), adhesion molecule (ICAM‐1), and angiogenic factor (VEGF) were all assessed using biochemical and histological techniques. Linalool treatment significantly reduced lesion size and oxidative stress in a dose‐dependent manner. The higher dose group (Lina 100) showed nearly normal histological features tissue histology. Linalool boosted antioxidant enzymes, reduced inflammatory signaling via NF‐κB and MAPK pathways, promoted apoptosis, and suppressed angiogenesis and cellular adhesion. These findings suggest that linalool exerts multi‐targeted effects to reduce EMS severity. The study highlights linalool's promise as a potential therapeutic agent for EMS, though further research in human models is needed to confirm its clinical relevance.

Linalool ameliorates endometriosis by suppressing oxidative stress and NF‐κB/MAPK‐mediated inflammatory signaling, promoting apoptosis, and inhibiting cell adhesion and angiogenesis. These effects are associated with reduced expression of TNF‐α, IL‐1β, NF‐κB, ERK1/2, p38, ICAM‐1, and VEGF, increased Bax, caspase‐3, and Beclin‐1 expression, enhanced antioxidant defenses, and attenuation of endometriotic lesion growth.

1. Introduction

Endometriosis (EMS) is a perplexing disease affecting women's reproductive systems. Characterized by the presence of endometrial tissue outside the uterus, this chronic inflammatory condition is prevalent among 10%–15% of women of reproductive age, primarily affecting those between 25 and 45 years old. EMS often results in ulcers and adhesions within the peritoneal mesothelium, adversely affects oocytes and ovaries, and interferes with the pregnancy implantation process. Consequently, 30%–50% of those with EMS experience infertility. The disease incurs an annual cost ranging from €0.8 to €12.5 billion in European nations [1]. Beyond physical ailments, dealing with chronic pain over an extended period leads to feelings of depression, sexual dysfunction, and a reduced quality of life [2].

Multiple theories exist regarding the pathogenesis of EMS. The most trustworthy theory suggests retrograde menstruation expels blood and endometrial tissue from the fallopian tubes into the peritoneal and pelvic cavities. These tissues then grow and lead to the development of EMS [3]. The survival of ectopic tissue relies on the mesothelium's capacity to infiltrate, attach, and develop blood vessels. In individuals with EMS, heightened internal oxidative stress in cells within endometriotic lesions, increased macrophages and phagocytosis, and iron overload resulting from retrograde menstruation typically contribute to increased oxidative stress damage [4, 5]. When ROS levels increase and cause oxidative stress, nuclear factor erythroid 2‐related (Nrf2) translocates from the cytosol to the nucleus and elevates antioxidant gene expressions [6]. The number of macrophages increases in peritoneal fluid (PF) and release cytokines such as tumor necrosis factor‐alpha (TNF‐α), interleukin‐1ß (IL‐1ß), and nuclear factor kappa B (NF‐kB). TNF‐α, IL‐1ß effect on mitogen‐activated protein kinase/extracellular signal‐regulated kinase (MAPK/ERK) and mitogen‐activated protein kinase/p38 (MAPK/P38) pathways. The MAPK/ERK pathway promotes the survival and proliferation of ectopic endometriotic tissue. The activated forms of extracellular signal‐regulated kinase 1 and 2 (ERK1/2) has an antiapoptotic function, and it can affect B‐cell lymphoma 2 (Bcl‐2) for decreased apoptosis. MAPK/P38 pathway causes elevation inflammatory reactions, and the phosphorylated form of P38 increases in the EMS. NF‐kB has a vital role in inflammation, apoptosis, and angiogenesis. TNF‐α, IL‐1ß, and phosphoinositide3‐kinase/protein kinase B/mammalian target of rapamycin (PI3k/AKT/mTOR) elevate NF‐kB. NF‐kB has an addictive effect on Bcl‐2. Bcl‐2 suppresses BCL2‐associated X apoptosis regulator (Bax) and beclin1. Bax activates caspase‐3 (CAS3) and leads to apoptosis. Beclin1 is an autophagy‐related protein that may influence the progression of endometriosis. NF‐kB enhances vascular endothelial growth factor (VEGF); additionally, VEGF and AKT/mTOR have two‐way interaction by positive feedback and amplifying their action, resulting in angiogenesis development. TNF‐α and NF‐kB increase Intercellular adhesion molecule‐1 (ICAM‐1), which helps to elevation adhesion and angiogenesis [4, 5, 7, 8, 9].

Numerous treatments are available based on molecular cascades or stimulators [10]. Recent studies demonstrate that antioxidant treatments significantly affect regressing EMS lesions and decreasing associated complications [11]. Linalool, a monoterpene, is mainly sourced from aromatic plants, especially lavender. Several studies have shown that as a multifunctional compound, linalool possesses antioxidant, anti‐inflammatory, and apoptotic properties [12]. In a study comparing linalool with ascorbic acid, although it was less potent, linalool exhibited high antioxidant potential [13]. An advantage of linalool is its safe oral consumption, widely used as a food additive in the industry [14]. Because endometriosis involves multiple pathological pathways, the multitarget effects of linalool may be therapeutically relevant and may contribute to the regression of endometriotic lesions. Our aim of this study is to investigate the effect of linalool on OS, inflammation, apoptosis, and adhesion in the EMS rat model.

2. Materials and Methods

2.1. Animals and Groups

The treatment and care of rats and therapeutic procedures were conducted at the Laboratory Animal Care Center of Lorestan University of Medical Sciences. All experimental procedures were approved by the Ethics Committee for the Use and Care of Animals of Lorestan University (LU.ECRA.2023.25) and conducted in accordance with the National Research Council's Guide for the Care and Use of Laboratory Animals. Additionally, the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) were followed [15]. Twenty‐four unmated female Wistar‐Albino rats, each 8 weeks old and weighing approximately 200 ± 20 g, were obtained from the same university. These rats were housed in a controlled environment at a temperature of 24°C ± 2°C, with a relative humidity of 50% ± 10%, and they were subjected to a 12‐h light–dark cycle. They were given a week to acclimate to their new laboratory environment. The rats' health was monitored daily. The rats were evenly divided into four groups, each consisting of six rats [16, 17]: (1) Sham group: Rats in this group were anesthetized and underwent a mock surgery in which the abdominal area was opened, but no further interventions were performed. The incision was then sutured. (2) Control group: After being anesthetized, the rats in this group had endometriosis induced and were subsequently given a 0.9% saline solution at volume of 1 ml for 28 days. (3) Linalool 50 group (Lina 50): Similar to the control group, rats had endometriosis induced, but they were treated with linalool (97% purity; Sigma‐Aldrich, Merck KGaA, Darmstadt, Germany; catalog no. 8.18627.0250; CAS No. 78–70‐6) at a dose of 50 mg/kg for 28 days [18, 19, 20]. (4) Linalool 100 group (Lina 100): Also subjected to endometriosis induction, rats in this group received a linalool dose of 100 mg/kg for 28 days [18, 19, 20]. The sample size in each group was selected based on the previous comparable experimental research and in accordance with the 3Rs principle for animal research. The linalool doses were selected based on the previously published studies revealing their biological efficacy and acceptable safety profile in experimental rat models. These two doses were employed to examine a potential dose‐dependent therapeutic response while remaining within a range reported to be well tolerated in rodents. Nevertheless, the present study was designed to investigate the pharmacodynamic effects of linalool rather than to establish its pharmacokinetic profile or maximum tolerated dose.

