Search Results (104)

Hormone-dependent breast cancer, particularly in its treatment-resistant forms, remains a significant therapeutic challenge. In this study, we applied a fully computational strategy to design steroid-based compounds capable of simultaneously targeting three key receptors involved in disease progression: progesterone receptor (PR), estrogen receptor alpha (ER-α), and HER2. Using a robust 3D-QSAR model (R² = 0.86; Q²_LOO = 0.86) built from 52 steroidal structures, we identified molecular features associated with high anticancer potential, specifically increased polarizability and reduced electronegativity. From a virtual library of 271 DFT-optimized analogs, 31 compounds were selected based on predicted potency (pIC₅₀ > 7.0) and screened via molecular docking against PR (PDB 2W8Y), HER2 (PDB 7JXH), and ER-α (PDB 6VJD). Seven candidates showed strong binding affinities (ΔG ≤ −9 kcal/mol for at least two targets), with Estero-255 emerging as the most promising. This compound demonstrated excellent conformational stability, a robust hydrogen-bonding network, and consistent multitarget engagement. Molecular dynamics simulations over 100 nanoseconds confirmed the structural integrity of the top ligands, with low RMSD values, compact radii of gyration, and stable binding energy profiles. Key interactions included hydrophobic contacts, π–π stacking, halogen–π interactions, and classical hydrogen bonds with conserved residues across all three targets. These findings highlight Estero-255, alongside Estero-261 and Estero-264, as strong multitarget candidates for further development. By potentially disrupting the PI3K/AKT/mTOR signaling pathway, these compounds offer a promising strategy for overcoming resistance in hormone-driven breast cancer. Experimental validation, including cytotoxicity assays and ADME/Tox profiling, is recommended to confirm their therapeutic potential. Full article

HMG-CoA reductase inhibitors, statins, are extensively used to treat hyperlipidemia, coronary artery disease, and other atherosclerotic disorders. However, one of the common side effects of statin therapy is a mild elevation in liver aminotransferases, observed in less than 3% of patients. Atorvastatin and simvastatin, in particular, are most frequently associated with statin-induced liver injury, leading to treatment discontinuation. Recent research has highlighted the antioxidant and anti-inflammatory properties of glucagon-like peptide-1 receptor (GLP-1R) activation in protecting against liver injury. Nonetheless, the potential protective effects of liraglutide (LIRA), a GLP-1R agonist, against atorvastatin (ATO)-induced liver dysfunction have not been fully elucidated. In this context, the present study aimed to investigate the protective role of LIRA in mitigating ATO-induced liver injury in rats, offering new insights into managing statin-associated hepatotoxicity. Indeed, LIRA treatment improved liver function enzymes and attenuated histopathological alterations. LIRA treatment enhanced antioxidant defenses by increasing Nrf2 content and superoxide dismutase (SOD) activity, while reducing NADPH oxidase. Additionally, LIRA suppressed inflammation by downregulating the HMGB1/TLR-4/RAGE axis and inhibiting the protein expression of pY323-MAPK p38 and pS635-NFκB p65 content resulting in decreased proinflammatory cytokines (TNF-α and IL-1β). Furthermore, LIRA upregulated GLP-1R gene expression and promoted autophagic influx via the activation of the pS473-Akt/pS486-AMPK/pS758-ULK1/Beclin-1 signaling cascade, along with inhibiting apoptosis by reducing caspase-3 content. In conclusion, LIRA attenuated ATO-induced oxidative stress and inflammation via activation of the Nrf-2/SOD cascade and inhibition of the HMGB1/TLR-4/RAGE /MAPK p38/NFκB p65 axis. In parallel, LIRA stimulated autophagy via the AMPK/ULK1/Beclin-1 axis and suppressed apoptosis, thus restoring the balance between autophagy and apoptosis. Full article

