Oxidative stress-mediated impairment of osteoblast function is a critical pathological basis for the development and progression of various bone metabolic diseases. BicC family RNA binding protein 1 (BICC1) plays important roles in osteoblast function, however, the underlying mechanism remains unclear. This study aimed to investigate the protective role of BICC1 in oxidative stress-induced osteogenic injury and the underlying mechanism. An oxidative injury model was established in MC3T3-E1 cells using 300 µM hydrogen peroxide (H2O2). Bicc1 over-expressing lentivirus and the pyruvate dehydrogenase kinase isozyme (PDK) inhibitor dichloroacetate (DCA) were used for intervention. The osteoblast differentiation and mitochondrial membrane potential and apoptosis were measured by biochemical analysis and Alizarin Red S staining. RT-qPCR and Western blot were used to detect mRNA and protein levels of key molecules. RNA sequencing was performed to identify core targets and signaling pathways. H₂O₂ treatment significantly suppressed MC3T3-E1 cell viability and osteogenic differentiation, enhanced apoptosis, and up-regulated PDK1 expression as well as pyruvate dehydrogenase complex (PDH) phosphorylation. Bicc1 over-expression markedly alleviated H₂O₂-induced mitochondrial dysfunction and apoptosis, restored osteogenic differentiation, and inhibited the activation of the PDK1/PDH axis. Transcriptomic analysis showed that PDK1 was a key target molecule in Bicc1-regulated apoptosis and oxidative stress responses. Inhibition of PDK1 recapitulates the protective effect of BICC1 to oxidative stress injury in MC3T3-E1 cells. Bicc1 attenuates oxidative stress injury and promotes osteogenic differentiation in MC3T3-E1 cells, and may serve as a potential therapeutic target for osteoporosis.
Dexamethasone (Dex) causes growth plate injury and growth retardation in children, yet the underlying epigenetic and post-transcriptional mechanisms remain poorly defined. This study addresses this gap by dissecting the MALAT1/miR-124-3p/LPCAT3 axis in Dex-induced chondrocyte ferroptosis. Primary rat growth plate chondrocytes and 2-week-old male Sprague-Dawley rats were treated with Dex. DNA methylation of the MALAT1 promoter was assessed by MSP analysis. Gain- and loss-of-function studies, CCK-8, ferroptosis markers and luciferase reporter assays were performed in vitro. In vivo, tibial growth plate morphology, zone-specific heights, systemic growth parameters and LPCAT3 expression were evaluated. The results showed that Dex reduced chondrocyte viability and suppressed MALAT1 expression in a dose-dependent manner, accompanied by hypermethylation of one CpG islands in the MALAT1 promoter. Treatment with 5-Aza-2’-deoxycytidine (5-Aza) restored MALAT1 expression and reduced methylation levels. MALAT1 overexpression attenuated Dex-induced ferroptosis, as evidenced by decreased Fe²⁺ and MDA levels and restored SLC7A11 and Gpx4 expression. Mechanistically, MALAT1 directly binds to miR-124-3p, which targets LPCAT3. miR-124-3p mimics abolished the protective effects of MALAT1, while LPCAT3 overexpression reversed the inhibitory effects of miR-124-3p mimics on cell viability and ferroptosis markers. In vivo, Dex reduced growth plate thickness, tibial length, and body weight gain, while both MALAT1 overexpression and 5-Aza treatment attenuated these deficits and restored LPCAT3 expression. In conclusion, DNA methylation-mediated MALAT1 downregulation promotes Dex-induced chondrocyte ferroptosis via the miR-124-3p/LPCAT3 axis. Targeting this pathway may represent a preclinically promising therapeutic strategy that warrants further investigation in glucocorticoid-induced growth retardation in children.
The reduced blood flow and energy substrates to the affected area during ischemia and sudden exposure to these molecules after ischemia is called reperfusion. Reperfusion can be as damaging to cells and tissues as ischemia. This study aims to examine the effects of brain Ischemia–Reperfusion (I/R) and 1-week 3',4'-Dihydroxyflavonol (DiOHF) treatment on Matrix Metalloproteinasease (MMP), occludin, claudin-5, and β-actin levels in frontal cortex and hippocampus tissue in male rats. In this study, twenty-eight male Wistar-Albino rats were allocated into four experimental groups: Control, Sham, Ischemia–Reperfusion (I/R), and Ischemia–Reperfusion + DiOHF. Under general anesthesia, bilateral carotid artery ligation was performed to induce ischemia for 30 min, followed by reperfusion. DiOHF supplementation (10 mg/kg) was administered for one week. At the end of the treatment period, the animals were sacrificed under general anesthesia, and the frontal cortex and hippocampus tissues were harvested. Gene expression levels of MMP-3, MMP-9, occludin, claudin-5, and β-actin in the collected tissues were analyzed using real-time PCR. While I/R suppressed the levels of claudin-5 and occludin in the frontal cortex and hippocampus, it increased the levels of β-actin, matrix metalloproteinase-3 (MMP-3), and matrix metalloproteinase-9 (MMP-9). 3',4'-Dihydroxyflavonol supplementation for 1 week corrected the deteriorations caused by I/R. The study results show that 1 week of 3',4'-Dihydroxyflavonol treatment after I/R corrects the changes in structural damage indicators caused by transient bilateral carotid occlusion-induced ischemia–reperfusion in the frontal cortex and hippocampus to a certain extent.