2.2. Surgical Procedure

EMS was induced using the technique outlined by Vernon and Wilson [21]. Anesthesia was induced in all rats using a combination of 10% ketamine hydrochloride (80 mg/kg, Bremer, Warburg, Nordrhein‐Westfalen, Germany) and 2% xylazine hydrochloride (10 mg/kg, Interchemie, Castenray, the Netherlands). Following aseptic preparation of the surgical area, a 3 cm midline incision was made to access the abdomen. To create the endometriosis model in both the control and treatment groups, the right horn of the uterus was exposed, ligated, and then divided into cranial and caudal parts. The right uterine horn was divided into three pieces, each measuring approximately 5 × 5 mm, kept moist in a sterile 0.9% saline solution. Two pieces were implanted into the right abdominal muscular wall, while one piece was attached to the mesentery of the jejunum using 5–0 nylon suture material. The linea alba was sutured with PGA 4–0 (Supa, Karaj, Iran) using a simple continuous pattern, and the skin was sutured with nylon 3–0 (Supa, Karaj, Iran) using a cruciate pattern. Fourteen days after endometriosis induction, a relaparotomy was performed to confirm the success of the procedure. Treatments began immediately after confirmation of lesion development, and continued for 28 days. Rats were euthanized 42 days after endometriosis induction to assess the effects of linalool on the endometriotic lesions. The endometriosis implants were kept at −80°C until they were used for western blotting analysis.

2.3. Oxidative Stress

To measure oxidative stress parameters, tissue samples were homogenized and a supernatant was obtained. After that, malondialdehyde (MDA, Zell Bio, Lonsee, Germany), catalase (CAT, Zell Bio, Lonsee, Germany), superoxide dismutases (SOD, Randox, Crumlin, Northern Ireland), and glutathione peroxidase (GPx, Crumlin, Northern Ireland) levels were measured according to the instructions of the kits.

2.4. Western Blot

In this study, protein extraction was achieved by employing a lysis buffer, followed by centrifugation of the samples at 12,000 rpm for 10 min at 4°C. The resulting supernatant was harvested and stored at −20°C. Protein concentrations were subsequently quantified via the Bradford assay, assessing absorbance differences at 630 nm, with bovine serum albumin (BSA) utilized as the reference standard. Prior to loading into wells, protein samples were combined with sample buffer and heated at 100°C for 5–10 min. Electrophoresis was then conducted on an SDS‐PAGE gel at 120 V for 45 min. Proteins were transferred from the gel to a PVDF membrane using a western blot apparatus set at 120 V for 1.5 h. To minimize nonspecific binding of the primary antibody, the membrane was immersed in a blocking solution—consisting of 2% non‐fat dry milk in TBS‐T buffer—and agitated for 75 min at room temperature. The PVDF membrane was then incubated with diluted primary antibody solutions for 16–18 h. The antibodies included β‐actin (sc‐517 582, Santa Cruz Biotechnology, Texas, USA, 1:300), p38 α (sc‐535, Santa Cruz Biotechnology, Texas, USA, 1:200), Akt (E‐AB‐30471, Elabscience, Wuhan, China, 1:1000), Phospho‐Akt (Ser473, Cell Signaling Technology, Danvers, Massachusetts, USA, 1:1000), ICAM‐1 (sc‐8439, Santa Cruz Biotechnology, 1:500), mTOR (sc‐517 464, Santa Cruz Biotechnology, 1:500), p‐mTOR (sc‐293 133, Santa Cruz Biotechnology, 1:500), p ERK1/ERK2 (MAB1018, R&D Systems Minneapolis, USA, 0.5 μg/mL), NFκB p65 (sc‐8008, Santa Cruz Biotechnology, 1:500), caspase 3 rabbit mAb (D3R6Y, Cell Signaling Technology, 1:1000), Nrf2 (sc‐365 949, Santa Cruz Biotechnology, 1:200), TNFα (sc‐130 349, Santa Cruz Biotechnology, 1:200), and VEGF (E‐AB‐67255, Elabscience, 1:500). Following incubation, the membrane was rinsed three times with TBS‐T buffer and then exposed to species‐specific secondary antibody (sc‐516 102, Santa Cruz Biotechnology, Texas, USA, 1:1000) for 75 min at room temperature. The membrane underwent three 15‐min washes with a washing solution. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection kit (Thermo Scientific, USA), and the relative protein/β‐actin density was analyzed with ImageJ software. Full‐length gel of western blot for all evaluated markers are provided in Supporting Information Data S1.

2.5. Histopathological Evaluation

2.5.1. Hematoxylin and Eosin

Tissue samples were collected primarily from the 10% buffered formalin solution, and dehydration was performed at increasing ethylene alcohol concentrations. Tissue samples were then cleaned with xylene and embedded in paraffin. Sections, measuring 4 μm in thickness, were generated using a rotary microtome. Sections were then affixed on slides and stained with hematoxylin and eosin (H&E).

2.5.2. Immunofluorescence

After deparaffinization of the samples in xylene and rehydration in ethanol and water, sections were treated in a microwave oven at 95°C for 12 min in sodium citrate buffer (pH 7.4). Briefly, sample sections were incubated overnight with a combination of ERK antibody (1:250; Rabbit monoclonal [EPR17526] to ERK1 + ERK2) ab184699 Abcam UK, Rabbit polyclonal to Nrf2 (1:100, ab31163), and p38α (1:50 [C‐20]: sc‐535) Santa Cruz US, mouse monoclonal antibody to p‐Akt1/2/3 (1:50, B‐5): sc‐271 966, and p‐mTOR (1:50, 59. Ser 2448): sc‐293 133 Slides were then washed with PBS and incubated for 1 h with an appropriate combination of secondary antibodies Goat Anti‐Mouse IgG (H + L) (CY3 conjugated) E‐AB‐1011 1:50 Elabscience and Goat Anti‐Rabbit IgG (H + L) (FITC conjugated) E‐AB‐1014 1:50 Elabscience. Nuclei were then counterstained with DAPI, and sections were placed under a coverslip (Immuno‐Mount, ThermoShandon, Pittsburgh, PA, USA). Microscopic analysis of the sections was then performed using an Olympus BX50 (Tokyo, Japan) microscope equipped with a DP72 camera (SPOT Insight, Diagnostic Instruments, USA) using the Olympus Cell A software and assembled using Adobe Photoshop (Adobe Systems, San Jose, CA, USA).

