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Results for “Membrane Potential, Mitochondrial”

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PubMed1979

Cellular energy metabolism, trans-plasma and trans-mitochondrial membrane potentials, and pH gradients in mouse neuroblastoma.

A method for quantitative evaluation of transmembrane electrical potential and pH gradients across a subcellular compartment in an intact cell is presented. This approach has been applied in studies of mouse neuroblastoma C-1300 clone NB41A3, in which the transmembrane electrical potential and pH gradients and the mitochondrial volume percent have been determined. Membrane potentials and pH gradients were measured by two different methods. Equilibrium distributions of [(3)H]triphenylmethyl phosphonium and [(14)C]-thiocyanate ions gave calculated apparent membrane potentials of -77.0 and -29.6 mV, respectively, at 20-25 degrees C; a value of -60.8 mV was obtained from microelectrode measurements. Equilibrium distributions of weak acids ([(14)C]trimethylacetic acid and 5,5-di[(14)C]methyl-2,4-oxazolidine-dione) and of weak bases ([(14)C]dimethylamine and [(14)C]trimethylamine) gave calculated upper and lower limits of the pH gradient (Delta pH = pH(e) - pH(i)) of -0.14 and -0.21 pH unit, respectively. The microelectrode measurements showed that the intracellular pH is within 0.1 of a pH unit or less of the extracellular pH over the extracellular pH range of 7.35-6.85. The mitochondrial volume percent calculated on the basis of the measured cytochrome c content is 5.6 +/- 1.2% and compares well with estimates of 5.4 +/- 1.1% obtained from 25 electron micrographs. Measurements of the cellular energetic parameters gave values within the range found in other cells and perfused organs. Comparison of the results of the microelectrode and equilibrium measurements permits estimates of the electrical potential and pH gradients across the mitochondrial membrane (mitochondria-to-cytoplasm gradients) to be made and suggests that the trans-mitochondrial membrane protonmotive force in the intact cell cannot be greater than -143 mV.

Animals↗

PubMed1977

Relation between the gradient of the ATP/ADP ratio and the membrane potential across the mitochondrial membrane.

The relation between the intramitochondrial and extramitochondrial ratio ATP/ADP, the transmembrane potential and pH gradient is investigated in the present communication. For this purpose mitochondria are equilibrated with added [14C]ATP in the presence of substrate and oligomycin for eliminating phosphate transfer by ATPase. The membrane potential was measured by the distribution of 86Rb+ in the presence of valinomycin, the deltapH by the distribution of [14C]acetate. In the energized state by varying deltapsi between 60 and 160 mV, the internal (ATP/ADP)i is decreased 30-fold, the external (ATP/ADP)e remains largely constant. As a result, the deltalog (ATP/ADP)e/(ATP/ADP)i = deltalogphi is increased linerly with deltapsi according to the following relation: deltalogphi = 0.85 deltapsi - 0.35. The deltapH was changed between 0.1 and 0.8 by increasing the Pi concentration causing only a minor decrease of deltalogphi would be expected if the ATP-ADP exchange has a significant electroneutral portion. Also in the uncoupled and respiration-inhibited state the same function between deltalogphi and deltapsi is found as in the energized states. It is concluded that under these conditions the ATP-ADP exchange is largely electrical.

Adenosine Diphosphate↗

PubMed2026

Alterations of endothelial cell bioenergetics in congenital diaphragmatic hernia.

