Nutraceuticals known to promote hair growth do not interfere with the inhibitory action of tamoxifen in MCF7, T47D and BT483 breast cancer cell lines.

BACKGROUND: Hair loss/thinning is a common side effect of tamoxifen in estrogen receptor (ER) positive breast cancer therapy. Some nutraceuticals known to promote hair growth are avoided during breast cancer therapy for fear of phytoestrogenic activity. However, not all botanical ingredients have similarities to estrogens, and in fact, no information exists as to the true interaction of these ingredients with tamoxifen. Therefore, this study sought to ascertain the effect of nutraceuticals (+/- estrogen/tamoxifen), on proliferation of breast cancer cells and the relative expression of ERα/ß. METHODS: Kelp, Astaxanthin, Saw Palmetto, Tocotrienols, Maca, Horsetail, Resveratrol, Curcumin and Ashwagandha were assessed on proliferation of MCF7, T47D and BT483 breast cancer cell lines +/- 17ß-estradiol and tamoxifen. Each extract was analysed by high performance liquid chromatography (HPLC) prior to use. Cellular ERα and ERß expression was assessed by qRT-PCR and western blot. Changes in the cellular localisation of ERα:ERß and their ratio following incubation with the nutraceuticals was confirmed by immunocytochemistry. RESULTS: Estradiol stimulated DNA synthesis in three different breast cancer cell lines: MCF7, T47D and BT483, which was inhibited by tamoxifen; this was mirrored by a specific ERa agonist in T47D and BT483 cells. Overall, nutraceuticals did not interfere with tamoxifen inhibition of estrogen; some even induced further inhibition when combined with tamoxifen. The ERα:ERß ratio was higher at mRNA and protein level in all cell lines. However, incubation with nutraceuticals induced a shift to higher ERß expression and a localization of ERs around the nuclear periphery. CONCLUSIONS: As ERα is the key driver of estrogen-dependent breast cancer, if nutraceuticals have a higher affinity for ERß they may offer a protective effect, particularly if they synergize and augment the actions of tamoxifen. Since ERß is the predominant ER in the hair follicle, further studies confirming whether nutraceuticals can shift the ratio towards ERß in hair follicle cells would support a role for them in hair growth. Although more research is needed to assess safety and efficacy, this promising data suggests the potential of nutraceuticals as adjuvant therapy for hair loss in breast cancer patients receiving endocrine therapy.

Effects and mechanisms of frehmaglutin D and rehmaionoside C improve LPS-induced acute kidney injury through the estrogen receptor-mediated TLR4 pathway in vivo and in vitro.

BACKGROUND: Sepsis-induced acute kidney injury (S-AKI) is an inflammatory disease with sex differences and there has no effective drugs to cure it. Frehmaglutin D (Fre D) and rehmaionoside C (Reh C) are two violetone compounds with estrogenic activity isolated from Rehmannia glutinosa. However, whether these two drugs exert protective effects on S-AKI through their estrogen-like activity are unclear. PURPOSE: This study aimed to explore the effects and mechanisms of Fre D and Reh C on lipopolysaccharide (LPS)-induced S-AKI through the estrogen receptor pathway in vivo and in vitro and to explore the interaction between ER and TLR4 for the first time. METHODS: The LPS-induced female BALB/c mice S-AKI mouse model was established by adding the estrogen receptor antagonist ICI182,780. Renal function, inflammation, oxidative stress, apoptosis, immune cells, and expression of key proteins of the ER-TLR4-IL-1ß pathway were tested. The affinity of Fre D and Reh C for the ER was investigated by molecular docking. Then, an in vitro S-AKI model was established, and ERα/ERß antagonists (MPP/PHTPP) were added and combined with gene overexpression techniques. The interaction between ER and TLR4 was further explored by Co-IP, GST pull-down and SPR techniques. RESULTS: Fre D and Reh C ameliorated LPS-induced renal damage, inflammation in mice, regulated the immune cells, decreased ROS levels, increased ERα and ERß protein expression, and decreased TLR4, caspase 11 and IL-1ß protein expression. These effects were blocked by ICI182,780. Molecular docking results showed that Fre D and Reh C bound ERα and ERß with similar potency. The results of in vitro suggested that Fre D and Reh C reduced the levels of inflammation, ROS and apoptosis, TLR4, caspase 11, and IL-1ß protein expression and increased ERα/ERß protein expression in cells. All of these effects were reversed by the addition of MPP/PHTPP and further enhanced after ERα/ERß gene overexpression with no significant difference in effects. Moreover, there was an indirect or direct interaction between ER and TLR4, and the binding of ERα and ERß to TLR4 was concentration dependent. CONCLUSION: Fre D and Reh C may improve S-AKI through the ER-TLR4-IL-1ß pathway and may act on both ERα and ERß receptors. Moreover, ERα and ERß may interact directly or indirectly with TLR4, which was studied for the first time.

