ERβ Levels Research Articles

P0141 Estrogen receptor β alleviates colitis in intestinal epithelial cells by activating HIF-1α and ATG-9a mediated autophagy

Abstract Background Ulcerative colitis (UC) manifests as excessive inflammation and compromised integrity of the intestinal epithelial barrier. Estrogen receptor β (ERβ) exerts anti-inflammatory effects in the colon. However, the mechanism remains unclear. This study aims to explore the role and mechanism of ERβ in alleviating colitis, and provide a novel therapeutic target for UC. Methods The level of ERβ and autophagy in the inflammatory and non-inflammatory colons of patients with UC were assessed. Mice with dextran sulfate sodium (DSS)-induced colitis were used to establish an in vivo model. The body weight change, colon length, disease activity index (DAI) and histological score of mice in each group were compared. In vitro, HT-29 cell inflammation was induced by lipopolysaccharide (LPS). ERB041, an ERβ specific agonist, was administered to DSS-induced mice and LPS-induced HT-29 cells. Autophagy in vivo and in vitro was evaluated by western blot, transmission electron microscope, and immunofluorescence. Co-immunoprecipitation, chromatin immunoprecipitation, and dual-luciferase reporter assay were conducted to further delineate the mechanism between ERβ and autophagy. Results Compared with non-inflammatory mucosa, the expression of ERβ in the inflammatory mucosa in patients with UC was down-regulated, with autophagy inhibited significantly. ERβ activation attenuated the intestinal epithelial barrier disruption, decreased the level of pro-inflammatory cytokines, and significantly attenuated DSS-induced experimental colitis. Mechanically, ERB041 intervention activated autophagy, which was manifested as up-regulation of LC3BII expression, decreased p62 level and increased number of autolysosomes. ERβ interacted with hypoxia-inducible factor 1α (HIF-1α) and activated autophagy. Furthermore, HIF-1α bound to the promoter region of ATG- 9a, and positively regulates its transcription. Conclusion ERβ exerts anti-inflammatory effect by up-regulating HIF-1α and activating ATG-9a mediated autophagy in intestinal epithelial cells, and may be a promising therapeutic target for UC.

Peroxisome Proliferator-Activated Receptor Gamma Coactivator-1-Alpha in Endometriosis: Expression, Regulation, and Potential Role.

The present study was designed to comprehensively analyze the expression profiles of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), estrogen-related receptor-α (ERRα), estrogen receptor-β (ERβ), interleukin-6 (IL-6), cysteinyl-aspartic acid-specific protease-3 (caspase-3), and cysteinyl-aspartic acid-specific protease-9 (caspase-9) in endometriosis tissues. It also aimed to elucidate the hitherto unclarified role of PGC-1α in the processes of proliferation, apoptosis, and gene expression regulation of human endometrial stromal cells, thereby providing novel insights and identifying potential molecular targets for advancing endometriosis treatment modalities. A total of 49 ectopic endometrial tissue samples and 50 normal endometrial tissue samples were meticulously collected from patients who underwent gynecological surgeries in the People's Hospital Affiliated to Fujian University of Traditional Chinese Medicine in Fuzhou, China, between January 2022 and January 2023. Immunohistochemistry was used to detect protein expression levels. Human primary endometrial stromal cells were transfected and grouped for in-depth analysis. Real-time quantitative polymerase chain reaction (RT-qPCR) was utilized to measure gene expression, the cell counting kit 8 (CCK-8) assay was employed to evaluate cell proliferation, and the lactate dehydrogenase (LDH) method was used to determine cell death. In endometriosis tissues, compared with normal endometrial tissues, the expression levels of PGC-1α, ERRα, and IL-6 were significantly increased. The expression of ERβ was also elevated, while the expression levels of caspase-3 and caspase-9 were decreased. In cell experiments, after transfection with interference plasmids to reduce PGC-1α expression, the expression levels of ERRα and ERβ were decreased, the expression of IL-6 was increased, and the expression levels of caspase-3 and caspase-9 were augmented. Conversely, after transfection with overexpression plasmids to enhance PGC-1α expression, the expression levels of ERRα and ERβ were elevated, the expression of IL-6 was diminished, and the expression levels of caspase-3 and caspase-9 were decreased. Moreover, when PGC-1α expression was interfered with by siRNA, cell proliferation was attenuated (albeit not statistically significant), and cell apoptosis was enhanced. Overexpression of PGC-1α promoted cell proliferation and inhibited cell apoptosis. PGC-1α potentially plays a crucial role in the pathogenesis of endometriosis by regulating ERRα, ERβ, IL-6, and apoptosis-related factors. This study provides a strong theoretical foundation and a novel direction for developing endometriosis treatment strategies. To the best of our knowledge, it is the first to systematically explore the relationship between PGC-1α and these key factors in endometriosis, filling a knowledge gap. Future studies are needed to further dissect its mechanism and realize its clinical application potential.

17β-estradiol inhibits lipopolysaccharide-induced inflammation and pyroptosis of Leydig cells of the domestic yak (Bos grunniens) via the SIRT1/Nox4/ROS pathway.

