The protective effects of ritodrine against hypoxia/reoxygenation-induced injury in endometrial stromal cells

Abstract

Endometriosis (EMS) is often observed in women of childbearing age and significantly impacts patients’ quality of life. Ritodrine is a β2 receptor agonist applied for relaxing the uterine smooth muscle. Its inhibitory effects on inflammation have recently been noted. The present study explored the protective impact of Ritodrine on hypoxia/reoxygenation (H/R)- induced injury in endometrial stromal cells (ESCs). Human ESCs (HESCs) were treated with Ritodrine (0.1, 0.5 μM) for 24 h, followed by exposure to H/R for 6 h. Ritodrine ameliorated H/R-induced higher reactive oxygen species (ROS), declined glutathione (GSH) concentration and increased production of tumor necrosis factor-α (TNF-α), interleukin- 6 (IL-6), and monocyte chemotactic protein 1 (MCP-1) in HESCs. Furthermore, Ritodrine ameliorated the H/R-induced higher nuclear level of nuclear factor κ-B (NF-κB) p65 expression and increased luciferase activity of the NF-κB promoter. In addition, we show that Ritodrine mitigated H/R-induced higher estrogen receptor α (ER-α) expression in HESCs. Interestingly, overexpressing ER-α abolished the regulatory effects of Ritodrine on oxidative stress and the NF-κB pathway-mediated inflammation. Collectively, our data reveal that Ritodrine alleviated H/R-induced injury in ESCs by inhibiting the ER-α/NF-κB pathway.

Introduction

Endometriosis (EMS) is a common disease observed in women of childbearing age. It is mainly characterized by endometrial tissue implantation outside the uterine cavity, accompanied by dysmenorrhea, lower abdominal pain, dyspareunia, and even infertility. ¹ Although EMS has been recognized for several decades, its pathogenesis remains unclear. ² The lack of an adequate understanding of the pathophysiological changes of EMS contributes to the shortage of treatment strategies. In recent years, work has shown that oxidative stress plays an essential role in the pathogenesis of EMS.^3,4 Under healthy conditions, balancing reactive oxygen species (ROS) production and antioxidant function in endometrial tissue is critical to regulating oxidative stress in the uterus. However, during the progression of EMS, the abnormal production of ROS and reproductive toxins could lead to endometrial tissue injury, which ultimately disturbs menstruation and dislocation of injured endometrial tissue into the peritoneal cavity. The peritoneal production of ROS could further stimulate aberrant endometrium development. ⁵ Particularly, substantial reactive oxygen species (ROS) are released by endometrial stromal cells (ESCs) in ectopic endometrial tissues, which induces ectopic endometrial tissue in the abdominal cavity and other extrauterine locations. Moreover, the release of inflammatory cytokines and aggravation of the inflammatory response are triggered by the released ROS, which contributes to the ectopic endometrial tissues’ production of more ROS to form a vicious cycle. ⁵ Furthermore, it is well reported that the progression of EMS can be relieved by agents targeting oxidative stress. ⁶ NF-κB pathway-mediated inflammation is another crucial pathological mechanism of EMS reported in recent years. Nanda et al. ⁷ reported that the expressions of multiple cytokines in EMS were enhanced under oxidative stress, and the excessive degradation of the extracellular matrix promoted the invasion process of intima cells. These pathological changes are closely associated with NF-κB signaling under oxidative stress, further affecting the enhancement of matrix metalloproteinases (MMP) expression of the downstream target molecules. Furthermore, intercellular adhesion molecule1 (ICAM-1) which is secreted by ESCs, can be targeted by NF-κB to impact the endometrial invasion by regulating cell-cell interactions, cell-surface receptors interactions, and extracellular matrix interactions.^8,9 Further studies show that a considerable number of exosomes is involved in the development of EMS through the NF-κB pathway to affect adhesion and invasion responses. Exosomal miR-22–3p derived from peritoneal macrophages activates NF-κB and enhances the expression of MMP, which participates in the degradation process of extracellular matrix and enhances the invasion ability of ESCs. ¹⁰ Therefore, oxidative stress and NF-κB pathway-mediated inflammation might be effective targets for treating clinical EMS.