Ameliorative effects of androstenediol against acetic acid-induced colitis in male wistar rats via inhibiting TLR4-mediated PI3K/Akt and NF-κB pathways through estrogen receptor ß activation.

5-androstenediol (ADIOL) functions as a selective estrogen receptor ß (ERß) ligand with a protective effect against many diseases. So, we conducted a novel insight into its role in acetic acid (AA)-induced colitis and investigated its effect on TLR4-Mediated PI3K/Akt and NF-κB Pathways and the potential role of ERß as contributing mechanisms. METHODS: Rats were randomized into 5 Groups; Control, Colitis, Colitis + mesalazine (MLZ), Colitis + ADIOL, and Colitis + ADIOL + PHTPP (ER-ß antagonist). The colitis was induced through a rectal enema of acetic acid (AA) on the 8th day. At the end of treatment, colons were collected for macroscopic assessment. Tissue levels of malondialdehyde (MDA), superoxide dismutase (SOD), nuclear factor kappa b (NF-κB), toll-like receptor (TLR4), and phosphorylated Protein kinase B (pAKT) were measured. Besides, Gene expression of interleukin-1beta (IL-1ß), metalloproteases 9 (Mmp9), inositol 3 phosphate kinase (PI3K), Neutrophil gelatinase-associated lipocalin (NGAL), ERß and NLRP6 were assessed. Histopathological and immunohistochemical studies were also investigated. RESULTS: Compared to the untreated AA group, the disease activity index (DAI) and macroscopic assessment indicators significantly decreased with ADIOL injections. Indeed, ADIOL significantly decreased colonic tissue levels of MDA, TLR4, pAKT, and NF-κB immunostainig while increased SOD activity and ß catenin immunostainig. ADIOL mitigated the high genetic expressions of IL1ß, NGAL, MMP9, and PI3K while increased ERß and NLRP6 gene expression. Also, the pathological changes detected in AA groups were markedly ameliorated with ADIOL. The specific ERß antagonist, PHTPP, largely diminished these protective effects of ADIOL. CONCLUSION: ADIOL could be beneficial against AA-induced colitis mostly through activating ERß.

Sex and interspecies differences in ESR2-expressing cell distributions in mouse and rat brains.

BACKGROUND: ESR2, a nuclear estrogen receptor also known as estrogen receptor ß, is expressed in the brain and contributes to the actions of estrogen in various physiological phenomena. However, its expression profiles in the brain have long been debated because of difficulties in detecting ESR2-expressing cells. In the present study, we aimed to determine the distribution of ESR2 in rodent brains, as well as its sex and interspecies differences, using immunohistochemical detection with a well-validated anti-ESR2 antibody (PPZ0506). METHODS: To determine the expression profiles of ESR2 protein in rodent brains, whole brain sections from mice and rats of both sexes were subjected to immunostaining for ESR2. In addition, to evaluate the effects of circulating estrogen on ESR2 expression profiles, ovariectomized female mice and rats were treated with low or high doses of estrogen, and the resulting numbers of ESR2-immunopositive cells were analyzed. Welch's t-test was used for comparisons between two groups for sex differences, and one-way analysis of variance followed by the Tukey-Kramer test were used for comparisons among multiple groups with different estrogen treatments. RESULTS: ESR2-immunopositive cells were observed in several subregions of mouse and rat brains, including the preoptic area, extended amygdala, hypothalamus, mesencephalon, and cerebral cortex. Their distribution profiles exhibited sex and interspecies differences. In addition, low-dose estrogen treatment in ovariectomized female mice and rats tended to increase the numbers of ESR2-immunopositive cells, whereas high-dose estrogen treatment tended to decrease these numbers. CONCLUSIONS: Immunohistochemistry using the well-validated PPZ0506 antibody revealed a more localized expression of ESR2 protein in rodent brains than has previously been reported. Furthermore, there were marked sex and interspecies differences in its distribution. Our histological analyses also revealed estrogen-dependent changes in ESR2 expression levels in female brains. These findings will be helpful for understanding the ESR2-mediated actions of estrogen in the brain.

