RESUMEN

This study aimed to evaluate the localised effects of intrauterine ozone therapy on endometrial recovery in mares with endometritis. Our investigation assessed changes in gene expression profiles of anti-inflammatory (IL-1RA and IL-10), proinflammatory (IL-R1B3i and TNFα) and pleiotropic (IL-6) cytokines, along with detailed histological measurements of epithelial and endometrial thickness and the glandular area ratio. Twenty mares were assigned to a 2 × 2 factorial design based on endometritis diagnosis and treatment (control or 42 µg/mL ozone insufflation), resulting in four groups: NC (negative for endometritis/control), NO (negative/ozone), PC (positive/control) and PO (positive/ozone). Oestrus was induced with 2 mg of oestradiol benzoate on Days -1, 1 and 3, plus 1 mg on Day 5. Day 0 marked the initial uterine treatment, followed by insufflations on Days 1 and 2 with O3 (ozone) or O2 (control). Uterine biopsies were taken before treatment on Day 0 and Day 6 for histological analysis and gene expression assessment. Data were analysed using a statistical model that included endometritis status, treatment type, biopsy times (D0 and D6) and their interactions, analysed with Proc Glimmix. Regardless of treatment or endometritis status, significant biopsy effects (p < 0.01) indicated increased epithelial height and endometrial thickness in Day 6 samples. Analysis of IL-1 and TNFα revealed a significant interaction (p < 0.05) among endometritis, treatment and biopsy, with higher IL-1B3i expression on Day 6 in the PC group. The treatment effect (p < 0.04) showed a higher frequency (p < 0.01) of animals with positive modulation in the PC group (66.7%) versus the PO group (0.0%). An interaction effect (p = 0.08) between endometritis and treatment resulted from higher IL-1RA expression on Day 6 in the PC group compared to the PO group. Biopsy effect was significant for IL-10 (p < 0.01), indicating higher values in the second sample associated with tissue repair. In the short-term evaluation, ozone therapy did not influence endometrial morphology and may modulate cytokine expression, specifically the reduction in IL-1 and TNFα levels. Therefore, this therapy appears to be a safe and potentially effective treatment for modulating the inflammatory response in mares with endometritis.

Asunto(s)

Citocinas , Endometritis , Enfermedades de los Caballos , Ozono , Útero , Animales , Femenino , Ozono/farmacología , Endometritis/veterinaria , Endometritis/tratamiento farmacológico , Caballos , Enfermedades de los Caballos/tratamiento farmacológico , Útero/patología , Citocinas/genética , Citocinas/metabolismo , Endometrio/efectos de los fármacos , Endometrio/metabolismo , Endometrio/patología , Estradiol/farmacología , Estradiol/análogos & derivados , Transcriptoma

RESUMEN

The objectives of this study were (1) to investigate the effects of the preovulatory follicle (POF) size on the accuracy of Doppler-based early pregnancy detection, and (2) to determine whether the removal of PGF2α (PGF) treatment during the resynchronisation protocol would affect fertility in beef cows. In Experiment 1, Nelore suckling cows (n = 224) were enrolled in an estradiol-progesterone-based timed artificial insemination (TAI) protocol. At TAI, cows were separated based on the range of POF diameters, as follows: ≤11.0 mm (n = 50), 11.1-12.9 mm (n = 64), 13.0-14.4 mm (n = 62) and ≥14.5 mm (n = 48). On day 22 after TAI, the corpus luteum (CL) blood flow (CLBF) of all cows was examined by colour Doppler ultrasonography to diagnose nonpregnant cows. The cows with the largest POF had the greatest positive predictive value (88.6%; 31 of 35) and diagnostic accuracy (91.7%; 44 of 48). In Experiment 2, Nelore cows (n = 233) were subjected to the same TAI protocol. Fourteen days after TAI, all cows were started on a resynchronisation protocol. Cows diagnosed as nonpregnant based on CLBF, on day 22, received 0.5 mg estradiol cypionate intramuscular (im) and were assigned to receive either 150 µg of PGF (PGF; n = 50) or 2 mL of saline (control; n = 47). Cows treated with PGF had a P/AI of 30.0% compared with a 48.9% P/AI in controls (p = 0.06). Our findings demonstrate that the POF size affects the accuracy of a CLBF-based early pregnancy diagnosis and that the removal of PGF treatment from the resynchronisation protocol tended to increase P/AI of the second TAI.

Asunto(s)

Cuerpo Lúteo , Dinoprost , Estradiol , Sincronización del Estro , Inseminación Artificial , Folículo Ovárico , Progesterona , Animales , Femenino , Bovinos , Embarazo , Inseminación Artificial/veterinaria , Sincronización del Estro/métodos , Progesterona/administración & dosificación , Progesterona/sangre , Progesterona/farmacología , Estradiol/análogos & derivados , Estradiol/administración & dosificación , Estradiol/farmacología , Cuerpo Lúteo/efectos de los fármacos , Dinoprost/administración & dosificación , Dinoprost/farmacología , Dinoprost/análogos & derivados , Fertilidad