BACKGROUND:Non-small cell lung cancer (NSCLC) remains a leading cause of cancer-related mortality worldwide, largely due to the supportive role of the tumor microenvironment (TME). Tumor-associated macrophages, particularly the M2 phenotype, are pivotal in promoting NSCLC progression. Exosomes, key mediators of intercellular communication, can transfer functional cargo from M2 macrophages to cancer cells, thereby regulating malignant behaviors. However, the specific mechanisms by which M2 macrophage-derived exosomes modulate NSCLC progression are not fully understood. METHODS:Bioinformatics analyses were initially performed to identify differentially expressed genes (DEGs) in M2 macrophages and NSCLC tissues using the GSE159112 and GSE268175 datasets. THP-1 cells were induced to differentiate into M2 macrophages, from which exosomes were isolated and characterized via nanoparticle tracking analysis, transmission electron microscopy, and western blotting. NSCLC cell lines (A549 and H23) were co-cultured with these exosomes to assess the transfer of ETS homologous factor (EHF). Functional assays, including 5-Ethynyl-2'-deoxyuridine (EdU), Transwell, flow cytometry, and sphere formation assays, were conducted to evaluate cell proliferation, migration, invasion, apoptosis, and stemness. Glycolytic capacity was determined by measuring glucose uptake, lactate production, and ATP levels. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays were employed to verify the transcriptional regulation of FGFR1 by EHF. Rescue experiments involving FGFR1 overexpression were performed to validate the signaling axis, and a xenograft mouse model was established to confirm the in vivo findings. RESULTS:EHF was identified as a critical upregulated transcription factor in both M2 macrophages and NSCLC tissues. M2 macrophage-derived exosomes were efficiently internalized by NSCLC cells, resulting in subsequent upregulation of its expression in recipient cells. Mechanistically, EHF transcriptionally activated fibroblast growth factor receptor 1 (FGFR1) by binding to its promoter region. Functionally, exosomes derived from EHF-deficient M2 macrophages significantly suppressed NSCLC cell proliferation, migration, invasion, and sphere formation, while promoting apoptosis. Concurrently, the loss of exosomal EHF inhibited glycolysis, evidenced by reduced glucose uptake, lactate production, and ATP levels. Crucially, restoring FGFR1 expression reversed the suppressive effects induced by EHF-deficient exosomes, confirming that the EHF/FGFR1 axis drives these malignant phenotypes. In vivo experiments further demonstrated that exosomes from EHF-silenced M2 macrophages inhibited tumor growth and downregulated proliferation markers. CONCLUSION:M2 macrophage-derived exosomal EHF promoted NSCLC progression and glycolysis by transcriptionally activating FGFR1. These findings highlight the EHF/FGFR1 axis as a novel molecular link between macrophages and NSCLC cells.
Long-term excessive fluoride intake accumulates in brain tissue, causing neuronal degeneration and nervous system dysfunction. This mechanism is closely related to elevated oxidative stress. α-Synuclein (α-Syn) is expressed in neuronal presynaptic terminals; under environmental toxin exposure or oxidative stress, its expression changes and it misfolds into oligomers and aggregates that are cytotoxic and induce oxidative cellular damage. Meanwhile, the nuclear factor erythroid 2–related factor 2 (Nrf2)–NAD(P)H: quinone oxidoreductase 1 (NQO1)/heme oxygenase-1 (HO-1) pathway is an important cellular system for regulating oxidative damage, and its core kinase is mainly expressed in brain astrocytes.The aim of this study is to investigate the effects of fluoride exposure on oxidative stress levels in astrocytes and the expression of α-Syn within the Nrf2-NQO1/HO-1 pathway. Primary astrocytes were treated with extracellular α-synuclein pre-formed fibrils (αSP) in conjunction with fluoride exposure. The results demonstrated that fluoride exposure induced reactive activation of primary astrocytes, increased levels of reactive oxygen species (ROS), decreased activities of superoxide dismutase (SOD) and catalase (CAT), and up-regulated the protein expression within the Nrf2-NQO1/HO-1 pathway. αSP caused oxidative stress in primary astrocytes and exacerbated oxidative damage induced by fluoride exposure. These findings are significant for enhancing the understanding of the mechanisms underlying brain injury associated with chronic fluorosis.
