Characterization of ERα Signaling to Cell Proliferation Induced by Chronic and Pulsatile E2 Stimulation in 2D and 3D Cell Cultures.

17ß-estradiol is a hormone that plays a vital role in human physiology. It acts through estrogen receptors, specifically estrogen receptor α and estrogen receptor ß, and its action is determined by the pulsatile secretion in the bloodstream. 17ß-estradiol affects cell proliferation, and dysregulation of 17ß-estradiol:estrogen receptor α signaling contribute to the development of breast cancer. Previous research on 17ß-estradiol:estrogen receptor α signaling has primarily used two-dimensional cell cultures, which do not fully recapitulate the complexity of tumors that exist in a three-dimensional environment and do not consider the pulsatile nature of this hormone. To address these limitations, we studied 17ß-estradiol:estrogen receptor α signaling in cell proliferation using both two-dimensional and three-dimensional breast cancer cell culture models under continuous and pulsatile stimulation conditions. Results revealed that breast cancer cells grown in an alginate-based three-dimensional matrix exhibited similar responsiveness to 17ß-estradiol compared with cells grown in conventional two-dimensional culture plates. 17ß-estradiol induced the expression of proteins containing estrogen response element in the three-dimensional model. The efficacy of the antiestrogen drugs fulvestrant (ICI182,280) and 4OH-tamoxifen was also demonstrated in the three-dimensional model. These results support the use of the three-dimensional culture model for studying tumor response to drugs and provide a more realistic microenvironment for such studies. Furthermore, the study revealed that a brief 5-min exposure to 17ß-estradiol triggered a physiological response comparable with continuous hormone exposure, suggesting that the cellular response to 17ß-estradiol is more important than the continuous presence of the hormone. In conclusion, the study demonstrates that the alginate-based three-dimensional culture model is suitable for studying the effects of 17ß-estradiol and antiestrogen drugs on breast cancer cells, offering a more realistic representation of tumor-microenvironment interactions. The results also highlight the importance of considering the physiological importance of the temporal dynamics in studying 17ß-estradiol signaling and cellular responses.

17ß-Estradiol reduces inhibitory synaptic currents in entorhinal cortex neurons through G protein-coupled estrogen receptor-1 activation of extracellular signal-regulated kinase.

Estrogens are believed to modulate cognitive functions in part through the modulation of synaptic transmission in the cortex and hippocampus. Administration of 17ß-estradiol (E2) can rapidly enhance excitatory synaptic transmission in the hippocampus and facilitate excitatory synaptic transmission in rat lateral entorhinal cortex via activation of the G protein-coupled estrogen receptor-1 (GPER1). To assess the mechanisms through which GPER1 activation facilitates synaptic transmission, we assessed the effects of acute 10 nM E2 administration on pharmacologically isolated evoked excitatory and inhibitory synaptic currents in layer II/III entorhinal neurons. Female Long-Evans rats were ovariectomized between postnatal day (PD) 63 and 74 and implanted with a subdermal E2 capsule to maintain continuous low levels of E2. Electrophysiological recordings were obtained between 7 and 20 days after ovariectomy. Application of E2 for 20 min did not significantly affect AMPA or NMDA receptor-mediated excitatory synaptic currents. However, GABA receptor-mediated inhibitory synaptic currents (IPSCs) were markedly reduced by E2 and returned towards baseline levels during the 20-min washout period. The inhibition of GABA-mediated IPSCs was blocked in the presence of the GPER1 receptor antagonist G15. GPER1 can modulate protein kinase A (PKA), but blocking PKA with intracellular KT5720 did not prevent the E2-induced reduction in IPSCs. GPER1 can also stimulate extracellular signal-regulated kinase (ERK), a negative modulator of GABAA receptors, and blocking activation of ERK with PD90859 prevented the E2-induced reduction of IPSCs. E2 can therefore result in a rapid GPER1 and ERK signaling-mediated reduction in GABA-mediated IPSCs. This provides a novel mechanism through which E2 can rapidly modulate synaptic excitability in entorhinal layer II/III neurons and may also contribute to E2 and ERK-dependent alterations in synaptic transmission in other brain areas.

Using 17ß-estradiol heparin-poloxamer thermosensitive hydrogel to enhance the endometrial regeneration and functional recovery of intrauterine adhesions in a rat model.

