The immortalized cell line hEM15A provides a consistent and reproducible experimental system, allowing repeated experiments without the variability or limited lifespan inherent to primary cells. This reproducibility makes it an ideal model for studying EMs-related mechanisms, such as sensitivity to ferroptosis. In this study, the human ESCs line hEM15A (PN-H156A, Wuhan Punosei Life Technology, Wuhan, China) was cultured in a complete medium specifically formulated for hEM15A. Cells were maintained under standard conditions in a CO₂ incubator at 37 °C with 5% CO₂. When the cells in a T25 flask reached 90% confluency, the spent medium was removed and cells were washed twice with 2 mL of phosphate-buffered saline (PBS; PN-SH30256.01, Hyclone, Logan, UT, USA). After removing the PBS, 1 mL of 0.25% trypsin (PN-25200056, Gibco, Shanghai, China) was added and the cells examined through a microscope (Thermo Fisher, Hillsboro, OR, USA) for 1–3 minutes until they became rounded in shape. To neutralize the trypsin, 5 mL of complete culture medium (PN-CPM-HES5, Procell, Wuhan, China) was added and the cells were gently pipetted to ensure proper collection.

The cell mixture was spun at 800 rpm for 5 minutes at 4 °C and the medium was then removed. The cell pellet was resuspended in complete culture medium and transferred to culture flasks, with the medium replaced every other day. For cryopreservation, the cells were frozen in a mixture consisting of 95% complete culture medium and 5% DMSO (PN-CPS-HESD5, Procell, China) and stored in liquid nitrogen. Each experiment utilized cells during the exponential growth stage. The cell line was validated by short tandem repeat (STR) profiling and tested negative for mycoplasma.

For the Erastin group, hEM15A cells were treated with 20 µmol/L of the ferroptosis inducer Erastin (S7242, Selleck, Houston, TX, USA) for 24 h. The overexpression (OE)-SLC1A5+Erastin and OE-negative control (NC)+Erastin groups were generated by transfecting Erastin group cells with SLC1A5 OE adenovirus vector (G31424, GenePharma, Shanghai, China) and an empty (NC) vector (GenePharma, G00000), respectively. Similarly, transfection of the Erastin group with SLC1A5 knockdown (KD) vector (GenePharma, G31425) or an empty (NC) vector (GenePharma, G00000) created the KD-SLC1A5+Erastin and KD-NC+Erastin groups, which used siRNA. The siRNA sequences were as follows: Sense strand: 5^′-GUCGACCAUAUCUCCUUGAdTdT-3^′; Antisense strands: 5^′-UCAAGGAGAUAUGGUCGACdTdT-3^′. The negative control siRNA (si-NC), which does not target any human gene, was also provided by GenePharma with the following sequences: Sense strand: 5^′-UUCUCCGAACGUGUCACGUdTdT-3^′; Antisense strand: 5^′-ACGUGACACGUUCGGAGAAdTdT-3^′. The Erastin+N-acetylcysteine (NAC) group was established by treating Erastin group cells with 5 mmol/L NAC, a ROS scavenger (Beyotime, Shanghai, China, S0077), for 24 h [17].

hEM15A cells undergoing exponential growth were placed into 96-well plates at 5 $\times$ 10³ cells per well and grown in a 5% CO₂ environment at 37 °C for 24 h. The culture medium in each well was then replaced with fresh medium containing 20 µM Erastin and incubated for an additional 24 h. Following this, 10 µL of Cell Counting Kit-8 (CCK-8) solution was added to each well containing 100 µL of cell culture medium, and the cells incubated for 2 h. Absorbance at 450 nm was then measured using a microplate reader (Thermo Fisher, USA) [18].

hEM15A cells treated with Erastin were washed with 1 mL of PBS. A 200 nM working solution of Mito-Tracker Red CMXRos (mitochondrial ROS red fluorescent probe, Beyotime, China, C1035) was prepared. The cells were incubated with 1 mL of the working solution at 37 °C in a 5% CO₂ incubator for 30 minutes, followed by three washes with PBS. Fluorescence was observed under a fluorescence microscope (Thermo Fisher, USA) within 2 h of staining.