RESUMEN

BACKGROUND: Aging and comorbidities like type 2 diabetes and obesity contribute to the development of chronic systemic inflammation, which impacts the development of heart failure and vascular disease. Increasing evidence suggests a role of pro-inflammatory M1 macrophages in chronic inflammation. A shift of metabolism from mitochondrial oxidation to glycolysis is essential for the activation of the pro-inflammatory M1 phenotype. Thus, reprogramming the macrophage metabolism may alleviate the pro-inflammatory phenotype and protect against cardiovascular diseases. In the present study, we hypothesized that the activation of estrogen receptors leads to the elevation of the mitochondrial deacetylase Sirt3, which supports mitochondrial function and mitigates the pro-inflammatory phenotype in macrophages. MATERIALS AND METHODS: Experiments were performed using the mouse macrophage cell line RAW264.7, as well as primary male or female murine bone marrow macrophages (BMMs). Macrophages were treated for 24 h with estradiol (E2) or vehicle (dextrin). The effect of E2 on Sirt3 expression was investigated in pro-inflammatory M1, anti-inflammatory/immunoregulatory M2, and naïve M0 macrophages. Mitochondrial respiration was measured by Seahorse assay, and protein expression and acetylation were determined by western blotting. RESULTS: E2 treatment upregulated mitochondrial Sirt3, reduced mitochondrial protein acetylation, and increased basal mitochondrial respiration in naïve RAW264.7 macrophages. Similar effects on Sirt3 expression and mitochondrial protein acetylation were observed in primary female but not in male murine BMMs. Although E2 upregulated Sirt3 in naïve M0, pro-inflammatory M1, and anti-inflammatory/immunoregulatory M2 macrophages, it reduced superoxide dismutase 2 acetylation and suppressed mitochondrial reactive oxygen species formation only in pro-inflammatory M1 macrophages. E2 alleviated the pro-inflammatory phenotype in M1 RAW264.7 cells. CONCLUSIONS: The study suggests that E2 treatment upregulates Sirt3 expression in macrophages. In primary BMMs, female-specific Sirt3 upregulation was observed. The Sirt3 upregulation was accompanied by mitochondrial protein deacetylation and the alleviation of the oxidative and pro-inflammatory phenotype in M1 macrophages. Thus, the E2-Sirt3 axis might be used in a therapeutic strategy to fight chronic systemic inflammation and prevent the development of inflammation-linked diseases.

Asunto(s)

Estrógenos , Inflamación , Macrófagos , Mitocondrias , Sirtuina 3 , Regulación hacia Arriba , Animales , Femenino , Masculino , Ratones , Acetilación/efectos de los fármacos , Estradiol/farmacología , Estrógenos/farmacología , Inflamación/patología , Inflamación/metabolismo , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Fenotipo , Células RAW 264.7 , Sirtuina 3/metabolismo , Regulación hacia Arriba/efectos de los fármacos

RESUMEN

ρ-type γ-aminobutyric acid-A (GABAA) receptors are widely distributed in the retina and brain, and are potential drug targets for the treatment of visual, sleep and cognitive disorders. Endogenous neuroactive steroids including ß-estradiol and pregnenolone sulfate negatively modulate the function of ρ1 GABAA receptors, but their inhibitory mechanisms are not clear. By combining five cryo-EM structures with electrophysiology and molecular dynamics simulations, we characterize binding sites and negative modulation mechanisms of ß-estradiol and pregnenolone sulfate at the human ρ1 GABAA receptor. ß-estradiol binds in a pocket at the interface between extracellular and transmembrane domains, apparently specific to the ρ subfamily, and disturbs allosteric conformational transitions linking GABA binding to pore opening. In contrast, pregnenolone sulfate binds inside the pore to block ion permeation, with a preference for activated structures. These results illuminate contrasting mechanisms of ρ1 inhibition by two different neuroactive steroids, with potential implications for subtype-specific gating and pharmacological design.

Asunto(s)

Microscopía por Crioelectrón , Estradiol , Simulación de Dinámica Molecular , Pregnenolona , Receptores de GABA-A , Humanos , Sitios de Unión , Estradiol/metabolismo , Estradiol/farmacología , Células HEK293 , Pregnenolona/metabolismo , Pregnenolona/farmacología , Pregnenolona/química , Receptores de GABA-A/metabolismo , Receptores de GABA-A/química

RESUMEN

Benign prostatic hyperplasia (BPH) is a prevalent age-related condition often characterized by debilitating urinary symptoms. Its etiology is believed to stem from hormonal imbalance, particularly an elevated estradiol-to-testosterone ratio and chronic inflammation. Our previous studies using a mouse steroid hormone imbalance model identified a specific increase in macrophages that migrated and accumulated in the prostate lumen where they differentiated into lipid-laden foam cells in mice implanted with testosterone and estradiol pellets, but not in sham animals. The current study focused on further characterizing the cellular heterogeneity of the prostate in this model as well as identifying the specific transcriptomic signature of the recruited foam cells. Moreover, we aimed to identify epithelia-derived signals that drive macrophage infiltration and luminal translocation. Male C57BL/6J mice were implanted with slow-release testosterone and estradiol pellets (T + E2) or sham surgery was performed and the ventral prostates were harvested two weeks later for scRNA-seq analysis. We identified Ear2 + and Cd72 + macrophages that were elevated in response to steroid hormone imbalance, whereas a Mrc1 + resident macrophage population did not change. In addition, an Spp1 + foam cell cluster was almost exclusively found in T + E2 mice. Further markers of foam cells were also identified, including Gpnmb and Trem2, and GPNMB was confirmed as a novel histological marker with immunohistochemistry. Foam cells were also shown to express known pathological factors Vegf, Tgfb1, Ccl6, Cxcl16 and Mmp12. Intriguingly, a screen for chemokines identified the upregulation of epithelia-derived Cxcl17, a known monocyte attractant, in T + E2 prostates suggesting that it might be responsible for the elevated macrophage number as well as their translocation to the lumen. Our study identified macrophage subsets that responded to steroid hormone imbalance as well as further confirmed a potential pathological role of luminal foam cells in the prostate. These results underscore a potential pathological role of the identified prostate foam cells and suggests CXCL17-mediated macrophage migration as a critical initiating event.