Prolactin is a polypeptide hormone that plays a critical role in male reproduction. However, the underlying mechanisms of prolactin-regulated testosterone secretion and the roles of lncRNAs in this process remain unclear. We performed a comprehensive analysis of the testicular tissues of cashmere goats with different prolactin levels by means of RNA-sequencing. Then, we constructed a lncRNA–miRNA–mRNA interaction network by integrating previously submitted testicular mRNA sequencing data. We identified a novel lncRNA named lncRWDD3 and investigated its effects on testosterone synthesis in the Leydig cells of cashmere goat. The primary Leydig cells were used to explore the biological function of lncRWDD3/miR-1388-5p/NPY1R in vitro. This study found that 200 ng/mL of prolactin achieved the highest testosterone secretion in Leydig cells. LncRWDD3 or NPY1R overexpression promoted cAMP levels, testosterone secretion, and related gene expression, while lncRWDD3 or NPY1R interference had the opposite effect. It was found that lncRWDD3 acts on miR-1388-5p as a ceRNA, and neuropeptide Y receptor Y1 (NPY1R) was confirmed to be a target of chi-miR-1388-5p. Our research shows that prolactin regulates the testicular function of cashmere goats via the lncRNA–miRNA–mRNA ceRNA network, and lncRWDD3 acts as a ceRNA to activate NPY1R/cAMP signaling via the sponging of miR-1388-5p in order to govern testosterone synthesis in the Leydig cells of cashmere goats. Our results provide insights for future studies on the molecular mechanism of the prolactin regulation of testicular function in goats. Full article

Synbiotics can be used to reduce intestinal inflammation and mitigate dysbiosis in dogs with chronic inflammatory enteropathy (CIE). Prior research has not assessed the colonic mucosal ultrastructure of dogs with active CIE treated with synbiotics, nor has it determined a possible association between morphologic injury and signaling pathways. Twenty client-owned dogs diagnosed with CIE were randomized to receive either a hydrolyzed diet (placebo; PL) or a hydrolyzed diet supplemented with synbiotic-IgY (SYN) for 6 weeks. Endoscopic biopsies of the colon were obtained for histopathologic, ultrastructural, and molecular analyses and were compared before and after treatment. Using transmission electron microscopy (TEM), an analysis of the ultrastructural alterations in microvilli length (MVL), mitochondria (MITO), and rough endoplasmic reticulum (ER) was compared between treatment groups. To explore potential signaling pathways that might modulate MITO and ER stress, a transcriptomic analysis was also performed. The degree of mucosal ultrastructural pathology differed among individual dogs before and after treatment. Morphologic alterations in enterocytes, MVL, MITO, and ER were detected without significant differences between PL and SYN dogs prior to treatment. Notable changes in ultrastructural alterations were identified post-treatment, with SYN-treated dogs exhibiting significant improvement in MVL, MITO, and ER injury scores compared to PL-treated dogs. Transcriptomic profiling showed many pathways and key genes to be associated with MITO and ER injury. Multiple signaling pathways and their associated genes with protective effects, including fibroblast growth factor 2 (FGF2), fibroblast growth factor 7 (FGF7), fibroblast growth factor 10 (FGF10), synaptic Ras GTPase activating protein 1 (SynGAP1), RAS guanyl releasing protein 2 (RASGRP2), RAS guanyl releasing protein 3 (RASGRP3), thrombospondin 1 (THBS1), colony stimulating factor 1 (CSF1), colony stimulating factor 3 (CSF3), interleukin 21 receptor (IL21R), collagen type VI alpha 6 chain (COL6A6), ectodysplasin A receptor (EDAR), forkhead box P3 (FoxP3), follistatin (FST), gremlin 1 (GREM1), myocyte enhancer factor 2B (MEF2B), neuregulin 1 (NRG1), collagen type I alpha 1 chain (COL1A1), hepatocyte growth factor (HGF), 5-hydroxytryptamine receptor 7 (HTR7), and platelet derived growth factor receptor beta (PDGFR-β), were upregulated with SYN treatment. Differential gene expression was associated with improved MITO and ER ultrastructural integrity and a reduction in oxidative stress. Conversely, other genes, such as protein kinase cAMP-activated catalytic subunit beta (PRKACB), phospholipase A2 group XIIB (PLA2G12B), calmodulin 1 (CALM1), calmodulin 2 (CALM2), and interleukin-18 (IL18), which have harmful effects, were downregulated following SYN treatment. In dogs treated with PL, genes including PRKACB and CALM2 were upregulated, while other genes, such as FGF2, FGF10, SynGAP1, RASGRP2, RASGRP3, and IL21R, were downregulated. Dogs with CIE have colonic ultrastructural pathology at diagnosis, which improves following synbiotic treatment. Ultrastructural improvement is associated with an upregulation of protective genes and a downregulation of harmful genes that mediate their effects through multiple signaling pathways. Full article