Ritodrine is a drug used to prevent premature labor. Ritodrine is a β2 receptor agonist that binds to the β2 receptors on the membrane of uterine myocytes to inhibit contractions. In endometrial tissue, recent work demonstrates that β2 receptors signaling plays important roles in the development of endometriosis and is potentially a target for intervention.^11,12 Hypoxia occurs during pregnancy and the uterus itself is sensitive to hypoxia/ischemia events. ¹³ In the mouse model, Ritodrine treatment suppresses uterine contraction, which is required for endometrial regeneration, indicating the potential action of this drug on endometrial tissue. ¹³ Recently, a promising inhibitory effect on the release of inflammatory factors has been observed during clinical treatment using Ritodrine. ¹⁴ These data suggest that Ritodrine treatment could influence endometrial tissue and have anti-inflammatory effects.

Endometrial stromal cells (ESCs) are exposed to hypoxia during placentation in the uterus. Thus, hypoxia/regeneration-exposed endometrial stromal cells can be used as an in vitro model to investigate the mechanism of endometriosis. ¹⁵ Estrogen receptors (ERα and ERβ) are essential for female reproduction. ERα mediates endometrial growth and implantation. ERα -deficient or -overexpressing stromal cells have been used to investigate its role in HESCs. ¹⁶ We established a hypoxia/regeneration (H/R) model and introduced ERα into cultured ESCs. In this study, we investigate the protective impact of Ritodrine on H/R-induced injury in ESCs to excavate the possibility of treating EMS with Ritodrine.

Materials and methods

Cell culture, treatment, and transduction

Primary human ESCs (HESCs) were obtained from the Beijing Crisprbio Biotechnology Co., Ltd (Beijing, China) and cultured in RPMI1640 medium supplemented with 5% FBS under 37°C and 5% CO₂. To achieve ER-α overexpressed HESCs, cells were transduced with (lentiviral ER-α) for 48 h, followed by verification using the western blotting assay. The treatment reagent Ritodrine (Sigma-Aldrich, USA) was dissolved in phosphate-buffered saline (PBS). Dose responsive cells were challenged with Ritodrine at the concentrations of 0.01, 0.05, 0.1, 0.5, 1.0, 2.0 μM for 24 h. For all other experiments, the cells were treated with Ritodrine (0.1, 0.5 μM) for 24 h, followed by exposure to H/R.

Establishment of the H/R model

HESCs were plated in a 60 mm cultural dish and allowed to reach confluence. The hypoxia treatment of the HESCs model was described as previously documented. ¹⁷ In brief, the culture dish was incubated in a modular chamber (Thermo Scientific, USA) containing humidified hypoxic air (1% Oxygen, 5% Carbon Dioxide, and 94% Nitrogen) for 6 h. Subsequently, cells were subjected to a normoxic condition (20% O₂, 5% CO₂, and 75% N₂) of reoxygenation for 24 h.

3-(45)-dimethylthiahiazo (-z-y1)-35-di- phenytetrazoliumromide (MTT) assay

HESCs were implanted in a 96-well plate for 24 h, followed by adding the MTT solution. After incubation for 4 h, cells were introduced with 150 μL DMSO. Lastly, the spectrophotometer (Shanghai Metash Instruments Co., Ltd, Shanghai, China) was utilized to measure the OD value at 570 nm.

2,7-Dichlorodi -hydrofluorescein diacetate (DCFH-DA) assay

The ROS method of measurement in HSECs was described as previously documented. ¹⁸ Briefly, the culture medium was removed and rinsed with warm PBS twice. Diluted DCFH-DA (1:1000) was added and incubated at 37°C for 20 min in the dark. Cells were digested with 0.25% trypsin for 1 min, and the digestion was terminated by adding a serum-containing medium. The collected cell precipitate was suspended again, and the fluorescence intensity (excitation wavelength was 488 nm, emission wavelength was 525 nm) was detected using a fluorescence microplate analyzer (Molecular Devices, Sunnyvale, USA).