BACKGROUND: Pulmonary vascular development in congenital diaphragmatic hernia (CDH) is characterized by impaired angiogenesis and pathologic remodeling that contribute to pulmonary hypertension/hypoplasia. Mitochondria regulate endothelial energy, redox balance, and angiogenic signaling, suggesting a role in CDH vascular disease. METHODS: Endothelial cells (ECs) were isolated from umbilical veins of healthy and CDH newborns. Mitochondrial bioenergetics and glycolytic acidification were assessed by extracellular flux. Oxidative stress, mitochondrial membrane potential, and mitochondrial mass were measured by flow cytometry, while mitochondrial DNA copy number (mtDNA-CN) and morphology were evaluated by qPCR and microscopy. RESULTS: CDH ECs exhibited increased maximal respiratory capacity with elevated proton leak and reduced ATP coupling efficiency. Basal glycolytic activity was elevated. These changes were accompanied by increased mitochondrial superoxide and cellular reactive oxygen species and by severity-associated loss of membrane potential. Despite reduced MitoTracker Green, mtDNA-CN was unchanged, and confocal imaging revealed a highly branched, peripherally distributed network. CONCLUSIONS: These data define a distinct endothelial mitochondrial phenotype marked by metabolic activation, bioenergetic inefficiency, and oxidative stress, with concurrent upregulation of glycolysis and oxidative phosphorylation rather than a glycolytic shift. Structural remodeling with preserved mitochondrial content further indicates qualitative changes. Collectively, these findings link mitochondrial dysfunction to vascular pathology in CDH. IMPACT: Defines a distinct mitochondrial state in CDH endothelium, characterized by metabolic activation with inefficient oxidative phosphorylation, redox imbalance, and structural reorganization in primary human cells. Demonstrates that mitochondrial alterations in CDH occur without changes in mitochondrial content, supporting a model of qualitative remodeling. Provides rare human, cell-based data in CDH, addressing a major gap in a field largely reliant on animal models and indirect measures. Links mitochondrial alterations to clinical severity, supporting relevance to disease burden and heterogeneity. Establishes a framework for mitochondrial involvement in CDH vascular disease, with potential implications for future biomarker development and therapeutic targeting.

Journal Article↗

PubMed2026

Mitochondrial uncoupler BAM15 attenuates cryopreservation-induced damage in human sperm by stabilizing mitochondrial homeostasis†.

Human sperm cryopreservation is essential for sperm banking and assisted reproduction, yet freeze-thaw stress promotes oxidative injury that reduces motility and damages the acrosome and nuclear DNA. Here, we tested whether the mitochondrial uncoupler BAM15 improves post-thaw human sperm quality and examined mechanisms linked to mitochondrial homeostasis. Ejaculates were cryopreserved using a standard protocol supplemented with graded concentrations of BAM15. After thawing, total and progressive motility and viability were assessed. Flow cytometry quantified the DNA fragmentation index and the proportion of high DNA stainability cells. Mitochondrial membrane potential, intracellular reactive oxygen species, and lipid peroxidation were measured to evaluate mitochondrial function and oxidative status. Ultrastructural preservation of the acrosome, plasma membrane, midpiece mitochondria, and flagellar axoneme was examined by transmission electron microscopy. Compared with untreated controls, BAM15 increased total and progressive motility and improved viability. BAM15 reduced DNA fragmentation and decreased high DNA stainability, indicating enhanced genomic integrity. Consistently, BAM15 improved mitochondrial membrane potential while suppressing intracellular reactive oxygen species and lipid peroxidation, supporting attenuation of freeze-thaw oxidative damage. Transmission electron microscopy further revealed more continuous acrosomal and plasma membranes, fewer swollen or vacuolated midpiece mitochondria, and improved preservation of axonemal architecture. Collectively, these findings identify BAM15 as a promising cryopreservation supplement that stabilizes mitochondrial homeostasis and improves the functional and structural quality of human sperm after thawing.

Humans↗

PubMed2026

Protective effects of seminal exosomes on cryopreserved sperm via inhibiting oxidative damage.

This study aimed to explore the protective effect of seminal plasma exosomes (SPEs) on human sperm structure and function during cryopreservation and its potential mechanism. The samples were divided into two groups: the control group was treated solely with sperm cryoprotectant before freezing, while the exosome group was supplemented with SPEs. After cryopreservation and thawing, sperm progressive motility, normal morphological rate, and survival rate were evaluated. Furthermore, PKH67 labeling experiments were performed, and oxidative stress markers as well as energy metabolism indicators in sperm were detected. Subsequent mechanism exploration was conducted via proteomic analysis and protein validation assays. This work reveals that adding SPEs at a concentration of 1 or 2 mg/ml effectively improves sperm progressive motility after cryopreservation. After supplementing with SPEs, sperm glucose levels are reduced and mitochondrial membrane potential is enhanced. Simultaneously, SPEs alleviate oxidative stress by decreasing reactive oxygen species (ROS) and DNA fragment index (DFI) while increasing superoxide dismutase (SOD) activity. Functional annotation of proteomics reveals that 14 of the differentially expressed proteins (DEPs) are associated with sperm motility. Enriched metabolic pathways related to sperm motility and sperm protein validation experiments indicate that the expression of MAPK, p-MAPK, and p-JNK proteins in sperm is higher in the Exosome group than in the Control group. This study provides important theoretical support for the application of SPEs in mitigating cryopreservation damage to sperm by enhancing antioxidant capacity. The specific mechanism may be mediated by the MAPK/p-JNK pathway.