Mechanical damage or infection to the endometrium can lead to the formation of adhesions in the uterine cavity, which may result in reduced reproductive outcome and/or pregnancy complications. The prognosis of this disease is poor due to few effective treatments and the complex environment of endometrium. Heparin-Poloxamer Hydrogel (HP hydrogel) is a nontoxic and biodegradable biomaterial, which has been commonly used as a sustained-release delivery system. In this study, we applied a mini-endometrial curette to scrape the endometrium of rats to mimic the process of curettage in patients. After the establishment of IUA model in rats, we injected the thermo-sensitive hydrogel(E2-HP hydrogel) into the injured uterine cavity and evaluated the therapeutic effect of E2-HP hydrogel on the recovery of IUA. Our results showed that E2-HP hydrogel can significantly facilitate the regeneration of injured endometrium along with inhibiting the cell apoptosis in IUA model. Furthermore, we revealed that E2-HP hydrogel on the recovery of IUA was closely associated with the upregulation of kisspeptin through activating the ERK1/2 and MAPKs p38 pathways. In conclusion, E2-HP hydrogel can effectively transfer E2 into the injured endometrium and it can be considered as a promising therapeutic method for the women with intrauterine adhesions.

Effects of estradiol on in vitro maturation of buffalo and goat oocytes.

PURPOSE: The effects of estradiol on oocyte development seem to be varied among species. The present study investigated the effects of 17ß-estradiol on in vitro maturation of buffalo and goat oocytes. METHODS: Cumulus oocyte complexes (COCs) were aspirated from large antral follicles of slaughtered buffalo and goat ovaries. COCs were cultured in TCM-199 medium supplemented with 0, 0.5, 1, and 1.5 µg/mL of 17ß-estradiol for in vitro maturation. Then, oocytes were used for the examination of state of nuclear maturation and cumulus expansion. RESULTS: In both species, oocytes treated with 17ß-estradiol showed higher cumulus expansion rate than control (0 µg/mL treated). In buffalo, the percentage of oocytes matured to the metaphase II (MII) stage increased in the concentration-dependent manner of 17ß-estradiol. Similarly, estradiol positively influenced nuclear maturation of goat oocytes in vitro. CONCLUSIONS: Estradiol has promoting effects on normalprogress of in vitro oocyte meiosis in buffalos and goats.

17ß-estradiol Attenuates the Middle Ear Inflammatory Response to Nontypeable Haemophilus influenzae.

OBJECTIVES: 17ß-estradiol (E2) is a steroidal hormone with immunomodulatory functions that play a role in infectious and inflammatory diseases. E2 was recently identified as the leading upstream regulator of differentially expressed genes in a comparative RNA sequencing study of pediatric patients with otitis media (OM) versus OM-free counterparts and may therefore play a role in the inflammatory response to bacterial otopathogens during pediatric OM. This study examined the effect of E2 on bacterial-induced inflammatory cytokine expression in an in vitro pediatric OM model. METHODS: An immortalized middle ear (ME) epithelial cell line, ROM-SV40, was developed from a pediatric recurrent OM patient. The culture was exposed to E2 at physiological levels for 1-48 h prior to 6 h-stimulation with nontypeable Haemophilus influenzae (NTHi) whole cell lysate. TNFA, IL1B, IL6, and IL8 were assayed by qPCR and ELISA. RESULTS: E2 pretreatment (24 h) abrogated NTHi induction of IL6; a longer pretreatment (1-10 nM, 48 h) abrogated IL1B induction (p < 0.05). E2 pretreatment (5 nM, 48 h) abrogated NTHi-induced IL8 secretion (p = 0.017). CONCLUSION: E2 pretreatment partially rescued NTHi-induced cytokine production by ME epithelia. These data support a role for E2 in moderating the excessive inflammatory response to middle ear infection that contributes to OM pathophysiology. LEVELS OF EVIDENCE: NA Laryngoscope, 134:3815-3819, 2024.

An integrated network pharmacology and molecular docking approach to reveal the role of Arctigenin against Cutibacterium acnes-induced skin inflammation by targeting the CYP19A1.