Cervical cancer remains a leading cause of cancer related mortality in women worldwide. Cisplatin (CP) is a cornerstone chemotherapeutic agent; however, its clinical utility is constrained by dose-limiting toxicity and the emergence of drug resistance, necessitating novel combinatorial strategies. This study investigated the therapeutic potential of combining CP with Aloe vera extract (AVE) against HeLa cervical cancer cells and the normal human skin fibroblast cell line CCD-1072Sk. Cytotoxicity assays performed using the xCELLigence Real-Time Cell Analysis (RTCA) system determined IC50 values of 18.45 µM for CP and 18.88 µg/mL for AVE in HeLa cells, compared with 53.31 µM and 19.09 µg/mL, respectively, in CCD-1072Sk cells. The CP + AVE combination exhibited markedly enhanced antiproliferative activity in HeLa cells compared with CP alone, while CCD-1072Sk cells exhibited continued but decelerated proliferation, suggesting a differential cellular response. Seahorse metabolic analysis revealed that AVE, both alone and in combination with CP, markedly increased mitochondrial oxygen consumption rate (OCR) and ATP-linked respiration in HeLa cells, indicating induction of sustained bioenergetic stress beyond the cells’ adaptive capacity. Transcriptomic profiling of HeLa cells identified 453 differentially expressed genes (DEGs) upon combination treatment, with significant downregulation of oxidative phosphorylation (OXPHOS) components, mitochondrial electron transport chain subunits, and the fatty acid metabolism gene CPT1B, collectively indicating a severe disruption of cellular energy metabolism. DepMap database analysis confirmed that downregulated targets including CPT1B and CHN2 are functionally important for HeLa cell survival. Collectively, these data suggest that AVE drives HeLa cells into a hypermetabolic state that overloads mitochondrial capacity; in combination with CP, this converging bioenergetic stress produces enhanced cytotoxicity descriptively consistent with an additive interaction (a term used here in its dose-response sense, as no formal combination-index or Bliss/Loewe synergy analysis was performed) at the IC50 doses employed. This study highlights the potential of AVE as a metabolically active combinatorial agent capable of potentiating CP efficacy by targeting cancer cell energy metabolism, and provides a mechanistic basis for further investigation. Schematic summary of the study design and principal findings.
Rheumatoid arthritis (RA) is associated with inflammation, oxidative stress and infiltration of immune cells ( 90
Chronic hepatitis B (CHB), caused by persistent hepatitis B virus (HBV) infection, continues to pose a major global health threat. Aurora kinase A (AURKA) has been implicated in fibrosis, yet its role in HBV-induced liver fibrosis remains unclear. This study investigated the function and mechanism of AURKA in hepatic stellate cell (HSC) activation and fibrogenesis during CHB. Bioinformatics screening of GSE83148 dataset and validation in human CHB liver specimens, LX-2 HSCs treated with conditioned medium (CM) from HBV-replicating cells, and a persistent HBV recombinant cccDNA (rcccDNA) mouse model established by tail-vein injection of Ad/rcccDNA were performed. Gene and protein expression was analyzed by quantitative PCR and immunoblotting, respectively. Cytokine levels were measured by ELISA. Mitochondrial function was assessed via ROS and JC-1 assays. Protein interactions and ubiquitination were evaluated by co-immunoprecipitation (Co-IP) and protein stability assays. In additional experiments, LX-2 cells were stimulated with purified HBV virions at varying MOIs to assess direct viral effects. AURKA was significantly upregulated in CHB patients and HBV-CM-treated LX-2 HSCs. In LX-2 HSCs, AURKA knockdown attenuated HBV-CM-induced inflammatory cytokine secretion (IL‑1β, IL‑6), mitochondrial dysfunction (mitochondrial ROS, mitochondrial membrane potential), HSC proliferation, and fibrotic marker expression (COL I, α-SMA). Mechanistically, AURKA interacted with TLR3 and was associated with its K48-linked polyubiquitination and degradation. TLR3 silencing abolished the protective effects of AURKA knockdown. In vivo, using a persistent HBV rcccDNA mouse model, AURKA knockdown ameliorated HBV-induced hepatic inflammation and fibrosis by upregulating TLR3. Notably, purified HBV virions alone were sufficient to dose-dependently induce AURKA expression and downstream fibrotic responses in HSCs. This study identifies a novel AURKA/TLR3 axis that regulates HBV-induced HSC activation and liver fibrosis, positioning AURKA as a potential therapeutic target for CHB-associated fibrosis.
