Flocoumafen (FCF), a second-generation anticoagulant rodenticide (SGAR), has limited toxicological data regarding its effects on aquatic organisms. In this study, we exposed zebrafish embryos to FCF solutions at concentrations of 0.2, 0.4, and 0.8 mg/L until 120 h post-fertilization (hpf). The results revealed a decrease in survival rates. Notably, FCF exposure significantly reduced the frequency of spontaneous tail coils at 24 hpf, while shortened body length and induced spinal curvature at 120 hpf. Furthermore, zebrafish larvae exhibited craniofacial abnormalities and incomplete bone mineralization at 120 hpf following FCF exposure. In Tg(HuC:EGFP) transgenic strains, neuronal loss was observed. Additionally, FCF-exposed zebrafish larvae showed a marked reduction in locomotor ability, activity levels, and turning capacity. The qPCR and enzyme activity assays revealed significant changes in gene expression associated with the Notch signaling pathway, accompanied by increased levels of reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA). Astaxanthin (ASTA) partially alleviated the toxicities induced by FCF. These findings suggest that FCF may induce skeletal and neurological toxicities by affecting oxidative stress, disrupting the normal expression of skeletal and nervous system-related genes in the Notch signaling pathway, and ultimately leading to behavioral abnormalities. Our findings may provide new insights into a comprehensive evaluation of FCF toxicology in aquatic organisms, and may assist the government in formulating and implementing regulatory policies regarding the application of FCF.

Wnt/β-catenin signaling pathway is the most frequently altered cascade in colorectal cancer (CRC), and the nucleus accumulation of β-catenin is the hallmark of the activation of Wnt/β-catenin signaling. However, despite extensive research efforts, the molecular regulators governing β-catenin nuclear translocation remain poorly understood. The deubiqutinase enzyme USP22 was involved closely in the tumorigenesis of CRC with the underlying mechanism remaining unclear. In our study, we identified USP22 was upregulated in CRC and was closely associated with the activation of Wnt/β-catenin signaling. In the mechanism study we demonstrated for the first time that USP22 deubiquitinated β-catenin in a deubiquitinase activity-dependent manner to facilitate its nuclear translocation and enhance Wnt pathway activation, thereby promoting CRC cell proliferation. Furthermore, we revealed USP22 auto-deubiquitylated and stabilized itself. Additionally, we identified Berberine (BBR), a potent USP22 inhibitor, effectively suppressed Wnt pathway activation and CRC cell proliferation by targeting USP22. Notably, BBR reduced tumor stemness, exhibited substantial toxicity towards cancer stem cells and overcame the chemoresistance. Together, our finding elucidated USP22 promoted CRC progression via deubiquitinating β-catenin and promoting its subsequent nucleus translocation, and highlighted BBR was a potential therapeutic agent for CRC treatment as a novel USP22 inhibitor.

The transcriptional alterations underlying epithelial-to-mesenchymal transition (EMT) in chemoresistant ovarian cancer remain a matter of debate, with emerging evidence pointing to tumour cell plasticity and subclone reprogramming. In this study, we developed a cisplatin-tolerant ovarian cancer model by treating the cisplatin-sensitive OVCAR-3 cell line with a single dose of cisplatin, generating the OVCAR-3 CP variant. These cisplatin-tolerant cells exhibited distinct EMT-related changes at both transcriptomic and protein levels, potentially regulated by epigenetic mechanisms. EMT profiling revealed that OVCAR-3 CP cells did not display a pronounced mesenchymal phenotype but rather retained epithelial characteristics and showed elevated expression of ALDH3A1. In contrast, the parental chemosensitive OVCAR-3 cells expressed canonical mesenchymal markers (CDH2, VIM, ZEB1/2, SNAIL, SLUG) and lacked stemness marker expression. Upon xenografting, both OVCAR-3 and OVCAR-3 CP cells demonstrated phenotypic plasticity, with parental OVCAR-3 xenografts acquiring EMT-like features resembling to those observed in cisplatin-tolerant tumours. These findings suggest a decoupling of EMT from cisplatin-tolerance and instead underscore a stronger association between stemness traits and cisplatin tolerance. Our data further indicate that xenografting can induce significant cellular reprogramming. Comprehensive characterization of ovarian cancer cell-derived xenograft is therefore essential, as they represent a valuable translational platform for investigating therapy adapted ovarian cancer cells.