Estradiol (E2) secreted by Leydig cells (LCs) can accumulate in the testes due to constriction of the reproductive lumen. Estrogen is not only important for reproduction, but also protects against inflammation. In this study, the role of pyroptosis in testicular inflammation and the effects of E2 against inflammation and pyroptosis of yak interstitial cells were investigated. Inflamed testes exhibited structural damage and pyroptosis with decreased E2, testosterone, and estrogen receptor β (ERβ) levels in testicular fluid. E2 alone inhibited testosterone secretion and increased ERβ expression in mature LCs. In LCs, lipopolysaccharide (LPS) causes inflammation by activation of TNF-α and IL-6, and pyroptosis via activation of the classical and non-classical pyroptosis pathways. LPS inhibits sex hormone secretion and ERβ expression in LCs. E2 inhibited the LPS-induced decrease of ER expression in LCs and also inhibited LPS-induced interstitial cell inflammation and pyroptosis, which was partially blocked by Selisistat (EX-527, SIRT1 inhibitor) or Fulvestrant (ICI 182,780, E2 non-genomic receptor inhibitors). In conclusion, E2 relieved LPS-induced inflammation and pyroptosis of yak LCs via the SIRT1/Nox4/ROS pathway. This finding provides new insights into the role of estrogen in male reproductive health and offers a potential therapeutic strategy to improve testicular immune and reproductive function by modulating hormonal homeostasis.

Single-Cell Transcriptome Sequencing and Analysis Provide a New Approach for the Treatment of Small Cell Neuroendocrine Carcinoma of the Cervix

Introduction: Small cell neuroendocrine carcinoma of the cervix (SCNECC) is a rare gynecologic malignant tumor, which has lack of systematic research. In order to investigate its molecular characteristics, origin, and pathogenesis, single-cell transcriptome sequencing (scRNA-Seq) of SCNECC was performed for the first time, the cellular and molecular landscape was revealed, and the key genes for clinical prognosis were screened. Methods: This article initially performed the scRNA-Seq on a tumor tissue sample from an SCNECC patient, combined with scRNA-Seq data from a healthy cervical tissue sample downloaded from a public database; the single-cell transcriptome landscape was constructed. Then, we investigated the cell types, intratumoral heterogeneity, characteristics of tumor microenvironment, and potential predictive markers of SCNECC. Results: We identified two malignant cell populations, tumor stem cells and malignant carcinoma cells, and revealed two tumor progression pathways of SCNECC. By analyzing gene expression levels in the pathophysiology of SCNECC, we found that the expression levels of ERBB4 and NRG1, as well as the expression profile of mTOR signaling pathway mediated by them, were significantly upregulated in malignant carcinoma cells. In addition, we also found that carcinoma cells were able to stimulate malignant cell proliferation through the FN1 signaling pathway. The immune cells were in a stress state, with T-cell depletion, macrophage polarization, and mast cell glycolysis. These results suggested that carcinoma cells could interfere with immune response and promote tumor escape through MIF, TGFb, and other immunosuppressive-related signaling pathways. Conclusion: This study revealed the mechanism of genesis and progression in SCNECC and the related important signaling pathways, such as mTOR, and provided new insights into the treatment of SCNECC.

Scleromitrion diffusum (Willd.) R. J. Wang Inhibits Gastric Cancer via ERBB2/ERBB3/PI3K/AKT Pathway.

This study aimed to evaluate the anticarcinogenic potential of Scleromitrion diffusum (Willd.) R. J. Wang (SD) extracts in vitro, along with exploring the underlying compatibility mechanisms. Scleromitrion diffusum (Willd.) R. J. Wang extract was prepared and gastric cancer (GC) cells were treated to detect the half maximal inhibitory concentration (IC50)/proliferation and migration/invasion by MTS method and transwell assay. The compatibility mechanisms of SD were analyzed by systems pharmacology strategy, combined with cellular experimental validation. Scleromitrion diffusum (Willd.) R. J. Wang extract showed inhibitory ability on the proliferation of the GC cell lines dose- and time-dependently. A total of 3 active ingredients are involved in anti-gastric cancer effects of SD, based on the top 50 pathways. The "herb-composition-target-pathway" network showed the multi-target and multi-pathway characteristics of SD. There were 52 related targets shared by SD and GC. The cellular experiments supported that SD significantly reduced ERBB2 and ERBB3 expression levels in GC cells. The overexpression of ERBB2 or ERBB3 partially offset the anti-tumor effects of SD. Scleromitrion diffusum (Willd.) R. J. Wang inhibited gastric cancer growth and metastasis in vitro, which may be related to the inhibition of the ERBB2/ERBB3/PI3K/AKT pathway.

Sex differences in contextual fear conditioning and extinction after acute and chronic nicotine treatment

