Search Results (225)

In familial Alzheimer’s disease (FAD), presenilin 1 (PSEN1) E280A cholinergic-like neurons (ChLNs) induce aberrant secretion of extracellular amyloid beta (eAβ). How PSEN1 E280A ChLNs-eAβ affects microglial activity is still unknown. We obtained induced microglia-like cells (iMG) from human peripheral blood cells (hPBCs) in a 15-day differentiation process to investigate the effect of bolus addition of Aβ42, PSEN1 E280A cholinergic-like neuron (ChLN)-derived culture supernatants, and PSEN1 E280A ChLNs on wild type (WT) iMG, PSEN1 E280A iMG, and sporadic Alzheimer’s disease (SAD) iMG. We found that WT iMG cells, when challenged with non-cellular (e.g., lipopolysaccharide, LPS) or cellular (e.g., Aβ42, PSEN1 E280A ChLN-derived culture supernatants) microenvironments, closely resemble primary human microglia in terms of morphology (resembling an “amoeboid-like phenotype”), expression of surface markers (Ionized calcium-binding adapter molecule 1, IBA-1; transmembrane protein 119, TMEM119), phagocytic ability (high pHrodo™ Red E. coli BioParticles™ phagocytic activity), immune metabolism (i.e., high generation of reactive oxygen species, ROS), increase in mitochondrial membrane potential (ΔΨm), response to ATP-induced transient intracellular Ca²⁺ influx, cell polarization (cluster of differentiation 68 (CD68)/CD206 ratio: M1 phenotype), cell migration activity according to the scratch wound assay, and especially in their inflammatory response (secretion of cytokine interleukin-6, IL-6; Tumor necrosis factor alpha, TNF-α). We also found that PSEN1 E280A and SAD iMG are physiologically unresponsive to ATP-induced Ca²⁺ influx, have reduced phagocytic activity, and diminished expression of Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) protein, but when co-cultured with PSEN1 E280A ChLNs, iMG shows an increase in pro-inflammatory phenotype (M1) and secretes high levels of cytokines IL-6 and TNF-α. As a result, PSEN1 E280A and SAD iMG induce apoptosis in PSEN1 E280A ChLNs as evidenced by abnormal phosphorylation of protein TAU at residue T205 and cleaved caspase 3 (CC3). Taken together, these results suggest that PSEN1 E280A ChLNs initiate a vicious cycle between damaged neurons and M1 phenotype microglia, resulting in excessive ChLN death. Our findings provide a suitable platform for the exploration of novel therapeutic approaches for the fight against FAD. Full article

Background/Objectives: Chronic inflammation is recognized as an important risk factor for a variety of health disorders. Omega-6 polyunsaturated fatty acids (n-6 PUFAs), particularly linoleic (LA) and arachidonic acid (AA), have been shown to be either pro- or anti-inflammatory, and researchers have advocated both for and against reducing their dietary intake. This study sought to correlate the levels of ten inflammation-related biomarkers across multiple pathways with red blood cell (RBC) membrane levels of the major dietary and circulating n-6 PUFAs. Methods: We included 2777 participants (mean age: 66 ± 9 years, 54% women, 9.8% minorities) from the Framingham Offspring and minority-enriched Omni cohorts, and calculated partial correlation coefficients. Results: After multivariable adjustment, RBC LA was inversely correlated (all p ≤ 0.05) with five markers of inflammation, receptors, or pathways: C-reactive protein (r = −0.06); soluble interleukin-6 (r = −0.15); intercellular adhesion molecule-1 (r = −0.09); monocyte chemoattractant protein-1 (r = −0.07); and P-selectin (r = −0.07). RBC AA was inversely correlated (all p ≤ 0.05) with soluble interleukin-6 (r = −0.10); intercellular adhesion molecule-1 (r = −0.14); monocyte chemoattractant protein-1, and (r = −0.06); and osteoprotegerin (r = −0.07). Lipoprotein-associated phospholipase-A2 mass and activity, urinary isoprostanes, and tumor necrosis factor receptor-2 were not significantly correlated with LA or AA. Conclusions: In our large community-based study, we observed weak but statistically significant inverse associations between several types of inflammatory biomarkers with RBC n-6 PUFAs. Our findings do not support the hypothesis that omega-6 fatty acids are pro-inflammatory. Full article