Cell culture supernatants from each group were collected and analyzed using the human Malondialdehyde (MDA) ELISA Kit (Hnybio, Shanghai, China, HNY-E1035) according to the manufacturer’s instructions. Light absorption at 450 nm was measured with an RT-6000 plate reader (RT-6000, Rayto, Shenzhen, China) [19].

Total cellular RNA was extracted using Trizol reagent (9109, Takara Bio, Shiga, Japan) and purified by chloroform phase separation, isopropanol precipitation, and washing with 75% ethanol. The RNA concentration and purity were assessed with a NanoDrop 2000 device (Thermo Fisher Scientific, USA, ND-2000) by measuring the 260/280 absorbance ratio. A ratio between 1.9 and 2.1 was considered to indicate satisfactory purity.

The purified RNA was reverse transcribed into cDNA using the HiScript III First Strand cDNA Synthesis Kit (R312-01, Vazyme, Nanjing, China). The reverse transcription reaction was carried out by incubating at 25 °C for 5 minutes, 42 °C for 30 minutes, and 85 °C for 5 minutes. Quantitative real-time PCR (qPCR) was then conducted with the CFX96 Touch system (1855195, Bio-Rad, Hercules, CA, USA).

The qPCR process involved initial heating at 95 °C for 30 seconds, followed by 40 cycles of melting at 95 °C for 10 seconds, binding at 60 °C for 30 seconds, and lengthening at 72 °C for 15 seconds. Melting curve analysis was performed by holding at 60 °C for 60 seconds, then heating to 95 °C for 15 seconds. Experimental results were calculated using the 2^{-Δ⁢Δ⁢Ct} method [20]. Specific primers for PCR amplification were synthesized based on the sequences listed in Table 1.

Cells were washed three times with ice-cold PBS and lysed on ice for 30 min in RIPA buffer (Beyotime, China, P0013B) containing PMSF (Beyotime, ST506) and a cocktail of protease inhibitors (P8340, Sigma, Burlington, MA, USA). Lysates were centrifuged at 12,000 rpm for 15 min at 4 °C, and the supernatants collected. Protein concentrations were measured using the Enhanced BCA Protein Assay Kit (Beyotime, P0010), and 40 µg aliquots were stored at –80 °C. Samples were mixed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (Beyotime, P0015L), boiled at 95 °C for 10 min, separated on 10% SDS-PAGE gels, and transferred to 0.45 µm Polyvinylidene Fluoride (PVDF) membrane. Membranes were blocked with 5% nonfat milk in Tris-Buffered Saline with Tween 20 (TBST) for 1 h at room temperature, then incubated overnight at 4 °C with the following primary antibodies: anti-SLC1A5 (1:1000, Abcam, Cambridge, UK, ab237704), anti-c-Myc (1:1000, Abcam, UK, ab32072), and anti-GPX4 (1:1000, Abcam, UK, ab125066), and anti-$\beta$-actin (1:5000, Abcam, UK, ab8227), and anti-GAPDH (1:5000, Abcam, UK, ab181602) as an internal reference for loading control. After three 15-min washes with TBST, membranes were incubated with Horseradish Peroxidase (HRP)-conjugated secondary antibodies (Bioss, Beijing, China, bs-0295G-HRP) or 1–2 h at room temperature. Protein bands were detected using an ECL kit (NCM Biotech, Suzhou, China, P10300), quantified with ImageJ (v1.5, National Institutes of Health, Bethesda, MD, USA), and normalized to GAPDH and $\beta$-actin [21].