Enzyme linked immunosorbent assay (ELISA)

The level of reduced GSH, TNF-α, IL-6, and MCP-1 was determined using an ELISA assay (Mibio, Shanghai, China). In brief, the required lath was removed from the aluminum foil bag after balancing for 60 min at room temperature, and the remaining lath was sealed using a self-sealing bag at 4°C. After 50 μL adding different concentrations of the standard to each well and 50 μL test sample into each well; 50 μL biotin-labeled antibody was introduced, followed by incubating at 37°C for 30 min. After discarding the liquid and three washes, 100 μL horseradish peroxidase (HRP) labeled detection antibody was added to each well of standard and sample wells. The reaction wells were then sealed with a sealing plate membrane and incubated at 37°C for 30 min. After discarding the liquid and three washes, 50 μL substrate A and substrate B were added to each well and incubated at 37°C for 15 min in the dark. After 50 μL adding stop solution to each well, the optical density (OD) value of each well was measured at 450 nm wavelength within 15 min using a microplate analyzer (Molecular Devices, Sunnyvale, USA).

Real-time PCR

Following extracting RNAs from HESCs with the TRIZOL reagent (Leagene, Beijing, China), the cDNA transcription kit (MedChem Express, USA) was utilized to transcribe the isolated RNAs to cDNAs, followed by performing the PCR reaction with SYBR Green Real-time PCR Master Mix (Roche Diagnostics, Basel, Switzerland) using the ABI 7900 real-time PCR machine. Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) was utilized to normalize gene expressions, which were calculated with the 2^−ΔΔCt method.

Western blotting assay

Cells were lysed with cell lysate for 30 min, and the nuclear contents were obtained with nuclear lysate. The lysate was transferred to a 1.5 mL centrifuge tube for centrifugation at 12,000 r/min at 4°C for 5 min. The total protein concentration of the supernatant was determined with the bicinchoninic acid (BCA) method. After 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separation, proteins were transferred to a polyvinylidene fluoride (PVDF) membrane, which was sealed with 5% skim milk powder and shaken at room temperature for 1.5 h. The PVDF membrane was removed and rinsed with tris-buffered saline and tween (TBST) solution 3 times. NF-κB p65 (1:2000, Affinity, Melbourne, Australia), ER-α (1:1000, Affinity, Melbourne, Australia), and Tubulin (1:10,000, Affinity, Melbourne, Australia), and Lamin B1 (1:5000, Cell Signaling Technology, USA) primary antibodies were added overnight at 4°C. TBST solution was added for rinsing 3 times, and the second antibody was added and incubated at room temperature for 1.5 h. PVDF membrane was rinsed with TBST solution 3 times, and bands were obtained after exposure to enhanced chemiluminescence (ECL) solution, which was further quantified using the Image J software.

Luciferase activity of NF-κB promoter

NF-κB promoter-luciferase construct (pNF-κB-Luc) plasmid was obtained from a commercial source (Cat# 219,078; Agilent Technologies, USA). The transfection of the plasmid was performed using the FuGENE HD Transfection Reagent (Promega, USA) according to the manufacturer’s instructions. In brief, HESCs were transfected with 0.25 ×g pNF-κB-Luc and cultured for 48 h, followed by centrifugation at l 000 r/min for 5 min to collect the cells. Subsequently, cells were washed twice with PBS and 250 μL Reporter lysis buffer was added, followed by 15 min incubation at room temperature. After centrifugation at 4°C at 1000 r/min for 5 min, 20 μL cell lysates were collected and placed in the determination tube, followed by adding 100 μL luciferase reaction substrate. The luminescence intensity was lastly determined by a fluorescence analyzer (Molecular Devices, Sunnyvale, USA).

Statistical analysis

Achieved data were expressed as mean±standard deviation (SD), followed by analysis with the GraphPad software using the one-way analysis of variance (ANOVA) method with Bonferroni corrected post-hoc test. p < 0.05 was considered a significant difference in the present study.