2.5.3. Immunohistochemistry

Immunohistochemical procedures were performed on 3 μm thick paraffin‐embedded target sections using the following antibodies: polyclonal rabbit anti‐Casp‐3 (orb 10 237, Biorbyt, UK) at 1:50 dilution, Bcl‐2 (orb 10 173, Biorbyt, UK), TNF‐a (ab6671, Abcam, UK) at 1:100 dilution, Bax (sc 7480, Santa Cruz, USA) at 1:50 dilution, NF‐kB‐p65 (orb312399, Biorbyt, UK) at 1:100 dilution VEGF (LS‐B7747, LSBio, USA) at 1:100 dilution, IL‐1β (ab226918, Abcam, UK) at 1:100 dilution and Beclin (orb orb10182, Biorbyt, UK) at 1:100. After dewaxing and rehydrating, sections were immersed in target retrieval solution (Tris‐EDTA, pH 9.0) and heat‐mediated antigen retrieval was performed in a water bath for 20 min at 98°C to deliver unmasked antigens. Subsequently, the sections were treated with H2O 2 (3%) in PBS for 15 min to inhibit endogenous peroxidase, and nonspecific background staining was inhibited by incubating the sections with normal rabbit sera (5%) in PBS for 20 min. Sections were incubated 1 h with primary antibodies and then incubated for 20 min with biotinylated goat anti‐rabbit IgG (prediluted, Biocare, USA), followed by incubation with streptavidin horseradish peroxidase (sHRP) (prediluted, Biocare, USA) for 20 min. The antibody binding sites were visualized with DAB chromogen substrate. Eventually, sections were counterstained with Mayer's Hematoxylin (Bio Optica, Italy).

2.6. Statistical Analysis

For our statistical examination, we employed SPSS software version 26. Upon assessing the data's normality, we conducted either a parametric analysis (One‐way ANOVA accompanied by Tukey's post hoc test) or a non‐parametric analysis (Kruskal‐Wallis and Mann–Whitney U tests), depending on the results. This approach facilitated the comparison between groups.

3. Results

3.1. Macroscopic Evaluation

The sizes of endometriosis lesions in the abdominal wall and mesentery were assessed, and related results are provided at Figure 1. Our findings revealed that size of lesions in the control group was 10.54 and 8.18 mm in the parietal and visceral lesions respectively. In the Lina 50 and Lina 100 groups, lesion sizes were significantly reduced compared to the control group.

FIGURE 1.

Macroscopic appearance of endometriosis lesions in the abdominal and mesenteric part within various study groups.

3.2. Oxidative Stress Evaluation

In the analysis of oxidative stress parameters presented in Figure 2, the significance threshold was set at a p‐value below 0.05. Comparison between the control group and both the Sham group and the group treated with 100 mg/kg of linalool showed significant variations across all measured biomarkers (MDA, SOD, GPx, and CAT), with p‐values lower than 0.05. Meanwhile, administering 50 mg/kg of linalool resulted in notable differences in MDA and SOD levels when compared to the control group, also reflecting p‐values less than 0.05. Additionally, the evaluation highlighted a significant discrepancy in SOD levels across different treatment groups, with the highest dose yielding the most pronounced effect. No significant disparity was observed between the Sham and the linalool 100 mg/kg groups regarding any of the studied parameters, indicating comparable outcomes.

FIGURE 2.

The graphs indicate biomarker concentrations (MDA level and GPx, SOD, and CAT activity). Significant differences between the sham group and other groups are indicated by #, between the control group and treatment groups are indicated by *, and between treatment groups are indicated by †.

3.3. Hematoxylin and Eosin Findings

H&E results are presented at Figure 3. In the sham group, there was no evidence of inflammation or stromal thickening. The tissue integrity was preserved, and no pathological changes were observed. In the control group, there was clear evidence of ectopic endometrial glands and stromal tissue within the peritoneal cavity. The lesions were characterized by the presence of endometrial stroma (SM) surrounding glandular structures. Additionally, increased stromal proliferation and signs of inflammation were observed, as indicated by stromal thickening and leukocyte infiltration. The lesion size and stromal area were notably enlarged compared to the sham group. Treatment with linalool at a dose of 50 mg/kg showed a moderate reduction in the severity of endometriotic lesions. The ectopic endometrial glands were less pronounced, and the surrounding stroma exhibited reduced cellular proliferation. Notably, inflammatory infiltration was decreased compared to the control group. However, some pathological features, such as ectopic glands and stromal expansion, were still present, albeit at a lower degree. The administration of linalool at 100 mg/kg resulted in a marked improvement in histological features. The lesions displayed significantly reduced ectopic endometrial glands and stromal thickening. The endometrial stroma (SM) and epithelium (EP) were less prominent, and the number of inflammatory cells was substantially lower compared to both the control and linalool 50 groups. The tissue exhibited near‐normal histological characteristics, with minimal pathological changes.

FIGURE 3.

Sham group: (A, B) normal uterine tissue; the epithelium (arrows) and glands (g) are in normal condition. Control group: (C, D) the endometrial cyst has developed on peritoneal side. Epithelial cells are well demonstrated (arrow); a bundle of smooth muscle cells C. the endometrial cyst is developed on intestinal mesenteric surface with highly proliferated epithelial cells (arrows); (D) Linalool 50: (E, F) degenerative and necrotic epithelial cells are visible (arrows) with debris of necrotic cells sloughed into the lumen; (E) Higher magnification; (F) Linalool 100: (G, H) thinner layer of degenerated epithelial cells (arrow) with more copious debris are being shed in the center of lumen (arrow heads); (G) Higher magnification; (H) SM; smooth muscle cells layer, EP; epithelial degenerated cells, N; necrotic cells or debris.