The cell supernatant was collected and standard curves for Gln and glutamate (Glu) were generated using the Glutamine Assay Kit (Colorimetric, Abcam, UK, ab197011) and the Glutamate Assay Kit (Colorimetric, Abcam, UK, ab83389), respectively. For each measurement, 50 µL of either the standard or sample was pipetted into a 96-well plate. The reaction mix was prepared and added according to the respective kit instructions. The plates were then incubated at 37 °C for 30 minutes in the dark, and absorbance was measured at 450 nm using an enzyme labeling instrument (4376600, Thermo Fisher Scientific, Vantaa, Uusimaa, Finland) [22].

Cell culture supernatants from each group were analyzed using the human Glutathione (GSH) ELISA Kit (Hnybio, Shanghai, China, HNY-E1080) according to the manufacturer’s instructions, with absorbance measured at 450 nm using the RT-6000 microplate reader (Rayto, China, RT-6000). The E-BC-K773-M Ferrous Ion Colorimetric Assay Kit (Elabscience Biotechnology, Wuhan, China, E-BC-K773-M) was used to evaluate ferrous ion (Fe²⁺) content by measuring absorbance at 593 nm, as described in the kit instructions [22].

To prepare the working solution for evaluation of mitochondrial membrane potential, 30 µL of JC-1 (200X, Beyotime, China, C2006) was added to 4.8 mL of dilution buffer, followed by the addition of 1.2 mL of JC-1 staining buffer (5$\times$, Beyotime, China, C2006). One mL of the JC-1 staining working solution was then added to the hEM15A cell culture and incubated at 37 °C for 20 minutes. After incubation, the cells were washed with 1 mL of JC-1 staining buffer (1$\times$, Beyotime, China, C2006) and observed under a fluorescence microscope (Thermo Fisher, USA) [23].

To assess c-Myc binding to SLC1A5, cells were fixed with 1% formaldehyde (Beyotime, P0099) for 10 min, quenched with 0.125 M glycine (Sigma, G8898), and lysed in SDS buffer (Beyotime, P0013) with protease inhibitors (Beyotime, P2076). Chromatin was sheared by sonication and then centrifuged. The resulting supernatant was pre-cleared by incubating with 900 µL ChIP Dilution Buffer, 20 µL 50$\times$ Protease Inhibitor Cocktail (PIC) (78430, Thermo Fisher Scientific, Waltham, MA, USA), and 60 µL Protein Agarose/Salmon Sperm DNA (Beyotime, P2076) for 1 h at 4 °C. After adding 1 µL of antibody, the samples were incubated overnight at 4 °C. Immune complexes were washed (Beyotime, P2076) and eluted with 100 µL 10% SDS, 100 µL 1 M NaHCO₃, and 800 µL ddH₂O. Cross-links were reversed at 65 °C overnight with 20 µL 5 M NaCl. DNA was purified by incubation with 10 µL 0.5 M EDTA, 20 µL 1 M Tris-HCl (pH 6.5), and 2 µL proteinase K (Beyotime, ST506) at 45 °C for 2 h, then analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) [24].

The estrogen-driven reproductive cycle in rats aligns with the hormonal basis of human EMs, making it a relevant system for studying lesion dynamics and therapeutic responses. To establish the rat EMs model, specific pathogen free (SPF)-grade SD rats weighing 160–200 g aged 6–8 weeks were purchased from the Laboratory Animal Co., Ltd. (Guangzhou, Guangdong, China). The animals were housed at 20–25 °C with 40–70% humidity, a 10 h light cycle, and ad libitum access to food and water. After a one-week acclimation period, the rats were randomly assigned to the EMs model group (n = 15) and control group (sham surgery group) (n = 5). Daily oral administration of estradiol valerate (0.1 mg/kg, Sigma-Aldrich, USA, Cat#: E1631) for 3 days was used to synchronize the estrous cycle and stimulate endometrial proliferation. Following isoflurane anesthesia (3% induction and 1.5–2% maintenance, Lunan Better Pharmaceutical Co., Ltd., Guangzhou, China), the route of administration is inhalation, a 0.5 cm² piece of endometrial tissue from the left uterine horn was transplanted to the right abdominal wall in the EMs group. The sham surgery group underwent the same procedure but without endometrial tissue transplantation. Two weeks post-surgery, the abdominal tissues were harvested and hematoxylin and eosin (HE) histopathology was performed to confirm successful development of the EMs model. Rats were euthanized humanely in accordance with animal welfare guidelines using carbon dioxide (CO₂) inhalation followed by cervical dislocation.