Chemotherapy resistance is the primary cause of clinical treatment failure and unfavorable prognosis among breast cancer patients. Consequently, the exploration of novel molecular targets for chemotherapy resistance is warranted. Here, we demonstrated that Zinc Finger Protein 184 (ZNF184) facilitates chemoresistance in breast cancer. Through integrated bioinformatics and experimental validation, we identified that ZNF184 was highly expressed in paclitaxel-resistant breast cancer cells. Knockdown of ZNF184 inhibited cell proliferation and re-sensitized resistant cells to paclitaxel in vitro and in patients-derived organoids (PDOs). Mechanistically, ZNF184 regulates the expression of stemness-related genes CD44, OCT4, Nanog, SOX2, and ALDH1A1, thereby promoting the proliferation of breast cancer cells and subsequent paclitaxel resistance. Pan-cancer analysis revealed the potential of ZNF184 as a prognostic and predictive biomarker for adverse clinical outcomes. Collectively, these findings reveal a previously unknown role of ZNF184 in breast cancer progression and paclitaxel resistance, providing new insights into ZNF184 as a potential therapeutic target for cancer patients.

Non-small cell lung cancer (NSCLC) is a metabolism associated disease which mainly depends on anaerobic glycolysis to produce the macromolecules needed for biosynthesis and rapid cell proliferation. Since the cancer cells depend on glycolysis for energy production, the development of drug targets that inhibit the glyco-metabolism will be a promising approach for the management of NSCLC. Plant derived phytocompounds have demonstrated anti-NSCLC activity by modulating the glycolytic pathway, thus curbing the energy requirement essential for the proliferation of cancer cells. In the current study, we explored the efficacy of farnesol in A549 lung adenocarcinoma cells using in vitro assays. Farnesol inhibited the viability of A549 cells to 50% at 21.5 µg/mL. Relative proteomic profiling via nano LC-MS/MS analysis identified 277 differentially expressed proteins in control and farnesol treated samples. Notably, PKM (FC = -3.911819), TKT (FC = -2.857373), ALDOA (FC = -4.8557) and LDH (FC = -2.624372) were downregulated exhibiting a strong interacting network in STRING analysis indicating suppression of anaerobic glycolysis. Furthermore, a decrease in the expression of GluIIβ, FBKP1A and apoptotic regulators such as LAP2 and ATP5F subunits suggest initiation of autophagy and apoptosis. AO/EtBr staining confirmed a late apoptotic shift while, DAPI staining revealed nuclear fragmentation at this concentration. Additionally, farnesol impaired mitochondrial ATP synthesis by reducing mitochondrial membrane potential (MMP) to 66% and elevated ROS levels to 54% creating a disturbance in mitochondrial stability. Overall, Farnesol significantly disrupts anaerobic glycolysis in A549 cells promoting cell death through mitochondrial dysfunction, oxidative stress, apoptosis and reducing cellular acidosis.

Oxaliplatin resistance significantly impairs therapeutic outcomes in colorectal cancer. However, reliable diagnostic markers for early detection of resistance remain limited. This study aimed to identify novel diagnostic signatures through integrative bioinformatics and machine learning approaches.

To investigate the effects of fungal volatile organic compounds (FVOCs) on the mycelial growth of Ganoderma lucidum, and to elucidate the molecular mechanisms underlying the growth-promoting effect of 3-octanone. G. lucidum was cultivated with 1-octen-3-ol, 3-octanol and 3-octanone for 7 days, after which colony diameter and mycelial dry weight were measured to assess their effects on mycelial growth. RNA-seq was used to investigate gene expression changes following 3-octanone exposure. While 1-octen-3-ol or 3-octanol inhibited mycelial growth in G. lucidum, 3-octanone promoted it. In total, 590 differentially expressed genes (DEGs), including 162 upregulated and 428 downregulated genes, were identified following 3-octanone exposure. Functional annotation revealed that among the DEGs, 23 genes were related to fungal cell wall biosynthesis and remodeling, whereas 21 genes were involved in plant-derived polysaccharide degradation. Furthermore, significant expression changes were observed in genes related to secondary metabolism. Our results indicate that G. lucidum can use 3-octanone as a signal to recognize other fungi, potentially facilitating the expansion of its own territory within wood in nature.