BackgroundChronic cigarette smokers report withdrawal symptomology, including affective dysfunction and cognitive deficits. While there are studies demonstrating sex specific withdrawal symptomology in nicotine-dependent individuals, literature examining the underlying biological mediators of this is scant and not in complete agreement. Therefore, in this study, we evaluated the sex specific effects of nicotine and withdrawal on contextual fear memory, a hippocampally dependent aspect of cognition that is disrupted in nicotine withdrawal.MethodsMale and female B6/129F1 mice (8–13 weeks old) were used in all experiments. For the acute nicotine experiment, mice received intraperitoneal saline or nicotine (0.5 mg/kg) prior to contextual fear conditioning and test. For the chronic nicotine experiment, mice received nicotine (18 mg/kg/day) or saline for 11 days, then underwent contextual fear conditioning and test. Following the test, mice underwent minipump removal to elicit withdrawal or sham surgery, followed by the fear extinction assay. Bulk cortical tissue was used to determine nicotinic acetylcholine receptor levels via single point [3H]Epibatidine binding assay. Gene expression levels in the dorsal and ventral hippocampus were quantified via RT-PCR.ResultsWe found that female mice had a stronger expression of contextual fear memory than their male counterparts. Further, following acute nicotine treatment, male, but not female, subjects demonstrated augmented contextual fear memory expression. In contrast, no significant effects of chronic nicotine treatment on fear conditioning were observed in either sex. When examining extinction of fear learning, we observed that female mice withdrawn from nicotine displayed impaired extinction learning, but no effect was observed in males. Nicotine withdrawal caused similar suppression of fosb, cfos, and bdnf, our proxy for neuronal activation and plasticity changes, in the dorsal and ventral hippocampus of both sexes. Additionally, we found that ventral hippocampus erbb4 expression, a gene implicated in smoking cessation outcomes, was elevated in both sexes following nicotine withdrawal.ConclusionsDespite the similar impacts of nicotine withdrawal on gene expression levels, fosb, cfos, bdnf and erbb4 levels in the ventral hippocampus were predictive of delays in female extinction learning alone. This suggests sex specific dysfunction in hippocampal circuitry may contribute to female specific nicotine withdrawal induced deficits in extinction learning.

The influence of the NRG1/ERBB4 signaling pathway on pulmonary artery endothelial cells.

This study aimed to examine the influence of the Neuregulin-1 (NRG1)/ERBB4 signaling pathway on the function of human pulmonary artery endothelial cells (HPAECs) and investigate the underlying mechanisms. Enzyme-linked immunosorbent assay indicated that ERBB4 levels in the serum of patients with pulmonary embolism (PE) were significantly higher than those of healthy controls (p < 0.05). In cellular studies, thrombin stimulation for 6 h led to a significant decrease in cell viability and overexpression of ERBB4 compared to control (p < 0.05). In the NRG1 group, apoptosis of HPAECs was reduced (p < 0.05), accompanied by a decrease in ERBB4 expression and an increase in p-ERBB4, phosphorylated serine/threonine kinase proteins (Akt) (p-Akt), and p-phosphoinositide 3-kinase (PI3K) expression (p < 0.05). In the AG1478 group, there was a significant increase in HPAEC apoptosis and a significant decrease in p-ERBB4 and ERBB4 expression compared to the Con group (p < 0.05). In the AG1478 + NRG1 group, there was an increase in the apoptosis rate and a significant decrease in the expression of p-ERBB4, ERBB4, p-Akt, and phosphorylated PI3K compared to the NRG1 group (p < 0.05). In animal studies, the PE group showed an increase in the expression of ERBB4 and p-ERBB4 compared to the Con group (p < 0.05). NRG1 treatment led to a significant reduction in embolism severity with decreased ERBB4 expression and increased p-ERBB4 expression (p < 0.05). Gene set enrichment analysis identified five pathways that were significantly associated with high ERBB4 expression, including CHOLESTEROL HOMEOSTASIS, OXIDATIVE PHOSPHORYLATION, and FATTY ACID METABOLISM (p < 0.05). Therefore, NRG1 inhibits apoptosis of HPAECs, accompanied by a decrease in ERBB4 and an increase in p-ERBB4. NRG1 inhibition in HPAECs apoptosis can be partially reversed by inhibiting ERBB4 expression with AG1478. ERBB4 has the potential to be a novel biological marker of PE.

A new exploration: characterization of the differentiation trajectory of prostate cancer cells

Prostate cancer is one of the most common malignant tumors in men, and in-depth study of its gene expression patterns is crucial for understanding the formation and development of prostate cancer. Although single-cell transcriptomics has deeply explored the heterogeneous expression characteristics of prostate cancer, given that normal epithelial cells themselves have different states of differentiation, these normal differentiation characteristics may lead to confusion with heterogeneous tumor characteristics. In this study, we used single-cell data from the GEO database to analyze in detail the heterogeneity of prostate cancer tumor cells/tumor-associated epithelium cells (TAECs), with a particular focus on the differentiation state of epithelial cells in matching normal tissue. We found that after subtype pairing analysis of normal tissue and tumor tissue epithelium based on differentiation status, the characteristics identified later were not consistent with the general characteristics originally exhibited by different TAECs subpopulations. Among them, all TAECs subpopulations showed P53 enrichment and downregulation of the apoptotic pathway, and expressed higher levels of EGFR, ERBB2, interferon receptors, MIF, and cell adhesion-related signals; through transcription factor regulatory network analysis, we observed that YY1, NKX3-1, and EHF had higher transcriptional activity in TAECs subpopulations than normal epithelial cells at the same differentiation stage, while ATF3 was the opposite. Among them, YY1 may act as an upstream regulator of the MIF signaling pathway, and ATF3 is a key upstream transcriptional regulator of differentially expressed genes in the P53 and apoptotic pathways. Immune infiltration analysis showed that the above four transcription factors were significantly correlated with the infiltration of immune cells in prostate cancer, and pan-cancer analysis showed that their expression-related survival risks were widely present in different cancers. It is worth noting that this is merely a preliminary, exploratory study, which inevitably has some deficiencies and limitations. Despite this, this study is committed to bringing a novel and unique perspective to the field through this work, with the hope of opening up new levels of understanding and stimulating more in-depth research and discussion.