Atherosclerosis (AS) is a chronic vascular disease characterized by inflammatory response and lipid deposition in the arterial walls caused by endothelial injury. Sinomenine (SIN) could delay AS progression by attenuating endothelial inflammation. However, its role and mechanism in the oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVEC) injury remain unclear. An in vitro model of AS in HUVECs was established using ox-LDL. CCK-8 detected cell viability. ROS, SOD, MDA, TNF-α, IL-β, and IL-6 were evaluated using commercial kits. Western blot was used to detect NLRP3, apoptosis-associated speck-like protein containing ASC, activated caspase-1, GSDMD-N, Arg1, CD206, iNOS, CXCL14, and MEF2A protein levels. CXCL14 mRNA level was measured using RT-qPCR. Binding between MEF2A and CXCL14 promoter was validated using dual-luciferase reporter and ChIP. SIN exposure alleviated ox-LDL-caused oxidative stress, inflammation, pyroptosis, and M1 macrophage polarization in HUVECs. CXCL14 or MEF2A silencing abolished the protective effect of SIN on ox-LDL-caused HUVEC injury. Mechanistically, MEF2A directly interacts with CXCL14 promoter and promotes its transcription. SIN treatment restrained ox-LDL-evoked HUVEC injury and M1 macrophage polarization partly via targeting the MEF2A/CXCL14 axis, providing new insights for future research on the application of SIN in AS treatment. SIN treatment suppressed ox-LDL-induced oxidative stress, inflammation, pyroptosis, and M1 macrophage polarization in HUVECs. CXCL14 or MEF2A silencing abolished the protective effect of SIN on ox-LDL-caused HUVEC injury. MEF2A could induce CXCL14 transcription.
F-box protein 32 (FBXO32), an E3 ubiquitin ligase, has been implicated in various cellular processes, but its role and regulatory network in DR-associated endothelial dysfunction are unclear. Here, we aimed to investigate the function of FBXO32 in high glucose (HG)-induced vascular endothelial cell injury and the underlying molecular mechanisms mediated by DNMT1 and Myc. Human retinal microvascular endothelial cells (HRMECs) were exposed to HG to mimic DR in vitro. The expression of FBXO32 and DNMT1 was detected by qRT-PCR and Western blot. Gain-of-function and loss-of-function experiments were performed to manipulate FBXO32, DNMT1, and Myc expression. Cell migration was evaluated by wound healing assay. Inflammatory cytokines (IL-1β, IL-6, TNF-α) were measured by ELISA. Glycolytic metabolism was assessed by detecting glucose consumption, lactate production, ATP level, and extracellular acidification rate (ECAR). Co-immunoprecipitation (Co-IP), ubiquitination assay, protein stability assay and GST pull down assay were used to verify the interaction between FBXO32 and Myc. Methylation-specific PCR (MSP) was performed to detect the methylation status of FBXO32 promoter. HG treatment significantly downregulated FBXO32 expression in HRMECs. Overexpression of FBXO32 inhibited HG-induced cell migration, inflammation (decreased IL-6 and TNF-α levels), and glycolytic metabolism (reduced glucose consumption, lactate production, ATP level, and ECAR). Treatment with 2-deoxy-D-glucose (2-DG), a glycolysis inhibitor, abolished the effects of FBXO32 overexpression on cell migration and inflammation, indicating that FBXO32 exerts its function by regulating glycolysis. The interaction between FBXO32 and Myc were confirmed by Co-IP assay and GST pull down assay. Ubiquitination and protein stability assays showed that FBXO32 promotes the ubiquitination and degradation of Myc, thereby reducing Myc protein stability. Overexpression of Myc reversed the inhibitory effects of FBXO32 on HG-induced cell migration, inflammation, and glycolysis. Furthermore, we identified DNMT1 as an upstream regulator of FBXO32. DNMT1 binds to the promoter region of FBXO32 and induces its methylation, leading to the downregulation of FBXO32. Knockdown of DNMT1 upregulated FBXO32 expression, inhibited HG-induced cell migration, inflammation, and glycolysis, while co-knockdown of DNMT1 and FBXO32 reversed these effects. Our findings demonstrate that DNMT1-mediated methylation downregulates FBXO32 expression in HG-induced vascular endothelial cells. FBXO32 inhibits HG-induced endothelial cell migration, inflammation, and glycolytic reprogramming by promoting the ubiquitination and degradation of Myc. This DNMT1-FBXO32-Myc regulatory axis provides a novel therapeutic target for the treatment of DR.
