BBA Advances最新文献

{"title":"Lipidomic profiling of Arabidopsis chloroplast protein phosphatase SLP1 mutants reveals altered diurnal lipid remodeling","authors":"Chris White-Gloria ,&nbsp;Greg B. Moorhead ,&nbsp;Adriana Zardini Buzatto","doi":"10.1016/j.bbadva.2026.100180","DOIUrl":"10.1016/j.bbadva.2026.100180","url":null,"abstract":"<div><div>Lipids are fundamental to the biology of all organisms, including the functioning of subcellular organelles. The plant chloroplast is the site of photosynthesis and serves as a hub of metabolic events that are dynamically regulated between the light and dark periods. The regulation of chloroplast events, like most other biological processes, is often controlled by protein phosphorylation. Here, we explored the lipidome of <em>Arabidopsis thaliana</em> under both light and dark conditions, examining lipid changes upon overexpression or knockout of the chloroplast-specific phosphatase <em>Shewanella</em>-like protein phosphatase 1 (SLP1). To date, no substrates have been identified for the phosphatase SLP1. Results indicate that <em>Arabidopsis thaliana</em> possesses a dynamic lipidome that undergoes significant changes between light and dark conditions, and that protein phosphorylation, in part controlled by SLP1, plays a crucial role in regulating chloroplast metabolism and its associated lipidome. Alterations due to loss or over-expression of SLP1 in the chloroplast are transmitted to other cellular compartments, leading to altered lipids throughout the cell. We report that although both SLP1 mutant lines display perturbed glycerolipid metabolism, knockout and overexpression affect distinct pathways. In the three <em>Arabidopsis</em> lines studied, we also identify dramatic diurnal changes in the abundance of oxidized lipids. Together, these findings establish SLP1-dependent dephosphorylation as an upstream regulator of diel lipid remodelling, linking chloroplast activity to broader cellular lipid organization.</div></div>","PeriodicalId":34672,"journal":{"name":"BBA Advances","volume":"9 ","pages":"Article 100180"},"PeriodicalIF":3.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145977442","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Drying parameters and aging modulate protective properties of vitrified trehalose","authors":"U.G.V.S.S. Kumara,&nbsp;Thomas C. Boothby","doi":"10.1016/j.bbadva.2026.100188","DOIUrl":"10.1016/j.bbadva.2026.100188","url":null,"abstract":"<div><div>Storage of biological materials underpins medical, research, and biotechnological applications. Although cold-chain preservation is effective, it is costly, infrastructure-dependent, and vulnerable to disruption. Room-temperature dry storage, inspired by desiccation-tolerant organisms, offers an alternative by stabilizing biomolecules in vitrified matrices that limit molecular motion and degradation. Trehalose is widely used as a vitrifying agent, but its protective capacity depends on glassy properties shaped by drying conditions, environment, storage duration, and biomolecule type. However, systematic links between these factors and stability remain poorly defined. Here, we examine how drying conditions and storage duration influence the stability of DNA, RNA, and enzymes in trehalose-based vitrified systems. DNA remained stable across all conditions, independent of trehalose or drying parameters, reflecting intrinsic resistance to desiccation damage. RNA exhibited moderate sensitivity to drying without trehalose but was stabilized in its presence, although RNA integrity did not consistently correlate with measured glassy properties. In contrast, enzymes were highly sensitive to drying in the absence of trehalose and strongly protected under conditions that promoted favorable vitrified properties. Short-term enzyme protection (30 min) positively correlated with higher glass transition temperature (Tg). However, during prolonged dry storage, higher Tg was inversely correlated with enzyme stability and instead tracked detrimental physical aging of the vitrified matrix. These findings demonstrate that optimal glass properties depend on both biomolecule class and timescale, providing a framework for rationally designing room-temperature preservation strategies.</div></div>","PeriodicalId":34672,"journal":{"name":"BBA Advances","volume":"9 ","pages":"Article 100188"},"PeriodicalIF":3.