NLRC5 exerts anti-endometriosis effects through inhibiting ERβ-mediated inflammatory response

BackgroundEndometriosis is well known as a chronic inflammatory disease. The development of endometriosis is heavily influenced by the estrogen receptor β (ERβ), while NOD-like receptors (NLRs) family CARD domain-containing 5 (NLRC5) exhibits anti-inflammatory properties during endometriosis. However, whether NLRC5-mediated anti-inflammation is involved in the ERβ-mediated endometriosis is still uncertain. This study aimed to assess that relation.MethodsNine cases of eutopic endometrial tissue and ten cases of ectopic endometrial tissue were collected from patients with endometriosis, and endometrial samples from ten healthy fertile women were analyzed, and the expression levels of ERβ were quantified using immunohistochemistry (IHC). Subsequently, we constructed mouse model of endometriosis by intraperitoneal injection. We detected the expression of ERβ, NLRC5, tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and IL-10 and measured the volume of ectopic lesions in mice with endometriosis. In vitro, human endometrial stromal cells (hESCs) were transfected respectively with ERβ-overexpressing and NLRC5-overexpressing plasmids. We then assessed the expression of ERβ and NLRC5 using quantitative real-time PCR (qRT-PCR) and western blot analysis. Furthermore, we measured the concentrations of TNF-α, IL-6, and IL-10 in the cell culture supernatant through enzyme-linked immunosorbent assay (ELISA). Additionally, we evaluated the migration and invasion ability of hESCs using transwell and wound healing assays.ResultsInhibition of NLRC5 expression promotes the development of ectopic lesions in mice with endometriosis, upregulates the expression of pro-inflammatory factors TNF-α and IL-6, and downregulates the expression of anti-inflammatory factor IL-10. The high expression of NLRC5 in endometriosis depended on the ERβ overexpression. And ERβ promoted the migration of hESCs partially depend on inflammatory microenvironment. Lastly, NLRC5 overexpression inhibited ERβ-mediated development and inflammatory response of endometriosis.ConclusionsOur results suggest that the innate immune molecule NLRC5-mediated anti-inflammation participates in ERβ-mediated endometriosis development, and partly clarifies the pathological mechanism of endometriosis, expanding our knowledge of the specific molecules related to the inflammatory response involved in endometriosis and potentially providing a new therapeutic target for endometriosis.

Diarylpropionitrile-stimulated ERβ nuclear accumulation promotes MyoD-induced muscle regeneration in mdx mice by interacting with FOXO3A

Duchenne muscular dystrophy (DMD) is an X-linked recessive progressive degenerative disease of skeletal muscle, characterized by intramuscular inflammation, muscle regeneration disorder and replacement of muscle with fibroadipose tissue. DMD is caused by the absence of normal dystrophy. Impaired self-renew ability and limited differentiation capacity of satellite cells are proved as main reasons for muscle regeneration failure. The deficiency of estrogen impedes the process of muscle regeneration. However, the role of estrogen receptor β (ERβ) in muscle regeneration is still unclear. This study aims to investigate the role and the pharmacological effect of ERβ activation on muscle regeneration in mdx mice. This study showed that mRNA levels of ERβ and myogenic-related genes both witnessed increasing trends in dystrophic context. Our results revealed that treatment with selective ERβ agonist (DPN, diarylpropionitrile) significantly increased myogenic differentiation 1 (MyoD-1) level and promoted muscle regeneration in mdx mice. Similarly, in mdx mice with muscle-specific estrogen receptor α (ERα) ablation, DPN treatment still promoted muscle regeneration. Moreover, we demonstrated that myoblasts differentiation was accompanied by raised nuclear accumulation of ERβ. DPN treatment augmented the nuclear accumulation of ERβ and, thus, contributed to myotubes formation. One important finding was that forkhead box O3A (FOXO3A), as a pivotal transcription factor in Myod-1 transcription, participated in the ERβ-promoted muscle regeneration. Overall, we offered an interesting explanation about the crucial role of ERβ during myogenesis.

The effect of Mongolian Medicine Chagan Gaoyou-4 Powder on bone mineral density induced by retinoic acid in rats