Results

The cytotoxicity of Ritodrine in human endometrial stromal cells

Firstly, HESCs were challenged with Ritodrine at the concentrations of 0.01, 0.05, 0.1, 0.5, 1.0, 2.0 μM for 24 h to determine the optimized incubation concentration of Ritodrine. The molecular structure of Ritodrine is illustrated in Figure 1(a). We found the cell viability changed slightly as the concentration of Ritodrine elevated from 0.01 μM to 0.5 μM, but was greatly reduced when the concentration was higher than 1.0 μM (Figure 1(b)). Therefore, in the subsequent assays, 0.1 μM and 0.5 μM were utilized as the incubation concentrations.

Figure 1.

The cytotoxicity of Ritodrine in human endometrial stromal cells (HESCs). Cells were challenged with Ritodrine at the concentrations of 0.01, 0.05, 0.1, 0.5, 1.0, 2.0 μM for 24 h (A). Molecular structure of Ritodrine; (B). Cell viability in MTT assay (*, **, p < 0.01, 0.001 vs. vehicle group).

Ritodrine alleviated H/R-induced oxidative stress in HESCs

HESCs were treated with Ritodrine (0.1, 0.5 μM) for 24 h, followed by exposure to H/R for 6 h. A higher ROS level was observed in H/R-stimulated HESCs (Figure 2(a)), which was greatly repressed by 0.1 and 0.5 μM Ritodrine. Furthermore, the declined level of reduced GSH (Figure 2(b)) in H/R-stimulated HESCs was dramatically reversed by 0.1 and 0.5 μM Ritodrine. These results suggest that the H/R-induced oxidative stress in HESCs was alleviated by Ritodrine.

Figure 2.

Ritodrine alleviated hypoxia/reoxygenation (H/R)- induced oxidative stress in human endometrial stromal cells (hESCs). Cells were treated with Ritodrine (0.1, 0.5 μM) for 24 h, followed by exposure to H/R for 6 h (A). The levels of reactive oxygen species (ROS) were measured using 2,7-Dichlorodi -hydrofluorescein diacetate (DCFH-DA); (B). The levels of reduced glutathione (GSH) (※, p < 0.0001 vs. vehicle group; *, **, p < 0.05, 0.01 vs. H/R group).

Ritodrine repressed H/R-induced inflammation in HESCs

Inflammation along with oxidative stress in HESCs was reported to be closely associated with the development of EMS. ¹⁹ We found that TNF-α, IL-6, and MCP-1 (Figure 3(a)) were dramatically upregulated in H/R-stimulated HESCs, all of which were greatly downregulated by 0.1 and 0.5 μM Ritodrine. The protein levels of TNF-α, IL-6, and MCP-1 are shown in Figure 3(b). The level of TNF-α was promoted from 119.6 pg/mL to 322.7 pg/mL by the stimulation with H/R, then greatly reduced to 215.6 and 169.4 pg/mL by 0.1 and 0.5 μM Ritodrine, respectively. Furthermore, the concentrations of IL-6 in the control, H/R, 0.1, and 0.5 μM Ritodrine groups were 101.1, 255.9, 159.1, and 129.2 pg/mL, respectively. Lastly, the level of MCP-1 was increased from 77.5 pg/mL to 199.3 pg/mL by the stimulation with H/R, then greatly declined to 128.7 and 105.8 pg/mL by 0.1 and 0.5 μM Ritodrine, respectively. These data suggest that the inflammation induced by H/R in HESCs was alleviated by Ritodrine.

Figure 3.

Ritodrine repressed hypoxia/reoxygenation (H/R)- induced inflammation in human endometrial stromal cells (hESCs). Cells were treated with Ritodrine (0.1, 0.5 μM) for 24 h, followed by exposure to H/R for 6 h (A). mRNA of tumor necrosis factor-α (TNF-α), interleukin- 6 (IL-6), and monocyte chemotactic protein 1 (MCP-1); (B). Protein levels of tumor necrosis factor-α (TNF-α), interleukin- 6 (IL-6), and monocyte chemotactic protein 1 (MCP-1) (※, p < 0.0001 vs. vehicle group; *, **, p < 0.05, 0.01 vs. H/R group).