Male↗

PubMed2025

Correction of pathogenic mitochondrial DNA in patient-derived disease models using mitochondrial base editors.

Mutations in the mitochondrial genome can cause maternally inherited diseases, cancer, and aging-related conditions. Recent technological progress now enables the creation and correction of mutations in the mitochondrial genome, but it remains relatively unknown how patients with primary mitochondrial disease can benefit from this technology. Here, we demonstrate the potential of the double-stranded DNA deaminase toxin A-derived cytosine base editor (DdCBE) to develop disease models and therapeutic strategies for mitochondrial disease in primary human cells. Introduction of the m.15150G > A mutation in liver organoids resulted in organoid lines with varying degrees of heteroplasmy and correspondingly reduced ATP production, providing a unique model to study functional consequences of different levels of heteroplasmy of this mutation. Correction of the m.4291T > C mutation in patient-derived fibroblasts restored mitochondrial membrane potential. DdCBE generated sustainable edits with high specificity and product purity. To prepare for clinical application, we found that mRNA-mediated mitochondrial base editing resulted in increased efficiency and cellular viability compared to DNA-mediated editing. Moreover, we showed efficient delivery of the mRNA mitochondrial base editors using lipid nanoparticles, which is currently the most advanced non-viral in vivo delivery system for gene products. Our study thus demonstrates the potential of mitochondrial base editing to not only generate unique in vitro models to study these diseases, but also to functionally correct mitochondrial mutations in patient-derived cells for future therapeutic purposes.

Humans↗

PubMed2026

Ergothioneine Alleviated the Apoptosis of HK Cells by Regulating Ferroptosis.

INTRODUCTION/OBJECTIVE: Ergothioneine (EGT) is a naturally occurring antioxidant with protective effects on various human cell types. The impact of this substance on HK-2 cells, a human renal proximal tubular epithelial cell line, and the associated molecular mechanisms remain incompletely elucidated. METHODS: The present study aims to elucidate the effects of EGT on apoptosis induced by RAS-selective Lethal Molecule 3 (RSL3) and Erastin in HK-2 cells, as well as the potential mechanisms involved. The renal cortical proximal tubular epithelial HK-2 cells were cultured and exposed to RSL3 and Erastin, with or without EGT treatment. Cell viability and apoptosis were assessed using the Cell Counting Kit-8 (CCK-8) assay, while the detection of ferrous ion content and mitochondrial membrane potential changes was accomplished through the utilisation of flow cytometry and the JC-1 staining method, respectively. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, proteomic analysis, and western blotting were employed to explore the molecular pathways involved. RESULTS: The data demonstrated that RSL3 and Erastin exhibited a substantial inhibitory effect on HK-2 cell proliferation, concomitant with the accumulation of intracellular ferrous ions and a shift in mitochondrial membrane potential. The EGT treatment effectively mitigated RSL3- and Erastin-induced apoptosis in HK-2 cells. GO and KEGG enrichment analysis demonstrated that EGT suppressed pathways and functions associated with oxidative stress during ferroptosis. Proteomics analysis further demonstrated that EGT modulated various biological processes, molecular functions, and cellular components in HK-2 cells. The protective mechanism of EGT against RSL3- and Erastin-induced HK-2 cells was potentially mediated through regulation of ferroptosis. The results of the western blot analysis demonstrated that EGT modulated the expression levels of Glutathione Peroxidase 4 (GPX4) and Solute Carrier Family 7 member 11 (SLC7A11) in HK-2 cells. CONCLUSION: These data demonstrated that EGT exerts an alleviating effect on the apoptosis of HK-2 cells induced by RSL3 and Erastin by modulating ferroptosis. These findings suggest that EGT has the potential to serve as a therapeutic candidate for the treatment of kidney diseases in the future.

Ergothioneine↗

PubMed2026

Single-nucleus profiling of postmortem diffuse midline gliomas identifies mitochondrial biogenesis as a resistance mechanism to imipridone therapy.