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147802424","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Purinergic signaling modulates endothelial-to-mesenchymal transition in human aortic valve endothelial cells","authors":"Vera Schmidt ,&nbsp;Andreas Weber ,&nbsp;Artur Lichtenberg ,&nbsp;Jürgen Schrader ,&nbsp;Pascal Martsch ,&nbsp;Mareike Barth ,&nbsp;Payam Akhyari","doi":"10.1016/j.bbadva.2026.100190","DOIUrl":"10.1016/j.bbadva.2026.100190","url":null,"abstract":"<div><div>Endothelial-to-mesenchymal transition (EndMT) is a phenotypic switch in which endothelial cells acquire mesenchymal characteristics, involving both functional and morphological changes. While EndMT is essential for cardiac development, its aberrant activation contributes to adult cardiovascular pathologies, including calcific aortic valve disease (CAVD). Dysregulation of ectonucleotidases—membrane-bound enzymes that regulate extracellular ATP and adenosine metabolism—has been implicated in such diseases. Altered extracellular nucleotide signaling influences valvular interstitial cell (VIC) degeneration and may interact with valvular endothelial cells (VECs) undergoing EndMT. The objective of this study was to investigate the role of the purinergic signaling system in regulating EndMT in human aortic VECs.</div><div>Primary human VECs were cultured in vitro and treated with inhibitors of ectonucleoside triphosphate diphosphohydrolase 1 (CD39), ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), and 5′-nucleotidase (CD73), alongside adenosine and P2 purinergic receptor agonists. EndMT markers and signaling pathways were assessed via phosphorylation assays and mRNA expression analysis of key transcription factors, including SLUG, SNAIL, ZEB1, and ZEB2. Inhibition of ATP- and AMP-hydrolyzing enzymes (CD39, ENPP1, CD73) enhanced p38 phosphorylation and modulated SLUG expression. Activation of P2 and adenosine A2B receptors altered SNAIL levels, while A2A receptor signaling influenced ZEB1 and ZEB2 expression. These perturbations resulted in pronounced morphological changes consistent with EndMT.</div><div>In conclusion, dysregulation of the purinergic signaling system induces EndMT in human aortic VECs, highlighting a potential mechanistic link between extracellular nucleotide metabolism and valvular pathology. Targeting purinergic pathways may represent a therapeutic avenue for CAVD and related vascular disorders.</div></div>","PeriodicalId":34672,"journal":{"name":"BBA Advances","volume":"9 ","pages":"Article 100190"},"PeriodicalIF":3.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147749109","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Fluid shear stress activates c-Src and promotes RANKL localization in the plasma membrane in osteoblast-like MC3T3-E1 cells","authors":"Takuma Matsubara ,&nbsp;Anna Yoshimura ,&nbsp;Shoichiro Kokabu","doi":"10.1016/j.bbadva.2026.100185","DOIUrl":"10.1016/j.bbadva.2026.100185","url":null,"abstract":"<div><div>Receptor activator of NF-κB ligand (RANKL) produced by osteoblastic lineage cells is essential for osteoclastogenesis, yet RANKL can be sequestered in intracellular, lysosome-like compartments under basal conditions. How mechanical cues mobilize RANKL toward the cell surface remains poorly defined. Here, we tested whether fluid shear stress alters RANKL subcellular distribution in osteoblast-like MC3T3-E1 cells and examined the involvement of the non-receptor tyrosine kinase c-Src. MC3T3-E1 cells expressing fluorescently tagged RANKL were subjected to fluid shear stress, and RANKL localization was analyzed by microscopy and subcellular fractionation. Fluid shear stress increased c-Src activation (Tyr416 phosphorylation) and promoted redistribution of RANKL toward the cell periphery, accompanied by an increase of RANKL in the membrane fraction. Co-expression experiments showed spatial association of RANKL with c-Src at the cell periphery after shear stimulation. Moreover, constitutively active c-Src (Y527F) enhanced peripheral localization of RANKL even in the absence of shear stress. Together, these data support a model in which shear stress activates c-Src to facilitate RANKL localization from intracellular stores toward membrane-proximal regions, thereby providing a mechanistic link between mechanical cues and osteoblast-derived osteoclastogenic signaling.</div></div>","PeriodicalId":34672,"journal":{"name":"BBA Advances","volume":"9 ","pages":"Article 100185"},"PeriodicalIF":3.