Although the brain is a major target organ of estrogens, the distribution of estrogen receptors in the brain is not fully understood. ESR2, also known as estrogen receptor ß, is an estrogen receptor subtype; its localization in the brain has long been controversial because it has traditionally been difficult to detect. In the present study, we analyzed the expression sites of ESR2 in mouse and rat brains using immunohistochemistry with a well-validated antibody, PPZ0506. The immunohistochemical analysis revealed a more localized expression of ESR2 protein in brain subregions than has previously been reported. Additionally, there were clear sex and interspecies differences in the distribution of this protein. We also observed changes in ESR2 expression in the female brain in response to circulating estrogen levels. Our results, which show the precise expression profiles of ESR2 protein in rodent brains, will be helpful for understanding the ESR2-mediated actions of estrogen.

Prophylactic Effects of Hemp Seed Oil on Perimenopausal Depression: A Role of HPA Axis.

Hemp seed, the dried fruit of Cannabis sativa L. (Moraceae), has been extensively documented as a folk source of food due to its nutritional and functional value. This study evaluated the antidepressant effect of hemp seed oil (HSO) during its estrogen-like effect in Perimenopausal depression (PMD) rats induced by ovariectomy combined with chronic unpredictable mild stress (OVX-CUMS). Female SD rats (SPF, 10 weeks, sham operated group, ovariectomy (OVX) model group, ovariectomy - chronic unpredictable mild stress (OVX-CUMS) group, HSO + OVX-CUMS group, fluoxetine (FLU) + OVX-CUMS group, n=8) were subjected to treatment with HSO (4.32 g/kg) or fluoxetine (10 mg/kg) for 28 days (20 mL/kg by ig). Sucrose preference test (SPT), forced swimming test (FST), open field test (OFT), estrogen receptor α (ERα) and estrogen receptor ß (ERß) expression, estradiol (E2), follicle stimulating hormone (FSH), luteinizing hormone (LH), cortisol (CORT), adrenocorticotropic hormone (ACTH), corticotropin releasing hormone (CRH), norepinephrine (NE), 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5HIAA) levels are measured to evaluate the function of the hypothalamic-pituitary-ovarian (HPO) and hypothalamic-pituitary-adrenal (HPA) axis. The results showed that OVX-CUMS significantly decrease sucrose preference rate in SPT, increase immobility time in FST and OFT, and decrease movement distance and stand-up times in OFT. HSO treatment significantly improves depression-like behaviors, upregulates the expression of ERα and ERß, improves HPO axis function by increasing E2 levels and decreasing FSH and LH levels, reverses HPA axis hyperactivation by decreasing CORT, ACTH, and CRH levels, and upregulates NE, 5-HT, and 5HIAA levels in model rats. The findings suggested that HSO could improve depression-like behavior in OVX-CUMS rats by regulating HPO/HPA axis function and neurotransmitter disturbance.

The effect of acupuncture on oestrogen receptors in rats with benign prostatic hyperplasia.

The effects of acupuncture on the protein and gene expression of oestrogen receptors (ERs) alpha (α) and beta (ß) in testosterone-induced benign prostatic hyperplasia (BPH) in rats remains unclear. In this study, rats were randomly divided into four groups (n = 10 per group). The rats in the blank group did not receive any treatment, while the rats in the model group were injected intraperitoneally with testosterone propionate for 28 days to establish the BPH model and then randomly sub-divided into a control group, an acupuncture group and a finasteride group (positive control group). Dissections were performed after rats were anesthetized with isoflurane, and then the weight and volume of the prostate were then measured. The expression of ERs was detected via immunohistochemistry, western blot and real-time polymerase chain reaction. The results showed that ERα was discontinuously distributed in epithelial cells and expressed in large quantities in stromal cells, and ERß was aggregated and expressed in hyperplastic nodules. Acupuncture and finasteride could significantly improve the distribution of ERα and ERß which suggested that acupuncture and finasteride could improve BPH. There was no significant difference in ERα messenger ribonucleic acid (mRNA) expression among the groups, but the ERß mRNA expression in the finasteride group showed a significant difference compared with the control and acupuncture groups. The mechanism of the acupuncture treatment of BPH may be related to the increased transcription level of ERß mRNA in prostate tissues, the improved distribution of ERα expression in epithelial cells and the aggregation expression of ERs in hyperplastic nodules.

Complex Extract of Polygonatum sibiricum and Nelumbinis semen Improves Menopause Symptoms via Regulation of Estrogen Receptor Beta in an Ovariectomized Rat Model.

