{"paper_id":"d9c67217-f7cf-48a4-98f6-af5bd6e153e3","body_text":"The presence of endometrial tissue outside the uterine cavity is the defining characteristic of endometriosis. Epidemiological data indicate that 5%–10% of women of reproductive age are afflicted by endometriosis, resulting in various complications, such as discomfort, irregular menstruation, dysmenorrhea, and infertility ( Moradi  et al. , 2021 ;  Harder  et al ., 2024 ). Additionally, 75% of these women experience difficulties in conceiving, often due to disruptions in the process of folliculogenesis ( Smolarz  et al. , 2021 ;  Yin  et al. , 2021 ;  Cano  et al. , 2024 ). Seventy-five percent of these women experience difficulties in conceiving, occasionally due to issues with the folliculogenesis process. The pathophysiological mechanisms underlying endometriosis remain ambiguous.\nAdditionally, the peritoneal fluid (PF) exhibits increased concentrations of reactive oxygen species (ROS), interleukin-1β (IL-1β), and tumor necrosis factor (TNF)-α ( Oală  et al. , 2024 ). This increases the concentration of these chemicals in the ovaries. Increased oxidative stress and lipid peroxidation in follicular fluid can negatively impact oocyte quality and potentially contribute to infertility in endometriosis. The use of F2-isoprostanes (FiSOPs) through immunohistochemical examination can be used as a biomarker for lipid peroxidation and oxidative stress in tissues or cells ( Dymanowska  et al ., 2024 ;  Moretti  et al. , 2025 ).\nThe increase in inflammatory cytokines and ROS will accelerate apoptosis in the follicles via both extrinsic and intrinsic routes, thereby disrupting the process of folliculogenesis ( Smolarz  et al ., 2021 ;  Bonavina and Taylor, 2022 ;  Dymanowska  et al ., 2024 ). Elevated apoptosis in the ovaries, resulting in follicular abnormalities, contributes to a decline in oocyte quality ( Bonavina and Taylor, 2022 ;  Didziokaite  et al. , 2023 ). Treatment to address the disruption of the folliculogenesis process in endometriosis has not yet been successful ( Dymanowska  et al. , 2024 ).\nSaussurea lappa  is a significant medicinal plant commonly employed in traditional medicine in many developing Asian countries. It is used in its entirety, as rhizomes, or as roots. Various chemical constituents, including costunolide, dehydrocostus lactone, and santamarin, are present after extraction ( Hassan and Masoodi, 2020 ;  Okubo  et al. , 2021 ;  Fitrianingsih  et al. , 2024 ).  Saussurea lappa  is a widely used medicinal plant in traditional medicine across Asia. It contains bioactive compounds such as costunolide, dehydrocostus lactone, and santamarin. These compounds are believed to reduce the levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-6), ROS, and caspase-3, thereby suppressing apoptosis and potentially promoting folliculogenesis ( Hassan and Masoodi, 2020 ;  Jo  et al. , 2020 ;  Nadda  et al. , 2020 ;  Semwal  et al. , 2020 ;  Abdul  et al. , 2023 ;  Elshaer  et al. , 2024 ).\nHowever, the processes and effects of the promotion of folliculogenesis by  S. lappa  remain unknown, necessitating further investigation for a thorough understanding. This study aimed to elucidate the mechanism by which  S. lappa  extract influences ovarian follicle growth in a mouse model of endometriosis, with the potential to enhance folliculogenesis in ovaries affected by endometriosis.\n\nDry roots of  S. lappa  were obtained from local herbs in Indonesia, and taxonomic identification was performed using the deoxyribose nucleotide acid (DNA) sequencing method in the BRIN laboratory (Indonesian National Research and Innovation Agency) cleaned with running water to remove attached dirt. Samples were dried without being exposed to direct sunlight, cut into small pieces, and ground until they became powder. Then, 200 g of powder was dissolved in 1000 ml of 70% ethanol in a 1,000 ml Erlenmeyer flask, and sonicated with an ultrasonicator for 30 minutes at 45°C, and repeated three times. The sample was filtered using Whattman filter paper, concentrated using a rotary vacuum evaporator at 45°C, and macerated for 5 days at room temperature until it was colorless and a thick extract was obtained ( Abubakar and Haque, 2020 ).\nGas chromatography-mass spectrometry (GC-MS) has emerged as an essential analytical technique across various fields, including environmental science, food quality assessment, and pharmaceutical research. Components in the 70% ethanol extract of  S. lappa  were qualitatively identified based on peak areas from GC-MS analysis with the Agilent 5977B Series MSD ( Omer  et al. , 2019 ;  Mohsen  et al. , 2022 ;  Sukmawati and Muflihunna, 2024 ).