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147538470","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Understanding glycan structure and function through artificial intelligence","authors":"Lachlan Coff,&nbsp;Ujala Bashir,&nbsp;Daniel Bojar","doi":"10.1016/j.bbadva.2026.100196","DOIUrl":"10.1016/j.bbadva.2026.100196","url":null,"abstract":"<div><div>Glycoinformatics has entered the artificial intelligence era. Stymied by a lack of big data, high sequence complexity, and biosynthetic dependencies, the application of machine learning to glycomics data has largely emerged this decade. In this mini-review, we explore the latest groundbreaking computational approaches applied to glycan sequencing, classifying disease risk from glycan biomarkers, and predicting protein–glycan interactions. We detail advancements in the architectures of these models, concluding that they have matured to the stage of extracting predictive information from glycan sequences. We also discuss their challenges and limitations, and how to fully reap their rewards in the future.</div></div>","PeriodicalId":34672,"journal":{"name":"BBA Advances","volume":"10 ","pages":"Article 100196"},"PeriodicalIF":4.5,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13315649/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148362693","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"TCA cycle intermediates as an adjunct strategy for human iPSC-derived cardiomyocyte maturation","authors":"Keshav Narayan Alagarsamy ,&nbsp;Emilee Bueckert ,&nbsp;Mehak Gupta ,&nbsp;Michel Aliani ,&nbsp;Sanjiv Dhingra","doi":"10.1016/j.bbadva.2026.100183","DOIUrl":"10.1016/j.bbadva.2026.100183","url":null,"abstract":"<div><div>Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) are a valuable tool for modeling cardiac diseases, drug testing, and regenerative applications. However, their application is limited by the immature phenotype of iPSC-CM. During maturation from fetal to adult phenotype cardiomyocytes undergo a transition from glycolysis to oxidative phosphorylation for energy production, which is supported by efficient tricarboxylic acid (TCA) cycle activity. Our metabolomics data suggest that the level of intermediates of TCA cycle including succinate, malate, fumarate, and α-ketoglutarate was very low in iPSC-CM. Therefore, we investigated the effect of supplementation with these metabolites on the maturation of iPSC-CM. We cultured iPSC-CM in glucose (Glu), galactose (Gal), or galactose plus TCA cycle intermediates (Gal+TCA) and evaluated the incremental effect of TCA cycle intermediates supplementation relative to Glu and Gal. The treatment with these TCA cycle intermediates led to improved calcium handling and cellular morphology of iPSC-CM relative to Glu and Gal. Furthermore, the treatment with TCA cycle metabolites enhanced electrical activity, improved mitochondrial health, and the cells were shifting toward oxidative phosphorylation relative to Glu only. This shift in the energy metabolism was associated with an upregulation in the expression of cardiomyocyte maturation genes and downregulation in the expression of fetal genes in Gal + TCA group relative to Glu. Overall, the benefits of Gal+TCA supplementation were quite evident compared to Glu alone but generally modest relative to Gal supplementation, supporting that TCA cycle intermediates supplementation can be used as an adjunct strategy to promote iPSC-CM maturation.</div></div>","PeriodicalId":34672,"journal":{"name":"BBA Advances","volume":"9 ","pages":"Article 100183"},"PeriodicalIF":3.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147310829","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CEMIP promotes gefitinib resistance in lung cancer with EGFR-mutation possibly through forming a stable complex CEMIP/c-MYC","authors":"Li Li ,&nbsp;Jiaxiu Xie ,&nbsp;Jinxing Liang ,&nbsp;Hua Su ,&nbsp;Xiaoju Shen ,&nbsp;Xiaoxiang Mo ,&nbsp;Xinli Lin ,&nbsp;Weidan Tan ,&nbsp;Kun Wu ,&nbsp;Fucui Zhu ,&nbsp;Li Wang ,&nbsp;Ying Liu","doi":"10.1016/j.bbadva.2025.100176","DOIUrl":"10.1016/j.bbadva.2025.100176","url":null,"abstract":"<div><div>While gefitinib greatly improves the prognosis of lung cancer patients with epidermal growth factor receptor (EGFR) mutation, its inevitable resistance severely diminishes therapeutic efficacy. Cell migration-inducing protein (CEMIP) might interact with cellular MYC proto-oncogene (c-MYC) to synergistically drive gefitinib resistance. However, the mechanism by which CEMIP/c-MYC regulates gefitinib resistance in LUAD remains unknown. The gefitinib-acquired resistance (AR) cell line PC9GR was established by gradual dose-escalation induction. CEMIP expression was up-regulated in PC9GR cells, suggesting the positive corelationship might exsit between CEMIP expression and gefitinib resistance. Similarly, overexpressing CEMIP in PC9 cells not only increased IC₅₀ value of gefitinib but also enhanced the proliferation, migration and tolerance to gefitinib. In contrast, downregulating the expression of CEMIP in PC9GR cells partially restored gefitinib sensitivity and reduced malignant phenotypes. Furthermore, c-MYC promoted