Menopause is a hormone-deficiency state that causes facial flushing, vaginal dryness, depression, anxiety, insomnia, obesity, osteoporosis, and cardiovascular disease as ovarian function decreases. Hormone-replacement therapy is mainly used to treat menopause; however, its long-term use is accompanied by side effects such as breast cancer and endometriosis. To identify the effect of a complex extract of Polygonatum sibiricum (PS) and Nelumbinis semen (NS) on improving menopause without side effects, an ovariectomized rat model was established to analyze several menopause symptoms. Compared to single extracts, the complex extract restored vaginal epithelial cell thickness and decreased serotonin concentration by increasing the estrogen receptors ERα (ESR1) and ERß (ESR2), depending on the ratio. Although the complex extract exerted a lower weight-loss effect than the single extracts, improved blood-lipid metabolism was observed after increasing high-density lipoprotein cholesterol levels and decreasing low-density lipoprotein cholesterol and triglyceride levels, and ovariectomy-induced osteoporosis was alleviated by suppressing osteoclast production. Thus, by increasing only ERß expression without regulating ERα expression in the uterus, the complex extract of PS and NS may be a natural treatment for improving menopause symptoms without side effects, such as endometriosis.

11-Ethoxyviburtinal improves chronic restraint stress-induced anxiety-like behaviors in gender-specific mice via PI3K/Akt and E2 /ERß signaling pathways.

Anxiety disorder is a chronic and disabling psychiatric disorder that is more prevalent in females than in males. 11-Ethoxyviburtinal is an iridoid extracted from Valeriana jatamansi Jones, which has anxiolytic potential. The aim of the present work was to study the anxiolytic efficacy and mechanism of 11-ethoxyviburtinal in gender-specific mice. We first evaluated the anxiolytic-like efficacy of 11-ethoxyviburtinal in chronic restraint stress (CRS) mice of different sexes through behavioral experiments and biochemical indexes. In addition, network pharmacology and molecular docking were used to predict potential targets and important pathways for the treatment of anxiety disorder with 11-ethoxyviburtinal. Finally, the influence of 11-ethoxyviburtinal on phosphoinositide-3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, estrogen receptor ß (ERß) expression, and anxiety-like behavior in mice was verified by western blotting, immunohistochemistry staining, antagonist intervention methods, and behavioral experiments. 11-ethoxyviburtinal alleviated the anxiety-like behaviors induced by CRS and inhibited neurotransmitter dysregulation and HPA axis hyperactivity. It inhibited the abnormal activation of the PI3K/Akt signaling pathway, modulated estrogen production, and promoted ERß expression in mice. In addition, the female mice may be more sensitive to the pharmacological effects of 11-ethoxyviburtinal. 11-ethoxyviburtinal may exert its anxiolytic-like effects through PI3K/Akt and E2/ERß signaling pathways. Meanwhile, by comparing the male and female mice, gender differences may affect the therapy and development of anxiety disorder.

The Phytoestrogenic Effect of Hibiscus sabdariffa Involves Estrogen Receptor α in Ovariectomized Wistar Rats.

The calyxes of Hibiscus sabdariffa present multiple pharmacological effects primarily attributed to their high anthocyanin content; however, little is known about their phytoestrogenic effect. Ovarian hypofunction (OH) is a process characterized by the rapid detention of the production of ovarian hormones, which compromises reproductive and cognitive functions. Hormone replacement therapy (HRT) efficiently compensates for OH; nevertheless, questions have been raised on its secondary effects and safety. One of the alternatives to tackling OH involves using phytoestrogens such as anthocyanins for their structural similarity to natural estrogens. In a Wistar rat model of ovariectomy (OVX), we recently reported the beneficial properties of an anthocyanin-rich extract prepared from the calyces of H. sabdariffa (HSE) in hindering the adverse effects of OH on memory performance and highlighted a possible phytoestrogenic impact through the modulation of estrogen receptor (ER) expression. We now report that HSE and estradiol differentially affected the expression of ERα and ERß. ERα was more sensitive to HSE; meanwhile, estradiol preferentially modulated ERß. Thus, our study leads to further research on using H. sabdariffa as a nutrition-based alternative to HRT.

Status of estrogen receptor expression and epigenetic methylation in Leydig cells after exposure to metalloestrogen - selenium.