\nThe research methodology used is a true experimental study employing a post-test-only control group design. This study used 32 female mice (Mus musculus, BALB/c strain), aged approximately 12 weeks and weighing between 25 and 30 g, to determine the sample size using the resource equation method. This research was conducted in the research laboratory of the Faculty of Veterinary Medicine at Airlangga University. On the first day, the mice were administered an intramuscular injection of cyclosporine A (Sandimmune; Novartis, Basel, Switzerland) at a dose of 10 mg/kg, along with 5.4 g of estrogen (continued until day 5) at a dose of 5.4 µg. Subsequently, 0.1 ml of an endometrial isolate (human endometrial tissue) was intraperitoneally injected to establish an endometriosis model. The administration of cyclosporine A as an immunosuppressant during the first few days (up to day 5) was intended to provide time for the endometrial tissue to adhere and establish a blood supply before the immune response from the mice begins to work. The immune system of mice that reacts after the initial implantation period was expected to resemble the immune system changes that occur in endometriosis ( Burns  et al ., 2022 ). On day 14, the endometriosis model was finalized, characterized by the proliferation of endometriotic tissue in the peritoneum ( Dwiningsih  et al. , 2021 ;  Burjiah  et al ., 2022 ). This study included four groups: one control group (K1) and three treatment groups (K2, K3, and K4). On day 15,  S. lappa  extract was orally administered at doses of 200 (K2), 400 (K3), and 600 (K4) mg/kg for 21 days. This dose was obtained from several published studies that have some desired effects, and no toxicity was found in the animal models ( Mammate  et al ., 2023 ;  Elshaer  et al. , 2024 ). After treatment completion, the experimental animals underwent ovariectomy, with the specimens preserved in 10% formalin. A visual macroscopic examination was conducted to determine the occurrence of endometriosis in mice, which shows images such as nodules or implants and areas of bleeding or reddish discoloration, found on the peritoneal wall, uterus, and ovaries ( Yan  et al ., 2019 ;  Burns  et al. , 2022 ).\nImmunohistochemistry and TUNEL assay were conducted to assess the concentrations of FiSOP, TNF-α, and apoptosis in granulosa cell (GCs). A visual/manual scoring semi-quantitative method was used to assess the immunohistochemistry ( Taylor and Levenson, 2006 ;  Crowe and Yue, 2019 ).\nFurthermore, hematoxylin-eosin staining was used to quantify primary, secondary, and transcription factor across all groups. Subsequently, a statistical study was conducted to evaluate the impact of  S. lappa  extract on folliculogenesis.\nThe Research Ethics Committee of the Faculty of Medicine at Airlangga University in Surabaya, Indonesia, approved the experimental protocol no. 2.KEH.010.01.2024.\n\nAll documented data were meticulously analyzed over three trials. Data were analyzed using SPSS version 26.0 and Smart PLS version 3. A one-way analysis of variance (ANOVA) was conducted, followed by a Bonferroni post hoc test to minimize the occurrence of false positives. ANOVA showed a significant difference in this parameter with a  p -value of 0.000. The post hoc Bonferroni test indicated that K1 was significantly different from K3 and K4, with a  p -value of 0.000, as was the difference between K2 and K3 and K4, with  p -values of 0.003 and 0.000, respectively. The best treatment for the reduction of TNF-α, FiSOP, and apoptosis in GCs is K4, which showed the lowest results.\nThe normality test for FiSOP, TNF-α, and apoptosis data indicated a normal distribution ( p  > 0.05). Furthermore, statistical analysis revealed that  S. lappa  extract significantly affected the levels of FiSOP, TNF-α, and apoptosis in GCs at all stages of follicle development. The GC/MS analysis of the 70% ethanol extract from the dried root of  S. lappa  ( Fig. 1 ) showed a 99% concordance in the qualitative profile. The analysis revealed the following compounds: 1. dehydrocostus lactone, 2. dehydrosausurea lactone, 3. beta-cyclodihydrocostunolide, 4. beta-cyclocostunolide, 5. alpha-costol, 6. beta-costol, and 7. Santamarin ( Fig. 2 ). All constituent chemicals belong to the sesquiterpene lactone category.\nComparison of FiSOP levels ( Fig. 3 ) in GCs between the control group (K1) and treatment groups administered  with S. lappa  70% ethanol extract at dosages of 200 (K2), 400 (K3), and 600 (K4) mg/kg. The primary follicles in the groups that received 400 (K3) and 600 (K4) mg/kg had lower levels, while the secondary follicles in the 600 mg/kg group (K4) also had lower levels, and the tertiary follicles ( Fig. 4 ) at 400 (K3) and 600 mg/kg (K4) had lower levels compared to the control group (K1).