Hypertension and diabetes are two common causes of chronic kidney disease. Hypertension can induce renal vascular injury, glomerular damage, podocyte loss, and tubular injury, leading to tubulointerstitial fibrosis. A number of factors influence the regulation of hypertension, among which G-protein-coupled receptors (GPCRs) have been studied extensively because they are desirable targets for drug development. Compared to hypertension, the regulatory effects of GPCRs on hypertensive kidney disease (HKD) are less generalized. In this review, we discussed the GPCRs involved in hypertensive kidney disease, such as angiotensin II receptors (AT1R and AT2R), Mas receptor (MasR), Mas-related G-protein-coupled receptor member D (MrgD), relaxin family receptor 1 (RXFP1), adenosine receptors (A₁, A_2A, A_2B, and A₃), purinergic P2Y receptors, and endothelin receptors (ET_A and ET_B). The progression of HKD is rarely reversed but can be retarded by ameliorating the hypertensive microenvironment in the kidneys. However, simply reducing blood pressure cannot stop the progression of HKD. Diabetic nephropathy (DN) is the most common cause of end-stage renal disease (ESRD), which is a major cause of morbidity and mortality in diabetes. Many GPCRs are involved in DN. Here, we select some well-studied GPCRs that are directly associated with the pathogenesis of DN to illustrate their mechanisms. The main purpose of this review is to provide an overview of the GPCRs involved in the occurrence and progression of HKD and DN and their probable pathophysiological mechanisms, which we hope will help in developing new therapeutic strategies. Full article

Platelet activation processes begin when injury to blood vessels exposes the subendothelial matrix, leading platelets to attach to it, where they become activated and exert their hemostatic function. Excessive platelet aggregation is associated with thrombotic disorders such as arterial thrombosis. To manage such diseases, medications that inhibit thrombosis are continuously sought, despite potential drawbacks that include hemorrhage. This study described the development of a novel peptide-based vaccine that targets the purinergic ADP P2Y₁ receptor (abbreviated EL2Vac) and its pharmacological characterization. Thus, we designed and developed an EL2Vac that targets the ligand-binding domain of the P2Y₁ receptor protein, which is located in its second extracellular loop (EL2). We then evaluated the vaccine’s ability to trigger an immune response (antibody production) in immunized mice, modulate platelet function, its antithrombotic activity, and any effects on hemostasis, by employing a thrombosis model and the tail bleeding time assay. Results showed significant levels of antibody production in mice treated with EL2Vac, in comparison with the random peptide vaccine control (EL2rVac), which persisted at least up to six months post vaccination. Moreover, we observed significant inhibition of the ADP-induced aggregation response in platelets from EL2Vac-treated mice, relative to those from EL2rVac controls. This inhibition was selective for ADP, as other agonists, such as the thromboxane A₂ receptor (TPR) agonist U46619 or high-dose collagen, had no detectable effect on aggregation. As for its capacity to protect against thrombosis, our data showed a significant delay in the occlusion time of the EL2Vac mice when compared with the random peptide control vaccine, which was also observed (at least) six months post vaccination. Interestingly, EL2Vac did not appear to prolong the tail bleeding time, supporting the notion that it is devoid of a bleeding diathesis. As a conclusion, this study documents the design and evaluation of a novel peptide-based vaccine, EL2Vac, which appears to selectively target the P2Y₁ receptor and protect against thrombus formation without impairing hemostasis. Thus, EL2Vac may provide a promising clinical option to treat thromboembolic disorders. Full article