The trace element selenium (Se) is essential for the maintenance of spermatogenesis and fertility. A growing volume of evidence shows that Se is necessary for testosterone synthesis, and Se can stimulate Leydig cell proliferation. However, Se can also act as a metalloestrogen, which can mimic estrogen and activate the estrogen receptors. This study aimed to investigate Se effect on estrogen signaling and the epigenetic status of Leydig cells. Mouse Leydig cells (MA-10) were cultured in a medium supplemented with different Se concentrations (4, 8 µM) for 24 h. Next, cells were assessed for morphological and molecular (qRT PCR, western blot, immunofluorescence) analyses. Immunofluorescence revealed strong immunosignal for 5-methylcytosine in both control and treated cells, with a stronger signal in the 8 µM treated group. qRT-PCR confirmed an increased expression of methyltransferase 3 beta (Dnmt3b) in 8 µM cells. Analysis of the expression of γH2AX (a marker for double-stranded DNA breaks) revealed an increase in the DNA breaks in cells exposed to 8 µM Se. Selenium exposure did not affect the expression of canonical estrogen receptors (ERα and ERß), however, an increase in membrane estrogen receptor G-protein coupled (GPER) protein expression was observed.To sum up, in a high concentration (8 µM) Se affects GPER expression (non-genomic estrogen signaling) in Leydig cells possibly via acting on receptor protein and/or its binding. This causes DNA breaks and induces changes in Leydig cell methylation status, especially in de novo methylation which is mediated by Dnmt3b.

Breast cancer prevention with liquiritigenin from licorice through the inhibition of aromatase and protein biosynthesis in high-risk women's breast tissue.

Breast cancer risk continues to increase post menopause. Anti-estrogen therapies are available to prevent postmenopausal breast cancer in high-risk women. However, their adverse effects have reduced acceptability and overall success in cancer prevention. Natural products such as hops (Humulus lupulus) and three pharmacopeial licorice (Glycyrrhiza) species have demonstrated estrogenic and chemopreventive properties, but little is known regarding their effects on aromatase expression and activity as well as pro-proliferation pathways in human breast tissue. We show that Gycyrrhiza inflata (GI) has the highest aromatase inhibition potency among these plant extracts. Moreover, phytoestrogens such as liquiritigenin which is common in all licorice species have potent aromatase inhibitory activity, which is further supported by computational docking of their structures in the binding pocket of aromatase. In addition, GI extract and liquiritigenin suppress aromatase expression in the breast tissue of high-risk postmenopausal women. Although liquiritigenin has estrogenic effects in vitro, with preferential activity through estrogen receptor (ER)-ß, it reduces estradiol-induced uterine growth in vivo. It downregulates RNA translation, protein biosynthesis, and metabolism in high-risk women's breast tissue. Finally, it reduces the rate of MCF-7 cell proliferation, with repeated dosing. Collectively, these data suggest that liquiritigenin has breast cancer prevention potential for high-risk postmenopausal women.

miR-363-3p/PTEN is involved in the regulation of lipid metabolism by genistein in HepG2 cells via ERß.

BACKGROUND: Genistein (GEN) is one of the most well-known phytoestrogens identified in various legumes. Although increasing evidence shows GEN has a potential use in phytotherapy to regulate lipid metabolism, its therapeutic mechanisms have not yet been completely elucidated, especially epigenetic alterations of miRNAs to alleviate lipid accumulation in the liver remains unknown. PURPOSE: To clarify how GEN modulates the miRNA profile in HepG2 cells and investigate molecular mechanisms of the modulated miRNA on regulating hepatic lipid metabolism. METHODS: The miRNA microarray was performed to compare the miRNAs expression patterns, followed by determining principal miRNA and its target gene associated with hepatic lipid metabolism modulated by GEN. miR-363-3p mimics (mi) and phosphatase and tensin homolog (PTEN)-siRNA were transfected into HepG2 cells and GEN was further treated with the cells for 24 h RESULTS: GEN induced downregulation of miR-363-3p and upregulation of PTEN, which was a target mRNA of miR-363-3p. The miR-363-3p mi led to an upregulation of sterol-regulatory element-binding protein-1c (SREBP-1c) and its downstream lipid synthesis-related factors in HepG2 cells. In addition, the inhibition of PTEN led to an increase of lipogenesis, which was associated with the AKT/mTOR signal regulation. However, GEN treatment could abrogate the lipogenic effects of miR-363-3p mi or PTEN siRNA. The modulation was associated with estrogen receptor ß (ERß). CONCLUSION: We discerned a new mechanism that GEN regulated hepatic lipid metabolism by inhibiting miR-363-3p, which could be mediated via ERß and by targeting PTEN in HepG2 cells. Additionally, GEN reduced hepatic lipid accumulation by regulating PTEN-AKT/mTOR signal. It implicated a protective role of GEN by elucidating its epigenetic modification of the miRNA modulated by ERß on improving hepatic lipid metabolism and provided novel evidence of the mechanism on targeting miR-363-3p/PTEN in treating hepatic lipid disorders.

ERß1 Sensitizes and ERß2 Desensitizes ERα-Positive Breast Cancer Cells to the Inhibitory Effects of Tamoxifen, Fulvestrant and Their Combination with All-Trans Retinoic Acid.