Asunto(s)

Estradiol , Células Espumosas , Macrófagos , Ratones Endogámicos C57BL , Próstata , Testosterona , Animales , Masculino , Ratones , Testosterona/metabolismo , Macrófagos/metabolismo , Próstata/metabolismo , Próstata/patología , Estradiol/farmacología , Células Espumosas/metabolismo , Modelos Animales de Enfermedad , Quimiocinas CXC/metabolismo , Quimiocinas CXC/genética , Biomarcadores/metabolismo , Regulación hacia Arriba

RESUMEN

ETHNOPHARMACOLOGICAL RELEVANCE: Endometriosis (EMS) is a common gynecological disease that causes dysmenorrhea, chronic pelvic pain and infertility. Luoshi Neiyi Prescription (LSNYP), a traditional Chinese medicine (TCM) formula, is used to relieve EMS in the clinic. AIMS: This study aimed to examine the active components of LSNYP and the possible mechanism involved in its treatment of EMS. MATERIALS AND METHODS: Ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) was used to identify the chemical components of LSNYP. Human primary ectopic endometrial stromal cells (ecESCs) and eutopic endometrial stromal cells (euESCs) were isolated, and the expression levels of hypoxia inducible factor 1A (HIF1A), enhancer of zeste homolog 2 (EZH2) and steroidogenic factor 1 (SF-1) were detected by immunofluorescence and qPCR. Cobalt chloride (CoCl2) was utilized to construct an in vitro hypoxic environment, and lentiviruses were engineered to downregulate HIF1A and EZH2 and upregulate EZH2. Subsequently, the expression levels of HIF1A, EZH2, and SF-1 were measured using qPCR or western blotting. The binding of EZH2 to the SF-1 locus in ESCs was examined via ChIP. Furthermore, the effects of LSNYP on the HIF1A/EZH2/SF-1 pathway were evaluated both in vitro and in vivo. RESULTS: A total of 185 components were identified in LSNYP. The protein and gene expression levels of HIF1A and SF-1 were increased, whereas those of EZH2 were decreased in ecESCs. After treating euESCs with 50 µmol L-1 CoCl2 for 24 h, cell viability and estradiol (E2) production were enhanced. Hypoxia decreased EZH2 protein expression, while si-HIF1A increased it. SF-1 was increased when EZH2 was downregulated in normal and hypoxic environments, whereas the overexpression of EZH2 led to a decrease in SF-1 expression. ChIP revealed that hypoxia reduced EZH2 binding to the SF-1 locus in euESCs. In vitro, LSNYP-containing serum decreased E2 and prostaglandin E2 (PGE2) production, inhibited cell proliferation and invasion, and reduced the expression of HIF1A, SF-1, steroidogenic acute regulatory protein (StAR), and aromatase cytochrome P450 (P450arom). In vivo, LSNYP suppressed inflammation and adhesion and inhibited the HIF1A/EZH2/SF-1 pathway in endometriotic tissues. CONCLUSIONS: LSNYP may exert pharmacological effects on EMS by inhibiting E2 synthesis and inflammation through regulation of the HIF1A/EZH2/SF-1 pathway. These results suggest that LSNYP may be a promising candidate for the treatment of EMS.

Asunto(s)

Medicamentos Herbarios Chinos , Endometriosis , Proteína Potenciadora del Homólogo Zeste 2 , Estradiol , Subunidad alfa del Factor 1 Inducible por Hipoxia , Endometriosis/tratamiento farmacológico , Endometriosis/metabolismo , Femenino , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/genética , Medicamentos Herbarios Chinos/farmacología , Estradiol/farmacología , Animales , Factor Esteroidogénico 1/genética , Factor Esteroidogénico 1/metabolismo , Transducción de Señal/efectos de los fármacos , Ratones , Adulto , Células Cultivadas , Inflamación/tratamiento farmacológico , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo

RESUMEN

The aim of the present study was to determine whether adipokines monocyte chemoattractant protein-1 (MCP-1) and plasminogen activator inhibitor-1 (PAI-1) can affect the functions of ovarian cells in cats. The addition of either MCP-1 or PAI-1 increased viability; promoted the accumulation of proliferation markers and progesterone and estradiol release; and decreased the accumulation of apoptosis markers in cultured feline granulosa cells. The present observations suggest that MCP-1 or PAI-1 can be physiological stimulators of ovarian granulosa cell functions.