Chemo-resistance is a major challenge in rectal cancer treatment. This study investigates the therapeutic potential of MEK inhibitors, cobimetinib and trametinib, in 5-fluorouracil (5-FU)-resistant rectal cancer cells. High-throughput drug screening identified these inhibitors as top candidates based on their selective drug sensitivity scores (sDSS). Both drugs exhibited dose-dependent cytotoxicity against rectal cancer cells while sparing normal epithelial cells and showed synergistic interactions with 5-FU. MEK inhibition disrupted redox homeostasis, increasing reactive oxygen species (ROS) and oxidative damage markers, including protein carbonyl and malondialdehyde (MDA), while also decreasing mitochondrial respiration, as evidenced by reduced oxygen consumption rates (OCR). Apoptotic induction was significantly reduced in mitochondrial respiration-deficient p⁰ cells, supporting the role of mitochondrial respiration in MEK inhibitor activity. Genetic MEK1/2 knockdown mimicked these effects, confirming MEK1/2 as a key regulator of oxidative stress and mitochondrial respiration. In vivo, cobimetinib and trametinib suppressed tumor growth in a chemo-resistant colorectal cancer xenograft model without significant toxicity, inducing oxidative stress and decreasing mitochondrial respiration. These findings highlight MEK inhibitors as promising candidates for overcoming chemo-resistance in rectal cancer by targeting oxidative stress and mitochondrial respiration.
Liver ischemia/reperfusion injury (IRI) is a major complication of hemorrhagic shock, hepatectomy and liver transplantation. Intestinal microecology has momentous functions in various human diseases. The present study aimed to elucidate the role and underlying mechanism of intestinal microecology in liver IRI. A liver IRI mouse model was constructed and validated using hematoxylin and eosin staining, enzyme-linked immunosorbent assay and Naphthol AS-D chloroacetate esterase staining. The function of intestinal microecology in liver IRI was evaluated using flow cytometry and western blot analysis. Moreover, the mechanisms of intestinal microecology in liver IRI were assessed using a series of molecular experiments. The results revealed that liver IRI associated with intestinal microecology dysbiosis exhibited increased hepatic neutrophil infiltration, MAPK pathway activation and inflammatory cytokine production. The 16 S rRNA gene sequencing of fecal samples from Sham, IRI, IRI+antibiotic pre-treatment and fecal transplantation (FT) groups revealed microbial community alterations, with shifts in Bacteroidota and Firmicutes abundance associated with liver injury and neutrophil recruitment. PCA, PCoA and taxonomic profiling further confirmed group-dependent remodeling of the gut microbial community. FT using fecal microbiota from IRI donor mice exacerbated liver neutrophil infiltration, MAPK/CXCR2 activation and inflammatory responses, whereas TAK-242-mediated TLR4 blockade attenuated these effects. Overall, the present study suggests that gut microbiota dysbiosis may enhance liver IRI by promoting neutrophil recruitment, at least in part through the TLR4/MAPK/CXCR2 axis, revealing a novel microbe-immune-liver interaction that may be targeted therapeutically.