Ritodrine inhibited H/R-induced NF-κB activation in HESCs

NF-κB signaling is a critical inflammatory pathway, ²⁰ which is reportedly involved in the development of EMS. ⁷ An upregulated nuclear level of NF-κB p65 (Figure 4(a)) was observed in H/R-stimulated HESCs, which was significantly reversed by 0.1 μM and 0.5 μM Ritodrine. Furthermore, the luciferase activity of the NF-κB promoter (Figure 4(b)) was greatly activated by the stimulation with H/R, but greatly repressed by 0.1 μM and 0.5 μM Ritodrine. These data suggest that the H/R-induced NF-κB activation in HESCs was inhibited by Ritodrine.

Figure 4.

Ritodrine inhibited hypoxia/reoxygenation (H/R)- induced nuclear factor -κB (NF-κB) activation in human endometrial stromal cells (hESCs). Cells were treated with Ritodrine (0.1, 0.5 μM) for 24 h, followed by exposure to H/R for 6 h (A). Nuclear levels of NF-κB p65; (B). Luciferase activity of NF-κB promoter (※, p < 0.0001 vs. vehicle group; *, **, p < 0.05, 0.01 vs. H/R group).

Ritodrine downregulated the H/R-induced elevated expression level of ER-α in HESCs

ER-α is an important nuclear receptor regulating the NF-κB pathway ²¹ and pathological processing of EMS. ²² We found that the expression level of ER-α (Figure 5) was dramatically elevated by H/R, but greatly suppressed by 0.1 μM and 0.5 μM Ritodrine, indicating that ER-α might be involved in the functional mechanism of Ritodrine.

Figure 5.

Ritodrine downregulated hypoxia/reoxygenation (H/R) -induced elevated expression level of estrogen receptor α (ER-α) in human endometrial stromal cells (hESCs). Cells were treated with Ritodrine (0.1, 0.5 μM) for 24 h, followed by exposure to H/R for 6 h (A). mRNA of ER-α; (B). Protein of ER-α (※, p < 0.0001 vs. vehicle group; *, **, p < 0.05, 0.01 vs. H/R group).

Overexpression of ER-α abolished the effect of Ritodrine on H/R-induced oxidative stress in HESCs

To identify the involvement of ER-α in the functional mechanism of Ritodrine, HESCs were transduced with lentiviral ER-α with or without the treatment with Ritodrine (0.5 μM) for 24 h, followed by exposure to H/R for 6 h. The transfection efficacy was identified according to Western blotting results (Figure 6(a)). The increased ROS level (Figure 6(b)) in H/R-stimulated HESCs was greatly repressed by Ritodrine, which was significantly reversed by the overexpression of ER-α. Furthermore, the declined GSH level in H/R-stimulated HESCs (Figure 6(c)) was dramatically promoted by Ritodrine, which was pronouncedly reversed by the overexpression of ER-α. These data suggest that the effect of Ritodrine on H/R-induced oxidative stress in HESCs was abolished by the overexpression of ER-α.

Figure 6.

Overexpression of estrogen receptor α (ER-α) abolished the effect of Ritodrine on hypoxia/reoxygenation (H/R) -induced oxidative stress in HESCs. Cells were transduced with lentiviral ER-α with or without the treatment with Ritodrine (0.5 μM) for 24 h, followed by exposure to H/R for 6 h (A). The efficacy of transfection was confirmed by western blotting. (B). The levels of reactive oxygen species (ROS) were measured using 2,7-Dichlorodi -hydrofluorescein diacetate (DCFH-DA); (C). The levels of reduced glutathione (GSH) (※, p < 0.0001 vs. vehicle group; **, p < 0.01 vs. H/R group; &&, p < 0.01 vs. H/R+ Ritodrine group).