Two weeks post-surgery, EMs model rats were treated daily with intraperitoneal Erastin (40 mg/kg) [25] alone (n = 5) or in combination with NAC (250 mg/kg) (n = 5) for 28 consecutive days to establish the respective treatment groups. The experimental protocols were approved by the Medical Ethics Review Board of The Tenth Affiliated Hospital, Southern Medical University (Dongguan People’s Hospital) (Approval No. IACUC-AWEC-202410028) [26].

Tissue samples were fixed, rinsed, and preserved in 70%–80% ethanol. They underwent graded ethanol dehydration and xylene clearing before paraffin infiltration and embedding. Sections (4 µm) were prepared, mounted on slides, and subjected to HE staining [27].

For the evaluation of ROS in tissue sections, DCFH-DA (Thermo Fisher Scientific, D6883) was diluted 1:1000 in serum-free medium to 10 µmol/L. Tissue sections were incubated with 200 µL of this solution at 37 °C for 30 min in a Thermo Fisher Heracell VIOS 160i incubator, allowing DCFH-DA to react with intracellular ROS. After three washes with PBS (Thermo Fisher, 10010023), ROS fluorescence was imaged using an Olympus (Guangzhou, Guangdong, China) IX83 microscope (excitation 488 nm, emission 525 nm) [28].

The volume of abdominal ectopic EM lesions was measured using an ultrasound imaging system, followed by laparotomy under isoflurane anesthesia (3% induction, 1.5–2% maintenance) for lesion exposure. Pelvic adhesions were quantified on a scale from 0 (absent) to 10 (extensive), based on the degree of adhesion around the ectopic lesions and intestines. A score of 0 indicated no adhesions, while a score of 10 indicated severe adhesions involving the mesentery and peritoneum [29].

To quantify SLC1A5 and c-Myc expression in rat EM lesions, RT-qPCR was performed using the FastPure Tissue Total RNA Isolation Kit (Beyotime, China). Total RNA was extracted from 10 mg of fresh tissue with 500 µL Buffer RL, homogenized, and purified through FastPure RNA Columns III with sequential washes (700 µL Buffer RW1, 700 µL then 500 µL Buffer RW2 with ethanol). RNA was eluted with 30 µL RNase-free ddH₂O and quantified using a NanoDrop 2000 instrument (Thermo Fisher Scientific, USA). For cDNA synthesis, 500 ng RNA was reverse-transcribed in a 20 µL reaction volume (5$\times$ gDNA wiper mix, Hiscript III Enzyme Mix, Oligo(dT)20VN, Random hexamers) at 37 °C for 15 min and 85 °C for 5 s. RT-qPCR was conducted in a 20 µL system with 5 µL diluted cDNA, 2$\times$ Taq Pro Universal SYBR qPCR Master Mix, and specific primers (Table 2), using the CFX96 Touch system. The cycling conditions were: 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s, 60 °C for 30 s, and 95 °C for 15 s, with melt curve analysis from 65 °C to 95 °C. Expression was analyzed using the 2^{-Δ⁢Δ⁢Ct} method. Additional details can be found in the Supplementary Material.

The Glutamine Assay Kit (Colorimetric) (ab197011) and Glutamate Assay Kit (ab83389) (Abcam, UK) were used to measure Gln and Glu, respectively. Approximately 0.1 g of tissue was blended with 1 mL of Reagent 1 provided in the assay kit in a chilled bath. The mixture was spun at 10,000 rpm for 10 minutes at 4 °C, and the upper liquid collected for further testing. Absorbance at 450 nm was measured using an RT-6000 microplate reader (Rayto, China, RT-6000) [30].