transcriptional activity through binding to the promoter region of CEMIP. Rescue assays demonstrated that reducing the expression of c-MYC downregulated the IC₅₀ value of Gefitinib, migration and tolerance of gefitinib in PC9 OE cells, while overexpressing c-MYC reversed these malignant phenotypes in PC9GR shCEMIP cells. Subcutaneous xenograft model also supported that the expression of CEMIP positively correlated to c-MYC in tumor tissues. Mechanistically, protein docking simulations and Co-IP assays indicated that CEMIP directly interacted with c-MYC via its 1–177aa domain to form the CEMIP/c-MYC complex. Furthermore, CHX chase assays showed that CEMIP and c-MYC mutually stabilized the expression of both proteins. The elevated CEMIP/c-MYC axis accelerates EMT to enhance cell migration, thereby contributing to the acquisition of gefitinib resistance in LUAD.</div></div>","PeriodicalId":34672,"journal":{"name":"BBA Advances","volume":"9 ","pages":"Article 100176"},"PeriodicalIF":3.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145658878","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Role of recombinant S100A13 expression in regulating mitochondrial fission and fusion in lung epithelial cells","authors":"Somaya Alqattan ,&nbsp;Mona Alonazi ,&nbsp;Eid Almutairy ,&nbsp;Vineesh V. Raveendran ,&nbsp;Abdulaziz Alamri ,&nbsp;Futwan Al-mohanna","doi":"10.1016/j.bbadva.2026.100195","DOIUrl":"10.1016/j.bbadva.2026.100195","url":null,"abstract":"<div><div>S100A13, a calcium-binding protein containing two EF-hand motifs, contributes to intracellular calcium homeostasis, a key determinant of mitochondrial quality control. We previously showed that S100A13 modulates mitochondrial membrane potential (ΔΨm) in patient-derived skin fibroblasts carrying S100A13 (p.I80Gfs*13<em>)</em> and S100A3 (p.R77C) mutations. However, its role in regulating mitochondrial dynamics remains unclear. Here, we investigated whether S100A13 regulates mitochondrial fusion–fission balance in human bronchial epithelial cells (BEAS-2B) using Myc-tagged wild-type or p.I80Gfs*13 S100A13 constructs. The S100A13 p.I80Gfs*13 mutant markedly attenuated bradykinin- and ionophore-induced intracellular calcium transients and reduced ΔΨm compared with wild-type S100A13 (<em>p</em> &lt; 0.05). These alterations were associated with severe mitochondrial ultrastructural abnormalities, disrupted cristae organization, and increased mitochondrial fragmentation (<em>p</em> &lt; 0.05). Interestingly, both wild-type and p.I80Gfs*13 mutant S100A13 increased expression of the mitochondrial fusion-associated proteins MFN1/2 and OPA1 while reducing expression of the fission mediator MFF. However, despite these apparently pro-fusion molecular changes, the S100A13 p.I80Gfs*13 mutant failed to maintain mitochondrial fusion competency, suggesting a functional uncoupling between fusion protein abundance and mitochondrial fusion competency. Collectively, these findings identify S100A13 as an important regulator of intracellular calcium-dependent mitochondrial dynamics and demonstrate that C-terminal truncation disrupts calcium-dependent regulation of mitochondrial fusion and cristae integrity in lung epithelial cells.</div></div>","PeriodicalId":34672,"journal":{"name":"BBA Advances","volume":"10 ","pages":"Article 100195"},"PeriodicalIF":3.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148214728","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Neddylation-dependent RAB40-Cullin5 E3 ligase complex controls ubiquitin-mediated proteasomal degradation of DNAJC13","authors":"Kazuki Sato,&nbsp;Naoto Sugeno,&nbsp;Takafumi Hasegawa,&nbsp;Shun Yoshida,&nbsp;Mitsunori Fukuda,&nbsp;Akemi Fujibayashi,&nbsp;Kiyotoshi Sekiguchi,&nbsp;Shun Ishiyama,&nbsp;Takaaki Nakamura,&nbsp;Ako Miyata,&nbsp;Satoshi Watanabe,&nbsp;Kensho Ikeda,&nbsp;Atsushi Takeda,&nbsp;Masashi Aoki","doi":"10.1016/j.bbadva.2026.100200","DOIUrl":"10.1016/j.bbadva.2026.100200","url":null,"abstract":"<div><div>DNAJC13 is a member of DNAJ co-chaperone family and its missense mutation causes a rare familial form of Parkinson’s disease. DNAJC13 is indispensable for neuronal homeostasis by regulation of autophagy and the regulation of receptor expression on the cell surface. However, the regulatory mechanisms governing DNAJC13 expression remain poorly understood. We previously demonstrated that DNAJC13 binds to RAB40 small GTPase. Unlike other RABs, RAB40 proteins interact with activated Cullin-5 (CUL5) via the SOCS box, forming an Cullin-RING E3 ubiquitin ligase (CRL) complex that regulates ubiquitylation of target proteins. Thus, as a binding partner of RAB40s, DNAJC13 degradation may be regulated by this