Overexpression of ER-α reversed the effects of Ritodrine on H/R-induced NF-κB activation in HESCs

As shown in Figure 7(a), we found that the protein level of TNF-α in H/R-stimulated HESCs was increased from 125.1 pg/mL to 365.3 pg/mL and further decreased to 132.2 pg/mL by Ritodrine treatment, which was reversed to 235.1 pg/mL by the overexpression of ER-α. The levels of IL-6 in the control, H/R, Ritodrine, and Ritodrine+ER-α groups were 95.6, 276.3, 132.2, and 235.1 pg/mL, respectively. The protein level of MCP-1 in H/R-stimulated HESCs was elevated from 82.1 pg/mL to 205.6 pg/mL and further declined to 103.9 pg/mL by Ritodrine treatment, which was reversed to 175.1 pg/mL by the overexpression of ER-α. Furthermore, the promoted luciferase activity of the NF-κB promoter in H/R-stimulated HESCs (Figure 7(b)) was dramatically repressed by Ritodrine, which was greatly reversed by the overexpression of ER-α. These results suggest that the effects of Ritodrine on H/R-induced NF-κB activation in HESCs were abolished by the overexpression of ER-α Figure 8.

Figure 7.

Overexpression of estrogen receptor α (ER-α) reversed the effect of Ritodrine on hypoxia/reoxygenation (H/R) -induced NF-κB activation in HESCs. Cells were transduced with lentiviral ER-α with or without the treatment with Ritodrine (0.5 μM) for 24 h, followed by exposure to H/R for 6 h (A). Protein levels of tumor necrosis factor-α (TNF-α), interleukin- 6 (IL-6), and monocyte chemotactic protein 1 (MCP-1). (B). Luciferase activity of NF-κB promoter (※, p < 0.0001 vs. vehicle group; **, p < 0.01 vs. H/R group; &&, p < 0.01 vs. H/R+ Ritodrine group).

Figure 8.

The protective effects of Ritodrine against hypoxia/reoxygenation (H/R)- induced injury in human endometrial stromal cells (hESCs).

Discussion

Ischemia/Reperfusion (I/R) is an important mechanism that causes cell death and tissue injury. It is known that I/R-induced cardiac injury is characterized by mitochondrial permeability alteration, overproduction of reactive oxygen species (ROS), imbalance of intracellular ions, and an irregular immune response.^23–27 The endometrium experiences repeated regeneration during the estrous or menstrual cycle. The endometrium is exposed to hypoxia upon menstruation and during placentation in the uterus. ESCs have the capacity to differentiate into other cell lineages. The I/R-induced ESCs have been used as pharmaceutical targets of endometriosis.^15,28

Recent studies have found that oxidative stress is a prevalent factor involved in EMS patients. Oxidative stress refers to an imbalance of the redox reaction rate and excessive production of ROS, resulting in damage to DNA, proteins, and lipids, as well as influence on gene expression and protein function. ²⁹ A series of studies have pointed out that significantly increased levels of oxidation-related markers were observed in the peritoneal fluid, follicular fluid, and peripheral circulating blood of EMS patients,^30–33 accompanied by a decrease in antioxidant levels. As indicated in a prospective study, the concentration of advanced oxidized protein products in the peritoneal fluid is positively correlated with the total number of deep intestinal infiltrating EMS lesions. ³¹ Carvalho et al. ³⁴ detected 36 biomarkers related to oxidative stress and found that the expression levels of 23 biomarkers were significantly increased in EMS patients, suggesting that a peroxide state was observed in EMS patients. Ovarian endometriotic cyst is one of the most common types of EMS. Studies have found that when the ROS production rate is greater than the endogenous antioxidant system, the excessively produced ROS damage peritoneal mesothelium cells to promote ectopic endometrium growth. ⁶ Furthermore, as second messengers, ROS activate protein kinase B (AKT) and NF-κB signaling to facilitate the development of EMS.^35,36 We found that oxidative stress was significantly triggered in H/R-stimulated HESCs, which was consistent with the description reported by Li under the condition of hydrogen peroxide. ³⁷ After treatment with Ritodrine, oxidative stress was dramatically mitigated, suggesting a protective effect of Ritodrine on H/R-induced oxidative damage to HESCs.