Blood was collected from the eyeball of rats and allowed to stand at room temperature for 30 minutes to 2 h, avoiding vigorous shaking to prevent hemolysis. Once the blood clotted naturally and the serum separated, the sample was centrifuged for 10 min at 4 °C and 2000 rpm. The supernatant was collected and placed on ice for later use. Standards were prepared by diluting in a 96-well plate according to the instructions provided in the ELISA kit manuals. The following ELISA kits were used: rat TNF-$\alpha$ ELISA Kit (PT516, Beyotime, China), rat IL-6 ELISA Kit (PI328, Beyotime, China), and rat IL-1$\beta$ ELISA Kit (PI303, Beyotime, China). Light absorption at 450 nm was determined with a Rayto RT-6000 plate reader (China).

Results were shown as the mean $\pm$ SD, and calculated and graphed with GraphPad Prism 9 (Dotmatics, Boston, MA, USA). The t-test was used to compare two groups, while one-way ANOVA examined differences across multiple groups. In cases of non-normal data, the Kruskal-Wallis test was applied, followed by Dunn’s post hoc test. p-values below 0.05 indicated statistical significance: *p 0.05, **p 0.01, and ***p 0.001 [25].

We first investigated the mechanism by which Erastin induces ferroptosis in hEM15A cells. The CCK-8 assay revealed that treatment with Erastin reduced the cell proliferation rate (Fig. 2A), while RT-qPCR showed that it increased the expression of SLC1A5 mRNA (Fig. 2B). Additionally, Erastin was observed to increase ROS levels in hEM15A cells, as indicated by ROS fluorescent probes (Fig. 2C,D). These findings suggest that Erastin increases ROS levels in hEM15A cells, possibly via upregulation of SLC1A5.

Fig. 2.

Cellular responses, SLC1A5 expression, and metabolic and mitochondrial functions of hEM15A cells under different treatment conditions, including Erastin treatment. (A) The Cell Counting Kit-8 (CCK-8) assay revealed reduced cell viability after treatment of hEM15A cells with Erastin, indicating induction of ferroptosis. (B) RT-qPCR showed that Erastin treatment increased the expression of SLC1A5 mRNA. (C,D) Erastin treatment increased the reactive oxygen species (ROS) level in hEM15A cells, as indicated by the fluorescence intensity of ROS-specific probes (C1035). Scale bar: 50 µm. (E–G) Glu and Gln levels were significantly increased in hEM15A cells following Erastin treatment, with the overexpression (OE)-SLC1A5+Erastin group showing further upregulation and the knockdown (KD)-SLC1A5+Erastin group showing reduced levels (p 0.001). Additionally, Erastin decreased the GSH level in hEM15A cells. Overexpression of SLC1A5 also downregulated GSH, whereas SLC1A5 knockdown increased GSH (p 0.001). Experimental Groups: Control; Erastin; OE-SLC1A5+Erastin group, OE-SLC1A5: SLC1A5 overexpression; OE-negative control (NC)+Erastin group, OE-NC: empty (NC) vector; KD-SLC1A5+Erastin group, KD-SLC1A5: SLC1A5 KD; KD-NC+Erastin group, KD-NC: empty (NC) vector. (H,I) The mitochondrial membrane potential was evaluated using JC-1 dye, with results presented as histograms and fluorescent images. Erastin treatment decreased the mitochondrial membrane potential in hEM15A cells, with no change observed in empty vector-transfected cells. Overexpression of SLC1A5 lowered the mitochondrial membrane potential, whereas SLC1A5 knockdown increased the mitochondrial membrane potential, suggesting that Erastin regulates mitochondrial Gln metabolism via SLC1A5. Scale bar: 50 µm. **p 0.01, ***p 0.001, n = 3.