Neuroblastoma (NB) is the most common extracranial solid malignancy of children, and MYCN amplification defines a high-risk subtype with poor outcomes. Although widely used in preclinical drug discovery, NB cell lines are often selected based on availability rather than the molecular characteristics of patient-derived tumors, leading to a critical translational gap between experimental outcomes and clinical relevance. To address this, we developed a rank-based transcriptomic correlation framework to assess the concordance between patient-derived tumors (n = 642; combined from the SEQC/MAQC-III and TARGET cohorts) and publicly available NB cell lines (n = 39). This system-level analysis enabled the identification of cell model representatives (CMRs) that closely recapitulate the gene expression landscapes of clinical tumors. COG-N-557, SMS-KAN, and NB-SD emerged as the top CMRs for MYCN-amplified tumors, whereas COG-N-549, FELIX, and SK-N-SH were identified for MYCN-nonamplified tumors. Pathway enrichment analyses indicated that MYCN-amplified CMRs retain key transcriptional programs involved in neuronal development and tumor proliferation, supporting their biological relevance. Leveraging these models, we integrated pharmacogenomic connectivity mapping and drug-gene network analyses to uncover kinase inhibitors and epigenetic modulators as promising therapeutic candidates capable of targeting MYCN-driven transcriptional programs, despite MYCN being an undruggable oncogene. In conclusion, this study addresses a fundamental systems biology and translational research gap by establishing a data-driven framework for selecting NB cell lines that accurately reflect patient-derived tumor biology with direct implications for prioritizing therapeutically relevant drug candidates. Future studies should prioritize the top CMRs as in vitro models to enhance translational relevance and accelerate precision drug discovery in high-risk pediatric NB.

Acute pancreatitis (AP) remains a challenging clinical condition with limited therapeutic options and high mortality rates in severe cases. Traditional anti-inflammatory approaches have shown disappointing results in clinical trials, highlighting the urgent need for novel therapeutic strategies targeting the underlying pathophysiological mechanisms. The study by Jia et al presents compelling evidence for a previously unrecognized mechanism through which rutaecarpine, a bioactive alkaloid from traditional Chinese medicine, exerts protective effects against AP. This research demonstrates that rutaecarpine alleviates AP by targeting the epigenetic machinery, specifically through enhancer of EZH2-mediated suppression of FBXW11. The authors employed both in vitro cerulein-induced AR42J cell models and in vivo sodium taurocholate-induced rat models to establish the therapeutic efficacy of rutaecarpine and elucidate its molecular mechanisms. Their findings reveal that rutaecarpine upregulates EZH2 expression, leading to increased histone H3 methylation at the FBXW11 promoter region, thereby suppressing FBXW11 expression and consequently reducing inflammatory infiltration and oxidative stress. The significance of this work extends beyond demonstrating rutaecarpine's protective effects. It identifies FBXW11 as a novel therapeutic target in AP and provides the first evidence that traditional Chinese medicine compounds can modulate epigenetic reprogramming in pancreatic inflammation. Recent studies have confirmed FBXW11's role as an inflammatory biomarker in pancreatitis, supporting the clinical relevance of this pathway. The study's comprehensive approach, combining molecular docking, cellular thermal shift assays, and co-immunoprecipitation studies, strengthens the mechanistic insights. These findings open new avenues for AP treatment by targeting epigenetic regulators rather than relying solely on conventional anti-inflammatory strategies, potentially leading to more effective therapeutic interventions for this devastating condition.