Objective: Falciparum malaria is a major global health concern, affecting more than half of the world’s population and causing over half a million deaths annually. Red cell invasion is a crucial step in the parasite’s life cycle, where the parasite invade human erythrocytes to sustain infection and ensure survival. Two parasite proteins, Apical Membrane Antigen 1 (AMA-1) and Rhoptry Neck Protein 2 (RON2), are involved in tight junction formation, which is an essential step in parasite invasion of the red blood cell. Targeting the AMA-1 and RON2 interaction with inhibitors halts the formation of the tight junction, thereby preventing parasite invasion, which is detrimental to parasite survival. This study leverages machine learning (ML) to predict potential small molecule inhibitors of the AMA-1–RON2 interaction, providing putative antimalaria compounds for further chemotherapeutic exploration. Method: Data was retrieved from the PubChem database (AID 720542), comprising 364,447 inhibitors and non-inhibitors of the AMA-1–RON2 interaction. The data was processed by computing Morgan fingerprints and divided into training and testing with an 80:20 ratio, and the classes in the training data were balanced using the Synthetic Minority Oversampling Technique. Five ML models developed comprised Random Forest (RF), Gradient Boost Machines (GBMs), CatBoost (CB), AdaBoost (AB) and Support Vector Machine (SVM). The performances of the models were evaluated using accuracy, F1 score, and receiver operating characteristic—area under the curve (ROC-AUC) and validated using held-out data and a y-randomization test. An applicability domain analysis was carried out using the Tanimoto distance with a threshold set at 0.04 to ascertain the sample space where the models predict with confidence. Results: The GBMs model emerged as the best, achieving 89% accuracy and a ROC-AUC of 92%. CB and RF had accuracies of 88% and 87%, and ROC-AUC scores of 93% and 91%, respectively. Conclusions: Experimentally validated inhibitors of the AMA-1–RON2 interaction could serve as starting blocks for the next-generation antimalarial drugs. The models were deployed as a web-based application, known as PLASMOpred. Full article

K. alvarezii is a red macroalgae cultivated on a large scale in Asian countries. In Brazil, it is cultivated in states such as Piaui, Rio de Janeiro, and São Paulo due to the high economic value for the food industry given the high concentrations of carrageenan, a hydrocolloid formed mainly by carbohydrates, used as a gelling agent and emulsifier. Therefore, to aggregate value to its protein content, the goal was to identify the proteins from K. alvarezii and biotechnological potentials against human pathogens. The protein extract produced Na⁺-acetate buffer was the most efficient in inhibiting the growth of C. parapsilosis and C. krusei. The analysis of the mechanism of action revealed that proteins from K. alvarezii cause severe damage to cellular morphology, including the effect on the cell wall and membrane, as indicated by scanning electron microscopy (SEM). Fluorescence microscopy agreed with the SEM results, revealing an increase in membrane permeabilization and pore formation, in addition to high levels of ROS, followed by apoptosis triggered by caspase 3/7. Regarding the characterization of proteins, biochemical analysis revealed the presence of proteolytic enzymes and those involved in ROS metabolism. Proteomic analysis by LC-ESI-MS/MS identified 336 proteins involved in processes such as energetic and nucleotide metabolism, defense against (a)biotic stress, and protein folding. Our results revealed that K. alvarezii proteins presented potential against C. parapsilosis and C. krusei. Full article