\nFigure 5  contrasts the TNF-α levels in GCs from the control group (K1) with those in the treatment groups receiving  S. lappa  70% ethanol extract dosages of 200 (K2), 400 (K3), and 600 (K4) mg/kg. The primary follicles in the 600 mg/kg group (K4) exhibited reduced levels, and tertiary follicles ( Fig. 6 ) displayed decreased levels at 200 mg/kg (K2) compared with the control group (K1). The secondary follicles at the 600 mg/kg dosage (K4) demonstrated reduced levels relative to the control group (K1).\nApoptosis levels ( Fig. 7 ) in GCs were compared between the control group (K1) and the treatment groups with  S. lappa  70% ethanol extract at dosages of 200 (K2), 400 (K3), and 600 (K4) mg/kg. In all treatment groups (K2, K3, and K4) administered with  S. lappa  extract, the primary and secondary follicles exhibited reduced apoptosis levels compared with the control group (K1). The tertiary follicles ( Fig. 8 ) at the 600 mg/kg dosage (K4) demonstrated reduced levels relative to the control group (K1).\nThe number of primary, secondary, and tertiary follicles ( Fig. 9 ) was higher in all groups that received  S. lappa  extract (K2, K3, and K4) than in the control group (K1). The treatment group with a dose of 600 mg/kg (K4) had the highest number of primary, secondary, and tertiary follicles compared with the treatment groups receiving doses of 200 (K2) and 400 (K3) mg/kg, as well as the control group (K1).\n\nThe pathophysiology of endometriosis is still not fully understood, but increasing evidence suggests that a mix of hormonal, immunological, genetic, and environmental factors play a role in the initiation and progression of endometriosis ( Tian  et al. , 2021 ;  Bonavina and Taylor, 2022 ;  Esmaeilzadeh  et al. , 2022 ;  Fan  et al. , 2023 ;  Dymanowska  et al ., 2024 ;  Oală  et al ., 2024 ). Evidence indicates that individuals with endometriosis exhibit atypical immunological and inflammatory responses. Elevated levels of proinflammatory cytokines, such as TNF-α, IL-6, IL-1β, and IL-17A, have been observed in blood serum, PF, and ectopic lesions ( Bonavina and Taylor, 2022 ;  Dymanowska  et al. , 2024 ;  Oală  et al. , 2024 ).\nThe GC/MS analysis of  S. lappa  extract qualitatively identified several sesquiterpene lactone group compounds that have significant therapeutic effects, particularly in relation to anti-inflammatory and oxidative stress inhibition, both of which are crucial for managing various chronic diseases. This inhibition reduces the levels of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, indicating that it could be useful in treating inflammation-related issues. Sesquiterpene lactones, especially dehydrocostus lactone and costunolide, have anti-inflammatory, antioxidant, and anti-apoptotic effects ( Paço  et al ., 2022 ).\nIn the follicular fluid of women with endometriosis, vascular endothelial growth factor levels diminish, whereas TNF-α, IL-1, IL-8, IL-6, monocyte chemoattractant protein-1, and endothelin-1, along with various natural killer cells, B lymphocytes, and monocytes, exhibit elevated levels that impair folliculogenesis, resulting in infertility ( Prins  et al. , 2020 ;  Tian  et al. , 2021 ;  Kacem  et al. , 2023 ;  Brinca  et al. , 2024 ).\nHeme and iron compounds induce oxidative stress, which significantly contributes to endometriotic lesions during retrograde menstruation. Excessive oxidative stress can induce iron influx that modifies lipids and proteins, resulting in DNA damage and cellular apoptosis.\nConsequently, it will enhance the apoptosis process in the ovaries, especially within the follicles ( Galaris  et al. , 2019 ;  Yan  et al ., 2022 ;  Gensluckner  et al. , 2024 ).\nROS cause mitochondrial membrane depolarization and activate Bax/Bak channels on the outer mitochondrial membrane, promoting the release of cytochrome c and Smac/Diablo into the cytoplasm. Cytochrome c subsequently forms an apoptosome complex with APAF-1 in the cytosol and activates procaspase-9 into caspase-9, which is essential for the conversion of procaspase-3 into caspase-3 ( Mustafa  et al. , 2024 ;  Xu  et al. , 2024 ).\nFiSOPs are recognized as precise indicators of oxidative stress, reflecting the extent of lipid peroxidation in organisms. The measurement method is crucial for comprehending numerous diseases, particularly those resulting from oxidative stress that harms tissues and organs ( Saleem  et al. , 2023 ;  Ahmad  et al. , 2024 ). GCs in the primary, secondary, and tertiary follicles exhibited reduced levels of FiSOP upon treatment with a 70% ethanol extract of  S. lappa  ( Fig. 3 ). The treatment groups with doses of 400 (K3) and 600 (K4) mg/kg had the most pronounced decrease in FiSOP expression compared with the other groups. The impact of oxidative stress is an essential factor in GC apoptosis. An imbalance exists between ROS production and antioxidant defenses, leading to cellular damage. This occurrence is referred to as oxidative stress ( Regan  et al. , 2018 ).