Background: Sepsis is a life-threatening condition that is characterized by systemic inflammation and organ dysfunction, with adrenal dysfunction being a significant complication. This study aimed to investigate the role of necroptosis and hydrogen sulfide (H₂S) in sepsis-induced adrenal dysfunction. Methods: A cecal ligation and puncture (CLP)-induced sepsis mouse model was employed. Adrenocortical-specific mixed lineage kinase domain-like pseudokinase (MLKL) knockout (MLKL-KO) and cystathioneine β-synthase (CBS) knockout (CBS-KO) mice were generated using Cre-loxP technology and adrenocortical-specific Cre tool mice. In vitro experiments utilized TNFα-stimulated Y1 adrenocortical cells. The treatments included the H₂S donor NaHS, TNFα inhibitor R-7050, necroptosis inhibitor NSA and CBS inhibitor AOAA. Pathological assessment involved hematoxylin–eosin (H&E) staining and a Western blot analysis of necroptosis markers (the phosphorylation of MLKL (p-MLKL) and phosphorylation of receptor-interacting protein kinases 1 (p-RIPK1)). Results: Sepsis induced adrenal congestion, elevated TNFα levels, and activated necroptosis (increased p-MLKL/p-RIPK1) in wild-type mice. H₂S treatment attenuated adrenal damage, reduced TNFα, and suppressed necroptosis. MLKL knockout reduced septic adrenal dysfunction, whereas CBS knockout exacerbated septic adrenal dysfunction. In vitro, TNFα induced Y1 cell necroptosis, which was reversed by H₂S or NSA. AOAA exacerbated TNFα-induced necroptosis in Y1 cells. Conclusions: H₂S inhibits TNFα-mediated necroptosis, thereby preserving adrenal integrity in sepsis. Targeting the TNFα–necroptosis axis and enhancing endogenous H₂S production may represent novel therapeutic strategies for sepsis-associated adrenal dysfunction. Full article

(Proline3)PP, or (P³)PP, is an enzymatically stable, neuropeptide Y4 receptor (NPY4R)-selective, pancreatic polypeptide (PP) analogue with established weight-lowering and pancreatic islet morphology benefits in obesity-diabetes. In the current study, we now investigate the impact of twice-daily (P³)PP administration (25 nmol/kg) for 11 days on islet cell lineage, using streptozotocin (STZ) diabetic Ins1^Cre/+;Rosa26-eYFP and Glu^CreERT2;Rosa26-eYFP transgenic mice with enhanced yellow fluorescent protein (eYFP) labelling of beta-cell and alpha-cells, respectively. (P³)PP had no obvious impact on body weight or blood glucose levels in STZ-diabetic mice at the dose tested, but did return food intake towards control levels in Ins1^Cre/+;Rosa26-eYFP mice. Notably, pancreatic insulin content was augmented by (P³)PP treatment in both Ins1^Cre/+;Rosa26-eYFP and Glu^CreERT2;Rosa26-eYFP mice, alongside enhanced beta-cell area and reduced alpha-cell area. Beneficial (P³)PP-induced changes on islet morphology were consistently associated with decreased beta-cell apoptosis, while (P³)PP also augmented beta-cell proliferation in Ins1^Cre/+;Rosa26-eYFP mice. Alpha-cell turnover rates were returned towards healthy control levels by (P³)PP intervention in both mouse models. In terms of islet cell lineage, increased transition of alpha- to beta-cells as well as decreased beta- to alpha-cell differentiation were shown to contribute towards the enhancement of beta-cell area in (P³)PP-treated mice. Together these data reveal, for the first time, sustained NPY4R activation positively modulates beta-cell turnover, as well as islet cell plasticity, to help preserve pancreatic islet architecture following STZ-induced metabolic stress. Full article

P2X receptors are unspecific cation channels activated by ATP. They are expressed in various tissues and found in neuronal and immune cells. In mammals, seven subunits are described, which can assemble into homomeric and heteromeric trimers. P2X2 receptors play important roles in cochlear adaptation to elevated sound levels. Three mutations causing inherited progressive hearing loss have been identified. These mutations localize to the transmembrane domain 1 (V60L), the transmembrane domain 2 (G353R) and a β-sheet linking the ATP binding site to the pore (D273Y). Herein, mutations were studied in human homomeric P2X2 as well as in heteromeric P2X2/3 receptors. We measured their binding of a fluorescently labeled ATP derivative (fATP) and characterized the constructs using the patch-clamp technique. The conclusions from our results are as follows: 1. The mutations V60L and G353R show robust localization on the plasma membrane and binding of fATP, whereas the mutant D273Y has no binding to fATP. 2. The mutation V60L has an increased affinity to fATP compared with the wildtype. 3. The expression of hP2X2 V60L channels reduces cell viability, which may support its role in the pathogenesis of hearing loss. 4. All mutant P2X2 subunits can assemble into P2X2/3 heteromeric channels with distinct phenotypes. Full article