Parkinson's disease (PD) is characterized by mitochondrial dysfunction and impaired protein homeostasis, with the mitochondrial unfolded protein response (mtUPR) emerging as a key regulatory pathway in mitigating mitochondrial stress. This study aimed to explore the impact of shRNAs targeting CHCHD2 or FBXO7 on the mitochondrial unfolded protein response (mtUPR) in a Parkinson's disease (PD) cell model, clarify the mitochondrial-nuclear signaling pathways involving CHCHD2 and FBXO7, elucidate the mechanisms underlying mitochondrial dysfunction induced by these genes, and identify new therapeutic targets for early stage PD. An in vitro PD model was established by treating SH-SY5Y cells with MPP+; mitochondrial morphology was evaluated using transmission electron microscopy, and qRT-PCR and Western blot were employed to determine the expression levels of mRNAs and proteins associated with mtUPR, autophagy, CHCHD2, and FBXO7 under oxidative stress. In the MPP+-induced PD cell model, we knocked down CHCHD2 and FBXO7 via shRNA and treated the cells with JNK and AKT agonists to observe their effects on mtUPR protein expression. The results showed that mtUPR was activated in MPP+-exposed SH-SY5Y cells, and the expression of CHCHD2 and FBXO7 genes was significantly upregulated after MPP+ intervention; knockdown of CHCHD2 via shRNA resulted in a marked decrease in the expression of mtUPR-related proteins such as HSPA9, HSPD1, YME1L1, and CLPP, while shRNA targeting FBXO7 exerted only a minimal effect on these mtUPR proteins. Furthermore, the administration of JNK or AKT agonists significantly enhanced the expression of MPP+-induced mtUPR proteins, including HSPA9, HSPD1, YME1L1, and CLPP. Collectively, these findings indicate that CHCHD2, rather than FBXO7, plays an essential role in modulating the MPP+-induced mtUPR and suggest that CHCHD2 may regulate mitochondrial protein homeostasis by activating the mtUPR through the JNK/c-Jun and AKT/ERα pathways.

Polygonum cognatum (Madımak) is a plant traditionally consumed for medicinal purposes in Turkey. Unlike previous studies examining samples from different regions and seasons, this research presents the first comprehensive characterization of P. cognatum collected from the Central Black Sea Region (Tokat, 40°01'02″N, 36°28'15″E; 1210 m altitude) during the vegetative growth phase (June 2024), where geographical origin and collection time significantly influence secondary metabolite profiles. This study evaluates the phytochemical profile and multitarget biological activities of P. cognatum extracts obtained using solvents of different polarities (hexane, ethanol, and water). Advanced analytical techniques (liquid chromatography-tandem mass spectrometry, high-performance liquid chromatography-diode array detector, and gas chromatography-mass spectrometry) identified 28 phenolic compounds, with the ethanol extract showing the highest diversity (24 compounds) and total phenolic content (78.6 ± 2.3 mg GAE/g). Compounds identified for the first time in P. cognatum include isoquercetin-3-O-rhamnoside, apigenin-7-O-glucoside, and luteolin-4'-O-glucoside. The ethanol extract demonstrated superior multitarget bioactivity: potent antioxidant activity ( 2,2-diphenyl-1-picrylhydrazyl (DPPH) IC50: 76.4 ± 2.1 μg/mL), moderate but selective anti-inflammatory effects (COX-2 IC50: 145.3 ± 5.2 μg/mL; selectivity index: 2.06, indicating preferential COX-2 inhibition over COX-1) and significant antidiabetic potential (α-amylase IC50: 89.3 ± 3.1 μg/mL; α-glucosidase IC50: 76.8 ± 2.9 μg/mL), and antimicrobial activity (MIC: 62.5 μg/mL against S. aureus). Notably, this study demonstrates for the first time the histone deacetylase (HDAC) inhibitory activity of P. cognatum (IC50: 92.4 ± 3.8 μg/mL), revealing novel epigenetic modulation properties. Molecular docking studies showed strong correlations between binding affinities and experimental IC50 values (r = -0.87 to -0.91; p < 0.01). Cytotoxicity evaluation showed favorable safety profiles (CC50 > 500 μg/mL). Docking, IC50, and compositional data consistently indicate that quercetin, rutin, chlorogenic acid, and kaempferol are key contributors to the observed antioxidant, antidiabetic, anti-inflammatory, and HDAC inhibitory effects. These findings establish P. cognatum as a promising multitarget therapeutic agent with novel epigenetic regulatory mechanisms, supporting its potential development for inflammatory, metabolic, and epigenetic-related disorders.