We next investigated the role of SLC1A5 in Gln metabolism. The levels of Glu and Gln were significantly increased in hEM15A cells (p 0.001) following Erastin treatment (Fig. 2E,F). Compared to the OE-NC+Erastin group, the OE-SLC1A5+Erastin group showed significantly upregulated levels of Glu and Gln (p 0.001), while knockdown of SLC1A5 expression in the KD-SLC1A5+Erastin group reduced the levels of Glu and Gln (p 0.001). Erastin also decreased the level of GSH in hEM15A cells (p 0.001) (Fig. 2G). Overexpression of SLC1A5 downregulated the GSH level, while knockdown of SLC1A5 increased the level (p 0.001). GSH is a primary cellular antioxidant that plays a critical role in protecting cells from oxidative stress by neutralizing ROS and supporting the function of glutathione peroxidase 4 (GPX4), a key enzyme that inhibits ferroptosis [11].

Erastin treatment decreased the mitochondrial membrane potential in hEM15A cells, but it remained unchanged in empty vector-transfected cells (Fig. 2H,I). Overexpression of SLC1A5 lowered the mitochondrial membrane potential in hEM15A cells, whereas knockdown of SLC1A5 raised the potential. These findings indicate that Erastin regulates mitochondrial Gln metabolism via SLC1A5.

After modulating the expression of SLC1A5, ROS levels in each hEM15A cell group were assessed using ROS fluorescent probes. As shown in Fig. 3A,B, Following Erastin treatment, overexpression of SLC1A5 increased the ROS levels in hEM15A cells, which was reversed upon SLC1A5 knockdown (p 0.001). As shown in Fig. 3C,D, Erastin significantly increased the levels of Fe²⁺ and MDA (p 0.001). Overexpression of SLC1A5 further enhanced the levels of Fe²⁺ and MDA, whereas knockdown of SLC1A5 reduced these levels.

Fig. 3.

Oxidative stress markers and protein expression in hEM15A cells under different treatment conditions. (A,B) ROS levels in hEM15A cells with modulated SLC1A5 expression were assessed using fluorescent probes. No significant differences were observed between the empty vector and Erastin-treated groups, indicating that NC did not alter the mitochondrial ROS level. Scale bar: 50 µm. (C,D) Erastin treatment significantly increased Fe²⁺ and MDA levels in hEM15A cells (p 0.001). Overexpression of SLC1A5 further increased these levels, whereas SLC1A5 knockdown reduced them. (E) Western blot and quantitative analysis of c-Myc and GPX4 protein levels. Erastin treatment increased c-Myc expression and inhibited GPX4 expression in hEM15A cells. SLC1A5 overexpression significantly increased c-Myc (p 0.01) and decreased GPX4 (p 0.001), whereas SLC1A5 knockdown reduced c-Myc and increased GPX4 expression (both p 0.01). **p 0.01, ***p 0.001, n = 3.

Western blot analysis was employed to assess the expression levels of c-Myc and the ferroptosis-related protein GPX4. The latter is a key regulator of ferroptosis and acts as a potent inhibitor, thus playing a critical role in its modulation [31]. Erastin induced c-Myc expression and inhibited GPX4 expression in hEM15A cells (Fig. 3E). After transfection with the SLC1A5 overexpression plasmid, c-Myc expression increased significantly (p 0.001), while GPX4 expression decreased (p 0.01). Conversely, silencing of SLC1A5 in hEM15A cells reduced c-Myc expression and increased GPX4 expression (both p 0.001).