3.4. IHC and Western Blot Findings

Bax expression was slightly elevated in the control group (Panel B) compared to the sham group (Panel A). Treatment with linalool increased Bax expression, with greater increament in the linalool 100 group (Panel D) than in the linalool 50 group (Panel C). Bcl‐2 expression was highest in the control group (Panel F), while both linalool‐treated groups (Panels G and H) showed a dose‐dependent decrease, with the lowest levels in the linalool 100 group. Beclin 1 was markedly downregulated in the control group (Panel J) compared to the sham group (Panel I). Treatment with linalool increased Beclin 1 expression in a dose‐dependent manner, with the linalool 100 group (Panel L) demonstrating the most significant increment. The expression of cleaved caspase‐3 (an apoptosis execution marker) was significantly increased in the treatment groups (Panel O&P). Both linalool‐treated groups showed increased expression, with the linalool 100 group (Panel P) exhibiting a stronger suppression than the linalool 50 group (Panel O) (Figure 4). The western blot expression of Beclin1, an essential protein involved in autophagy, was significantly elevated in the groups receiving treatment compared to the control group (p < 0.05). Notably, the group treated with a high dose of linalool exhibited a significantly greater expression of Beclin1 than the group treated with a low dose (p < 0.05). Moreover, the expression levels of Beclin1 in the sham group were significantly higher than those in the control group (p < 0.05). Caspase3 is an important protein in molecular cascades related to cellular apoptosis, and Caspase3‐cleaved is an active form of caspase3‐pro. Evidence shows that caspase3‐pro is significantly lower in treatment groups than in sham and control groups (p < 0.05). There is a significant difference between the high‐dose and low‐dose linalool groups; the Lina 100 group was lower than the Lina 50 group (p < 0.05). The result demonstrated no significant difference between sham and control groups. Levels of cleaved caspase 3 significantly increased in both treatment groups compared to the control group and the sham group (p < 0.05) (Figure 4).

FIGURE 4.

Bax immunolabeling group (A–D); Sham: No evidence of immunostaining (A) Control: A few multifocal areas of staining in the wall of the cyst (B) inset; many cells are stained. b. Linalool 50: Immunopositive cells are scattered in the rim of the cyst (C) inset; few cells are weakly stained c. Linalool 100: Intensity of immunopositive cells is more prominent and higher than Lina 50 (D) inset; strong cytoplasmic staining in cells of stroma. d. Bcl2 immunostaining group (E–H); Sham: No cells are immunostained (E) Control: A few cells are stained in connective tissue of cyst (F) inset; many cells are labeling. f. Linalool 50: Weakly positive staining of few dispersed connective tissue cells (G) inset; pale immunopositive cells g. Linalool 100: Very few cells are immunolabeled (H) inset; a small cluster of cells have reacted h. Beclin immunopositive group (I–L); Sham: No reaction is presented (I) Control: No labeling is seen (J) Linalool 50: Scattered antibody staining is located in cellular cytoplasms (K) inset; weak stain k. Linalool 100: Diffuse intensive immunopositive cells (L) inset; strong staining l. Caspase3 immunoreacted group (M–P); Sham: Diffuse positive cells are obvious (M) inset; the cells are fairly faded reacted to antibody, m. Control: Multifocal immunostaining exclusively, in inner and outermost layers (N) inset; staining is also present in few of the epithelial cells n. Linalool 50: Minimal immunoreactivity is observed (O) inset; very few cells are stained. o. Linalool 100: Very strong reaction to antibody throughout the cyst (P) inset; highly labeling is dominant in the vicinity of the epithelial layer p. Graphs indicate western blot expressions of Beclin 1, caspase 3‐pro and cleaved. # indicates significant difference with the sham group, * indicates significant difference with the control group, and † indicates significant difference with the Lina 50 group. Color graphs indicate positive reaction intensity (pixel based) for BAX, Bcl‐2, Beclin, and Caspase‐3 in various study groups.

Levels of IL‐1β were approximately similar in the control and the sham group with a little higher expression in the control group (Panels A & B). In the treatment groups, Lina 100 (Panel D) highly increased the levels of IL‐1β compared to the other groups.

TNF‐α levels were significantly increased in the control group (Panel F) compared to the sham group (Panel E), reflecting heightened inflammation. Treatment with linalool reduced the expression of TNF‐α, with the linalool 100 group (Panel H) showing a more pronounced reduction than the linalool 50 group (Panel G). The control group (Panel J) exhibited elevated NF‐κB levels, indicative of inflammatory pathway activation. Linalool treatment resulted in a dose‐dependent decrease in NF‐κB expression, with the linalool 100 group (Panel L) demonstrating the greatest suppression compared to the linalool 50 group (Panel K). VEGF, a key angiogenic marker, was upregulated in the control group (Panel N). Both linalool‐treated groups displayed reduced VEGF expression, with the linalool 100 group (Panel P) achieving stronger suppression than the linalool 50 group (Panel O) (Figure 5). Western blot Analysis indicated that NF‐kB expression significantly differs between all groups (p < 0.05). The NF‐kB level was significantly higher in the control group than in the treatment groups (p < 0.05). NF‐kB level in the high‐dose group is significantly lower than in the low‐dose group (p < 0.05). TNF‐α expression in the control group was significantly higher than the sham group (p < 0.05). Comparison of TNF‐α between the control and treatment groups demonstrated significant differences and is higher in the control group (p < 0.05). TNF‐α in the high‐dose group was significantly lower than the low‐dose group (p < 0.05). VEGF demonstrated a significant difference between all groups (p < 0.05). The sham group was lower than all groups significantly (p < 0.05). Comparison between the control and treatment groups indicated a significant decrease for treatment groups (p < 0.05). Treatment with high doses was more effective than low doses to decrease VEGF expression. The result indicates that ICAM‐1 expression in treatment groups is significantly lower than in the control group (p < 0.05). There is no significant difference between the sham group and the control and low‐dose linalool groups (Figure 5).

FIGURE 5.