\nTNF-α will bind to tumor necrosis factor receptor (TNFR-1), thereby activating the TNFR-associated death domain (TRADD). The construction of the complex, including TNF-α, TNFR-1, and TRADD/Fas-associated protein with death domain (FADD), will trigger nuclear factor kappa-light-chain-enhancer of activated B cells activation. Cellular apoptotic signaling triggered by TNF-α is facilitated by TNFR-1. The engagement of TNFR-1 facilitates the assembly of the intracellular death-inducing signaling complex, which comprises TRADD, FADD, and TNFR-associated factor-1. The complex activates pro-caspase 8 into caspase 8 and influences the permeability of the mitochondrial membrane, resulting in the release of cytochrome c. Caspase 8 cleaves BH3-interacting death (BID) into truncated BID and activates pro-caspase 3 into caspase 3 ( Webster  et al. , 2020 ;  Vachliotis and Polyzos, 2023 ;  Guerrache and Micheau, 2024 ). GCs in the primary, secondary, and tertiary follicles had reduced TNF-α levels when exposed to a 70% ethanol extract of  S. lappa  ( Fig. 5 ). The GCs of primary follicles in the treatment group receiving a dosage of 600 mg/kg (K4) had the lowest levels of TNF-α. The GCs of secondary follicles exhibited no significant reduction, whereas the GCs of tertiary follicles demonstrated the lowest levels at 200 mg/kg (K2).\nGC apoptosis is essential for ovarian function, affecting follicular growth, hormone release, and general reproductive health. Apoptosis, or programed cell death in these cells, is a vital mechanism that can profoundly influence fertility. The mechanisms and conditions that affect apoptosis in GCs, emphasize the complexity of this phenomenon in both healthy and pathological circumstances ( Carlberg  et al. , 2000 ;  Regan  et al. , 2018 ). Apoptosis in GCs is a complex process by multiple molecular pathways, including caspases, miRNAs, and signaling systems. Malfunctioning or excessive apoptosis can result in follicular atresia and infertility ( Liu  et al. , 2023 ). The lowest level of GCs apoptosis was found in primary follicles receiving a dose of 600 mg/kg (K4); secondary follicles showed the lowest levels at doses of 200 (K2) and 400 (K3) mg/kg, while tertiary follicles showed the lowest levels at a dose of 600 mg/kg (K4). During the later stages of folliculogenesis, the oocyte is surrounded by several layers of GCs that differentiate into mural and cumulus GCs. GCs supply nutrients and substances essential for the maturation and developmental competence of the oocyte, while also safeguarding the oocyte from oxidative stress damage through their intrinsic antioxidant system throughout maturation ( Cavalcanti  et al. , 2023 ).\nThe GC/MS examination was a qualitative analysis. Therefore, to achieve reproducibility, an accurate determination of the material species was required, which was done through a DNA sequencing examination, followed by the preparation of extracts according to the established protocol, and then a qualitative GC/MS examination ( Monagas  et al ., 2022 ). The extract of  S. lappa contains  many constituents that can reduce ROS, TNF-α activity, and apoptosis ( El-Rahman  et al. , 2020 ;  Ashry  et al. , 2022 ). Active metabolites  of S. lappa , including costunolide and dehydrocostus lactone, exhibit anti-inflammatory properties. Sesquiterpene exhibits anti-inflammatory properties in vitro by facilitating the release of nitric oxide (NO) and TNF-α from their complexes, whereas santamarin demonstrates anti-inflammatory effects by promoting the expression of mRNA heme oxygenase, inhibiting NO reduction, which subsequently leads to the production of inducible NO synthase, suppressing cyclooxygenase-2, decreasing prostaglandin E2, and inhibiting the synthesis of TNF-α, IL-1β, and IL-6, which are critical mediators of inflammation ( El-Rahman  et al. , 2020 ,  Liu  et al. , 2021 ,  Gu  et al. , 2024 ,  Xu  et al ., 2024 ,  Vu  et al. , 2024 ). The application of  S. lappa  extract in endometriosis, particularly for enhancing folliculogenesis, remains unavailable. The results of this study are anticipated to facilitate the use of  S. lappa  extract as a treatment for GC disruption in follicles associated with endometriosis, which impairs follicle development.\n\nEndometriosis elevates the levels of FiSOP, TNF-α, and apoptosis in ovarian follicle GCs, thereby impairing folliculogenesis. Oral administration of a 70% ethanol extract from dried  S. lappa  root at doses of 400 (K3) and 600 (K4) mg/kg significantly decreased the levels of FiSOP, TNF-α, and apoptosis in the GCs of primary, secondary, and tertiary follicles, while simultaneously enhancing the follicle count, thus facilitating folliculogenesis.","source_license":"CC0","license_restricted":false}