BACKGROUND: Imipridone ONC201 is the first FDA-approved therapy for H3K27-altered diffuse midline glioma; however, clinical responses remain limited. Defining tumor-intrinsic determinants and microenvironmental, extrinsic factors that shape sensitivity or resistance to imipridones will identify actionable therapeutic opportunities and inform improved clinical strategies. METHODS: To identify mechanisms of imipridone resistance, we obtained postmortem brain tissue from DMG patients who had received imipridones and/or standard care. Single-nucleus RNA and open-chromatin sequencing were performed on N = 22 cases. Immunofluorescence-based myeloid phenotyping was performed on N = 46 cases. Mitochondrial copy-number analysis was performed on N = 19 cases. Validation of imipridone sensitivity, its effect on mitochondrial density, and its synergy with inhibition of mitochondrial biogenesis were assessed in DMG primary cells. RESULTS: We established a single-cell RNA/open-chromatin atlas from postmortem DMG cases and found imipridone treatment resulting in regressed mesenchymal transition, reduced myeloid-derived suppressive cells, and reversed aberrant H3K27-altered enhancer activity. Resistant tumors showed increased mitochondrial density, turnover, and membrane potential. Mitochondrial biogenesis and PPARGC1A emerged as resistance biomarkers and actionable targets. CONCLUSIONS: These studies implicate mitochondrial biogenesis as a biomarker of imipridone resistance and a focus for the development of combinatorial strategies to provide effective therapeutic options for a challenging pediatric brain tumor.

Humans↗

PubMed2026

EPS8 Differentially Regulates Antioxidant Defense and Mitochondrial Homeostatic Signaling in LNCaP and Enzalutamide-resistant LNCaP Cells.

BACKGROUND/AIM: Epidermal growth factor receptor pathway substrate 8 (EPS8) is an adaptor protein implicated in tumor progression and therapeutic resistance; however, its role in mitochondrial homeostatic signaling and antioxidant regulation remains unclear. This study examined the effects of EPS8 modulation in lymph node carcinoma of the prostate (LNCaP) and enzalutamide-resistant LNCaP (LNCaP-Enz) cells. MATERIALS AND METHODS: LNCaP-Enz cells were generated by long-term exposure to enzalutamide and maintained in 5 μM enzalutamide. EPS8 expression was modulated by plasmid-mediated overexpression or shRNA-mediated knockdown. Superoxide dismutase (SOD) activity and cellular adenosine triphosphate (ATP) levels were measured using colorimetric assays. Mitochondrial membrane potential (ΔΨm) was evaluated using JC-1 fluorescence, and mitochondrial staining patterns were qualitatively examined using MitoTracker Green staining. Protein expression associated with antioxidant defense, mitochondrial dynamics, mitochondrial stress response, mitochondrial biogenesis, and AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR) signaling was analyzed by western blotting. RESULTS: EPS8 overexpression increased SOD activity and the expression of SOD1 and SOD2, whereas EPS8 knockdown reduced these antioxidant parameters. Conversely, EPS8 silencing increased cellular ATP levels and enhanced JC-1 red fluorescence patterns. EPS8 silencing increased MFN1 and OPA1 expression and reduced DRP1 expression, consistent with a fusion-associated mitochondrial profile. EPS8 silencing also increased SIRT1, PGC-1α, NRF1, TFAM, p-AMPK/AMPK, and p-mTOR/mTOR, but reduced HSP60, LONP1, ATF5, and CEBPβ expression. CONCLUSION: EPS8 differentially regulates SOD-associated antioxidant capacity and mitochondrial homeostatic signaling in LNCaP-based cell models. Further studies are required to determine whether EPS8 modulation affects enzalutamide responsiveness.

Humans↗

PubMed2026

Single-cell profiling of mitochondrial phenotyping-coupled mtDNA genotyping.

Simultaneously profiling mitochondrial DNA (mtDNA) heteroplasmy and phenotypic variability at the single-cell level remains a challenge due to the absence of integrated methods that map mitochondrial genotypes alongside their functional states. We introduce human single-cell mitochondrial phenotype-coupled mtDNA sequencing (scMPCDS), a platform that quantifies mtDNA mutations and heteroplasmy together with mitochondrial membrane potential and reactive oxygen species within individual cells. Unlike bulk sequencing or separate single-omics techniques, scMPCDS directly correlates mitochondrial genomic instability with functional outcomes. Using this approach, we demonstrate that DdCBE-mediated mtDNA editing induces cell-specific off-target mutations in the mitochondrial genome, which coincide with diverse phenotypic changes. Applying scMPCDS to HeLa cells and clear cell renal cell carcinoma tissues, we identify single-cell subpopulations exhibiting distinct mtDNA mutation burdens and altered bioenergetic profiles, implicating potential mitochondrial heterogeneity-driven tumor evolution. Overall, scMPCDS serves as a versatile tool to unravel mitochondrial genotype-phenotype relationships at the single-cell level in both normal and disease states, thereby advancing precise mitochondrial diagnostics and therapeutics.