Acne caused by inflammation of hair follicles and sebaceous glands is a common chronic skin disease. Arctigenin (ATG) is an extract of Arctium lappa L., which has significant anti-inflammatory effects. However, the effect and mechanism of ATG in cutaneous inflammation mediated by Cutibacterium acnes (C. acnes) has not been fully evaluated. The purpose of this study was to explore the effect and potential mechanism of ATG in the treatment of acne through network pharmacology and experimental confirmation. An acne model was established by injected live C. acnes into living mice and treated with ATG. Our data showed that ATG effectively improved acne induced by live C. acnes, which was confirmed by determining ear swelling rate, estradiol concentration and hematoxylin and eosin (H&E) staining. In addition, ATG inhibited the NLRP3 inflammasome signaling pathway in mice ear tissues and reduced the secretion of pro-inflammatory cytokines IL-1ß to relieve inflammation. The results of network pharmacology and molecular docking confirmed that ATG can regulate 17ß-Estradiol (E2) levels through targeted to CYP19A1, and finally inhibited skin inflammation. Taken together, our results confirmed that ATG regulated E2 secretion by targeting CYP19A1, thereby inhibiting the NLRP3 inflammasome signaling pathway and improving inflammation levels in acne mice. This study provides a basis for the feasibility of ATG in treating acne in clinical practice.

Sex Differences of Neutrophil Extracellular Traps on Lipopolysaccharide-Stimulated Human Neutrophils.

Objective: Sex differences exist in sepsis, but the commitment of neutrophils to these differences remains unclear. Neutrophil extracellular traps (NETs) function to remove pathogens, yet excessive NETs release can contribute to organ damage. This study explores effects of the gender hormones on endotoxin-induced NETs using neutrophils from both male and female sources. Methods: Blood samples were collected from healthy volunteers. Isolated neutrophils were seeded in collagen-coated cell culture plates, and NETs were induced by lipopolysaccharide (LPS) treatment. After 15 minutes of LPS treatment, 17ß-estradiol (0.03-272.4 ng/mL), testosterone enanthate (0.01-10 ng/mL), dimethyl sulfoxide, or ethanol (vehicle control) was added to the plates. These were incubated for three hours at 37°C with 5% CO2. Neutrophil extracellular traps formation was assessed using immunofluorescence staining. Results: Lipopolysaccharide-induced NETs formation was significantly greater in females than in males. In male-derived neutrophils, 17ß-estradiol at above the blood concentrations significantly suppressed LPS-induced NETs. No effect was seen while using testosterone enanthate to NETs at any concentration. In female-derived neutrophils, 17ß-estradiol, which was near to the highest concentration of non-pregnant women's blood, tended to increase NETs. Testosterone enanthate, which was near to female blood concentration, significantly promoted NETs. Conclusions: Sex differences existed in LPS-induced NETs of human neutrophil. In males, high concentrations of 17ß-estradiol administration may have a suppressive effect on excessive NETs during infection. In females, endogenous gender hormones may promote NETs during infection. Sex differences in neutrophils may need to be considered in organ damage owing to NETs excess such as sepsis.

17ß-estradiol promotes the progression of temporomandibular joint osteoarthritis by regulating the FTO/IGF2BP1/m6A-NLRC5 axis.

BACKGROUND: Temporomandibular joint osteoarthritis (TMJOA) is a degenerative cartilage disease. 17ß-estradiol (E2) aggravates the pathological process of TMJOA; however, the mechanisms of its action have not been elucidated. Thus, we investigate the influence of E2 on the cellular biological behaviors of synoviocytes and the molecular mechanisms. METHODS: Primary fibroblast-like synoviocytes (FLSs) isolated from rats were treated with TNF-α to establish cell model, and phenotypes were evaluated using cell counting kit-8, EdU, Tanswell, enzyme-linked immunosorbent assay, and quantitative real-time PCR (qPCR). The underlying mechanism of E2, FTO-mediated NLRC5 m6A methylation, was assessed using microarray, methylated RNA immunoprecipitation, qPCR, and western blot. Moreover, TMJOA-like rat model was established by intra-articular injection of monosodium iodoacetate (MIA), and bone morphology and pathology were assessed using micro-CT and H&E staining. RESULTS: The results illustrated that E2 facilitated the proliferation, migration, invasion, and inflammation of TNF-α-treated FLSs. FTO expression was downregulated in TMJOA and was reduced by E2 in FLSs. Knockdown of FTO promoted m6A methylation of NLRC5 and enhanced NLRC5 stability by IGF2BP1 recognition. Moreover, E2 promoted TMJ pathology and condyle remodeling, and increased bone mineral density and trabecular bone volume fraction, which was rescued by NLRC5 knockdown. CONCLUSION: E2 promoted the progression of TMJOA.