Objective: To investigate the effects of Chagan-Gaoyou-4 powder on spine bone mineral density and serum estrogen and estrogen receptor alpha (ERa) and estrogen receptor beta (ERβ) protein expression in bone tissue induced by retinoic acid in rats. Methods: Sixty 3-month-old SPF female SD rats were randomly divided into model group (retinoin group), normal group (SHAM group), Mongolian medicine group (Chagan-Gaoyou 4 group), and control group (Gushukang granule group). After 14 days of administration, spinal Bone Mineral Density (BMD) was measured, and external manifestations were observed. Results: Compared with the SHAM group, the BMD values of all medication groups decreased (P &lt; 0.05, P &lt; 0.001). Compared with the model group, the spinal BMD value of the Chagan Gaoyou-4 group was increased after 21 days of treatment, and the difference was statistically significant (P &lt; 0.01, P &lt; 0.05). After 21 days, the spinal BMD value of all treatment groups was increased (P &lt; 0.05), the spinal BMD value was also increased, especially Chagan Gaoyou-4 group was more obviously increased (P &lt; 0.05). Compared with the normal group, ERa and ERβ levels in the model group were significantly decreased (P &lt; 0.001), and ERa and ERβ levels in all drug groups were significantly increased (P &lt; 0.001) compared with the model group. After 21 days of treatment, there was no significant difference among the three treatment groups (P &gt; 0.05). ERa and ERβ levels in the Chagan Gaoyou-4 group were higher than those in the model group (P &lt; 0.05), but there was no significant difference between the group and the Gushukang granule group (P &gt; 0.05). The E2 content of the Chagan Gaoyou-4 group was higher than that of the model group (P &lt; 0.05). The mRNA expression of estrogen receptor ERa and ERβ in bone tissue of the left proximal femur was detected by RT-PCR. Compared with the model group, the expression of ERa and ERβ mRNA in the Chagan Gaoyou-4 group and Gushukang granule group was increased. Compared with the model group, ERp mRNA expression was increased in the Chagan Gaoyou-4 powder medium-dose group. Compared with the Gushukang granule group, there was no significant difference in ERm RNA expression in the Chagan Gaoyou-4 dose groups. Chagan Gaoyou-4 can up-regulate the expression of ERa and ERβ induced by retinoic acid in rats, indicating that the Chagan Gaoyou-4 powder group may promote bone formation, regulate bone resorption, improve bone mineral density, and achieve the purpose of preventing and treating OP by increasing estrogen level, stimulating estrogen receptor, and increasing the expression of ERa and ERβ in bone tissue. Conclusion: Chagan-Gaoyu-4 powder may have estrogen-like effects on the bone tissue of rats induced by retinoic acid and may increase the level of serum estrogen, ERa, and ERβ protein expression, thereby improving the spinal BMD of experimental rats. The Chagan-Gaoyou-4 powder group could improve the general condition of osteoporosis induced by retinoic acid in rats.

Pen2/ErbB4 signaling regulates stemness of pancreatic ductal carcinoma

Cancer stem cells (CSCs) are critical for progression, invasion, metastasis, and chemotherapy resistance of pancreatic ductal adenocarcinoma (PDAC). Presenilin enhancer 2 (Pen2), a vital component of the gamma-secretase complex, is overexpressed in various cancers and plays a significant role in carcinogenesis. Here, we investigated the association between Pen2 expression and the stem-like properties of PDAC cells. We analyzed Pen2 and its downstream target, Erb-B2 Receptor Tyrosine Kinase 4 (ErbB4), using public databases. The expression of Pen2 in CSC populations, marked by CD133+, CD44+, or epithelial cell adhesion molecule (EpCAM)+, was evaluated. Pen2-positive cells were sorted from Pen2-negative ones in PDAC cells transduced with a vector designed to express green fluorescent protein (GFP) under the Pen2 promoter. Stemness was examined in vitro and in vivo in Pen2-positive versus Pen2-negative cells. Our results showed that Pen2 was significantly upregulated, while ErbB4 was significantly downregulated in PDAC tissues compared to adjacent non-tumorous tissues, with an inverse relationship between Pen2 and Erbb4 levels. PDACs with high Pen2 expression are associated with considerably poorer patient survival. The CSC populations identified by CD133+, CD44+, and EpCAM+ markers displayed significantly higher Pen2 and lower EpCAM levels. Compared to Pen2-negative PDAC cells, Pen2-positive cells formed more tumor spheres, were more invasive and migratory, and showed significantly increased resistance to chemotherapy-induced apoptosis. Altering Pen2 levels reversed these oncogenic effects. In vivo, Pen2-positive cells formed larger tumors in immunodeficient mice. Overall, our findings suggest that Pen2 is highly expressed in CSCs within PDAC cells, being a novel therapeutic target.

ERβ activation improves nonylphenol-induced depression and neurotransmitter secretion disruption via the TPH2/5-HT pathway

The aim of this study is to investigate the role of estrogen receptor β (ERβ) in nonylphenol (NP) - induced depression - like behavior in rats and its impact on the regulation of the TPH2/5-HT pathway. In the in vitro experiment, rat basophilic leukaemia cells (RBL-2H3) cells were divided into the four groups: blank group, NP group (20 μM), ERβ agonist group (0.01 μM), and NP＋ERβ agonist group (20 μM＋0.01 μM). For the in vivo experiment, 72 adult male Sprague-Dawley rats were randomly divided into following six groups: the Control, NP (40 mg/kg) group, ERβ agonist (2 mg/kg, Diarylpropionitrile (DPN)) group, ERβ inhibitor (0.1 mg/kg, 4-(2-phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl) phenol (PHTPP)) group, NP+ERβ agonist (40 mg/kg NP ＋ 2 mg/kg DPN) group, and NP+ERβ inhibitor (40 mg/kg NP + 0.1 mg/kg PHTPP) group, with 12 rats in each group. Each rat in drug group were given NP by gavage and/or received a single intraperitoneal injection of DPN 2 mg/kg or PHTPP 0.1 mg/kg. Both in vivo and in vitro, NP group showed a decrease in the expression levels of ERβ, tryptophan hydroxylase (TPH1), and tryptophan hydroxylase-2 (TPH2) genes and proteins, and reduced levels of DA, NE, and 5-hydroxytryptophan (5-HT) neurotransmitters. RBL-2H3 cells showed signs of cell shrinkage, with rounded cells, increased suspension and more loosely arranged cells. The effectiveness of the ERβ agonist stimulation exhibited an increase exceeding 60% in RBL-2H3 cells. The application of ERβ agonist resulted in an alleviation the aforementioned alterations. ERβ agonist activated the TPH2/5-HT signaling pathways. Compared to the control group, the NP content in the brain tissue of the NP group was significantly increased. The latency to eat for the rats was longer and the amount of food consumed was lower, and the rats had prolonged immobility time in the behavioral experiment of rats. The expression levels of ERβ, TPH1, TPH2, 5-HT and 5-HITT proteins were decreased in the NP group, suggesting NP-induced depression-like behaviours as well as disturbances in the secretion of serum hormones and monoamine neurotransmitters. In the NP group, the midline raphe nucleus showed an elongated nucleus with a dark purplish-blue colour, nuclear atrophy, displacement and pale cytoplasm. ERβ might ameliorate NP-induced depression-like behaviors, and secretion disorders of serum hormones and monoamine neurotransmitters via activating TPH2/5-HT signaling pathways.