Humans↗

PubMed2025

Caffeic acid phenethyl ester protects renal tubular epithelial cells against ferroptosis in diabetic kidney disease via restoring PINK1-mediated mitophagy.

Mounting evidence indicates that renal tubular ferroptosis plays a crucial role in the progression of diabetic kidney disease (DKD). Caffeic acid phenethyl ester (CAPE), derived from propolis, a precious resinous substance synthesized by various bee species, has garnered broad attention in biomedical research. This study aims to explore the mechanism by which CAPE protects renal tubular epithelial cells (TECs) against ferroptosis in DKD. DBA/2J mice were administered streptozotocin (STZ) by intraperitoneal injection, fed a high-fat diet (HFD) and treated with CAPE. The findings revealed significant changes in ferroptosis markers. In diabetic mice and TECs under high-glucose (HG) conditions, levels of glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) decreased, while transferrin receptor 1 (TFR1) increased. These changes were accompanied by a reduction in antioxidant capability and the accumulation of malondialdehyde (MDA). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that the intersection targets of CAPE and ferroptosis were mainly located in the mitochondria and exhibited high enrichment values in mitophagy. Further investigations revealed that HG induced a depolarization of mitochondrial membrane potential and an excessive level of mitochondrial reactive oxygen species (ROS), accompanied by defective mitophagy. The administration of CAPE inhibited exacerbated ferroptosis and rescued defective mitophagy induced by DKD. In addition, CAPE restored PTEN-induced putative kinase 1 (PINK1) levels, which were markedly diminished in the kidneys of DKD mice and TECs subjected to HG. Molecular docking simulation experiments suggested that CAPE is steadily bound to the PINK1 active pocket. Cellular Thermal Shift Assay (CETSA) and Drug Affinity Responsive Target Stability assay (DARTS) showed that CAPE enhances the thermal stability of the PINK1 protein within a specific temperature range and protects the PINK1 protein from degradation by proteolytic enzymes. These results confirm that CAPE interacts with PINK1 as its specific target. However, the positive outcomes of CAPE treatment on ferroptosis were nullified by the PINK1 siRNA. This research indicates that CAPE has potential therapeutic benefits for DKD by protecting renal TECs against ferroptosis via rescuing PINK1-mediated mitophagy. These findings suggest that CAPE shows potential as a therapeutic agent to prevent tubular injury in DKD.

Animals↗

PubMed2025

Convergent activation of the integrated stress response and ER-mitochondria uncoupling in VAPB-associated ALS.

Vesicle-associated membrane protein-associated protein-B (VAPB) is an endoplasmic reticulum (ER) membrane-bound protein. The P56S mutation in VAPB causes a dominant, familial form of amyotrophic lateral sclerosis (ALS). However, the mechanism by which this mutation leads to motor neuron (MN) degeneration remains unclear. Utilizing inducible pluripotent stem cell (iPSC)-derived MNs expressing either wild-type (WT) or P56S VAPB, we demonstrate that the mutant protein reduces neuronal firing and disrupts ER-mitochondria-associated membranes (ER MAMs), with a time-dependent decline in mitochondrial membrane potential (MMP), hallmarks of MN pathology. These findings were validated in patient-derived iPSC-MNs. Additionally, VAPB P56S MNs show increased susceptibility to ER stress, elevated expression of the Integrated Stress Response (ISR) regulator ATF4 under stress, and reduced global protein synthesis. Notably, pharmacological ISR inhibition using ISRIB rescued ALS-associated phenotypes in both VAPB P56S and patient-derived iPSC-MNs. We present the first evidence that the VAPB P56S mutation activates ISR signaling via mitochondrial dysfunction in human MNs. These findings support ISR modulation as a strategy for ALS intervention and highlight the need for patient stratification in clinical trials.

Amyotrophic Lateral Sclerosis↗

PubMed2024

Integrated multi-omics approaches reveal the neurotoxicity of triclocarban in mouse brain.