Epigenetic memory enables the propagation of gene expression patterns following transient stimuli. Although three-dimensional chromatin organization is emerging as a key regulator of genome function, it is unknown whether it contributes to cellular memory. Here we establish that acute perturbation of the epigenome can induce cellular memory of gene expression in mouse embryonic stem cells. We uncover how a pulse of histone deacetylase inhibition translates to changes in transcription, histone modifications and genome folding. While most epigenomic and transcriptional changes are initially reversed once the perturbation is removed, some loci remain transcriptionally deregulated and genome architecture partially maintains its perturbed conformation. Consequently, a second pulse of transient hyperacetylation induces stronger memory of transcriptional deregulation. Using ultradeep Micro-C, we associate memory of gene expression with repressive Polycomb-mediated chromatin topology. These results demonstrate how cells can record transient stresses in their genome architecture, thereby enabling an enhanced response to subsequent perturbations.

This study investigates the role of arachidonate 5-lipoxygenase (ALOX5) in upregulating programmed death-ligand 1 (PD-L1) expression via its metabolite 5-hydroxyeicosatetraenoic acid (5-HETE) in ovarian cancer cells, and explores the effects on lipid metabolism and ferroptosis. Additionally, the study examines the role of Tectorigenin (TEC) in this process. Using network pharmacology, bioinformatics analysis, molecular docking, and drug affinity reaction target stability (DARTS) experiments, we identified ALOX5 as a potential therapeutic target for TEC in ovarian cancer treatment, possibly through lipid metabolism and ferroptosis pathways. In vitro experiments showed that TEC inhibits proliferation and invasion and promotes apoptosis in ovarian cancer cells without significant cytotoxicity in normal ovarian epithelial cells. TEC also inhibits lipid metabolism and promotes ferroptosis, reducing ovarian cancer cell viability. Inhibition of ALOX5 similarly suppresses lipid metabolism and enhances ferroptosis, effects that can be reversed by exogenous 5-HETE. In vivo studies using a nude mouse model demonstrated that TEC inhibits tumor growth and downregulates ALOX5, 5-HETE, and PD-L1 expression in tumor tissues. The findings suggest that the ALOX5/5-HETE signaling pathway is crucial for regulating lipid metabolism and ferroptosis in ovarian cancer, and TEC may exert its anti-tumor effects, at least in part, by modulating this pathway (The graphical abstract was shown in Fig. 1).

Cancer signaling encompasses a wide array of entangled molecular cascades that promote oncogenic progression and counteract the effect of tumor suppressors. Transforming growth factor β (TGFβ) induces complex and stage-dependent effects throughout tumor progression. During pre-malignant hyperplastic growth, TGFβ restricts cell proliferation and inflammation, while on the other hand, TGFβ promotes migration and distal metastasis of cancer cells. To dissect the temporal chromatin-based transcriptional response to TGFβ, we employed 3D culture models of isogenic human breast epithelial cells, exemplified by non-oncogenic MCF-10A (MI) and their HRAS-transformed counterpart (MII). Genome-wide chromatin accessibility profiling revealed an extensive chromatin opening induced by TGFβ at transcription start sites and enhancer elements in both models, with a marked enrichment of SOX4 binding motifs in oncogenic cells. Transcriptomic analyses unexpectedly revealed the upregulation of DNA replication and DNA damage response pathways, following TGFβ stimulation of oncogenic MII 3D cultures. Canonical TGFβ-driven programs, including epithelial-mesenchymal transition and metabolic reprogramming, were activated in both models. Notably, single-cell RNA-seq of primary breast tumors confirmed co-expression of SOX4 and cell cycle regulators. Mechanistically, we show that TGFβ induces the interaction between the MH2 domain of SMAD3 and the intrinsically disordered regions of SOX4, co-activating downstream gene targets. Validating the genome-wide analyses, we found that resistance of breast cancer cells to the CDK4/6 inhibitor palbociclib conferred by TGFβ stimulation was functionally dependent on SOX4. Collectively, our findings reveal an apparent oncogenic function of TGFβ in promoting cell cycle progression and drug resistance through SOX4, highlighting the pro-tumorigenic role of TGFβ signaling in breast cancer progression.