Co-exposure to cadmium and triazophos induces variations at enzymatic and transcriptional levels in Opsariichthys bidens

Heavy metals and pesticides are significant pollutants in aquatic environments, often leading to combined pollution and exerting toxic effects on aquatic organisms. With the rapid growth of modern industry and agriculture, heavy metal cadmium (Cd) and pesticide triazophos (TRI) are frequently detected together in various water bodies, particularly in agricultural watersheds. However, the combined toxic mechanisms of these pollutants on fish remain poorly understood. This experiment involved a 21-day co-exposure of Cd and TRI to the hook snout carp Opsariichthys bidens to investigate the toxic effects on liver tissues at both enzymatic and transcriptional levels. Biochemical analysis revealed that both individual and combined exposures significantly increased the content or activity of caspase-3 (CASP-3) and malondialdehyde (MDA). Moreover, the impact on these parameters was greater in the combined exposure groups compared to the corresponding individual exposure groups. These findings suggested that both individual and combined exposures could induce mitochondrial dysfunction and lipid peroxidation damage, with combined exposure exacerbating the toxicological effects of each individual pollutant. Furthermore, at the molecular level, both individual and combined exposures upregulated the expression levels of cu-sod, cat, and erβ, while downregulating the expression of il-1. Similar to the patterns observed in the biochemical parameters, the combined exposure group exhibited a greater impact on the expression of these genes compared to the individual exposure groups. These results indicated that exposure to Cd, TRI, and their combination induced oxidative stress, endocrine disruption, and immunosuppression in fish livers, with more severe effects observed in the combined exposure group. Overall, the interaction between Cd and TRI appeared to be synergistic, shedding light on the toxic mechanisms by which fish livers responded to these pollutants. These findings contributed to the understanding of mixture risk assessment of pollutants and were valuable for the conservation of aquatic resources.

Defining low-HER2 cancers using RNA quantification by next generation sequencing.

e13044 Background: The recent success of treatment with HER2-directed antibody-drug conjugate T-DXd (trastuzumab-deruxtecan) in treating low-HER2 expression breast cancer raises the possibility of similar success in other tumors that express similarly low levels of HER2. HER2 amplification has been reported in various types of tumors at relatively low frequency. However, “low-HER2” expression in various types of tumors remains undefined. In this study, we utilized next generation sequencing to explore the levels of ERBB2 (HER2), ERBB3, ERBB4, PIK3CA, and ERS1 mRNA in various types of cancers and attempted to set limits reflecting distinct biology of tumors based of level of ERBB2 expression. Methods: RNA was extracted from FFPE samples from patients with cancers of breast (N=203), ovary (N=95), endometrium (N=75), and esophagus/stomach (N=46). RNA was sequenced via a 1408 gene panel using a standard hybrid capture approach. Results: Based on previous data correlating gene amplification with gene expression (DOI: 10.1002/imed.1051), we defined ERBB2 expression as high, low, and very-low when ERBB2 expression measured &gt;1500 TPM (transcripts per million), 800-1500 TPM, and &lt;800 TPM, respectively. ERBB2 high, low, and very-low were detected in 20%, 32%, and 48% of breast cancer; 5%, 29%, and 65% of endometrial cancer; 9%, 26%, and 65% of ovarian cancer; 12%, 16%, and 72% of esophageal/stomach cancer; and 8%, 19%, and 73% of lung cancer. With minor exceptions, cases classified as ERBB2 high and low from the five tumor types showed no correlation with levels of PIK3CA, ESR1, or ERBB4 mRNA. In contrast, cases with relatively very-low ERBB2 showed significant correlation with PIK3CA, ESR1, and ERBB4 mRNA. Endometrial cancer was an exception and showed no correlation with PIK3CA mRNA in very-low ERBB2 cases. Activation of PIK3CA has been reported to be associated with resistance to anti-HER2 therapy. These findings suggest that cases showing high or low ERBB2 are independent of PIK3CA and likely less influenced by the negative effects of PIK3CA on response to HER2 inhibitors. Cases classified as ERBB2 high or low showed significantly higher (P&lt;0.0001) PIK3CA mutations as compared with ERBB2 very-low cases (27% vs 14%). ERBB2 mutations were relatively rare, detected in only 7%, 8%, 14%, 1%, and 4% of breast, endometrial, esophageal/stomach, ovarian, and lung cancers, respectively. However, significantly (P=0.001) higher mutation rate is detected in ERBB2-high and -low as compared with very-low ERBB2 cases (8% vs 6%). Conclusions: ERBB2/HER2 RNA quantification in cancers can define a biologically distinct group of patients. Patients defined as very-low ERBB2 as classified in this approach appear to be biologically distinct and more likely to be resistant to anti-HER2 therapy. Clinical trials using this approach in selecting patients for treatment with antibody-drug conjugates are justified and needed.