Triclocarban (TCC) is an antimicrobial ingredient that commonly incorporated in many household and personal care products, raising public concerns about its potential health risks. Previous research has showed that TCC could cross the blood-brain barrier, but to date our understanding of its potential neurotoxicity at human-relevant concentrations remains lacking. In this study, we observed anxiety-like behaviors in mice with continuous percutaneous exposure to TCC. Subsequently, we combined lipidomic, proteomic, and metabolic landscapes to investigate the underlying mechanisms of TCC-related neurotoxicity. The results showed that TCC exposure dysregulated the proteins involved in endocytosis and neurodegenerative disorders in mouse cerebrum. Brain energy homeostasis was also altered, as evidenced by the perturbation of pyruvate metabolism, TCA cycle, and oxidative phosphorylation, which in turn caused mitochondrial dysfunction. Meanwhile, the changing trends of sphingolipid signaling pathway and overproduction of mitochondrial reactive oxygen species (mROS) could enhance the neural apoptosis. The in vitro approach further demonstrated that TCC exposure promoted apoptosis, accompanied by the overproduction of mROS and alteration in the mitochondrial membrane potential in N2A cells. Together, dysregulated endocytosis, mROS-related mitochondrial dysfunction and neural cell apoptosis are considered to be crucial factors for TCC-induced neurotoxicity, which may contribute to the occurrence and development of neurodegenerative disorders. Our findings provide novel perspectives for the mechanisms of TCC-triggered neurotoxicity.

Animals↗

PubMed2026

Dysregulated Ribonucleoprotein Granules Impair Mitochondrial Function in RBM20-Related Dilated Cardiomyopathy.

BACKGROUND: Pathogenic variants in RBM20 cause severe dilated cardiomyopathy. Loss-of-function variants disrupt splicing; neomorphic gain-of-function (GoF) variants also mislocalize RBM20 to cytoplasmic ribonucleoprotein granules and are associated with more aggressive disease. The mechanism by which RBM20 mislocalization drives cardiac dysfunction remains unknown. METHODS: We investigated the effects of Rbm20 GoF and loss-of-function (LoF) variants using proteomic profiling, protein solubility assays, mitochondrial respiration and calcium flux analyses, and ultrastructural imaging in mouse models. Human induced pluripotent stem cell-derived cardioids were used to validate variant-specific phenotypes. RESULTS: Rbm20 GoF, but not LoF, variants caused posttranscriptional downregulation of soluble mitochondrial proteins, including the calcium efflux regulator TMEM65 (transmembrane protein 65), and reduced solubility of mitochondrial membrane proteins. Electron microscopy revealed enlarged mitochondria with cristae disorganization. Functional assays confirmed impaired oxidative phosphorylation, reduced mitochondrial membrane potential, and abnormal calcium handling in Rbm20 GoF models. Human cardioids reproduced these findings, demonstrating that cytoplasmic mislocalization, rather than splicing deficiency, drives mitochondrial dysfunction. CONCLUSIONS: Cytoplasmic mislocalization of RBM20 disrupts mitochondrial function by reducing mitochondrial protein abundance, leading to oxidative phosphorylation failure and abnormal mitochondrial calcium handling. This mechanism distinguishes RBM20 GoF from LoF variants and may explain the more severe heart failure phenotype observed in patients with RBM20 GoF variants. These insights advance the mechanistic understanding of RBM20-related cardiomyopathy and identify mitochondrial mRNA/protein regulation as a key node in cardiac energetics.