The aim of this study was to achieve the high secretion and expression of thymosin β4 derived from Pinctada fucata in Pichia pastoris, as well as to investigate its antibacterial properties and biological effects in promoting wound healing. The recombinant thymosin β4 protein (rTβ4) exhibited no hemolytic activity on rabbit red blood cells and demonstrated significant antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), with a minimum inhibitory concentration (MIC) of 25 μg/mL. It effectively inhibited bacterial growth and disrupted the cell wall and membrane structure of the bacteria. In the Sprague Dawley (SD) rat wound healing model, the wound healing rate in the rTβ4 treatment groups (at concentrations of 12.5 and 25 μg/mL) was significantly higher than that in the control group (p < 0.05), and the healing effect was comparable to that of the positive control group (Kangfu Xin solution, KFX). The histopathological study demonstrated that rTβ4 could reduce the infiltration of inflammatory cells and promote the proliferation and re-epithelialization of granulation tissue. In conclusion, this study successfully achieved the high expression of thymosin β4 derived from Pinctada fucata in Pichia pastoris and validated its antibacterial and wound healing potential through both In vitro and In vivo experiments. Full article

In this study, we systematically investigated the mechanisms of the antioxidation and anti-lipid accumulation effects of antioxidant peptides from Litsea cubeba on a free fatty acid (FFA)-induced NAFLD model of HepG2 cells. The NAFLD cell model was constructed by inducing the HepG2 hepatocellular carcinoma cell line with 0.5 mmol/L FFAs, and AQRDAGLL, QEGPFVR, and DVPPPRGPL were given to the culture to study their lipid-lowering and antioxidant activities on NAFLD cells. The lipid-lowering activities of the three antioxidant peptides were evaluated by Oil Red O staining and TG and TC content assays, and the results showed that all three peptides had strong ameliorating effects on FFA-induced lipid accumulation in NAFLD cells. The intracellular antioxidant protease (CAT, GSH, and SOD) activity levels and lipid peroxidation (MDA) content were measured and intracellular ROS levels were detected. The results showed that after intervention with the antioxidant peptides, the intracellular ROS levels in the NAFLD model cells were significantly reduced, the SOD and CAT activities were increased, the GSH content was elevated, and the MDA content was reduced, which indicated that AQRDAGLL, QEGPFVR, and DVPPPRGPL were able to inhibit the oxidative stress of the cells effectively and to achieve the effect of intervening in NAFLD. JC-1 fluorescence staining experiments showed that the mitochondrial membrane potential function of NAFLD cells was restored under the effect of the antioxidant peptides. Molecular dynamics simulations revealed that the main driving force between QEGPFVR and Keap1 protein was van der Waals forces, ΔG = −62.11 kcal/mol, which indicated that QEGPFVR was capable of spontaneously binding to Keap1 protein. Full article

Background: Tay–Sachs disease (TSD) is an autosomal recessive lysosomal storage disorder characterized by the accumulation of GM2 ganglioside due to mutations in the HEXA gene, which encodes the α-subunit of β-Hexosaminidase A. This accumulation leads to significant neuropathological effects and premature death in affected individuals. No effective treatments exist, but enzyme replacement therapies are under investigation. In our previous work, we demonstrated the internalization and efficacy of human recombinant lysosomal β-hexosaminidase A (rhHex-A), produced in the methylotrophic yeast Pichia pastoris, in reducing lipids and lysosomal mass levels in fibroblasts and neural stem cells derived from patient-induced pluripotent stem cells (iPSCs). In this study, we further evaluated the potential of rhHex-A to prevent GM2 accumulation using fibroblast and neuroglia cells from a TSD patient alongside a relevant mouse model. Methods: Fibroblasts and neuroglial cell lines derived from a murine model and TSD patients were treated with 100 nM rhHexA for 72 h. After treatment, cells were stained by anti-GM2 (targeting GM2 ganglioside; KM966) and anti-LAMP1 (lysosomal-associated membrane protein 1) colocalization staining and incubated with 50 nM LysoTracker Red DND-99 to label lysosomes. In addition, GM2AP and HEXB expression were analyzed to assess whether rhHex-A treatment affected the levels of enzymes involved in GM2 ganglioside degradation. Results: Immunofluorescence staining for LysoTracker and colocalization studies of GM2 and Lamp1 indicated reduced lysosomal mass and GM2 levels. Notably, rhHex-A treatment also affected the expression of the HEXB gene, which is involved in GM2 ganglioside metabolism, highlighting a potential regulatory interaction within the metabolic pathway. Conclusions: Here, we report that rhHex-A produced in yeast can efficiently degrade GM2 ganglioside and rescue lysosomal accumulation in TSD cells. Full article