Adjuvant endocrine therapy (AET) is the treatment of choice for early-stage estrogen receptor alpha (ERα)-positive breast cancer (BC). However, almost 40% of tamoxifen-treated cases display no response or a partial response to AET, thus increasing the need for new treatment options and strong predictors of the therapeutic response of patients at high risk of relapse. In addition to ERα, BC research has focused on ERß1 and ERß2 (isoforms of ERß), the second ER isotype. At present, the impact of ERß isoforms on ERα-positive BC prognosis and treatment remains elusive. In the present study, we established clones of MCF7 cells constitutively expressing human ERß1 or ERß2 and investigated their role in the response of MCF7 cells to antiestrogens [4-hydroxytamoxifen (OHΤ) and fulvestrant (ICI182,780)] and retinoids [all-trans retinoic acid (ATRA)]. We show that, compared to MCF7 cells, MCF7-ERß1 and MCF7-ERß2 cells were sensitized and desensitized, respectively, to the antiproliferative effect of the antiestrogens, ATRA and their combination and to the cytocidal effect of the combination of OHT and ATRA. Analysis of the global transcriptional changes upon OHT-ATRA combinatorial treatment revealed uniquely regulated genes associated with anticancer effects in MCF7-ERß1 cells and cancer-promoting effects in MCF7-ERß2 cells. Our data are favorable to ERß1 being a marker of responsiveness and ERß2 being a marker of resistance of MCF7 cells to antiestrogens alone and in combination with ATRA.

Understanding the Phytoestrogen Genistein Actions on Breast Cancer: Insights on Estrogen Receptor Equivalence, Pleiotropic Essence and Emerging Paradigms in Bioavailability Modulation.

Prevalent as a major phenolic ingredient of soy and soy products, genistein is recognized as an eminent phytoestrogen owing to its interacting ability with estrogen receptors (ERs). The metabolic conversion of plant-derived genistin to genistein by gut microbes and intestinal enzymes enhances its absorption at intestinal pH of ~7.5-7.8. Genistein interferes in breast cancer (BC) development via pleiotropic actions on cell proliferation, survival, angiogenesis, and apoptosis. Though multiple investigations have demonstrated genistein intake-driven reduced BC risk, similar efficacy has not been replicated in clinical trials. Furthermore, multiple studies have structurally and functionally equated genistein extents with 17-ß-estradiol (E2), the most available physiological estrogen in females, culminating in aggravated BC growth. Of note, both genistein and E2 function via interacting with ERs (ERα and ERß). However, although E2 shows almost equal affinity towards both ERα and ERß, genistein shows more affinity towards ERß than ERα. Our cautious literature survey revealed typical intake mode, ER expression pattern and the ratio of ERα and ERß, transactivators/ regulators of ERα and ERß expression and activities, patient age, and menopausal status as decisive factors affecting genistein BC activities. Of further interest are the mechanisms by which genistein inhibits triple-negative breast cancers (TNBCs), which lack ERs, progesterone receptors (PRs), and human epidermal growth factor receptors (HER2). Herein, we attempt to understand the dosage-specific genistein actions in BC cells and patients with an insight into its better response via derivative development, nanocarrier-assisted, and combinatorial delivery with chemotherapeutic drugs.

Sex and age differences in the distribution of estrogen receptors in mice.

Estrogen receptors (ERα and ERß) are crucial for the regulation of socio-sexual behaviors and the organization of sex-specific neural networks in the developing brain. However, how the distribution patterns of ERα and ERß change throughout life is unclear. Using genetically modified ERß-RFPtg mice, we investigated the distribution of ERα, ERß, and their colocalization in the ventromedial nucleus of the hypothalamus (VMH), anteroventral periventricular nucleus (AVPV), and bed nucleus of stria terminalis (BNST) from postnatal days (PD) 0 to 56. ERα expression was higher in females that showed an increase after PD14 in all brain regions, whereas ERß-RFP expression pattern was markedly different among the regions. In the VMH, ERß-RFP was highly expressed on PD0 and PD7 but decreased drastically to very low expression afterward in both sexes. In contrast, ERß-RFP expression was higher in females compared to males in the AVPV but lower in the BNST throughout life especially late- and post-pubertal periods. Our results demonstrating that ERα and ERß-RFP expression changed in a sex-, age- and region-specific manner contribute to further clarification of the mechanisms underlying estrogen-dependent organization of the brain in both sexes.

Equol exerts a protective effect on postmenopausal osteoporosis by upregulating OPG/RANKL pathway.