NF-κB binds to IκB in most resting cells, and IκB binds to the nuclear localization signal (NLS) at the end region of NF-κB through its anchor protein, which causes the NLS to induce the inactivated NF-κB remaining in the cytoplasm. ³⁸ In response to TNF-α, IL-1, LPS, growth factors, and viral infection, phosphorylation of IκB induces its degradation by specific kinases, which induces the transfer of free p50/p65 dimer to the nucleus. Subsequently, NF-κB p65 regulates the expression of target genes by binding to the κB site in the promoter region of the target gene. ³⁹ Both mRNA and protein expressions of NF-κB p65 were increased both in situ and in the ectopic endometrium of EMS patients. NF-κB directly or indirectly participates in the expression and regulation of multiple genes, especially inflammatory and immune-related genes. The present study found that NF-κB signaling was dramatically activated in HESCs by stimulation with H/R, accompanied by the increased production of inflammatory factors, which conformed to the observation under the condition of hypoxia reported by Li ¹⁹ this year. The NF-κB-mediated inflammation in H/R-stimulated HESCs was greatly ameliorated by Ritodrine, implying its inhibitory effect against H/R-induced inflammation in HESCs by repressing NF-κB signaling.

In recent years, Guzeloglu-Kayisli et al. ⁴⁰ reported the interaction between the estrogen receptor (ER) and NF-κB. Kitawaki et al. ⁴¹ showed in their study in 2003 that endometriosis is an estrogen-dependent disease. Clinically, it was found that ectopic lesions gradually disappeared in patients after menopause or oophorectomy, and pregnancy or ER inhibitors could temporarily delay the development of EMS. ⁴² Estrogen plays an important role in the occurrence and development of EMS. It impacts the process of endometrial implantation, invasion, and distant metastasis by binding with ER. ⁴³ As one of the subunits of ER, ER-α is evidently involved in the development of EMS. ²² Furthermore, ER-α is reported to regulate NF-κB pathway-mediated inflammation. ²¹ We found that ER-α was greatly upregulated in H/R-stimulated HESCs, which was dramatically alleviated by Ritodrine. In addition, the regulatory effects of Ritodrine on oxidative stress and NF-κB pathway-mediated inflammation were dramatically abolished by overexpressing ER-α, implying that ER-α was involved in the regulatory function of Ritodrine. In future work, the alleviation effect of Ritodrine on EMS will be further verified by introducing an EMS animal model with an appropriate dosage of Ritodrine.

The limitations of the current study have to be mentioned. Firstly, the findings from the current study have to be validated in in vivo experimentally induced endometriosis models. Endometrial stem cells are involved in the response to tissue injury and endometriosis, and the efficacy of Ritodrine must be confirmed in the evaluation of the uterus tissue. Since the current experiments were only performed in cultured HESCs, the in vivo experiment to study the therapeutic effect of Ritodrine in alleviating endometriosis is warranted. Secondly, the therapeutic effect and toxicity of Ritodrine have to be fully evaluated. The uterus has the endometrium located in the inner lining and the myometrium located outer layer, and the administration of Ritodrine will have an effect on endometrial cells and endometrial stem cells. Clinically, Ritodrine is mainly used for smooth muscle relaxation, and its side effects remains to be fully understood. The most severe side effects of Ritodrine are the severe maternal side effects such as cardiac side effects, and even death. ⁴⁴ Therefore, the future investigation has to be focused on the beneficial and side effects of Ritodrine on both endometrium and myometrium.

Collectively, our data reveal that Ritodrine alleviated H/R-induced injury in ESCs by regulating the ER-α/NF-κB pathway. A more in‐depth understanding of its molecular mechanism is needed to support its potential as a new treatment approach for endometriosis.