Analysis of the sensitivity to endocrine therapy (SET) assay in the PALLAS adjuvant trial of palbociclib in HR+/HER2- breast cancer (ABCSG-42/AFT-05/BIG-14-13).

538 Background: The phase 3 PALLAS trial compared 2 years of the CDK4/6 inhibitor palbociclib with endocrine therapy of provider choice, versus endocrine therapy alone, as adjuvant treatment for patients (pts) with Stage II-III hormone receptor-positive HER2-negative (HR+/HER2-) breast cancer. The SET2,3 index adjusts the measurement of SETER/PR index of endocrine receptor-related transcriptional activity with a baseline prognostic index (BPI) derived from tumor size (T), number of involved lymph nodes (N) and gene expression levels of ESR1, PGR, ERBB2 and AURKA (RNA4 subtype score). High SET2,3 (index ≥ 2.10) represents high endocrine sensitivity with favorable prognosis. Clinical outcome results from PALLAS were previously published, and the trial now has 5-year median follow-up. Methods: Participants in PALLAS provided a tumor tissue block prior to randomization. RNA was extracted from samples with sufficient tumor tissue and cellularity (&gt;25mm2 with ≥20% cancer nuclei). SET2,3 index was measured from 300ng total RNA using the QuantiGene Plex platform (ThermoFisher, Waltham, MA) following the manufacturer’s protocol and using pre-defined quality control (QC) parameters and cut point. Results from primary tumor samples prior to systemic therapy were analyzed. Invasive disease-free survival (iDFS) was summarized with Kaplan-Meier plots using log-rank test. Cox models were adjusted for treatment arm assignment, with a treatment arm by SET index value interaction term to test for prediction of palbociclib benefit. The pre-defined level of significance was a two-sided 0.05. Results: There were 4075 pts from the total PALLAS population (N=5796) who provided a primary tumor sample that met pre-analytical requirements to perform the SET assay, from which 3388 (83%) results passed quality control, including 3093 from a sample obtained prior to receiving systemic therapy (1559 in palbociclib arm and 1534 in the control arm). SET2,3 index could not be calculated for 12 pts due to missing tumor or nodal information. There was no evidence of an interaction between treatment arm and SET indices (continuous) for iDFS: SET2,3 index interaction p=0.12 and SETER/PR index interaction p=0.92. Estimated 5-year iDFS was superior for those with high SET2,3: iDFS was 91.4% (95%CI 89.9–92.8) in 1719 (55.8%) pts with high SET2,3, versus 78.5% (95%CI 76.0–80.8) in 1362 (44.2%) pts with low SET2,3 (HR 0.38, 95%CI 0.31–0.47, p&lt;0.0001). The SETER/PR index was also significantly associated with iDFS (HR 0.70 per 0.5 units, 95%CI 0.65–0.77, p&lt;0.0001). Conclusions: There was no evidence that the SET2,3 or SETER/PR index of endocrine-related transcriptional activity predicts benefit from palbociclib treatment. SET2,3 index was strongly prognostic for pts with Stage II-III breast cancer, with excellent 5-year IDFS for pts classified as SET2,3 high. Clinical trial information: NCT02513394 .

Prediction of response to neoadjuvant chemotherapy by MammaTyper® across breast cancer subtypes: A retrospective cross-sectional study

BackgroundNeoadjuvant chemotherapy (NACT) is widely used in the treatment of triple-negative and HER2-positive breast cancer (BC), but its use in estrogen receptor (ER) and/or progesterone receptor (PR) positive/HER2-negative BC is questioned because of the low pathologic complete response (pCR) rates. This retrospective study assessed the mRNA-based MammaTyper® assay's capability of predicting pCR with NACT, and ER, PR, Ki67, and HER2 status at immunohistochemical (IHC) through transcriptomics. MethodsDiagnostic biopsies from 76 BC patients treated at the Cremona Hospital between 2012-2018 were analyzed. Relative mRNA expression levels of ERBB2, ESR1, PGR, and MKI67 were measured using the MammaTyper® kit and integrated into a pCR score. Predicting capability of pCR and standard IHC biomarkers could be assessed with ROC curves in 75 and 76 patients, respectively. ResultsOverall, 68.0% patients obtained a MammaTyper® high-score and 32.0% a MammaTyper® low-score. Among high-score patients, 62.7% achieved pCR, compared to 16.7% in the low-score group (p = 0.0003). The binary MammaTyper® score showed good prediction of pCR in the overall cohort (area under curve [AUC] = 0.756) and in HR+/HER2-negative cases (AUC = 0.774). In cases with residual disease, the continuous MammaTyper® score correlated moderately with residual tumor size and decrease in tumor size. MammaTyper® showed substantial agreement with IHC for ESR1/ER and ERBB2/HER2, and moderate agreement for PGR/PR and MKI67/Ki67. ConclusionOverall, MammaTyper® pCR score may serve as a standardized tool for predicting NACT response in HR+/HER2-negative BC, potentially guiding treatment strategies. Additionally, it could provide a more standardized and reproducible assessment of ER, PR, HER2, and Ki67 status.