IL‐1B Immunopositive group (A–D); Sham: Strong and diffuse staining of normal stromal and glandular epithelial cells (A) The glands are strikingly stained a. Control: Multifocal positive cells are focused in both inner and outermost layers (B) inset; proliferated epithelial cells have reaction b. Linalool 50: Weak staining is evident (C) inset; only a few cells are stained c. Linalool 100: Diffuse, broadly staining of cells across the cyst (D) inset; intensive cellular labeling d. TNFa immunolabeling group (E–H); Sham: No positive staining in normal uterine tissue (E) Control: A few cells are stained sparsely (F) inset; intensive dispersed cells are stained f. Linalool 50: Weak to moderate reaction of small population of cells to antibody (G) inset; only few cells are faintly stained g. Linalool 100: Nearly, large population of cells are labeled with antibody (H) inset; the positive stained cells are focused at the center h. NF‐kB immunoreacted group (I–L); Sham: No immunoreaction (I) Control: A few cells are reacting strongly in middle layer (J) inset; cytoplasmic staining j. Linalool 50: Very few cells are cytoplasmically stained (K) inset; the cells are focused at the center k. Linalool 100: Low population of positive cells (L) inset; only weak staining is observed l. VEGF Immunostaining group (M–P); Sham: Cells have no labeling (M) Control: Many cells have intensive reaction (N) inset; scattered cells are stained n. Linalool 50: Sporadic cells have labeling (O) inset; a few scattered cells are positive in cyst wall o. Linalool 100: Moderate population of cells are demonstrated as stained cells (P) inset; focal staining of small number of cells is appearing at the center p. Graphs indicate western blot expressions of ICAM 1, TNF‐α, NF‐κB, and VEGF. # indicates significant difference with the sham group, * indicates significant difference with the control group, and † indicates significant difference with the Lina 50 group. Color graphs indicate positive reaction intensity (pixel based) for IL‐1B, TNF‐α, NF‐κB, and VEGF in various study groups.

3.5. Immunofluorescence and Western Blot Findings

The immunofluorescence analysis revealed minimal expression of p‐ERK 1/2 in the sham group. On the other hand, the control group showed increased expression of p‐ERK 1/2, indicating an increased activation of the ERK signaling pathway. Linalool treatment resulted in a dose‐related reduction of p‐ERK 1/2 expression. Linalool 50 group showed moderate fluorescence density, while linalool 100 group showed the most significant reduction, with results close to the sham group (Figure 6).

FIGURE 6.

Immunofluorescence expression of p‐ERK 1/2 among different experimental groups of the study. Bars indicate 100 μm in all slides.

Results for p‐AKT are shown at Figure 7. Immunofluorescence detection revealed faint p‐Akt expression in the sham group, with sparse red fluorescence. The control group showed significantly increased p‐Akt expression, suggesting activation of the Akt signaling pathway in endometriotic lesions. The linalool 50 group showed moderate fluorescence intensity, and the linalool 100 group showed a notable decrease in p‐Akt levels, with values close to those of the sham group (Figure 7). Western blot analysis indicated that AKT decreased in the control group, and sham and high‐dose treatment groups revealed significantly higher levels (p < 0.05). There was no significant difference between the sham group and the treatment groups. In line with our expectation, the result for p‐AKT was the reverse of AKT. P‐AKT for the control group was significantly higher than other groups (p < 0.05). In the low‐dose linalool group, P‐AKT was lower than in the high‐dose group (p < 0.05). The high‐dose linalool group, in contrast to the low‐dose linalool group, had no significant difference with the sham group (p < 0.05) (Figure 7).

FIGURE 7.

Immunofluorescence expression of p‐AKT among different experimental groups of the study. Bars indicate 100 μm in all slides. Graphs indicate western blot expressions of AKT and p‐AKT. # indicates significant difference with the sham group, * indicates significant difference with the control group, and † indicates significant difference with the Lina 50 group.

Intense expression of Nrf2 in the sham group was observed and there was a remarkably decreased Nrf2 expression in the control group, reflecting the inhibition of antioxidant defense mechanisms within endometriotic lesions. Linalool treatment successfully recovered Nrf2 expression in a dose‐dependent manner. The Lina 50 group showed moderate fluorescence intensity, while the Lina 100 group showed Nrf2 levels similar to the sham group (Figure 8). In the western blot assay, the sham group had the significantly highest level of Nrf2 compared to other groups (p < 0.05). In treatment groups, the Nrf2 level was significantly higher than in the control group (p < 0.05) (Figure 8).

FIGURE 8.

Immunofluorescence expression of Nrf2 among different experimental groups of the study. Bars indicate 100 μm in all slides. Graphs indicate western blot expressions of Nrf2. # indicates significant difference with the sham group, * indicates significant difference with the control group, and † indicates significant difference with the Lina 50 group.

In the sham group, p‐mTOR expression at a reduced level signified baseline values under a disease‐free state. In contrast, in the control group, p‐mTOR expression at a high level in comparison with that in the sham group signifies that disease initiation triggers p‐mTOR signaling processes. In the Lina 50 group, a reduced expression level of p‐mTOR when compared with that in the control group was seen. In contrast, in the Lina 100 group, a significant drop in p‐mTOR expression level was seen (Figure 9). In the western blot assessment, the level of mTOR in the sham group was significantly higher than in the control and high‐dose linalool groups (p < 0.05). The active form of mTOR, known as P‐mTOR, showed significant differences among all groups. P‐mTOR was significantly lower in the sham group compared to the other groups (p < 0.05). Additionally, it was significantly lower in the treatment groups compared to the control group (p < 0.05), and the high‐dose group had significantly lower levels compared to the low‐dose group (p < 0.05) (Figure 9).

FIGURE 9.

Immunofluorescence expression of p‐mTOR among different experimental groups of the study. Bars indicate 100 μm in all slides. Graphs indicate western blot expressions of mTOR and p‐mTOR. # indicates significant difference with the sham group, * indicates significant difference with the control group, and † indicates significant difference with the Lina 50 group.

Immunofluorescence analysis revealed low p‐P38 expression in the sham group with faint green fluorescence; whereas, on the other hand, in the control group, the expression of p‐P38 was greater with enhanced activation of MAPK in endometriotic lesions. Linalool administration resulted in dose‐dependent reduction in p‐P38 expression. In Lina 50, mild reduction in fluorescence intensity; and in Lina 100, significant inhibition in p‐P38 expression, with values almost similar to that of the sham group, was noted (Figure 10). Western blot results related to P‐38 show that there is a significant difference between all groups (p < 0.05). P‐38 levels in the treatment groups are significantly lower than in the control group (p < 0.05). P‐38 in the high‐dose linalool group was significantly lower than the low‐dose linalool group (p < 0.05). Expression of p‐38 was significantly lowest in the sham group (p < 0.05) (Figure 10).

FIGURE 10.

Immunofluorescence expression of p–P 38 among different experimental groups of the study. Bars indicate 100 μm in all slides. Graphs indicate western blot expressions of P 38 and p–P 38. # indicates significant difference with the sham group, * indicates significant difference with the control group, and † indicates significant difference with the Lina 50 group.