Chemotherapy-induced ncRNA-mediated plasticity is an emerging concept in cancer research. To that end, we observed a cisplatin-responsive regulatory program centered on piRNA activation. OSCC cells exposed to cisplatin markedly promoted the piRNA expression, with piR-hsa-30937 showing the most prominent upregulation. Mechanistically, cisplatin disrupts the OCT1-DNMT1 repressive complex that mediates DNA methylation of transcription factor binding sites of piR-hsa-30937, to derepress its expression. Functionally, piR-hsa-30937 targets GAB2 and sensitizes OSCC cells to cisplatin by suppressing proliferation, enhancing apoptosis, and γ-H2AX accumulation. Furthermore, GAB2 overexpression reversed these effects and desensitized OSCC cells to cisplatin by activating NF-κB-mediated JNK suppression. Overall, cisplatin actively remodels the OCT1-DNMT1-piR-hsa-30937 axis regulating piRNA expression, which in turn potentiates cisplatin cytotoxicity by attenuating GAB2-mediated survival signaling in OSCC.

Small cell lung cancer (SCLC) is an aggressive malignancy characterized by limited therapeutic options. In this study, we identified GL-V9 as a potent anti-SCLC agent that induces apoptosis through oxidative stress. GL-V9 significantly reduced SCLC cell viability in a dose-dependent manner and triggered apoptosis both in vitro and in xenograft models. Mechanistically, GL-V9 increased reactive oxygen species (ROS) levels and lipid peroxidation while impairing mitochondrial function, suggesting that its cytotoxic effects are mediated by oxidative stress. Drug-target interaction analyses revealed that GL-V9 directly binds to STEAP3, a key regulator of iron metabolism, and promotes its degradation via the ubiquitin-proteasome pathway. The loss of STEAP3 disrupted iron homeostasis and exacerbated oxidative stress. In contrast, STEAP3 overexpression attenuated ROS accumulation, mitochondrial damage, and apoptosis both in vitro and in vivo. Further investigation demonstrated that STEAP3 degradation decreased the stability of CISD2, a [2Fe-2S] cluster-containing mitochondrial protein essential for redox balance. GL-V9 downregulated CISD2 in a STEAP3-dependent manner, and restoring CISD2 expression significantly rescued cells from GL-V9-induced oxidative stress and apoptosis. Clinically, both STEAP3 and CISD2 are upregulated in SCLC tumors, and their elevated expression correlates with poor patient survival. Co-expression analysis associated these proteins with pathways involved in oxidative stress and mitochondrial dysfunction. Overall, these findings suggest that GL-V9 induces apoptosis in SCLC by targeting STEAP3 for proteasomal degradation, thereby disrupting the STEAP3-CISD2 axis and promoting oxidative stress-driven cell death. This study identifies a previously unrecognized redox regulatory pathway in SCLC and proposes a potential therapeutic strategy centered on selective induction of oxidative stress.

The aberrant modification level of Histone H3 lysine 9 trimethylation (H3K9me3) is a major barrier for somatic cell nuclear transfer (SCNT) embryo development. The removal of H3K9me3 from the embryonic genome via microinjection of KDM4 mRNA can improve the developmental efficiency. However, this strategy is limiting because of its invasive and time-consuming. In the present study, through comparative transcriptomic analysis with in vivo embryos, we found that porcine SCNT embryos exhibit insufficient zygotic genome activation (ZGA) transcription, along with aberrantly elevated expression of the SUV39H2 and SUV39H1. Then we investigated an alternative approach by inhibiting SUV39H2 and SUV39H1 via their specific inhibitor OTS186935 (targeting SUV39H2) and F5446 (targeting SUV39H1). Individual treatment with either inhibitor significantly reduced H3K9me3 levels and improved blastocyst formation rates. Notably, combined treatment with OTS186935 and F5446 synergistically further enhanced developmental outcomes. This combination treatment potently decreased H3K9me3 levels, rescued transcriptional activity during ZGA, and improved gene expression profiles, making them more closely resemble those of in vivo embryos. Consequently, compared to the control group, the combined treatment significantly increased the blastocyst rate (Treatment: 25.49 ± 4.47% vs. Control: 15.15 ± 2.69%, P < 0.001), blastocyst diameter (Treatment: 116.9 ± 4.37 μm vs. Control: 75.4 ± 2.87 μm, P < 0.001), and total cell number (Treatment: 33.00 ± 3.74 vs. Control: 28.75 ± 6.55, P < 0.05). Our findings demonstrate that dual inhibition of SUV39H1 and SUV39H2 effectively corrects aberrant H3K9me3 modification, ameliorates ZGA defects, and provides a simplified and efficient strategy to enhance the developmental competence of porcine SCNT embryos.