Temporal lobe epilepsy (TLE) is one of the most common types of epilepsy, with frequent seizures often leading to cognitive, emotional, and psychiatric issues. A prominent pathological change associated with TLE is hippocampal sclerosis (HS), characterized by neuronal loss, gliosis, and increased neuron fibre density. However, the pathogenesis of Temporal lobe epilepsy with hippocampal sclerosis (TLE-HS) remains unclear. This study aimed to investigate the abnormal expression and regulatory mechanism of hub genes in TLE-HS. The source data were obtained from the epilepsy dataset (GSE256068) of the Gene Expression Omnibus GEO database. Then, differential expression gene (DEG) analysis and weighted gene coexpression network analysis (WGCNA) were employed to screen for module-related DEGs in TLE-HS, followed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Subsequently, these intersected targets were subjected to cross-validation using three machine learning algorithms- LASSO regression, SVM-RFE, and RF, ultimately identifying three hub genes. Finally, CIBERSORT and ssGSEA algorithms were used to analyze the infiltration status of different immune cell populations in TLE-HS patients, followed by assessing the association between hub genes and immune cell populations. The expression of hub genes was determined using RT-qPCR and western blot. Functional experiments were performed using CCK-8, flow cytometry, and special kits. Results indicated that three hub genes, NADH dehydrogenase (ubiquinone) 1 alpha subcomplex subunit 4-like 2 (NDUFA4L2), Protein-tyrosine Phosphatase 4A3 (PTP4A3), and Zinc-alpha-2-glycoprotein (AZGP1), were identified in TLE-HS, which are associated with the infiltration of specific immune cells. Besides, NDUFA4L2 expression was reduced in kainic acid (KA)-induced HT22 cells compared to the other two hub genes. Thus, NDUFA4L2 was selected for this research. Moreover, NDUFA4L2 overexpression alleviated KA‑induced HT22 cell neurotoxicity, apoptosis, oxidative stress, and mitochondrial dysfunction. In conclusion, NDUFA4L2 upregulation could alleviate KA-induced neurotoxicity oxidative stress, which provided a theoretical foundation and a potential therapeutic target for epilepsy. Screening of module-related DEGs in TLE-HS from Transcriptome GSE256068 database. Hub genes were analyzed using machine learning and immune infiltration. NDUFA4L2 was a potential target of KA-induced neuronal cells in TLE-HS.
Impaired osteogenic differentiation in bone marrow-derived mesenchymal stem cells (BMSCs) is involved in the pathogenesis of osteoporosis. ZC3H13, a crucial m6A writer, promotes BMSC osteogenic differentiation by inhibiting ferroptosis. However, its molecular underpinnings remain largely unexplored. Expression analysis was performed by quantitative PCR and immunoblotting. Cell proliferation was assessed by EdU assay. Osteogenic differentiation was assessed using alkaline phosphatase (ALP) expression/activity assays and Alizarin Red staining. Ferroptosis alteration was evaluated by detecting ROS, Fe2+, and MDA levels. RNA immunoprecipitation (RIP), methylated RIP (MeRIP), luciferase, and mRNA stability assays were used to validate the regulation of ZC3H13 in SLC3A2. The relationship between FOXO3 and ZC3H13 was confirmed by chromatin immunoprecipitation (ChIP) and luciferase experiments. ZC3H13 was downregulated in the bone marrow of osteoporosis patients. ZC3H13 deficiency inhibited BMSC proliferation and osteogenic differentiation while enhancing ferroptosis. Conversely, ZC3H13 overexpression promoted proliferation, differentiation and suppressed ferroptosis. Mechanistically, ZC3H13 mediated the m6A modification and stability of SLC3A2 in an IGF2BP2-dependent manner. ZC3H13 silencing suppressed BMSC proliferation and osteogenic differentiation while enhancing ferroptosis through SLC3A2 reduction. Furthermore, FOXO3 acted as a transcriptional activator of ZC3H13. Depletion of FOXO3 suppressed BMSC proliferation and osteogenic differentiation while enhancing ferroptosis through downregulation of ZC3H13. Additionally, FOXO3 regulated SLC3A2 expression via ZC3H13. This study delineates a novel FOXO3/ZC3H13/SLC3A2 regulatory cascade that safeguards BMSCs from ferroptosis and is compromised in osteoporosis, providing a new conceptual framework for therapeutic development.
Cerebral ischemia–reperfusion (I/R) occurs when blood flow is restored after a temporary interruption and may lead to brain dysfunction through oxidative stress and neurotransmitter imbalance. This study aimed to determine the effect of cerebral I/R and 1-week 3',4'-Dihydroxyflavonol (DiOHF) treatment on the antioxidant/antioxidant system and glutamate, GABA, and BDNF levels in cerebellum tissue in male rats. This study was performed on 28 Wistar-albino type male rats, which were formed as follows: 1-Control Group: No anesthesia or surgical procedure was applied to the animals in this group. 2-Sham Group: After general anesthesia was induced in the animals in this group, the carotid artery regions were opened and closed. After the application, solvent application was performed for 1 week (1 ml DiOHF vehicle). 3-Ischemia–Reperfusion Group: After general anesthesia, the carotid arteries were isolated in rats and ligated for 30 min, ischemia was induced, and then reperfusion was performed. 4-Ischemia–Reperfusion + DiOHF Group: After general anesthesia, the carotid arteries were ligated for 30 min and ischemia was performed in rats, then reperfusion was performed, and then DiOHF supplementation was performed for 1 week. After one week of treatment, the animals were sacrificed under general anaesthesia, and cerebellum tissues were taken. Malondialdehyde (MDA), glutathione peroxidase (GPx), catalase (CAT), superoxide dismutase (SOD), thioredoxin (TRx-2), glutamate, GABA, and brain-derived neurotrophic factor (BDNF) levels were evaluated in the relevant tissue. I/R increased MDA, glutamate, and GABA levels, while GPx, catalase, SOD, thioredoxin and BDNF levels were decreased. However, 1 week of 3',4'-Dihydroxyflavonol treatment corrected the changes that occurred with I/R. The study results show that 1 week of 3',4'-Dihydroxyflavonol treatment after I/R has a positive effect on cerebellum changes that occur in global brain I/R.