4. Discussion

Endometriosis is a complex disease involving multiple molecular pathways and factors in its pathogenesis. An increase in inflammatory and angiogenic factors, adhesion, oxidative stress, and the inhibition of factors involved in autophagy and apoptosis leads to the development and progression of this disease [22]. Given the complex and multifaceted nature of this disease, using multi‐functional compounds that impact the expression of factors related to inflammation, angiogenesis, apoptosis, and autophagy, among others, can be effective in controlling and treating this condition. Limonene and linalool, which have been shown in various studies involving human and animal models to have properties related to inflammation, angiogenesis, apoptosis, and autophagy, etc. [12, 23] are considered suitable options for controlling and treating endometriosis in this research.

In ectopic endometrial tissue, the balance between oxidants and antioxidants is disrupted due to the disruption of the ROS neutralization pathway [24]. Human studies have demonstrated impaired antioxidant defenses and increased lipid oxidation in the peritoneal fluid of patients with EMS [25, 26]. According to our results, linalool exhibited significant antioxidant properties by neutralizing reactive oxygen species (ROS) and restoring the oxidant‐antioxidant balance in treatment groups. It reduces lipid peroxidation, as evidenced by decreased malondialdehyde (MDA) levels, thereby protecting cellular integrity from oxidative damage. Additionally, linalool enhances the activity of key antioxidant enzymes like superoxide dismutase (SOD) and glutathione peroxidase (GPx), further strengthening the antioxidant defense system against oxidative stress. Parallel to our study, boswellia serrate resin gum in the rat model of EMS could decrease MDA levels and increase SOD and GPx activity significantly; this result agrees with our finding [27]. In another study, methyl ester of 2‐cyano‐3,12‐dioxooleana‐1,9‐dien‐28‐oic acid (CDDO‐Me) decreased MDA levels and increased SOD activity; this finding is consistent with our result [6].

Redox‐sensitive nuclear factor erythroid‐derived 2‐like 2 (Nrf2) controls endogenous antioxidant enzymes' transcription and protects against oxidative damage which is triggered by oxidative stress and inflammation, thereby facilitating the progression of endometriosis [28]. Overexpressed Nrf2 affects cell proliferation by directing glucose and glutamine towards anabolic pathways augmenting purine synthesis and influencing the pentose phosphate pathway to promote cell proliferation [29, 30]. However, the exact mechanism involving oxidative stress and its transcriptional factor Nrf2 and associated genes in endometriosis remains to be explored. During condition of oxidative stress, Keap1 cysteines become oxidized leading to a disruption of the Keap1‐Nrf2 complex and the release of the Nrf2 peptide. Nrf2 then translocates to the nucleus to transcribe genes encoding various antioxidant proteins and metabolic enzymes (Phase II detoxifying enzymes) [31]. With the elevated Nrf2 level, the expression of its downstream molecules or cytoprotective proteins like NQO1 and HO1 is enhanced, facilitating the survival of cells [28, 29, 31]. Activation of Nrf2 and its downstream protein is reported in various cancers and even in endometriosis [28, 32]. Nrf2 and its downregulated cytoprotective genes HO1 and NQO1 were found to be over expressed in endometriosis suggesting that Nrf2/HO1/NQO1 is involved in the pathogenesis of endometriosis [28, 33, 34]. As previously noted, linalool demonstrated the ability to diminish OS and neutralize ROS in the treatment groups. By mitigating oxidative stress and reducing the levels of ROS, the translocation of Nrf2 to the nucleus—where it regulates the transcription of downstream genes involved in antioxidant defense and cellular protection—was diminished. Consequently, the levels of Nrf2 remained elevated in the treatment groups due to its reduced nuclear translocation compared to the control group. By reducing the translocation of Nrf2 to the nucleus, the expression of its downstream molecules (that have cell protective role) are reduced, this is in contrast to the survival of ectopic endometrial cells. Similar results were observed in studies involving Hydrox and CDDO_ME effects on the rat model of EMS, where Nrf2 and oxidative stress parameters improved in treatment groups [6, 35].

Chronic inflammation in the abdominal cavity plays a significant role in the progression of endometriosis. Inflammatory mediators like IL‐1β and TNF‐α activate the transcription factor NF‐kB, and on the flip side, NF‐kB also increases their expression, which creates a positive feedback loop that boosts various inflammatory mediators such as TNF‐α, IL‐1, IL‐6, and IL‐12 [36, 37]. The NF‐kB signaling pathway has a major role in many chronic inflammatory diseases like endometriosis. Increased activation of NF‐kB in endometriosis leads to the maintenance and development of endometriotic lesions, making NF‐kB a potential drug target for this condition. Factors that reduce NF‐kB expression can disrupt the development of endometriosis [38]. The transcriptional activity of several pre‐inflammatory cytokines and chemokines like TNF‐α, IL‐1β, IL‐8, IL‐6, ICAM‐1, etc., is activated through NF‐kB signaling, highlighting NF‐kB's key role in the inflammatory responses in endometriosis [39]. Significant evidence suggests that signaling from NF‐kB and TNF‐α has the greatest impact on the development of endometriosis [40]. TNF‐α helps induce inflammation by activating several transcription factors like NF‐kB [41]. Studies have shown that ectopic endometrial tissues express higher levels of TNF‐α and NF‐kB compared to eutopic endometrial tissue, which confirms the formation of a self‐amplifying cycle by these two that maintains and increases the inflammatory response, leading to adhesion of misplaced tissues to areas outside the uterus, new blood vessel formation, inhibition of apoptosis, and increased proliferation [5]. When NF‐kB signaling is activated, its inhibitor IkB is phosphorylated by kinases (IkK) and degraded by the proteasome. This event allows NF‐kB dimers to enter the nucleus and trigger downstream gene transcription [4]. NF‐kB proteins usually separate in the cytoplasm along with their inhibitors, known as the IkB family. Pre‐inflammatory cytokines like IL‐6, IL‐1β, TNF‐α, and IL‐8 can cause the degradation of IkB, resulting in the relocation of NF‐kB from the cytoplasm to the nucleus [38, 42]. Additionally, the connection of NF‐kB to the DNA promoter leads to the expression of pro‐inflammatory cytokines, intercellular adhesion molecules, and angiogenesis factors [38, 43]. (MAPKs), intracellular signal transducers, mediate some of the effects exerted by proinflammatory cytokines. To date, three types of MAPKs have been well characterized, that is, extracellular signalregulated kinase (ERK), c Jun N‐terminal kinase (JNK) and p38 MAPK (p38). Phosphorylation of MAPKs induces their activities to phosphorylate downstream substrates, thus regulating cellular functions including gene expression, mitosis, movement, metabolism and apoptosis [44]. Evidence indicates that ERK, p38, and JNK are present in endometriotic cells and become activated by proinflammatory agents. Additionally, the secretion of inflammatory cytokines and COX‐2 expression induced by IL‐1b in these cells can be inhibited to varying degrees by MAPK inhibitors. Since IL‐1b plays a pivotal role in the progression of endometriosis, it suggests that MAPKs may have significant pathophysiological roles in the condition, acting as intracellular signal transducers [45]. In this study, linalool exhibited notable anti‐inflammatory effects primarily by inhibiting the NF‐kB signaling pathway and modulating MAPK/p38 and MAPK/ERK1/2 activation. It reduced the expression of inflammatory cytokines such as TNF‐α, disrupting the feedback loop that sustains inflammation in conditions like endometriosis. By lowering levels of NF‐kB, linalool prevents the translocation of NF‐kB into the nucleus, thereby decreasing gene transcription for pro‐inflammatory mediators. Additionally, linalool inhibits the activation of MAPKs/P38 and MAPK/ERK1/2, which further diminishes the secretion of inflammatory mediators, contributing to its anti‐inflammatory profile. The impact of linalool on ectopic endometrial tissues, which exhibit elevated TNF‐α and NF‐kB levels in control group, suggests it may alleviate the chronic inflammation associated with endometriosis. Overall, linalool's multifaceted action on key inflammatory pathways highlights its promise in managing inflammation and symptoms related to endometriosis. Similar results were noted with compounds like flavokawine A and CDDO‐ME in an experimental model of endometriosis, in treatment groups with these compounds, levels of NF‐kB, TNF‐α and IL‐1β were decreased [6, 46]. Studies on SB203580 and Quercetin also reported similar reductions in P38 and ERK1/2 [8, 47]. Interestingly, in IHC assay of our study, the expression of IL‐1β, was diffused and intensive in sham group, which slightly increased in the control group due to inflammatory reaction in this group. However, it was soared again in the Lina 100 group. It is well‐figured out that IL‐1β has a potential role in establishment of pregnancy for embryos in human, bovine and mouse. The IL‐1β has been identified in human, mouse and bovine endometrium and embryos of human, mouse, bovine and porcine. It is assumed that diminish secretion of IL‐1β can bring on to a reduction in NF‐kB expression and maternal innate immune responses impairment [48]. This finding is difficult to reconcile with our results. As we said IL‐1β was highly expressed in sham and lin100 groups and therefore it couldn't be related to inflammatory responses provoking. However, decreased expression of MAPK, P38, and ERK1/2 in treatment groups probably consistent to the other pathways of anti‐inflammatory effects of linalool.