Asunto(s)

Quimiocina CCL2 , Células de la Granulosa , Inhibidor 1 de Activador Plasminogénico , Animales , Gatos , Femenino , Inhibidor 1 de Activador Plasminogénico/metabolismo , Células de la Granulosa/metabolismo , Células de la Granulosa/fisiología , Células de la Granulosa/efectos de los fármacos , Quimiocina CCL2/metabolismo , Células Cultivadas , Proliferación Celular/fisiología , Estradiol/metabolismo , Estradiol/farmacología , Progesterona/metabolismo , Progesterona/farmacología , Apoptosis , Supervivencia Celular

RESUMEN

Salt-inducible kinases (SIKs), a family of serine/threonine kinases, were found to be critical determinants of female fertility. SIK2 silencing results in increased ovulatory response to gonadotropins. In contrast, SIK3 knockout results in infertility, gonadotropin insensitivity, and ovaries devoid of antral and preovulatory follicles. This study hypothesizes that SIK2 and SIK3 differentially regulate follicle growth and fertility via contrasting actions in the granulosa cells (GCs), the somatic cells of the follicle. Therefore, SIK2 or SIK3 GC-specific knockdown (SIK2GCKD and SIK3GCKD, respectively) mice were generated by crossing SIK floxed mice with Cyp19a1pII-Cre mice. Fertility studies revealed that pup accumulation over 6 months and the average litter size of SIK2GCKD mice were similar to controls, although in SIK3GCKD mice were significantly lower compared to controls. Compared to controls, gonadotropin stimulation of prepubertal SIK2GCKD mice resulted in significantly higher serum estradiol levels, whereas SIK3GCKD mice produced significantly less estradiol. Cyp11a1, Cyp19a1, and StAR were significantly increased in the GCs of gonadotropin-stimulated SIK2GCKD mice. However, Cyp11a1 and StAR remained significantly lower than controls in SIK3GCKD mice. Interestingly, Cyp19a1 stimulation in SIK3GCKD was not statistically different compared to controls. Superovulation resulted in SIK2GCKD mice ovulating significantly more oocytes, whereas SIK3GCKD mice ovulated significantly fewer oocytes than controls. Remarkably, SIK3GCKD superovulated ovaries contained significantly more preantral follicles than controls. SIK3GCKD ovaries contained significantly more apoptotic cells and fewer proliferating cells than controls. These data point to the differential regulation of GC function and follicle development by SIK2 and SIK3 and supports the therapeutic potential of targeting these kinases for treating infertility or developing new contraceptives.

Asunto(s)

Gonadotropinas , Células de la Granulosa , Ratones Noqueados , Proteínas Serina-Treonina Quinasas , Animales , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Femenino , Células de la Granulosa/efectos de los fármacos , Células de la Granulosa/metabolismo , Ratones , Gonadotropinas/metabolismo , Folículo Ovárico/efectos de los fármacos , Folículo Ovárico/metabolismo , Aromatasa/genética , Aromatasa/metabolismo , Fertilidad/genética , Fertilidad/efectos de los fármacos , Estradiol/farmacología

RESUMEN

The clinical studies for breast cancer (BC) are now assessing the efficacy of 2-Methoxyestradiol (2-ME), a naturally occurring derivative of estradiol. Our study aimed to explore the potential of combining the 2-ME and tamoxifen (TAM) on sensitization of TAM-resistant cells using LCC2 the TAM-resistant cells as a model and comparing the results to the sensitive cells MCF-7. Sulphorhodamine-B (SRB) assay is used to examine the 2-ME chemo-sensitizing impact on the cytotoxicity of TAM on LCC2 cells. Colorimetric assay kits were used to assess the level of the apoptosis-related markers caspases 3, Bcl2, and Bax in cell lysate. Hypoxia-inducible factor 1 alpha (HIF-1α) expression was measured using western blotting. Total cholesterol and triglyceride (TG) levels were examined colorimetrically, using the BIOLABO kit. The use of 2-ME enhanced the cytotoxic effects of TAM and effectively reversed TAM resistance. This was achieved by inhibiting the expression of HIF-1α, while concurrently increasing the levels of apoptotic marker caspase-3, as well as the pro-apoptotic protein Bax. Additionally, there was a reduction in the levels of Bcl2, an anti-apoptotic protein. Furthermore, a reduction in TG and cholesterol levels was noted. Our findings show that HIF-1α plays an important role in TAM resistance and that suppression of HIF-1α by 2-ME-mediated sensitization of BC-resistant cells to TAM. Therefore, the concurrent administration of TAM/2-ME might potentially serve as a viable therapeutic approach to address TAM resistance and enhance the overall therapy efficacy for patients with BC.

Asunto(s)

2-Metoxiestradiol , Neoplasias de la Mama , Regulación hacia Abajo , Resistencia a Antineoplásicos , Subunidad alfa del Factor 1 Inducible por Hipoxia , Tamoxifeno , Humanos , 2-Metoxiestradiol/farmacología , Tamoxifeno/farmacología , Tamoxifeno/análogos & derivados , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Células MCF-7 , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Femenino , Regulación hacia Abajo/efectos de los fármacos , Apoptosis/efectos de los fármacos , Antineoplásicos Hormonales/farmacología , Estradiol/farmacología , Estradiol/análogos & derivados