The rising demand for alternative solutions to diabetes mellitus has prompted significant interest in the exploration of plant-derived anti-diabetic compounds, especially within a circular economy framework that seeks sustainable and profitable reuse options. In this context, red (RSGO) and white (WGSO) grape seed oils, by-products of Sicilian vineyards, were prepared, analyzed for their fatty acid, polyphenol, carotenoid, and chlorophyll content, and evaluated for their glucose-lowering ability on HepG2 cells. Utilizing cytochemical techniques, flow cytometry, and protein blotting, we explored the effects of non-toxic oil dilutions on (i) glycogen storage, (ii) glucose consumption/uptake, (iii) GLUT-2, GLUT-4, and hepatocyte nuclear factor-1α (HNF1α) expression levels, and (iv) AMP-activated protein kinase (AMPK), insulin receptor substrate-1 (IRS-1), AKT, and PKCζ phosphorylation states, which are involved in insulin-mediated and -independent regulation of GLUT-4 membrane exposure. RGSO and WGSO, despite adopting slightly varying molecular strategies, were both proven to be effective stimulators of glucose absorption and glycogenesis. Specifically, RSGO promoted GLUT-2 and GLUT-4 up-regulation, whereas the WGSO-induced effect was associated with an increase in GLUT-4 levels alone. Moreover, the oils activated both pathways responsible for GLUT-4 translocation. Therefore, these wine-making residues have substantial potential as anti-diabetic solutions, holding promise for integration into the biomedical and food sectors. Full article

Resveratrol is a naturally occurring phenolic compound found in various foods such as red wine, chocolate, peanuts, and blueberries. Both in-vitro and in-vivo studies have shown that it has a broad spectrum of pharmacological effects such as providing cellular protection and promoting longevity. These effects include antioxidant, anti-inflammatory, neuroprotective, and anti-viral properties, as well as improvements in cardio-metabolic health and anti-aging benefits. Additionally, resveratrol has demonstrated the ability to induce cell death and inhibit tumor growth across different types and stages of cancer. However, the dual effects of resveratrol—acting to support cell survival in some contexts, while inducing cell death in others—is still not fully understood. In this study, we identify a novel target for resveratrol: the voltage-dependent anion channel 1 (VDAC1), a multi-functional outer mitochondrial membrane protein that plays a key role in regulating both cell survival and death. Our findings show that resveratrol increased VDAC1 expression levels and promoted its oligomerization, leading to apoptotic cell death. Additionally, resveratrol elevated intracellular Ca²⁺ levels and enhanced the production of reactive oxygen species (ROS). Resveratrol also induced the detachment of hexokinase I from VDAC1, a key enzyme in metabolism, and regulating apoptosis. When VDAC1 expression was silenced using specific siRNA, resveratrol-induced cell death was significantly reduced, indicating that VDAC1 is essential for its pro-apoptotic effects. Additionally, both resveratrol and its analog, trans-2,3,5,4′-tetrahydroxystilbene-2-O-glucoside (TSG), directly interacted with purified VDAC1, as revealed by microscale thermophoresis, with similar binding affinities. However, unlike resveratrol, TSG did not induce VDAC1 overexpression or apoptosis. These results demonstrate that resveratrol-induced apoptosis is linked to increased VDAC1 expression and its oligomerization. This positions resveratrol not only as a protective agent, but also as a pro-apoptotic compound. Consequently, resveratrol offers a promising therapeutic approach for cancer, with potentially fewer side effects compared to conventional treatments, due to its natural origins in plants and food products. Full article