One of the effective factors in the pathogenesis of endometriosis is the prevention of apoptosis and autophagy. When the ratio of pro‐apoptotic to anti‐apoptotic factors (BAX/BcL‐2) increases, it disrupts the mitochondrial membrane, which in turn leads to the release of cytochrome C from the mitochondria into the cytosol. Cytochrome C then forms a complex with Apaf1 and procaspase‐9 in the presence of dATP or ATP. This complex subsequently activates caspase‐9, an initiator caspase that can then activate an effector caspase, namely caspase‐3, which then induces apoptosis in the cell [49]. In the human endometrial epithelium, the BcL‐2 protein is active during the proliferation phase and gradually disappears in the secretory phase. Meanwhile, the BAX protein increases when the endometrium is in the secretory phase, so menstruation occurs due to the increased BAX/BcL‐2 ratio. Lower amounts of the BAX/BcL‐2 ratio are observed in the secretory phase of patients with endometriosis. The low level of this ratio can serve as a warning sign for the progression of endometriosis. This indicates that endometriosis tissue is associated with resistance to apoptosis [50, 51]. In the context of treatment groups, results indicated that linalool modulated the apoptotic process by altering the equilibrium between pro‐apoptotic and anti‐apoptotic factors. Specifically, linalool has the potential to enhance the expression of pro‐apoptotic proteins such as Bax while concurrently reducing levels of anti‐apoptotic proteins like BcL‐2. This alteration would result in a favorable shift in the BAX/BcL‐2 ratio, promoting apoptosis. Such a shift could compromise mitochondrial membrane integrity, leading to the release of cytochrome c into the cytosol and finally activating procaspase 3 and changing it to cleaved caspase 3 that results in the execution of ectopic endometrial cells. Additionally, existing studies have indicated that linalool possesses anti‐inflammatory properties, which may further contribute to its efficacy in managing endometriosis. Chronic inflammation is a recognized factor in the pathogenesis of endometriosis; thus, by attenuating inflammatory responses, linalool may foster a more conducive environment for apoptosis to occur [52]. Comparable findings were reported with investigations on flavokawine A and naringenin effects on EMS models, emphasizing the role of these compounds in modulating apoptotic markers [46, 53].

Molecular regulators, with a correlation‐based approach between autophagy and apoptosis, act as key points for the final outcome of the cell [54, 55]. The process of autophagy is related to several cellular signaling pathways, including the PI3K/AKT/mTOR pathway, hypoxia‐inducible factor (HIF)‐dependent pathways, and downstream molecule‐dependent pathways. mTOR is a serine–threonine kinase that acts as a negative regulator of autophagy. The inductive effects of activators in the mTOR pathway, such as PI3K or AKT, as well as mitogen‐activated protein kinases (MAPK), suppress autophagy, while inhibitors of the mTOR pathway (AMPK signaling and P53) promote autophagy. The ATG proteins that are downstream of mTOR, as mentioned, promote autophagy. mTOR inhibits the function of these downstream molecules, including ULK1, which is located in autophagosomes and is responsible for activating Beclin1. Beclin1 interacts with Ambra1, which facilitates the formation of autophagy. Beclin1 interacts with Ambra1, which facilitates the formation of autophagosomes and pushes the cell towards autophagy. The inhibitory effect of mTOR blocks the function of all downstream molecules, including Beclin1. Regarding the relationship between anti‐apoptotic proteins like Bcl2 and their connection to autophagy, it's important to note that this protein binds to Beclin1 and prevents it from attaching to the Class III PI3K complex, which inhibits the autophagy process through this molecule. In addition to Bcl2, another protein called BNIP3, a homologue of Bcl2, also binds to Beclin1 and can alter its function [56, 57, 58, 59, 60, 61, 62, 63]. Linalool promotes autophagy in treatment groups by inhibiting the mTOR pathway, which negatively regulates autophagy. This inhibition leads to decreased levels of phosphorylated AKT and mTOR, enhancing the expression of Beclin1, a key protein that initiates autophagy by forming complexes necessary for autophagosome formation. Additionally, linalool reduces levels of the anti‐apoptotic protein Bcl‐2, shifting the balance towards autophagy and facilitating Beclin1 activity. Overall, linalool enhances cellular processes essential for managing endometriosis by promoting the removal of dysfunctional cells, highlighting its therapeutic potential. Studies on Acai berry, Quercetin, and Metformin corroborate these findings regarding the PI3K/AKT/mTOR pathway and result in increasing autophagy in treatment groups [64, 65].