RESUMEN

An imbalance in estrogen signaling is a critical event in breast tumorigenesis. The majority of breast cancers (BCs) are hormone-sensitive; they majorly express the estrogen receptor (ER+) and are activated by 17ß-estradiol (E2). The steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step in steroid biosynthesis. The dysregulation of the epigenetic machinery, modulating E2 levels, is a primary occurrence for promoting breast tumorigenesis. StAR expression, concomitant with E2 synthesis, was reported to be aberrantly high in human and mouse hormone-dependent BC cells compared with their non-cancerous counterparts. However, the mechanism of action of StAR remains poorly understood. We discovered StAR as an acetylated protein and have identified a number of lysine (K) residues that are putatively acetylated in malignant and non-malignant breast cells, using LC-MS/MS (liquid chromatography-tandem mass spectrometry), suggesting they differently influence E2 synthesis in mammary tissue. The treatment of hormone-sensitive MCF7 cells with a variety of histone deacetylase inhibitors (HDACIs), at therapeutically and clinically relevant doses, identified a few additional StAR acetylated lysine residues. Among a total of fourteen StAR acetylomes undergoing acetylation and deacetylation, K111 and K253 were frequently recognized either endogenously or in response to HDACIs. Site-directed mutagenesis studies of these two StAR acetylomes, pertaining to K111Q and K253Q acetylation mimetic states, resulted in increases in E2 levels in ER+ MCF7 and triple negative MB-231 BC cells, compared with their values seen with human StAR. Conversely, these cells carrying K111R and K253R deacetylation mutants diminished E2 biosynthesis. These findings provide novel and mechanistic insights into intra-tumoral E2 regulation by elucidating the functional importance of this uncovered StAR post-translational modification (PTM), involving acetylation and deacetylation events, underscoring the potential of StAR as a therapeutic target for hormone-sensitive BC.

Asunto(s)

Neoplasias de la Mama , Estradiol , Fosfoproteínas , Humanos , Acetilación/efectos de los fármacos , Estradiol/farmacología , Estradiol/metabolismo , Femenino , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Células MCF-7 , Inhibidores de Histona Desacetilasas/farmacología , Procesamiento Proteico-Postraduccional , Lisina/metabolismo , Línea Celular Tumoral

RESUMEN

The lysosomal Ca2+ channel TRPML1 was found to be responsible for gastric acid secretion in murine gastric parietal cells by inducing the trafficking of H+/K+-ATPase containing tubulovesicles to the apical membrane. Therefore, we hypothesized a similar role of TRPML1 in regulating proton secretion in the immortalized human parietal cell line HGT-1. The primary focus was to investigate the involvement of TRPML1 in proton secretion using the known synthetic agonists ML-SA1 and ML-SA5 and the antagonist ML-SI3 and, furthermore, to identify food-derived compounds that target the channel. Proton secretion stimulated by ML-SA1 was reduced by 122.2 ± 22.7% by the antagonist ML-SI3. The steroid hormone 17ß-estradiol, present in animal-derived foods, diminished the proton secretory effect of ML-SA1 by 63.4 ± 14.5%. We also demonstrated a reduction in the proton secretory effects of ML-SA1 and ML-SA5 on TRPML1 knock-down cells. The food-derived compounds sulforaphane and trehalose promoted proton secretion in HGT-1 cells but may act independently of TRPML1. Also, histamine- and caffeine-induced proton secretion were affected by neither the TRPML1 antagonist ML-SI3 nor the TRPML1 knock-down. In summary, the results obtained suggest that the activation of TRPML1 promotes proton secretion in HGT-1 cells, but the channel may not participate in canonical signaling pathways.

Asunto(s)

Células Parietales Gástricas , Protones , Canales de Potencial de Receptor Transitorio , Humanos , Células Parietales Gástricas/metabolismo , Células Parietales Gástricas/efectos de los fármacos , Canales de Potencial de Receptor Transitorio/metabolismo , Canales de Potencial de Receptor Transitorio/genética , Línea Celular , Isotiocianatos/farmacología , Estradiol/farmacología , Estradiol/metabolismo , Sulfóxidos

RESUMEN

The G-protein-coupled estrogen receptor (GPER) has been described to exert several cardioprotective effects. However, the exact mechanism involved in cardiac protection remains unclear. The aim of this study is to investigate the role of GPER activation on excitation-contraction coupling (ECC) and the possibility that such effect participates in cardioprotection. The cardiac myocytes of male Wistar rats were isolated with a digestive buffer and loaded with Fura-2-AM for the measurement of intracellular calcium transient (CaT). Sarcomere shortening (SS) and L-type calcium current (ICaL) were also registered. The confocal technique was used to measure nitric oxide (NO) production in cells loaded with DAF-FM-diacetate. Cardiac myocytes exposed to 17-ß-estradiol (E2, 10 nM) or G-1 (1 µM) for fifteen minutes decreased CaT, SS, and ICaL. These effects were prevented using G-36 (antagonist of GPER, 1 µM), L-Name (NO synthase -NOS- inhibitor, 100 nM), or wortmannin (phosphoinositide-3-kinase -PI3K- inhibitor, 100 nM). Moreover, G1 increased NO production, and this effect was abolished in the presence of wortmannin. We concluded that the selective activation of GPER with E2 or G1 in the isolated cardiac myocytes of male rats induced a negative inotropic effect due to the reduction in ICaL and the decrease in CaT. Finally, the pathway that we proposed to be implicated in these effects is PI3K-NOS-NO.