Background: Blueberry anthocyanin such as Cyanidin-3-O-glucoside may help prevent Alzheimer’s disease. We aimed to investigate the preventive and therapeutic effects of Cyanidin-3-O-glucoside against Aβ_1–42-induced apoptosis of SH-SY5Y cells as well as the underlying mechanisms. Methods: Cell viability and intracellular and mitochondrial reactive oxygen species were detected by MTT, a reactive oxygen species detection kit, and a MitoSOX red mitochondrial superoxide indicator. The mitochondrial membrane potential, intracellular calcium ion content, and adenotriphophate (ATP) were identified via a mitochondrial membrane potential detection kit, calcium ion detection kit, and ATP detection kit, and apoptosis was detected via flow cytometry. Transcription of apoptosis-related genes was detected using real-time fluorescence quantitative polymerase chain reaction, and expression of apoptosis-related proteins was identified using Western blot. Results: We found that Cyanidin-3-O-glucoside could downregulate the expression of cytochrome c, caspase 9, caspase 3, and other genes and proteins, which consequently reduced the rate of apoptosis. Additionally, it could upregulate Bcl-2 gene and protein expression, downregulate Bax gene and protein expression, regulate mitochondrial membrane permeability and calcium-release channels, reduce calcium influx into mitochondria, maintain intracellular calcium ion levels, reduce intracellular levels of reactive oxygen species and increase ATP levels, maintain the mitochondrial membrane potential at a normal level, maintain normal mitochondrial functioning, and prevent apoptosis. Discussion: Taken together, Cyanidin-3-O-glucoside showed dose-dependent preventive and therapeutic effects against Aβ_1–42-induced apoptosis of SH-SY5Y cells. Conclusions: Cyanidin 3-O-glucoside showed a better preventive effect than therapeutic effect against Aβ_1–42-induced apoptosis in SH-SY5Y cells. Full article

The cytotoxic and apoptotic properties of four bioactive natural compounds, the prenylated α-pyronephloroglucinol heterodimer arzanol (ARZ), the methoxylated flavones eupatilin (EUP) and xanthomicrol (XAN), and the sesquiterpene zerumbone (ZER), were compared in SH-SY5Y human neuroblastoma cells to assess their potential as neuroblastoma-specific therapeutics. EUP, XAN, and ZER (2.5–100 μM) exerted marked significant cytotoxicity (MTT assay) and morphological changes after 24 h of incubation, following the order XAN > ZER > EUP > ARZ (no toxic effect). The propidium iodide fluorescence assay (PI, red fluorescence) and NucView^® 488 assay (NV, green fluorescence) evidenced a significant increase in the apoptotic cell number, vs. controls, in SH-SY5Y cells pre-incubated for 2 h with the compounds, in the following order of apoptotic potency: XAN > EUP > ZER > ARZ. The PubChem database and freely accessible web tools SwissADME, pkCSM-pharmacokinetics, and SwissTargetPrediction were used to assess the physicochemical/pharmacokinetic properties and potential protein targets of the compounds. At 50 μM, a positive correlation (r = 0.917) between values of % viability reduction and % human intestinal absorption (bioavailability) was observed, indicating a marked contribution of compound membrane permeability to cytotoxicity in SH-SY5Y cells. The capacity of compounds to induce apoptosis emerged as inversely correlated to the computed lipophilicity (r = −0.885). Full article