BACKGROUD: Estrogen deficiency is the leading cause of postmenopausal osteoporosis(PMOP) and phytoestrogens soy isoflavones (SI) have been shown to improve PMOP. Equol (Eq), an in vivo metabolite of phytoestrogens soy isoflavones (SI), has a more stable structure and stronger biological activity than its parent compound and has the greatest estrogenic activity. However, there are few studies on the therapeutic effect of Eq on PMOP. PURPOSE: To explore the therapeutic effect and mechanisms of Eq on POMP. METHODS: Osteoblast-like cells ROS1728 were cultured with different doses of Eq, estradiol (E2), separately. The effect of Eq on the proliferation, apoptosis, cell cycle of osteoblasts were detected by CCK-8 and flow cytometry, and the expression of OPG/RANK/RANKL signaling pathway of osteoblasts was detected by Quantitative real-time PCR (qRT-PCR) and Western blot (WB), and RNA silencing technology were carried out to explore the receptors through which Eq plays a role. Then PMOP rat model was established and treated by Eq or E2 to further verification of the effect and mechanism of Eq on PMOP. RESULT: Eq promoted the proliferation and inhibited the apoptosis of osteoblasts and increased the proportion of osteoblasts in the S phase and G2/M phase in a dose-dependent manner. Mechanistically, Eq treatment upregulated the expression of OPG and OPG/RANKL ratio in osteoblasts and this regulatory effect was mainly mediated through the ERß receptor. Furthermore, in vivo study, Eq improved microstructure and BMD of the femur of PMOP rat model, which imitated the osteoprotective effect of E2. Moreover, the Eq or E2 treatment increased serum levels of Ca, 1,25(OH)2D3, bone Gla-protein(BGP), and Type I procollagen (PC1), and reduced serum levels of phosphorus (P), parathyroid hormone(PTH), pyridinol (PYD), tartrate-resistant acid phosphatase (TRAP) and urinary level of deoxypyridinoline (DPD) in the treatment OVX group compared with the untreated OVX group. Meanwhile, Eq or E2 markedly induced the mRNA and protein expression of OPG and OPG/RANKL ratio. CONCLUSION: Eq can combine with ERß and exert a protective effect on PMOP by upregulating OPG/RANKL pathway.

[Effects of electroacupuncture on sexual development and ovarian estrogen receptor ß expression in female adolescent obese rats].

OBJECTIVE: To observe the effect of electroacupuncture on sexual development and ovarian estrogen receptor ß(ER-ß) expression in female adolescent obese rats induced by high-fat diet, so as to explore its underlying mechanisms of improving adolescent obesity. METHODS: Female SD rats (age of 21 days) were randomly divided into control, model and acupuncture groups, with 6 rats in each group. The obese model was established by feeding high-fat diet for 6 weeks. Rats of the acupuncture group received electroacupuncture(2 Hz, 0.5-1.2 mA)stimulation at bilateral "Sanyinjiao"(SP6), "Fenglong"(ST40) and "Zusanli"(ST36) for 30 min, once a day for 14 days. The body mass and abdominal circumference of rats were measured before and after treatment. The contents of serum follicle stimulating hormone (FSH), luteinizing hormone (LH) and estradiol (E2) were detected by ELISA. The number of corpus luteum and follicle were observed by HE staining. The expression levels of ER-ß mRNA and protein in ovary were detected by fluorescence quantitative PCR and Western blot, respectively. RESULTS: Compared with the control group, the body mass and abdominal circumference, the contents of serum FSH and E2, and the expression levels of ER-ß mRNA and protein in ovary were significantly increased (P<0.05)in the model group, while the number of mature follicles and corpus luteum increased significantly. Compared with the model group, the body mass and abdominal circumference, the contents of serum FSH and E2, and the expression levels of ER-ß mRNA and protein in ovary were significantly decreased (P<0.05) in the acupuncture group, while the number of mature follicles and corpus luteum decreased significantly. CONCLUSION: Electroacupuncture can effectively improve the levels of sex hormone and the development of ovary, down-regulate the expression levels of ER-ß mRNA and protein in ovary, so as to regulate the process of sexual development of female adolescent obese rats induced by high-fat diet.

Overexpression of estrogen receptor ß inhibits cellular functions of human hepatic stellate cells and promotes the anti-fibrosis effect of calycosin via inhibiting STAT3 phosphorylation.