Asunto(s)

Acoplamiento Excitación-Contracción , Miocitos Cardíacos , Óxido Nítrico , Fosfatidilinositol 3-Quinasas , Receptores Acoplados a Proteínas G , Animales , Masculino , Ratas , Estradiol/farmacología , Estradiol/metabolismo , Acoplamiento Excitación-Contracción/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Ratas Wistar , Receptores de Estrógenos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos

RESUMEN

It is well established that pyruvate kinase M2 (PKM2) activity contributes to metabolic reprogramming in various cancers, including colorectal cancer (CRC). Estrogen or 17ß-estradiol (E2) signaling is also known to modulate glycolysis markers in cancer cells. However, whether the inhibition of PKM2 combined with E2 treatment could adversely affect glucose metabolism in CRC cells remains to be investigated. First, we confirmed the metabolic plasticity of CRC cells under varying environmental conditions. Next, we identified glycolysis markers that were upregulated in CRC patients and assessed in vitro mRNA levels following E2 treatment. We found that PKM2 expression, which is highly upregulated in CRC clinical samples, is not altered by E2 treatment in CRC cells. In this study, glucose uptake, generation of reactive oxygen species (ROS), lactate production, cell viability, and apoptosis were evaluated in CRC cells following E2 treatment, PKM2 silencing, or a combination of both. Compared to individual treatments, combination therapy resulted in a significant reduction in cell viability and enhanced apoptosis. Glucose uptake and ROS production were markedly reduced in PKM2-silenced E2-treated cells. The data presented here suggest that E2 signaling combined with PKM2 inhibition cumulatively targets glucose metabolism in a manner that negatively impacts CRC cell growth. These findings hold promise for novel therapeutic strategies targeting altered metabolic pathways in CRC.

Asunto(s)

Neoplasias Colorrectales , Humanos , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/genética , Hormonas Tiroideas/metabolismo , Línea Celular Tumoral , Especies Reactivas de Oxígeno/metabolismo , Estrógenos/farmacología , Proteínas de Unión a Hormona Tiroide , Estradiol/farmacología , Apoptosis/efectos de los fármacos , Glucosa/metabolismo , Proteínas Portadoras/metabolismo , Piruvato Quinasa/metabolismo , Piruvato Quinasa/antagonistas & inhibidores , Piruvato Quinasa/genética , Glucólisis/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Femenino

RESUMEN

Dermal papilla cells (DPCs) exhibit self-recovery ability, which may be involved in hair growth. Therefore, we tested whether DPCs subjected to temporary growth-inhibiting stress (testosterone, 17ß-estradiol, mitomycin C, or undernutrition) treatments exhibit self-recovery behavior that can activate hair follicle growth, and examined the changes in cell proliferation capacity and gene expression. Related proteins were identified and their relationships with the hair cycle was examined using a mouse model. Recovery-period DPCs (i.e., from day 3 after loading) were subjected to microarray analysis to detect genetic variations common to each stress treatment. Co-culture of recovery-period DPCs and outer root sheath cells (ORSCs) confirmed the promotion of ORSC proliferation, suggesting that the activation of hair follicle growth is promoted via signal transduction. Chitinase 3-like 1 (CHI3L1) and C-X-C motif chemokine 5 (CXCL5) exhibited ORSC proliferation-promoting effects. Measurement of protein content in the skin during each phase of the hair cycle in mice revealed that CHI3L1 and CXCL5 secretion increased immediately after anagen transition. In a hair-loss mouse model treated with testosterone or 17ß-estradiol, CHI3L1 and CXCL5 secretion was lower in treated telogen skin than in untreated skin. Our results suggest that CHI3L1 and CXCL5 secreted by recovery-state DPCs promote hair growth.

Asunto(s)

Proteína 1 Similar a Quitinasa-3 , Folículo Piloso , Cabello , Animales , Humanos , Masculino , Ratones , Alopecia/metabolismo , Alopecia/patología , Proliferación Celular , Células Cultivadas , Quimiocina CXCL5/metabolismo , Proteína 1 Similar a Quitinasa-3/metabolismo , Proteína 1 Similar a Quitinasa-3/genética , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Estradiol/metabolismo , Estradiol/farmacología , Cabello/crecimiento & desarrollo , Folículo Piloso/metabolismo , Ratones Endogámicos C57BL , Mitomicina/farmacología , Transducción de Señal , Testosterona/metabolismo , Testosterona/farmacología

RESUMEN

Pregnancy and motherhood can have long-term effects on cognition and brain aging in both humans and rodents. Estrogens are related to cognitive function and neuroplasticity. Estrogens can improve cognition in postmenopausal women, but the evidence is mixed, partly due to differences in age of initiation, type of menopause, dose, formulation and route of administration. Additionally, past pregnancy influences brain aging and cognition as a younger age of first pregnancy in humans is associated with poorer aging outcomes. However, few animal studies have examined specific features of pregnancy history or the possible mechanisms underlying these changes. We examined whether maternal age at first pregnancy and estradiol differentially affected hippocampal neuroplasticity, inflammation, spatial reference cognition, and immediate early gene activation in response to spatial memory retrieval in middle-age. Thirteen-month-old rats (who were nulliparous (never mothered) or previously primiparous (had a litter) at three or seven months) received daily injections of estradiol (or vehicle) for sixteen days and were tested on the Morris Water Maze. An older age of first pregnancy was associated with impaired spatial memory but improved performance on reversal training, and increased number of new neurons in the ventral hippocampus. Estradiol decreased activation of new neurons in the dorsal hippocampus, regardless of parity history. Estradiol also decreased the production of anti-inflammatory cytokines based on age of first pregnancy. This work suggests that estradiol affects neuroplasticity and neuroinflammation in middle age, and that age of first pregnancy can have long lasting effects on hippocampus structure and function.