cardiomyopathy, dilated↗

PubMed2026

A model of cellular proliferation and mitochondrial biogenesis predicts prognosis and immunotherapy response in lung adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LUAD), which is the leading subtype of non-small cell lung cancer (NSCLC), poses considerable difficulties in accurate prognostic assessment and targeted therapeutic options. Cell proliferation-related genes (CPGs) and mitochondrial biogenesis-related genes (MBGs) play critical roles in tumor metabolic reprogramming; however, their prognostic value and molecular mechanisms in LUAD are poorly understood. This study aims to construct a CPG/MBG-based prognostic risk model for LUAD, evaluate its clinical utility in predicting prognosis and immunotherapy response, and experimentally validate the functional role of key model genes in LUAD progression. METHODS: By utilizing The Cancer Genome Atlas (TCGA)-LUAD and GSE72094 datasets, this investigation formulated a risk scoring model through differential expression screening combined with least absolute shrinkage and selection operator (LASSO)-Cox regression analysis. The molecular characteristics and clinical implications of the risk model were investigated via immune microenvironment evaluation, genomic alteration analysis, and drug sensitivity prediction. The functional contributions of key genes were further substantiated using quantitative reverse transcription polymerase chain reaction (qRT-PCR), commercial assay kits, the JC-1 fluorescent probe, the Cell Counting Kit-8 (CCK-8), Transwell invasion assays, and wound healing assays. RESULTS: A risk model based on seven CPGs and MBGs (PLK1, HMMR, CYP27A1, LDHA, NPAS2, KRT17, CIDEC) showed reliable predictive performance in both GSE72094 and the TCGA-LUAD cohorts. Enhanced tumor heterogeneity and an immunosuppressive microenvironment were observed in the high-risk group. Drug sensitivity analysis indicated that the risk model could guide personalized treatment strategies; for instance, high-risk patients showed increased susceptibility to agents such as docetaxel and 5-fluorouracil. In vitro experiments demonstrated that the key gene CIDEC exhibited upregulated expression in LUAD tissues and cells. Knockdown of CIDEC led to enhanced cellular energy metabolism and increased mitochondrial membrane potential, while also effectively suppressing cell invasion, proliferation, and migration. CONCLUSIONS: The established MBGs/CPGs prognostic model provides a novel tool for stratified treatment planning in LUAD, underscoring the crucial roles of cellular proliferation and mitochondrial biogenesis in tumor progression. Functional validation of CIDEC offers experimental support for the development of potential therapeutic strategies.

Lung adenocarcinoma (LUAD)↗

PubMed2026

Role of Sanqi Baiji San in Mitigating Ethanol-Induced Gastric Epithelial Cell Injury via PI3K/AKT-related Signaling.

This work aimed to clarify the protective mechanism of Sanqi Baiji San (SQBJ) against ethanol-induced gastric epithelial cell injury and to explore its potential relevance to gastric ulcer (GU). Network pharmacology was used to screen SQBJ's active components (Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform [TCMSP], Oral Bioavailability [OB] ≥ 20%, Drug-Likeness [DL] ≥ 0.1), map their targets (Universal Protein Resource [UniProt]), collect GU-related targets (GeneCards/OMIM/DrugBank), and analyze overlapping targets via Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment and molecular docking. Ethanol-injured Gastric Epithelial Cell Line-1 (GES-1) cells were treated with SQBJ or the PI3K inhibitor LY294002. CCK-8 was used to determine the optimal SQBJ concentration. Network pharmacology analyses identified 126 common targets enriched within PI3K/AKT/MAPK-related cascades and suggested potential interactions between principal SQBJ constituents and PI3K/AKT-related proteins. In ethanol-challenged cells, SQBJ alleviated cell injury by reducing inflammatory mediator release and oxidative stress, as evidenced by decreased intracellular reactive oxygen species and malondialdehyde levels. SQBJ restored mitochondrial membrane potential and ATP content and reduced apoptosis-associated changes in B-cell lymphoma-2, cleaved caspase-3, and Bcl-2-associated X protein levels. SQBJ also modulated PI3K/AKT- and MAPK-associated signaling markers. These protective effects were largely weakened by LY294002, suggesting that PI3K/AKT-related signaling is involved in SQBJ-mediated cytoprotection. These findings provide an in vitro mechanistic basis for the potential application of SQBJ in GU, although further validation in animal models of ethanol-induced gastric ulcer is required.

Ethanol↗

PubMed2026

Apolipoprotein E promotes papillary thyroid carcinoma progression by activating PINK1/Parkin-mediated mitophagy.