BACKGROUND: Estrogen receptor ß (ERß) is the major ER subtype in hepatic stellate cells (HSCs). Previously we reported phytoestrogen calycosin suppressed liver fibrosis progression and inhibited HSC-T6 cell functions, suggesting the effects may be related to ERß. Here, we explore the effect of overexpressed ERß on human HSCs and the role of ERß in pharmacological action of calycosin. METHODS: LX-2 cells were transfected with lentivirus to overexpress ERß. In the presence or absence of overexpressed ERß, the effects of ERß and calycosin on proliferation, migration, activation, collagen production and degradation of TGF-ß1-induced LX-2 cells and the role of ERß in the inhibition effect of calycosin were investigated. LX-2 cells overexpressed with ERß or treated with ER non-selective antagonist ICI182,780 were used to investigate the regulation of ERß on JAK2/STAT3 signaling pathway. CCK-8 method was used to screen effective doses of calycosin and investigate cell proliferation. The cell migration was detected by transwell chamber assay. The expression of α-SMA was detected by immunofluorescence and western blot. The protein expressions of Col-I, MMP1, TIMP1, JAK2, p-JAK2, STAT3 and p-STAT3 were detected by western blot. RESULTS: ERß overexpressed lentivirus was successfully transfected into LX-2 cells with high efficiency. Overexpressed ERß or calycosin alone inhibited the TGF-ß1-induced LX-2 cell proliferation and migration, downregulated the protein expressions of α-SMA, Col-I, TIMP-1, p-STAT3 and upregulated MMP-1. Both overexpressed ERß and calycosin had no significant effect on JAK2, p-JAK2 and STAT3 expressions. ERß overexpression further enhanced the above effects of calycosin. However, after the cells were treated with ICI182,780, downregulation of STAT3 phosphorylation induced by calycosin was reversed. CONCLUSIONS: ERß mediated the inhibition of major functions of LX-2 cell possibly by inhibiting the phosphorylation of STAT3, and was an important pathway through which calycosin exerted anti-liver fibrosis effect.

Hypothalamic Expression of Estrogen Receptor Isoforms Underlies Estradiol Control of Luteinizing Hormone in Female Rats.

Luteinizing hormone (LH) secretion during the ovarian cycle is governed by fluctuations in circulating estradiol (E2) that oppositely regulate kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC) of the hypothalamus. However, how these effects are orchestrated to achieve fertility is unknown. Here, we have tested the hypothesis that AVPV and ARC neurons have different sensitivities to E2 to coordinate changes in LH secretion. Cycling and ovariectomized rats with low and high E2 levels were used. As an index of E2 responsiveness, progesterone receptor (PR) was expressed only in the AVPV of rats with high E2, showing the preovulatory LH surge. On the other hand, kisspeptin neurons in the ARC responded to low E2 levels sufficient to suppress LH release. Notably, the Esr1/Esr2 ratio of gene expression was higher in the ARC than AVPV, regardless of E2 levels. Accordingly, the selective pharmacological activation of estrogen receptor α (ERα) required lower doses to induce PR in the ARC. The activation of ERß, in turn, amplified E2-induced PR expression in the AVPV and the LH surge. Thus, ARC and AVPV neurons are differently responsive to E2. Lower E2 levels activate ERα in the ARC, whereas ERß potentiates the E2 positive feedback in the AVPV, which appears related to the differential Esr1/Esr2 ratio in these 2 brain areas. Our findings provide evidence that the distinct expression of ER isoforms in the AVPV and ARC plays a key role in the control of periodic secretion of LH required for fertility in females.

Phytoestrogen arctigenin preserves the mucus barrier in inflammatory bowel diseases by inhibiting goblet cell apoptosis via the ERß/TRIM21/PHB1 pathway.

Intestinal mucus barrier dysfunction is closely involved in the pathogenesis of inflammatory bowel diseases (IBD). To investigate the protective effect and underlying mechanism of arctigenin, a phytoestrogen isolated from the fruits of Arctium lappa L., on the intestinal mucus barrier under colitis condition. The role of arctigenin on the intestinal mucus barrier and the apoptosis of goblet cells were examined by using both in vitro and in vivo assays. Arctigenin was demonstrated to promote the mucus secretion and maintain the integrity of mucus barrier, which might be achieved by an increase in the number of goblet cells via inhibiting apoptosis. Arctigenin selectively inhibited the mitochondrial pathway-mediated apoptosis. Moreover, arctigenin elevated the protein level of prohibitin 1 (PHB1) through blocking the ubiquitination via activation of estrogen receptor ß (ERß) to competitively interact with PHB1 and disrupt the binding of tripartite motif 21 (TRIM21) with PHB1. ERß knock down in the colons of mice with DSS-induced colitis resulted in significant reduction of the protection of arctigenin and DPN against the mucosal barrier. Arctigenin can maintain the integrity of the mucus barrier by inhibiting the apoptosis of goblet cells through the ERß/TRIM21/PHB1 pathway.