Purinergic P2 receptors are crucial in energy utilization and cellular signaling, making them key targets for stroke therapies. This study examines the temporal mRNA expression of all P2 receptors in rats and mice. Both species exhibited a common subset of P2X and P2Y receptors with elevated expression following cerebral ischemia and reperfusion (I/R), highlighting conserved mechanisms across these species. The receptors with upregulated expression in both species were P2X3, P2X4, P2X7, P2Y2, and P2Y6. While these similarities were observed, notable differences in receptor expression emerged between rats and mice. Rats exhibited a broader receptor profile, with five additional receptors (P2X1, P2Y1, P2Y12, P2Y13, and P2Y14) significantly upregulated compared to only two receptors (P2X2 and P2Y4) in mice, highlighting species-specific regulation of receptor expression distinct from the shared receptors. Following cerebral I/R, P2Y12 was the most upregulated receptor in rats, while P2Y2 was the most upregulated in mice. These findings reveal both conserved and species-specific changes in P2 receptor expression following cerebral I/R. Targeting purinergic receptors, particularly those conserved and upregulated in response to stroke, may represent a promising therapeutic approach. Full article

Background/Objective: Osteocytes are the most abundant cell type in the skeleton, with key endocrine functions, particularly in regulating osteoblast and osteoclast activity to maintain bone quality. Angiotensin II (Ang II), a critical component of the renin–angiotensin–aldosterone system, is well-known for its role in vasoconstriction during hypertension. Beyond its cardiovascular functions, Ang II participates in various biological processes, including bone metabolism. While its influence on osteoblast proliferation, differentiation, and osteoclastogenesis has been documented, its effects on osteocytes remain unexplored. This study hypothesized that Ang II enhances the osteoclastogenic activity of osteocytes. Methods: Mouse calvariae were cultured ex vivo in an Ang II-containing medium, analyzed via immunohistochemistry, and evaluated for osteoclastogenic gene expression through real-time PCR. Western blotting was employed to assess protein levels and signaling pathway activation in the MLO-Y4 osteocytic cell line in vitro. Results: Ang II significantly increased the expression of receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). These effects were abrogated by azilsartan, a blocker targeting Ang II type 1 receptors (AT1R). p38 and ERK1/2 in the MAPK pathway were also activated by Ang II. Conclusions: Ang II enhances osteocyte-mediated osteoclastogenesis via AT1R activation, highlighting its potential as a therapeutic target for bone diseases. Full article

G protein-coupled receptors (GPCRs) exist in the conformational equilibrium between inactive state and active state, where the proportion of active state in the absence of a ligand determines the basal activity of GPCRs. Although many GPCRs have different basal activity, it is still unclear whether physiological stresses such as substrate stiffness affect the basal activity of GPCRs. In this study, we identified that purinergic P2Y₆ receptor (P2Y₆R) induced spontaneous Ca²⁺ oscillation without a nucleotide ligand when cells were cultured in a silicon chamber. This P2Y₆R-dependent Ca²⁺ oscillation was absent in cells cultured in glass dishes. Coating substrates, including collagen, laminin, and fibronectin, did not affect the P2Y₆R spontaneous activity. Mutation of the extracellular Arg-Gly-Asp (RGD) motif of P2Y₆R inhibited spontaneous activity. Additionally, extracellular Ca²⁺ was required for P2Y₆R-dependent spontaneous Ca²⁺ oscillation. The GPCR screening assay identified cells expressing 10 GPCRs, including purinergic P2Y₁R, P2Y₂R, and P2Y₆R, that exhibited spontaneous Ca²⁺ oscillation under cell culture soft substrate. Our results suggest that stiffness of the cell adhesion surface modulates spontaneous activities of several GPCRs, including P2Y₆R, through a ligand-independent mechanism. Full article