Myocarditis, often triggered by infections such as sepsis, involves complex mechanisms including mitochondrial dysfunction, oxidative stress and inflammation. The role of activating transcription factor 5 (ATF5) in myocarditis remains unclear. This study aimed to investigate the role of ATF5 in LPS-induced cardiac injury and its potential mechanism involving mitophagy and pyroptosis. An LPS-induced septic cardiomyopathy model was established in H9c2 cardiomyocytes and rats. ATF5 was overexpressed via plasmid transfection in vitro and in vivo. Cell viability, apoptosis, inflammatory cytokine levels, oxidative stress markers, mitophagy-related proteins (LC3-II, Parkin, PINK1, p62), and pyroptosis-related proteins (NLRP3, Caspase-1, GSDMD) were assessed using CCK-8, flow cytometry, ELISA, Western blot, and biochemical assays. Autophagy inhibitor chloroquine (CQ) and GSDMD-targeted siRNA were applied to explore mechanistic interactions. The results showed that ATF5 overexpression alleviated LPS-induced cardiomyocyte injury by enhancing cell viability, reducing apoptosis, and suppressing inflammatory cytokine release (IL-1β, IL-6 and IL-18) and myocardial injury markers (CK-MB and cTn-I). ATF5 promoted mitophagy, reduced oxidative stress (ROS, LDH, GSH-Px and SOD), and inhibited pyroptosis. Mechanistically, CQ abolished the ATF5-mediated protection against cardiomyocyte injury, which was then restored by si-GSDMD. In vivo, ATF5 overexpression improved cardiac function, attenuated fibrosis and decreased serum injury markers in LPS-treated rats. In conclusion, ATF5 enhanced mitophagy, effectively cleared damaged mitochondria, reduced ROS generation, and subsequently inhibited the pyroptosis pathway, ultimately alleviating myocardial injury.
Thyroid eye disease (TED) is the most common extra-thyroidal complication of Graves’ disease, but its molecular pathogenesis is not fully understood. This study explored the mechanism associated with methyltransferase like 3 (METTL3), NOP2/Sun RNA methyltransferase 4 (NSUN4), and solute carrier family 2 member 3 (SLC2A3) in an in vitro TED model. Human normal orbital fibroblasts were stimulated with platelet-derived growth factor BB (PDGF-BB) to establish in vitro TED model. Differentially expressed genes were screened using online databases. Western blotting was performed for protein detection, and qPCR was used for mRNA quantification. CCK-8 assay and EdU assay were conducted to examine metabolic viability and proliferation, respectively. Hyaluronan (HA) production and glycolysis metabolism were assessed via commercial kits. Methylated RNA immunoprecipitation (MeRIP), RIP, dual-luciferase reporter assay, and RNA-protein pull-down assay were utilized for interaction analysis. Bioinformatics screening obtained 1139 differential genes and 45 glycolytic genes, yielding 2 overlapping genes that were considered as glycolysis-related genes in TED. SLC2A3 was highly expressed in TED and PDGF-BB-stimulated orbital fibroblasts, and silencing SLC2A3 suppressed PDGF-BB-induced cell proliferation, HA production and glycolysis. NSUN4 mediated the m5C methylation modification to increase SLC2A3 expression in an YBX1-dependent manner. NSUN4 inhibition restrained PDGF-BB-induced effects via downregulating SLC2A3. METTL3 mediated m6A methylation modification to reduce NSUN4 expression, with YTHDF2 as a reader protein. The influences of PDGF-BB on human orbital fibroblasts were significantly alleviated by METTL3 overexpression to down-regulate NSUN4. NSUN4 downregulation caused by METTL3-induced m6A modification of NSUN4 mRNA regulated m5C modification of SLC2A3 mRNA, thereby ameliorating PDGF-BB-induced proliferation, HA generation and glycolysis of human orbital fibroblasts in TED. The evidence uncovered the specific molecular mechanism underlying TED pathogenesis.