Asunto(s)

Estradiol , Hipocampo , Plasticidad Neuronal , Memoria Espacial , Animales , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Embarazo , Memoria Espacial/efectos de los fármacos , Memoria Espacial/fisiología , Estradiol/farmacología , Ratas , Inflamación/metabolismo , Aprendizaje por Laberinto/efectos de los fármacos , Aprendizaje por Laberinto/fisiología , Envejecimiento/fisiología , Paridad/fisiología

RESUMEN

Infusion of 17ß-estradiol (E2) into the dorsal hippocampus (DH) of ovariectomized (OVX) mice enhances memory consolidation, an effect that depends on rapid phosphorylation of extracellular signal-regulated kinase (ERK) and Akt. Astrocytic glutamate transporter 1 (GLT-1) modulates neurotransmission via glutamate uptake from the synaptic cleft. However, little is known about the contribution of DH astrocytes, and astrocytic glutamate transport, to the memory-enhancing effects of E2. This study was designed to test whether DH astrocytes contribute to estrogenic modulation of memory consolidation by determining the extent to which DH GLT-1 is necessary for E2 to enhance memory in object recognition and object placement tasks and trigger rapid phosphorylation events in DH astrocytes. OVX female mice were bilaterally cannulated into the DH or the DH and dorsal third ventricle (ICV). Post-training DH infusion of the GLT-1 inhibitor dihydrokainic acid (DHK) dose-dependently impaired memory consolidation in both tasks. Moreover, the memory-enhancing effects of ICV-infused E2 in each task were blocked by DH DHK infusion. E2 increased p42 ERK and Akt phosphorylation in DH astrocytes, and these effects were blocked by DHK. Results suggest the necessity of DH GLT-1 activity for object and spatial memory consolidation, and for E2 to enhance consolidation of these memories and to rapidly activate cell signaling in DH astrocytes. Findings indicate that astrocytic function in the DH of OVX females is necessary for memory formation and is regulated by E2, and suggest an essential role for DH astrocytic GLT-1 activity in the memory-enhancing effects of E2.

Asunto(s)

Astrocitos , Estradiol , Transportador 2 de Aminoácidos Excitadores , Ácido Glutámico , Hipocampo , Ovariectomía , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Femenino , Estradiol/farmacología , Ratones , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Ácido Glutámico/metabolismo , Transportador 2 de Aminoácidos Excitadores/metabolismo , Consolidación de la Memoria/efectos de los fármacos , Consolidación de la Memoria/fisiología , Fosforilación/efectos de los fármacos , Memoria/efectos de los fármacos , Memoria/fisiología , Ratones Endogámicos C57BL , Reconocimiento en Psicología/efectos de los fármacos , Reconocimiento en Psicología/fisiología , Ácido Kaínico/análogos & derivados

RESUMEN

Background: Spinal cord injury (SCI) results in lesions that destroy tissue and spinal tracts, leading to deficits in locomotor and autonomic function. We have previously shown that after SCI, surviving motoneurons innervating hindlimb muscles exhibit extensive dendritic atrophy, which can be attenuated by treadmill training or treatment with gonadal hormones post-injury. We have also shown that following SCI, both exercise and treatment with gonadal hormones improve urinary function. Animals exercised with forced running wheel training show improved urinary function as measured by bladder cystometry and sphincter electromyography, and treatment with gonadal hormones improves voiding patterns as measured by metabolic cage testing. Objective: The objective of the current study was to examine the potential protective effects of exercise or hormone treatment on the structure and function of motoneurons innervating the external urethral sphincter (EUS) after contusive SCI. Methods: Gonadally intact young adult male rats received either a sham or a thoracic contusion injury. Immediately after injury, one cohort of animals was implanted with subcutaneous Silastic capsules filled with estradiol (E) and dihydrotestosterone (D) or left blank; continuous hormone treatment occurred for 4 weeks post-injury. A separate cohort of SCI-animals received either 12 weeks of forced wheel running exercise or no exercise treatment starting two weeks after injury. At the end of treatment, urinary void volume was measured using metabolic cages and EUS motoneurons were labeled with cholera toxin-conjugated horseradish peroxidase, allowing for assessment of dendritic morphology in three dimensions. Results: Locomotor performance was improved in exercised animals after SCI. Void volumes increased after SCI in all animals; void volume was unaffected by treatment with exercise, but was dramatically improved by treatment with E + D. Similar to what we have previously reported for hindlimb motoneurons after SCI, dendritic length of EUS motoneurons was significantly decreased after SCI compared to sham animals. Exercise did not reverse injury-induced atrophy, however E + D treatment significantly protected dendritic length. Conclusions: These results suggest that some aspects of urinary dysfunction after SCI can be improved through treatment with gonadal hormones, potentially through their effects on EUS motoneurons. Moreover, a more comprehensive treatment regime that addresses multiple SCI-induced sequelae, i.e., locomotor and voiding deficits, would include both hormones and exercise.