Background: Antidepressants are a class of pharmaceuticals utilized for the management of many psychiatric disorders, including depression. A considerable number of antidepressants, particularly selective serotonin reuptake inhibitors (SSRIs), have been documented to demonstrate significant anticancer properties in various cancer cell lines. Objectives: The aim of this study was to evaluate the selective cytotoxic and apoptotic effects of escitalopram oxalate (ES) alone and in combination with etoposide (ET) on ET-resistant A549 (A549/90E) lung cancer cells. Methods: The cytotoxic effects of the drugs were determined by CCK-8, trypan blue, and neutral red assays. Apoptosis was observed by Annexin V fluorescein isothiocyanate (FITC)/PI and mitochondrial membrane potential (ΔΨm) assays. Moreover, the effects of the drugs, alone and in combination, on apoptosis-related proteins, caspase-3, PTEN, and resistance-related P-gP were determined by ELISA. The relationship between drugs and lung cancer was determined with protein–protein interaction (PPI) network analysis. Results: Our results revealed that ES significantly exerted cytotoxic effects on both wild-type and A549/90E cells compared with BEAS-2B cells. The IC₅₀ values of 48.67 and 51.6 μg/mL obtained for ET and ES, respectively, at the end of 24 h of incubation for A549 cells were applied reciprocally for each cell by including BEAS-2B together with the 2xIC₅₀ and ½ IC₅₀ values. The results of each combination were statistically evaluated with combination indices (CIs) obtained using the Compusyn synergistic effect analysis program. Combination doses with a synergistic effect in A549 and A549/90E cells and an antagonistic effect in BEAS-2B cells have been determined as ½ IC₅₀ for ET and ½ IC₅₀ for ES. ET ½ IC₅₀, ES ½ IC₅₀, and an ET ½ IC₅₀ + ES ½ IC₅₀ combination caused 18.37%, 55.19%, and 57.55% death in A549 cells, whereas they caused 44.9%, 22.4%, and 51.94% death in A549/90E cells, respectively. In A549 cells, the combination of ES ½ IC₅₀ and ET ½ IC₅₀ caused increased levels of caspase-3 (p < 0.01) and P-gP (p < 0.001), while PTEN levels remained unchanged. The combination resulted in an increase in caspase-3 (p < 0.001) and PTEN (p < 0.001) amounts, alongside a decrease in P-gP (p < 0.01) levels in A549/90E cells. The death mechanism induced by the combination was found to be apoptotic by Annexin V-FITC and ΔΨm assays. Conclusions: Based on our findings, ES was observed to induce cytotoxic and apoptotic activities in A549/90E cells in vitro. ES in combination therapy is considered to be effective to overcome ET resistance by reducing the amount of P-gP in A549/90E cells. Full article

Background: Besides conventional norms that recognize synovial fluid (SF) as a joint lubricant, nutritional channel, and a diagnostic tool in knee osteoarthritis (kOA), based on the authors previous studies, this study aims to define functional role of SF in kOA. Methods: U937, a monocytic, human myeloid cell line, was induced with progressive grades of kOA SF, and the induction response was assessed on various pro-inflammatory parameters. This ‘SF challenge test model’ was further extended to determine the impact of SF on U937 differentiation using macrophage-specific markers and associated transcription factor genes. Mitochondrial membrane potential changes in SF-treated cells were evaluated with fluorescent JC-1 probe. Results: a significant increase in nitric oxide, matrix metalloproteinase (MMP) 1, 13, and vascular endothelial growth factor (VEGF)-1 was noted in the induced cells. A marked increase was seen in CD68, CD86, and the transcription factors –activator protein (AP)-1, interferon regulatory factor (IRF)-1, and signal transducer and activator of transcription (STAT)-6 in the SF-treated cells indicating active monocytes to macrophage differentiation. Reduced mitochondrial membrane potential was reflected by a reduced red-to-green ratio in JC-1 staining. Conclusions: these results underline the active role of OA SF in stimulating and maintaining inflammation in joint cells, fostering monocyte differentiation into pro-inflammatory macrophages. The decline in the membrane potential suggestive of additional inflammatory pathway in OA via the release of pro-apoptotic factors and damaged associated molecular patterns (DAMPs) within the cells. Overall, biochemical modulation of SF warrants a potential approach to intervene inflammatory cascade in OA and mitigate its progression. Full article