Chlorophycean algae of the genera Chlamydomonas and Polytomella share a common photosynthetic ancestor. However, members of the Polytomella lineage have adopted a heterotrophic lifestyle, having lost the photosynthetic apparatus and relying instead on acetate or ethanol as carbon sources, with energy production centered on oxidative phosphorylation (OXPHOS). In this study, we investigated the composition of the mitochondrial supercomplexes of the colorless alga Polytomella parva. Mitochondrial membranes were solubilized using mild detergents such as glycol-diosgenin and digitonin, followed by separation of OXPHOS complexes supramolecular assemblies via Blue Native electrophoresis and Fast Protein Liquid Chromatography (FPLC). Additionally, complexome profiling of solubilized algal mitochondria resolved by Blue Native Gel Electrophoresis was carried out (data are available via ProteomeXchange with identifier PXD075371). The resulting data indicate that the OXPHOS supercomplexes of Polytomella closely resemble those observed in situ in the mitochondria of its green relative Chlamydomonas reinhardtii, as revealed by electron cryo-tomography and subtomogram averaging.
Cerebral infarction is a leading cause of severe long-term disability and functional and cognitive impairment. With the advancement of acute stroke treatment, more patients are now experiencing stroke with varying degrees of impairment. The present study was conducted to determine the effects of 1-week 3,4-dihydroxyflavonol (DiOHF) administration during transient experimental cerebral ischemia–reperfusion (I/R) in rats on NeuN, Tuba1a, Tubb3, and calbindin were evaluated as markers of neuronal phenotype and cytoskeletal organization, while ICAM and BDNF were assessed in relation to inflammatory and neurotrophic processes. Changes in these markers indicate alterations in neuronal marker expression, cytoskeletal integrity, and neurotrophic and inflammatory status. In this study, a total of 28 male Wistar albino rats, aged 10–12 weeks and weighing 300–400 g, were used. 1-Control Group (n = 6): The animals in this group received no anaesthesia or surgical procedures. 2-Sham Group (n = 6): After general anaesthesia was induced in the animals in this group, the carotid artery regions were opened and closed. 3- Ischemia–Reperfusion Group (n = 8): Under general anaesthesia, the carotid arteries of the rats were isolated and ligated for 30 min, followed by ischemia. 4- Ischemia–Reperfusion + DiOHF Group (n = 8): Under general anaesthesia, the carotid arteries of the rats were ligated for 30 min, followed by ischemia. Reperfusion was then allowed. Tuba1A, Tubb3, ICAM and Calbindin were analyzed by real-time PCR, BDNF by a commercial ELISA kit, and NeuN by immunohistochemistry. I/R decreased the levels of Neu N, Tuba1a, Tubb3, calbindin and BDNF in the striatum tissues and increased ICAM levels. DiOHF supplementation halted the decrease in expression level in Tuba1a, Tubb3, Calbindin, BDNF; the Increase in Icam level.Also DiOHF supplementation prevented the decrease in the level of anti-NeuN antibody and led to an increase. The study results revealed that one week of transient I/R in rats suppressed NeuN, TUBA1A, TUBB3, calbindin and BDNF levels, which are important in neuronal phenotype and cytoskeletal organization inflammatory and neurotrophic processes. However, a week DiOHF treatment significantly corrected the distortions caused by I/R.
Alterations in mitochondrial function and in reactive oxygen species generation have been associated with physiological aging. In this study, mice aged 3, 10, 20, and 24 months were utilized to investigate the changes in mitochondrial function and reactive oxygen species (ROS) at synapses. Mitochondrial membrane potential was 21% decreased in 20 months-old animals, while it increased (24%) at advanced age (24 months), compared with young mice. Coupling efficiency and ATP synthesis decreased in synaptosomes from 24-months old mice. Regarding mitochondrial respiratory complex activity, reductions in complex II-III and IV activity were observed (42% and 47%, respectively) at 10 months of age. A significant increase in complex I-III activity (48%) was found at 20 months, with no changes in complexes II-III or complex IV enzymatic activities. Likewise, complex II-III activity showed an increase (100%) at 24 months, while complex I-III significantly decreased (37%). An age-related increase in superoxide generation was observed, consistent with impaired respiratory chain function. Interestingly, H2O2 production rates were 43% decreased in 20-months old mice, compared to young animals. This study presents evidence that the aging process leads to changes in mitochondrial function in brain cortex synaptosomes, which become significantly impaired at the age of 20 months. Even though at more advanced ages, compensatory mechanisms might appear to counteract the impact of mitochondrial dysfunction and oxidative damage, mitochondrial bioenergetics seems to be severely compromised.