Monocytes Do Not Contribute to Sex Differences Seen in the Pharmacokinetics of Pegylated Liposomal Doxorubicin.

Pegylated liposomal doxorubicin (PLD) is widely utilised in cancer chemotherapy, but exhibits large inter-individual pharmacokinetic variability and sex differences in plasma clearance. Population pharmacokinetic modelling has suggested PLD plasma clearance correlates with peripheral monocyte counts, while sex hormones are known to affect endocytosis and phagocytosis in mononuclear cells. This study investigated whether sex hormones affect the uptake of PLD by human monocytes and macrophages in vitro. 17ß-estradiol, but not testosterone, inhibited the uptake of PLD in a concentration dependent manner in undifferentiated (but not differentiated) THP-1 cells, and primary monocytes obtained from women, but not men. Effects of estradiol were only evident at very high concentrations seen during pregnancy. No differences were observed in monocyte count or monocyte subtypes between males and females. These data show that monocytes do not contribute to sex differences seen in PLD clearance in humans of reproductive age.

Estrogen 17ß­estradiol accelerates the proliferation of uterine junctional zone smooth muscle cells via the let­7a/Lin28B axis in adenomyosis.

The estrogen 17ß­estradiol has been proven to serve an indispensable role in the occurrence and development of adenomyosis (ADS). The let­7a/Lin28B axis can control cell proliferation by acting as a tumor­inhibiting axis in numerous types of cancer. However, its role in ADS remains unknown. The present study aimed i) to elucidate the role of let­7a in regulating the proliferation of human uterine junctional zone (JZ) smooth muscle cells (SMCs) in ADS, ii) to evaluate whether 17ß­estradiol modifies the expression levels of let­7a and Lin28B in JZ SMCs in ADS, and iii) to establish how 17ß­estradiol affects the function of the let­7a/Lin28B axis in the proliferation of JZ SMCs in ADS. A total of 36 premenopausal women with ADS were enrolled as the experimental group and 34 women without ADS were recruited as the control group. Reverse transcription­quantitative PCR was used to evaluate the expression level of let­7a, and western blotting was performed to determine the Lin28B expression levels. Lentiviral null vector, let­7a overexpression lentiviral vector GV280 and let­7a inhibition lentiviral vector GV369 were used to infect cells to alter the expression of let­7a for further functional experiments. 17ß­estradiol and Cell Counting Kit­8 assays were conducted to determine how 17ß­estradiol affects the function of the let­7a/Lin28B axis in the proliferation of JZ SMCs in ADS. The results demonstrated that let­7a was downregulated and Lin28B was upregulated in the JZ SMCs of ADS compared with the control cells (P<0.0001). Moreover, a lower expression of let­7a led to faster proliferation of JZ SMCs (P<0.05), and 17ß­estradiol affected the let­7a/Lin28B axis to accelerate the proliferation of JZ SMCs in ADS (P<0.05). These data suggested that 17ß­estradiol collaborates with the let­7a/Lin28B axis to affect the development of ADS.

Conversion of Estrone to 17ß-Estradiol: A Potential Confounding Factor in Assessing Risks of Environmental Estrogens to Fish.

Feminization of male fish and the role of endocrine-active chemicals in this phenomenon has been an area of intense study for many years. Estrone (E1), a natural steroid, is found in aquatic environments sometimes at high concentrations relative to the estrogenic steroids 17ß-estradiol (E2) and 17α-ethynylestradiol. However, E1 has been less thoroughly studied than E2 or 17α-ethynylestradiol due in part to a relatively lower potency in metabolically limited estrogen receptor (ER) binding/activation assays. Recent evidence suggests that in vivo biotransformation of E1 to E2 may occur in fathead minnows (Pimephales promelas) residing in environments with high concentrations of E1, such as near wastewater treatment plants. The enzymes likely responsible for this biotransformation, 17ß-hydroxysteroid dehydrogenases (17ßHSDs), have been well characterized in mammals but to a lesser extent in fish species. In the present study, a novel systematic analysis of amino acid sequence data from the National Center for Biotechnology Information database demonstrated that multiple 17ßHSD isoforms are conserved across different fish species. Experimentally, we showed that metabolically active hepatic cytosolic preparations from 2 commercially important salmonid species, rainbow trout and lake trout, biotransformed E1 to E2 to a degree sufficient to alter results of competitive ER binding assays. These results from in silico and in vitro analyses indicate that E1 and biotransformation may play a significant role in adverse effects on development and reproduction of a variety of fish species in contaminated aquatic environments. Environ Toxicol Chem 2020;39:2028-2040. Published 2020. This article is a US Government work and is in the public domain in the USA.