Adhesion between a red blood cell and substrates is essential to many biophysical processes and has significant implications for medical applications. This study derived a theoretical formula for the adhesive force between a red blood cell and a bulk substrate, incorporating the Hamaker constant to account for van der Waals interactions. The derivation is based on a biconcave shape of an RBC, described by the well-known Ouyang–Helfrich equation and its analytical solution developed by Ouyang. The theoretical predictions align with experimental observations and the empirical spherical model, revealing a $F\propto D^{-2.5}$ relationship for biconcave RBCs versus $F\propto D^{-2}$ for spheres. While the current study focuses on idealized geometries and static conditions, future work will extend these findings to more complex environmental conditions, such as dynamic flow and interactions with plasma proteins, thereby broadening the applicability of the model. This work bridges foundational research in cell membrane mechanics with practical applications in hemostatic materials, platelet adhesion, and biomaterials engineering. The findings provide insights for designing advanced biological sensors, surgical tools, and innovative medical materials with enhanced biocompatibility and performance. Full article

Dysregulated iron metabolism-induced ferroptosis is considered a key pathological mechanism in the development of osteoporosis (OP). G protein-coupled receptor 30 (GPR30, also known as Gper1) is an estrogen-binding receptor that has shown therapeutic benefits in patients with certain degenerative diseases. Moreover, several studies have demonstrated the anti-ferroptotic effects of estrogen receptor activation. However, its role in the prevention and treatment of OP remains unclear, and there are currently no reports on the anti-ferroptotic function of GPR30 in OP. Therefore, this study aimed to investigate the ferroptosis-related effects and mechanisms of GPR30 in the context of OP. In vivo and in vitro experiments were conducted using wild-type (WT) C57BL/6 female mice and GPR30-knockout (GPR30-KO) C57BL/6J female mice. The microarchitecture of the distal femur was assessed using micro-computed tomography (micro-CT), and histomorphological changes were analyzed via hematoxylin and eosin (H&E) staining. Bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured to establish an iron overload model using ferric ammonium citrate (FAC). Interventions included GPR30 overexpression via transfection and BMP-6 inhibition using LDN-214117. Cell viability was evaluated with the CCK-8 assay, while osteogenic differentiation and mineralization levels were assessed using ALP and Alizarin Red S (ARS) staining. Iron accumulation was detected via Prussian blue staining, oxidative stress levels were evaluated using ROS staining, and mitochondrial membrane potential changes were analyzed using JC-1 staining. Transmission electron microscopy (TEM) was employed to observe mitochondrial ultrastructural changes. Additionally, key gene and protein expression levels were measured using immunofluorescence and Western blotting. The micro-CT analysis revealed significant bone microarchitecture deterioration and bone loss in the GPR30-KO mouse model. At the cellular level, GPR30 overexpression markedly reduced iron accumulation and oxidative stress in BMSCs, restored the mitochondrial membrane potential, and improved the mitochondrial ultrastructure. Furthermore, GPR30 enhanced osteogenic differentiation in BMSCs by promoting the activation of the BMP-6/HEP/FPN signaling pathway, leading to increased expression of osteogenic markers. The protective effects of GPR30 were reversed by the BMP-6 inhibitor LDN-214117, indicating that BMP-6 is a critical mediator in GPR30-regulated iron metabolism and ferroptosis inhibition. GPR30 inhibits ferroptosis in BMSCs and enhances osteogenic differentiation by activating the BMP-6/HEP/FPN signaling pathway. This provides new insights and potential therapeutic targets for the treatment of osteoporosis OP. Full article