Abnormal tissue adhesion of the endometrium in places outside the reproductive system is the first step in the development of endometriosis. Activation of NF‐kB signaling leads to the expression of key adhesion molecules in the abnormal endometrium, including molecules like ICAM‐1, VCAM‐1, and CD44 [66, 67, 68]. Research has shown that TNF‐α can significantly enhance the expression of genes related to cell adhesion, namely ICAM and VCAM, in the stromal cells of the endometrium by inducing NF‐kB signaling during the early stages of inflammation [66, 69, 70]. Linalool showed potential as a therapeutic agent for inflammatory conditions in endometriosis by modulating NF‐kB signaling and reducing the expression of adhesion molecules such as ICAM‐1. By inhibiting NF‐kB activation, linalool could lower levels of pro‐inflammatory cytokines like TNF‐α, which are known to enhance adhesion molecule expression in endometrial tissues. This reduction in adhesion molecules could mitigate abnormal tissue adhesion associated with endometriosis. Research on aloe‐emodin and aspirin showed similar reductions in ICAM‐1 levels in endometriosis models [66].

Angiogenesis is considered a key process in the pathogenesis of endometriosis. Many factors are involved in this complex mechanism, and vascular endothelial growth factor (VEGF) is a crucial mediator of angiogenesis [71, 72]. Macrophages have a major role in the expression of VEGF, and in macrophages, VEGF production can occur through multiple pathways. It seems that peritoneal macrophages and activated lymphocytes play a significant role in the secretion of pro‐inflammatory/angiogenic cytokines. For instance, in patients with endometriosis, IL‐1β is produced by activated macrophages, leading to increased expression of VEGF [73]. In a mouse model of endometriosis, it was reported that IL‐6 along with TNF‐α is secreted by macrophages, resulting in positive regulation of VEGF from neutrophils and infiltrating macrophages [74]. Another pathway that regulates VEGF expression is the mitogen‐activated protein kinases (MAPKs). MAPKs induce local transcription factors in the nucleus by activating the expression of specific target genes that need to be modified. MAPK signaling pathways can play various roles in inflammatory responses mediated by immunity, cell proliferation, cell differentiation, angiogenesis, and apoptosis, which are cellular events associated with endometriosis [75]. This approach has been demonstrated by Leconte and colleagues, showing that the MAPK/ERK signaling pathway can be activated via VEGFR and influence angiogenesis, promoting the growth of endometrial lesions [76]. Studies have shown that increased activation of NF‐kB is associated with upregulation of COX‐2 levels, which plays an important role in the synthesis of prostaglandin E2 (PGE2). Increased expression of PGE2 can, through the upregulation of VEGF and estrogen alongside inhibiting apoptosis and lymphocyte proliferation, contribute to the development of vascularization [77, 78, 79]. Linalool exerted anti‐angiogenic effects in endometriosis by modulating pro‐inflammatory cytokines like TNF‐α, which are produced by activated macrophages and enhance VEGF expression. Linalool's anti‐inflammatory properties could lead to reduced levels of these cytokines, thereby lowering VEGF and angiogenesis associated with ectopic endometrial tissue. Additionally, linalool may influence macrophage activity, disrupt MAPK signaling pathways, and inhibit the NF‐kB pathway, which is linked to COX‐2 and PGE2 upregulation. By targeting these multiple pathways, linalool presents a multifaceted approach to mitigating angiogenesis in endometriosis, aligning with our findings. Consistent with our results, studies on cannabidiol and thalidomide reported comparable decreases in VEGF levels [80, 81].

Although the rat uterine autotransplantation model provides a reproducible platform for evaluating lesion development and therapeutic interventions, it represents only selected aspects of human endometriosis. Human endometriosis is a highly heterogeneous disorder characterized by distinct lesion subtypes, variable hormonal responsiveness, complex immune interactions, and diverse clinical manifestations. Therefore, while the present findings demonstrate promising biological effects of linalool in an experimental setting, caution is required when extrapolating these results directly to clinical practice. Validation in human‐derived experimental systems and clinical studies will be necessary to establish the translational relevance of these findings.

5. Conclusion

This study set out to determine the treatment effects of linalool on endometriosis in rats. The results of this investigation show that linalool as a potent antioxidant agent could significantly reduce endometriosis lesions by decreasing oxidative stress, inflammatory signaling, angiogenic markers, and changes in apoptosis and autophagy‐related proteins, suggesting potential involvement of these pathways in its protective effects. Taken together, these results suggest that linalool could be used as an agent to diminish endometriosis lesions in rats. A limitation of this study is that the sample size is relatively small and a prior statistical analysis for sample size choosing was not performed. Moreover, gene expression of markers was not evaluated. Since no pathway‐specific inhibitors, genetic interventions, or autophagy flux assays were performed, these results should be interpreted as correlative rather than causal. In addition, although the selected linalool doses led to a significant biological effect, pharmacokinetic parameters, tissue bioavailability, or comprehensive toxicity profiles were not assessed in the present study. Hence, the optimal therapeutic dose, safety margin, and systemic exposure are suggested to be evaluated in future studies. More research using controlled trials is needed to examine the effects of linalool in other species such as humans.

Author Contributions

Reza Tadayonfar, Mahdi Bahrami, and Elahe Sobhani: conceptualization, methodology, investigation, writing – original draft. Abbas Raisi: project administration, methodology, investigation, visualization, data curation. Omid Dezfoulian: validation, software, writing – review and editing, supervision. Farshid Davoodi: conceptualization, methodology, investigation, writing – review and editing, formal analysis. Arash Kheradmand: investigation, writing – review and editing. Mohammad Kamalpour: methodology, writing – review and editing.

Funding

This work was supported by Lorestan University, grant no. LU‐9611511010‐2024.

Ethics Statement

This study was carried out at Lorestan University and was approved by the Animal Ethics Committee of the Faculty of Veterinary Medicine, Lorestan University (LU.ECRA.2023.25).

Consent

The authors have nothing to report.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Data S1: fsb272247‐sup‐0001‐Supinfo.pdf.

Data Availability Statement

All data generated or analyzed during this study are included in this published article.