The above findings indicate that Erastin increases ROS, c-Myc, and SLC1A5 expression. To explore the mechanism involved, the ROS scavenger NAC was used to intervene in Erastin-treated hEM15A cells. ChIP-PCR experiments revealed that c-Myc binds to the promoter region of SLC1A5, thereby increasing its transcription. SLC1A5 mRNA expression was suppressed after ROS levels were reduced by NAC (Fig. 4A,B) (p 0.01). Additionally, Western blot analysis showed that NAC treatment downregulated c-Myc expression in hEM15A cells (Fig. 4C; p 0.001). These results indicate the presence of a “ROS/c-Myc/SLC1A5” pathway in hEM15A cells.

Fig. 4.

Regulation of SLC1A5 expression and its impact on cellular metabolites in Erastin-treated hEM15A cells. (A,B) ChIP analysis showed that c-Myc binds to the promoter region of SLC1A5, thereby promoting its transcription. Treatment with the ROS scavenger N-acetylcysteine (NAC) reduced ROS levels and suppressed SLC1A5 mRNA expression (p 0.01). (C) Western blot analysis showed that treatment with NAC downregulated c-Myc expression in hEM15A cells (p 0.001). (D–H) Treatment of hEM15A cells with NAC significantly downregulated Glu, Gln, Fe²⁺, and MDA levels, while upregulating the GSH level (p 0.001). (I) Western blot analysis showed that ROS clearance with NAC resulted in upregulation of GPX4 expression in hEM15A cells. *p 0.05, **p 0.01, ***p 0.001, n = 3.

To investigate whether this pathway promotes ferroptosis, hEM15A cells were treated with Erastin, and ROS was neutralized with NAC. Key markers of Gln metabolism and ferroptosis were then evaluated. Significant downregulation of Glu, Gln, Fe²⁺, and MDA levels were observed, while GSH levels increased (Fig. 4D–H; p 0.001; Fig. 4). Western blot analysis also revealed upregulation of GPX4 expression following ROS clearance (Fig. 4I). These findings further confirm that Erastin enhances the sensitivity of hEM15A cells to ferroptosis via the ROS/c-Myc/SLC1A5 pathway.

HE staining revealed ectopic endometrial tissue in the rat EM model, with glandular structures resembling normal endometrium but displaying an irregular morphology. The glands were surrounded by stromal tissue and displayed fibrotic changes and signs of tissue repair. Extravasation of red blood cells and hemosiderin deposition were also observed. No such pathological changes were observed in the Control group (Fig. 5A), thus confirming the establishment of a rat EMs model. The volume of ectopic EMs lesions was measured using an ultrasound imaging system, and pelvic adhesions were scored according to the Haber scale. The lesion volume in the EMs group was 89.5 mm³ and the adhesion score was 8.3. This indicates a severe degree of pelvic adhesion, which is a common complication of EMs (Fig. 5B). SLC1A5 and c-Myc expression in the ectopic lesions of the EMs model were significantly lower compared to the Control group (Fig. 5C,D; p 0.01). Furthermore, serum Gln and Glu levels were significantly lower in the EMs lesions (Fig. 5E; p 0.01; Fig. 5). These findings suggest that SLC1A5 and c-Myc are involved in the pathogenesis of EMs.

Fig. 5.

Histopathological characteristics, lesion volume, adhesion score, and molecular characteristics of a rat endometriosis (EMs) model. (A) Hematoxylin and eosin (HE) staining and pathological changes in ectopic EMs lesions. These revealed disordered heterotopic tissue with signs of repair and fibrosis. No such changes were observed in the Control group, thus confirming successful establishment of the rat EMs model. Scale bar: 100 µm. (B) Ectopic lesion volume and pelvic adhesion were assessed using an ultrasound imaging system, with scoring based on the Haber criteria. The boundaries of the lesions are marked using white dots. The average lesion volume in the EMs group was 89.5 $\pm$ 13.3 mm³ and the average adhesion score was 8.3 $\pm$ 0.6, indicating severe pelvic adhesion. This is a common complication of EMs. (C,D) Detection of SLC1A5 and c-Myc expression by RT-qPCR and Western blot. SLC1A5 and c-Myc expression were significantly lower in the EMs group compared to the Control group (p 0.01). (E) Measurement of Gln and Glu levels using an ELISA kit. Serum Gln and Glu levels were significantly lower in the EMs model compared to the Control. **p 0.01, ***p 0.001, n = 3.