Asunto(s)

Neuronas Motoras , Condicionamiento Físico Animal , Ratas Sprague-Dawley , Traumatismos de la Médula Espinal , Uretra , Micción , Animales , Traumatismos de la Médula Espinal/terapia , Traumatismos de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/patología , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/fisiología , Neuronas Motoras/patología , Masculino , Uretra/efectos de los fármacos , Uretra/patología , Micción/efectos de los fármacos , Micción/fisiología , Ratas , Condicionamiento Físico Animal/fisiología , Estradiol/farmacología , Modelos Animales de Enfermedad , Terapia por Ejercicio/métodos

RESUMEN

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.

Asunto(s)

Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Estradiol , Osteoartritis , Animales , Ratas , Estradiol/farmacología , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Osteoartritis/metabolismo , Osteoartritis/patología , Osteoartritis/genética , Progresión de la Enfermedad , Sinoviocitos/metabolismo , Sinoviocitos/efectos de los fármacos , Sinoviocitos/patología , Ratas Sprague-Dawley , Modelos Animales de Enfermedad , Articulación Temporomandibular/patología , Articulación Temporomandibular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Células Cultivadas , Masculino , Adenosina/metabolismo , Adenosina/análogos & derivados , Proliferación Celular/efectos de los fármacos

RESUMEN

Sex steroids modulate the distribution of mammalian white adipose tissues. Moreover, WAT remodeling requires adipocyte progenitor cells. Nevertheless, the sex-dependent mechanisms regulating adipocyte progenitors remain undetermined. Here, we uncover Cxcr4 acting in a sexually dimorphic manner to affect a pool of proliferating cells leading to restriction of female fat mass. We find that deletion of Cxcr4 in Pparγ-expressing cells results in female, not male, lipodystrophy, which cannot be restored by high-fat diet consumption. Additionally, Cxcr4 deletion is associated with a loss of a pool of proliferating adipocyte progenitors. Cxcr4 loss is accompanied by the upregulation of estrogen receptor alpha in adipose-derived PPARγ-labelled cells related to estradiol hypersensitivity and stalled adipogenesis. Estrogen removal or administration of antiestrogens restores WAT accumulation and dynamics of adipose-derived cells in Cxcr4-deficient mice. These findings implicate Cxcr4 as a female adipogenic rheostat, which may inform strategies to target female adiposity.

Asunto(s)

Adipocitos , Adipogénesis , Adiposidad , PPAR gamma , Receptores CXCR4 , Células Madre , Animales , Receptores CXCR4/metabolismo , Receptores CXCR4/genética , Femenino , Masculino , Ratones , Adipocitos/metabolismo , Adipocitos/citología , Células Madre/metabolismo , Células Madre/citología , PPAR gamma/metabolismo , PPAR gamma/genética , Ratones Noqueados , Tejido Adiposo Blanco/metabolismo , Tejido Adiposo Blanco/citología , Dieta Alta en Grasa/efectos adversos , Receptor alfa de Estrógeno/metabolismo , Receptor alfa de Estrógeno/genética , Ratones Endogámicos C57BL , Estradiol/farmacología , Estradiol/metabolismo , Proliferación Celular , Factores Sexuales , Caracteres Sexuales

RESUMEN

Previous studies have suggested that the effects of androgens on body weight (BW) and appetite are affected by the estrogen milieu in females; however, the mechanism underlying these effects remains unclear. We hypothesized that androgens may affect endogenous oxytocin (OT), which is a hypothalamic anorectic factor, and that these effects of androgens may be altered by the estrogen milieu in females. To investigate this hypothesis, in the present study, we examined the effects of testosterone on peripheral and central OT levels in ovariectomized female rats that did or did not receive estradiol supplementation. Ovariectomized female rats were randomly divided into non-estradiol-supplemented or estradiol-supplemented groups, and half of the rats in each group were concurrently supplemented with testosterone (i.e., rats were divided into four groups, n = 7 per each group). We also measured peripheral and central OT receptor (OTR) gene expression levels. As a result, we found that testosterone increased serum and hypothalamic OT levels and OT receptor mRNA levels in non-estradiol-supplemented rats, whereas it had no effects on these factors in estradiol-supplemented rats. In addition, testosterone reduced food intake, BW gain, and fat weight in non-estradiol-supplemented rats, whereas it did not have any effects on BW, appetite, or fat weight in estradiol-supplemented rats. These findings indicate that the effects of androgens on OT may be affected by the estrogen milieu, and elevated OT levels may be related to the blunting of appetite and prevention of obesity under estrogen-deficient conditions.

Asunto(s)

Estradiol , Hipotálamo , Ovariectomía , Oxitocina , Receptores de Oxitocina , Testosterona , Animales , Oxitocina/sangre , Oxitocina/farmacología , Femenino , Testosterona/sangre , Hipotálamo/metabolismo , Hipotálamo/efectos de los fármacos , Estradiol/sangre , Estradiol/farmacología , Ratas , Receptores de Oxitocina/metabolismo , Receptores de Oxitocina/genética , Estrógenos/sangre , Estrógenos/farmacología , Peso Corporal/efectos de los fármacos , Ingestión de Alimentos/efectos de los fármacos , Ingestión de Alimentos/fisiología , Ratas Sprague-Dawley , Apetito/efectos de los fármacos , ARN Mensajero/metabolismo