The microbial transformation of 17ß­estradiol in an anaerobic aqueous environment is mediated by changes in the biological properties of natural dissolved organic matter.

Dissolved organic matter (DOM) is shown to act as an electron shuttle mediator which enhances the microbial degradation of steroid estrogens in natural water. Batch studies were conducted with 17ß­estradiol (E2), quinone-reducing bacteria, DOM, and Fe(III) as a terminal electron acceptor. The results show that anthraquinone­2­disulfonate (AQS) approximately doubles the microbial degradation of E2 by DOM alone. The effect decreases sharply at AQS concentrations above 1.0mmol·L-1. Over three oxidation-reduction cycles, the electron-shuttling ability of AQS and the E2 biodegradation rate decreased step by step. Changes in the biological properties of the dissolved organic matter increased its aromaticity, its quinone content, and its fulvic-like fluorescence while significantly improving the electron transfer between DOM and the microbes, which made the degradation more effective. This explains why steroid estrogens do not accumulate in natural aqueous environments. Moreover, the estrogenic activity of steroid estrogens is inhibited at low concentrations through the activity of DOM.

Nongenomic modulation of the large conductance voltage- and Ca2+-activated K+ channels by estrogen: A novel regulatory mechanism in human detrusor smooth muscle.

Estrogens have an important role in regulating detrusor smooth muscle (DSM) function. However, the underlying molecular and cellular mechanisms by which estrogens control human DSM excitability and contractility are not well known. Here, we used human DSM specimens from open bladder surgeries on 27 patients to elucidate the mechanism by which 17ß-estradiol regulates large conductance voltage- and Ca2+-activated K+ (BK) channels, the most prominent K+ channels in human DSM We employed single BK channel recordings on inside-out excised membrane patches, perforated whole-cell patch-clamp on freshly isolated DSM cells, and isometric tension recordings on DSM-isolated strips to investigate the mechanism by which 17ß-estradiol activates BK channels. 17ß-Estradiol (100 nmol/L) rapidly increased depolarization-induced whole-cell K+ currents in DSM cells. The 17ß-estradiol stimulatory effects on whole-cell BK currents were completely abolished by the selective BK channel inhibitor paxilline (1 µmol/L), clearly indicating that 17ß-estradiol specifically activates BK channels. 17ß-Estradiol also increased the frequency of ryanodine receptor-mediated transient BK currents. Single BK channel recordings showed that 17ß-estradiol (100 nmol/L) significantly increased the BK channel open probability of inside-out excised membrane patches, revealing that 17ß-estradiol activates BK channels directly. 17ß-Estradiol reduced spontaneous phasic contractions of human DSM-isolated strips in a concentration-dependent manner (100 nmol/L-1 µmol/L), and this effect was blocked by paxilline (1 µmol/L). 17ß-Estradiol (100 nmol/L) also reduced nerve-evoked contractions of human DSM-isolated strips. Collectively, our results reveal that 17ß-estradiol plays a critical role in regulating human DSM function through a direct nongenomic activation of BK channels.

Features of diagnostics and treatment of partial androgen deficiency of aging men.

INTRODUCTION: Decreasing of testosterone level is an important part of a male elderly. MATERIAL AND METHODS: To describe this phenomenon a PubMed and national databases were searched for 17ß-dehydrotestosterone, common testosterone, free testosterone, 17ß-estradiol, luteinizing hormone, partial androgen deficiency of aging men. RESULTS: The reduction in intensity of the processes of tissue renewal of the testicles results in a partial androgen deficiency of aging men. A decrease in the levels of total and free testosterone and an increase in the levels of 5α-dihydrotestosterone, 17ß-estradiol, sex hormone binding globulin, LH and FSH, along with a decrease in the amplitude of the rhythm of incretion of LH, FSH and total testosterone will testify to a deficiency of testosterone. CONCLUSIONS: It is very important to make an individualized selection of the dose of testosterone preparation which enters the blood plasma.