functional complex. Here, we found that both N-and C-terminal regions of DNAJC13, including IWN1 and IWN3 domains, were essential for its binding to RAB40 A/B/C. Moreover, DNAJC13 degradation of relies predominantly on the ubiquitin-proteasome system rather than on autophagy, and upregulated DNAJC13 ubiquitination in the presence of RAB40A/B/C. Intriguingly, RNAi-mediated silencing of the RAB40 subfamily caused a significant increase in DNAJC13 expression, accompanied by higher autophagic flux. Furthermore, co-immunoprecipitation experiments demonstrated that RAB40C, which is predominantly expressed in midbrain dopaminergic neurons, binds to CUL5 and its activator NEDD8 as well as RBX2 (E2). Finally, DNAJC13 cellular levels were regulated by NEDD8 activity. Taken together, these findings indicate that NEDD8-mediated neddylation plays a key role in RAB40-CUL5-mediated DNAJC13 degradation.</div></div>","PeriodicalId":34672,"journal":{"name":"BBA Advances","volume":"10 ","pages":"Article 100200"},"PeriodicalIF":4.5,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148738372","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Isonardosinone suppresses osteoclastogenesis through TRAF2/TRAF6-mediated disruption of calcium signaling and coordinate inhibition of MAPK/NF-κB pathways","authors":"Jielong Huang ,&nbsp;Yang LV ,&nbsp;Chujie Wang ,&nbsp;Jinfang Wang ,&nbsp;Wei Deng ,&nbsp;Jiyao Luan ,&nbsp;Zitong Tang","doi":"10.1016/j.bbadva.2025.100173","DOIUrl":"10.1016/j.bbadva.2025.100173","url":null,"abstract":"<div><h3>Background</h3><div>Osteoporosis (OP), a prevalent age-related skeletal disorder, is characterized by increased osteoclast (OC) activity, leading to bone loss, reduced bone mineral density, and elevated fracture risk. Through combined in vivo and in vitro experiments, this study aimed to elucidate the mechanisms by which isonardosinone (INS) inhibits OC differentiation, thereby providing new insights for potential therapeutic applications.</div></div><div><h3>Methods</h3><div>Initially, the optimal non-cytotoxic concentration of INS for bone marrow-derived macrophages (BMMs) was determined. The dose-dependent inhibitory effects of INS on OC differentiation were quantitatively assessed using tartrate-resistant acid phosphatase (TRAP) staining. Osteoclast-specific gene and protein expression profiles under INS treatment were analyzed via reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. Furthermore, fluorescence assays demonstrated that INS substantially inhibited the formation of F-actin rings and the nuclear translocation of phosphorylated p65 (p-p65), while also profoundly modulating the mitochondrial membrane potential in OC precursors and promoting the induction of apoptosis. Notably, these effects were accentuated with increasing concentrations of INS. To investigate the underlying mechanisms of INS-mediated suppression of mature OC formation, Western blot analysis was employed to evaluate the modulation of relevant signaling pathways. For translational validation, an ovariectomy (OVX)-induced osteoporosis murine model was established, with bone microarchitecture and molecular markers subsequently characterized using micro-computed tomography (Micro-CT) and immunohistochemistry.</div></div><div><h3>Results</h3><div>INS at concentrations ≤20 μM demonstrated no cytotoxic effects on BMMs. TRAP staining revealed dose-dependent inhibition of mature OC formation, with maximal suppression observed at the highest tested concentration. RT-PCR and Western blot analyses confirmed significant reductions in OC differentiation markers, including CTSK, MMP9, NFATc1, and c-Fos.Concurrently, INS upregulates antioxidant defense enzymes (Nrf2, HO-1, SOD-1, catalase), contributing to its anti-osteoclastogenic effects. Mechanistically, INS exerted bidirectional regulation of the TRAF2-RIPK1-Ca²⁺ signaling axis, concurrently modulating intracellular calcium flux and extracellular calcium-sensing pathways to disrupt OC maturation. Micro-CT quantification and immunohistochemical profiling demonstrated that INS administration significantly preserved trabecular bone mass and reduced osteoclast activity in OVX mice, thereby corroborating the in vitro findings.</div></div><div><h3>Conclusion</h3><div>INS aptly mitigates the process of osteoclastogenesis, leading to a notable enhancement in bone density, effectively exemplifying its pivotal role in the management of OP.</div></div>","PeriodicalId":34672,"journal":{"name":"BBA Advances","volume":"8 ","pages":"Article 100173"},"PeriodicalIF":3.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145519281","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}