Cenobamate is a new and highly effective antiseizure compound used for the treatment of adults with focal onset seizures and particularly for epilepsy resistant to other antiepileptic drugs. It acts on multiple targets, as it is a positive allosteric activator of γ-aminobutyric acid type A (GABA_A) receptors and an inhibitor of neuronal sodium channels, particularly of the late or persistent Na⁺ current. We recently evidenced the inhibitory effects of cenobamate on the peak and late current component of the human cardiac isoform hNav1.5. The determined apparent IC₅₀ values of 87.6 µM (peak) and 46.5 µM (late current) are within a clinically relevant range of concentrations (the maximal plasma therapeutic effective concentration for a daily dose of 400 mg in humans is 170 µM). In this study, we built a 3D model of the canonical hNav1.5 channel (UniProt Q14524-1) in open conformation using AlphaFold2, embedded it in a DPPC lipid bilayer, corrected the residue protonation state (pH 7.2) with H++, and added 2 Na⁺ ions in the selectivity filter. By molecular docking, we found the cenobamate binding site in the central cavity. We identified 10-point mutant variants in the binding site region and explored them via docking and MD. Mutants N1462K/Y (rs1064795922, rs199473614) and M1765R (rs752476527) (by docking) and N932S (rs2061582195) (by MD) featured higher predicted affinity than wild-type. Full article

Extracellular ATP plays an important role in renal physiology as well as the pathogenesis of acute kidney injury induced by renal ischemia and reperfusion (IR). Expression of the purinergic P2Y2 receptor has been shown on inflammatory and structural cells of the kidney, and P2Y2R is preferably activated by ATP (or UTP). Here, we investigated the molecular mechanism of P2Y2R during IR injury by using P2Y2R knockout (KO) mice and a selective P2Y2R agonist, MRS2768. After renal IR, P2Y2R KO mice showed greater increases in plasma creatinine, tubular damage and neutrophil infiltration, and significant induction of proinflammatory cytokines and apoptotic markers than wild-type (WT) mice. In contrast, treatment with MRS2768 reduced plasma creatinine levels, tubular damage and inflammation, and renal apoptosis in mice subjected to renal IR. In cultured human proximal tubular HK-2 cells, MRS2768 upregulated P2Y2R mRNA levels and decreased TNF-α/cycloheximide-induced apoptosis and inflammation. Importantly, P2Y2R activation by MRS2768 increased the phosphorylation of protein kinase C (PKC), Src, and phosphatidylinositol 3-kinase (PI3K)/Akt. In addition, the inhibition of PI3K/Akt abolished the protective effects of MRS2768 against TNF-α/cycloheximide-induced apoptosis and inflammation in HK-2 cells. In conclusion, activation of P2Y2R protects against tubular apoptosis and inflammation during renal IR via the PKC/Src/Akt pathway, suggesting P2Y2R is a promising therapeutic target for acute kidney injury. Full article

Ovarian development significantly influences the laying performance of geese. In this study, the transcriptome analysis was conducted on the ovarian tissues of Wanxi White Geese during the pre-laying (KL), laying (CL), and ceased-laying period (XL). Short Time-series Expression Miner (STEM) analysis and miRNA–mRNA regulatory network construction were performed to identify the key genes and miRNAs regulating laying traits. Comparative analysis of KL vs. CL, CL vs. XL, and XL vs. KL groups resulted in the identification of 337, 136, and 525 differentially expressed genes (DEGs), and 258, 1131, and 909 differentially expressed miRNAs (DEMs), respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (p < 0.05) revealed that the main enrichment pathways of DEGs and DEMs at different breeding periods were Neuroactive ligand–receptor interaction, GnRH signaling pathway and Wnt signaling pathway, all associated with ovarian development. According to the three groups of common pathways, four DEGs were screened out, including INHBB, BMP5, PRL, and CGA, along with five DEMs, including let-7-x, miR-124-y, miR-1-y, and miR-10926-z, all of them may affect ovarian development. A miRNA–mRNA regulatory network was constructed through integrated analysis of DEGs and DEMs, revealing nine miRNAs highly associated with ovarian development: miR-101-y, let-7-x, miR-1-x, miR-17-y, miR-103-z, miR-204-x, miR-101-x, miR-301-y, and miR-151-x. The dual-luciferase reporter gene verified the target relationship between WIF1 and miR-204-x, suggesting that these miRNAs may influence ovarian development in Wanxi White Goose by regulating the expression levels of their target genes within ovarian tissue. This study provides a theoretical foundation for analyzing the mechanisms of ovarian development across different breeding periods and accelerating the cultivation of new breeds through post-transcriptional regulation levels. Full article