To investigate the effect of Erastin on rats with EMs, Erastin alone or in combination with NAC was administered intraperitoneally. Ectopic EMs lesions in the Erastin group exhibited structural abnormalities, interstitial porosity, and epithelial cell necrosis (Fig. 6A). The improvement in ectopic lesions in the Erastin+NAC group was less pronounced than in the Erastin alone group. Moreover, the lesion volume and pelvic adhesion score in the Erastin only group (21.2 $\pm$ 2.0 mm³ and 2.7 $\pm$ 0.2, respectively) were significantly lower compared to the EMs Model group (94.8 $\pm$ 13.5 mm³ and 8.3 $\pm$ 0.6) (Fig. 6B; p 0.01). When NAC was used to clear ROS from the ectopic tissue, the lesion size increased from 21.2 $\pm$ 2.0 mm³ in the Erastin only group to 34.9 $\pm$ 2.6 mm³ in the Erastin+NAC group, while the pelvic adhesion score increased from 2.7 $\pm$ 0.2 to 4.3 $\pm$ 0.4 (p 0.05).

Fig. 6.

Evaluation of the therapeutic effect of Erastin in a rat model of EMs, and the effect of ROS. (A) Erastin treatment induced structural abnormalities, interstitial porosity, and epithelial cell necrosis in ectopic EMs lesions. This improvement was less pronounced in the Erastin+NAC group compared to the Erastin group. Scale bar: 100 µm. (B) Ectopic lesion volume and pelvic adhesion were assessed using an ultrasound imaging system, with scoring based on the Haber criteria. The boundaries of the lesions are marked using white dots. The lesion volume and pelvic adhesion score were significantly reduced in the Erastin group compared to the EM Model and Erastin+NAC groups (p 0.01). (C) ROS levels were assessed using a fluorescent probe. Erastin significantly increased ROS levels in ectopic EM lesions compared to the Control group, while the Erastin+NAC group showed a substantial decrease in ROS, indicating effective ROS clearance by NAC (p 0.001). Scale bar: 50 µm. (D,E) SLC1A5 and c-Myc expression were measured by RT-qPCR and Western blot. The Erastin group showed significantly higher mRNA and protein expression of SLC1A5 and c-Myc compared to the EMs Model group, while the Erastin+NAC group exhibited significantly lower levels (p 0.05). (F) Measurement of serum TNF-$\alpha$, IL-6, and IL-1$\beta$ levels using an ELISA kit. The Erastin group showed significantly higher levels of TNF-$\alpha$, IL-6, and IL-1$\beta$ compared to the EMs Model group, while NAC treatment significantly reduced these inflammatory factors by eliminating ROS (p 0.001). *p 0.05, **p 0.01, ***p 0.001, n = 3.

Fluorescent probes showed that Erastin significantly increased ROS levels in ectopic tissues from the EMs Model compared to the Control group (Fig. 6C). Conversely, the Erastin+NAC group exhibited a substantial decrease in ROS level, indicating that NAC effectively cleared ROS (p 0.001; Fig. 6). The Erastin group displayed notably elevated SLC1A5 and c-Myc mRNA and protein levels compared to the EM Model group (Fig. 6D,E). However, these levels were significantly lower in the Erastin+NAC group compared to the Erastin group (p 0.05; Fig. 6). The Erastin group exhibited significantly higher serum levels of TNF-$\alpha$, IL-6, and IL-1$\beta$ than the EMs Model group (Fig. 6F). NAC significantly reduced the levels of these inflammatory factors by eliminating ROS (p 0.001; Fig. 6). The above results suggest that Erastin improves EMs in a rat model by modulating the ROS/c-Myc/SLC1A5 pathway.