Mechanistic insights into the parental co-exposure of T-2 toxin and epoxiconazole on the F1 generation of zebrafish (Danio rerio)

Mycotoxins and pesticides frequently coexist in agricultural commodities on a global scale. The potential transgenerational consequences induced by these substances pose a significant threat to human health. However, there is a lack of data concerning the effects of co-contamination by these chemicals in the F1 generation following parental exposure. This investigation delved into the mixture effects of T-2 toxin (T-2) and epoxiconazole (EPO) on the offspring of zebrafish (Danio rerio). The findings revealed that exposure across generations to a combination of T-2 and EPO resulted in toxicity in the larvae of the F1 generation. This was demonstrated by a significant increase in the levels or activities of malondialdehyde (MDA), thyroxine (T4), Caspase3, and cas9, along with a decrease in the levels of cyp19a, ERα, and ERβ. These outcomes suggested that cross-generational exposure to T-2 and EPO in D. rerio disrupted oxidative balance, induced cell apoptosis, and affected the endocrine system. Moreover, these effects were magnified when the F1 generation was continuously exposed to these compounds. Notably, these adverse effects could persist in subsequent generations without additional exposure. This study underscored the potential dangers associated with the simultaneous presence of T-2 and EPO on the development of fish offspring and the resulting environmental hazards to aquatic ecosystems. These findings emphasized the significant health risks posed by cross-generational exposure and highlighted the need for additional legislative measures to address these concerns.

Changes in ovarian tissue structure and distribution of oestrogen receptors in Huanghuai goats at different ages.

To observe developmental changes in the ovarian tissue structure and distribution characteristics of oestrogen receptors (ERs) in the ovaries of Huanghuai goats at different ages, we selected healthy Huanghuai goats ewes and divided them into five groups (i.e. 3-, 30-, 60-, 90- and 120-day-old groups), with 10 animals in each group. The serum was separated after blood collection through the jugular vein, and the contents of oestrogen (E) and progesterone (P) in the serum of Huanghuai goats at each age were determined. Three goats were randomly selected from each group and sacrificed after anaesthesia, and the ovarian tissue was quickly obtained and placed in 4% paraformaldehyde fixative to prepare the tissue sections. Using HE, oestrogen receptors were immunohistochemically stained and observed. These results showed many primordial follicles and occasional secondary follicles in the ovaries of 3-day-old Huanghuai goats. Ovarian reticular structures were observed in 30-day-old ovarian medulla, with occasional near-mature growing follicles. Mature follicles and corpus luteum were occasionally detected in 60-day-old ovarian cortex. The 90-120-day-old ovarian cortices contained growing and mature follicles, and the number of mature follicles and corpora lutea increased, implying a significant luteal involution period. The E and P contents in the 120-day-old group were significantly higher than those in the 3-, 30-, 60- and 90-day-old groups. The levels of ERα and ERβ in the 3- and 30-day-old groups were mainly distributed in the granulosa cells of ovarian reproductive epithelial cells, primordial follicles, atretic follicles, and primary and secondary follicles. The ERα and ERβ levels of the 60-, 90- and 120-day-old groups were also distributed in the granulosa cells and luteal cells of mature follicles, especially in the 120-day-old endometrial cells of mature follicles, where ERβ was distributed significantly. The overall expression of ERβ in the ovary was higher than that of ERα. The results of this study provide basic data on the ovarian development and the specific expression of ERs and PRs in the ovaries of Huanghuai white goats, which play an important role in ovarian development and precocity.

25-hydroxycholesterol promotes proliferation and metastasis of lung adenocarcinoma cells by regulating ERβ/TNFRSF17 axis.

Lung adenocarcinoma is the main type of lung cancer in women. Our previous findings have evidenced that 25-hydroxycholesterol (25-HC) promotes migration and invasion of lung adenocarcinoma cells (LAC), during which LXR as a 25-HC receptor plays an important role. Estrogen receptor beta (ERβ) is a receptor of 27-hydroxycholesterol that is structurally analogous to 25-HC, but its role in the functional actions of 25-HC remained largely unknown. In this study, we demonstrated that 25-HC treatment triggered ERβ expression in LAC. Knockdown of ERβ inhibited 25-HC-mediated proliferation, migration and invasion, and reduced 25-HC-induced LAC metastasis in vivo. Further investigation revealed that ERβ knockdown restrained the expression of TNFRSF17 (BCMA). In vivo experiments also confirmed that ERβ knockdown blocked 25-HC-induced TNFRSF17 expression. TNFRSF17 knockdown also restrained 25-HC-induced proliferation, migration and invasion. Bioinformatic analysis showed that the levels of ERβ and TNFRSF17 were elevated in lung adenocarcinoma, and were closely related to tumor stages and nodal metastasis status. These results suggested that 25-HC promoted the proliferation and metastasis of LAC by regulating ERβ/TNFRSF17 axis.