BACKGROUND: Increasing evidence supports a progression-related role of apolipoprotein E (APOE) in papillary thyroid carcinoma (PTC), yet a clear mechanistic explanation for this association is still lacking. Considering the pivotal role of mitochondrial homeostasis in tumorigenesis, the potential role of APOE in promoting PTC progression through mitophagy regulation was investigated. Additionally, the involvement of the PINK1/Parkin-associated pathway in this process was examined to provide insights into its contribution to tumor progression. METHODS: APOE in thyroid carcinoma was characterized in terms of its expression profile, diagnostic relevance, and potential biological functions, based on integrative evidence derived from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets. APOE and mitophagy-related protein expression were further examined in PTC tissues by immunohistochemistry. Further evaluation of APOE in PTC cell lines focused on its association with proliferation, apoptosis, and mitophagy, with bidirectional functional perturbation serving as the basis for assessment. Pharmacological inhibitors were used to assess the involvement of mitophagy-related signaling in the observed APOE-dependent phenotypes. Additionally, the in vivo impact of APOE on PTC tumor growth and mitophagy was further investigated through a nude mouse xenograft model, providing insight into its potential role in tumor progression. RESULTS: A significant upregulation of APOE was observed in thyroid carcinoma tissues and PTC cell lines, supporting its potential relevance as a diagnostic biomarker. The modulation of APOE expression significantly influenced PTC cell proliferation and apoptosis, with overexpression promoting cell proliferation and inhibiting apoptosis, while knockdown led to the opposite effects. Mechanistically, APOE overexpression increased AMP-activated protein kinase (AMPK) phosphorylation and decreased mammalian target of rapamycin (mTOR) phosphorylation, accompanied by increased PINK1 and Parkin expression and mitophagy-related changes, including altered mitochondrial membrane potential, reduced overall reactive oxygen species levels, and increased autophagosome formation. Pharmacological inhibition of mitophagy attenuated the proliferative and antiapoptotic effects of APOE. CONCLUSIONS: These findings demonstrate that APOE promotes PTC progression in association with PINK1/Parkin-related mitophagy and modulation of the AMPK/mTOR axis. The APOE-associated mitophagy axis may provide a rationale for future preclinical investigation in PTC.

Apolipoprotein E (APOE)↗

PubMed2025

Suppression of OTUD4 protects against myocardial ischemia-reperfusion injury by increasing autophagic flux and inhibiting apoptosis in cardiomyocytes.

Dysregulated autophagic flux plays a critical role in myocardial ischemia-reperfusion injury (MIRI), complicating cardiac reperfusion therapy. In this study, we identified OTUD4 as a potential regulator of autophagic flux in MIRI using CRISPR/Cas9 sgRNA sequencing. However, the underlying mechanism is poorly understood. The purpose of this study is to investigate the effects of OTUD4 on autophagic flux in OGD-R treated AC16 cells (IRI model in vitro) and LAD artery ligation induced myocardial ischemia-reperfusion mice (MIRI model in vivo). In the in vitro IRI cell model, OTUD4 knockdown significantly reversed impaired autophagic flux, increased mitochondrial membrane potential, and decreased LDH activity, ROS production, autophagy and apoptosis. Overexpression of OTUD4 showed the opposite result. In the in vivo MIRI model, OTUD4 knockdown also significantly decreased infarct area, improved cardiac structure and function, reduced serum BNP and LDH levels, attenuated cardiac tissue injury/fibrosis/myocardial hypertrophy, and ultimately exerted myocardial protective effects against ischemia-reperfusion injury. Importantly, OTUD4 knockdown inhibited autophagosome-associated markers (LC3II/LC3I, Beclin1, ATG9), autophagy substrate p62, increased lysosomal activity marker LAMP2, and activated the autophagy pathway (AKT/mTOR), thereby promoting the recovery of impaired autophagic flux in the MIRI model. Moreover, OTUD4 showed strong interaction with UBAC1, and OTUD4 deficiency decreases UBAC1 protein expression by impairing its deubiquitination, thereby regulating autophagy. In short, blocking OTUD4 restored damaged autophagic flux in I/R induced myocardial injury both in vivo and in vitro, inhibited myocardial cell apoptosis, and greatly improved cardiac function in ischemia-reperfusion mice. KEY MESSAGES: OTUD4 was identified as a key negative regulator of autophagy flux in myocardial ischemia-reperfusion injury (MIRI) via genome-wide CRISPR/Cas9 screening. OTUD4 knockdown exerts cardioprotective effects by reducing apoptosis and ROS generation and improving heart function in both in vitro and in vivo models. The interaction between OTUD4 and UBAC1 was confirmed, and OTUD4 maintains UBAC1 stability through deubiquitination, providing new insights into the ubiquitination regulatory mechanism in myocardial injury. Targeting OTUD4 has therapeutic potential for MIRI, as OTUD4 knockdown alleviated MIRI in both in vitro and in vivo models, suggesting the possibility of developing OTUD4 inhibitors for cardiac reperfusion treatment.

Animals↗