Diabetic kidney disease (DKD) is a leading cause of end-stage renal disease, driven by hyperglycemia-induced mitochondrial apoptosis in renal tubular epithelial cells. Death-associated protein kinase 1 (DAPK1) is a key mediator of cell death, but its regulation in DKD remains unclear. Here, we investigated the mechanisms underlying DAPK1 upregulation and its role in mitochondrial apoptosis under high glucose (HG) conditions in HK-2 cells and db/db mouse models. In db/db mice, renal DAPK1 protein levels were elevated, while KLHL20 levels were reduced, correlating with glomerular and tubular injury. In HK-2 cells, HG (33 mM, 48 h) significantly increased DAPK1 mRNA and protein levels while prolonging its half-life. Mechanistically, HG transcriptionally suppressed KLHL20, an E3 ubiquitin ligase adaptor that targets DAPK1 for proteasomal degradation. Co-immunoprecipitation confirmed KLHL20-DAPK1 interaction and showed reduced DAPK1 ubiquitination under HG. Overexpression of KLHL20 restored DAPK1 ubiquitination and reduced its protein levels without affecting mRNA, confirming post-translational regulation. Functionally, DAPK1 knockdown attenuated HG-induced mitochondrial apoptosis. KLHL20 overexpression similarly protected against HG-induced apoptosis, but this effect was abrogated by DAPK1 co-overexpression, establishing DAPK1 as a critical downstream effector. These findings reveal a novel KLHL20-DAPK1 axis where HG stabilizes DAPK1 by downregulating KLHL20, promoting mitochondrial apoptosis in renal tubular cells. Targeting this pathway may offer therapeutic strategies for DKD.

Senescence marker protein-30 (SMP-30; regucalcin) plays a crucial role in intracellular calcium homeostasis. This study investigated hepatic SMP-30 expression during non-alcoholic fatty liver disease (NAFLD) progression and evaluated the therapeutic potential of laminarin (LAM), a brown algae-derived polysaccharide, using high-fat diet (HFD)-fed mice and palmitic acid (PA)-treated Huh7 cells. Mice fed an HFD for 20 weeks developed NAFLD, characterized by elevated ALT/AST levels, hepatic steatosis, and significantly reduced SMP-30 expression. However, LAM treatment administered via drinking water (1%) or intraperitoneal injection (50 mg/kg) significantly attenuated lipid accumulation and restored hepatic SMP-30 expression. LAM reversed PA-induced lipid accumulation and SMP-30 downregulation in Huh7 cells. Mechanistically, LAM modulated the expression of SMP-30 and antioxidant proteins associated with activation of AKT/GSK3β/NRF2 signaling pathway, thereby mitigating the adverse effects of PA-induced toxicity. In conclusion, hepatic SMP-30 expression decreases during NAFLD progression, and LAM treatment restores these levels while alleviating lipid accumulation. These findings suggest that LAM may represent a promising therapeutic agent for NAFLD by improving lipid metabolism and reducing oxidative stress through the regulation of SMP-30.

Exogenous Glutathione S-transferase Mu 2 (GSTM2) supplementation has emerged as a promising strategy to counteract aging. However, approaches to enhance endogenous GSTM2 expression remain underexplored. Here, we identify HCY-NBD, an SO2-targeting small molecular that binds GSTM2 and stabilizes GSTM2 protein levels under high-glucose (HG)-induced vascular endothelial senescence. Mechanistically, HCY-NBD promotes sulfenylation at Cys174 of GSTM2 and inhibits its K48-linked ubiquitination at this residue, thereby stabilizing GSTM2 protein. In cellular studies, we observe that HCY-NBD upregulates GSTM2 and inhibits HG-induced senescence and calcification in vascular endothelial cells. Consistent with this, in vivo administration of HCY-NBD in db/db mice increases GSTM2 levels and mitigates senescence and calcification in the thoracic aorta. Collectively, our findings demonstrate that HCY-NBD inhibits HG-induced vascular senescence and calcification by stabilizing GSTM2 protein levels via enhancing Cys174 sulfenylation and suppression of site-specific ubiquitination-mediated degradation. Here, we first develop a new strategy to enhance endogenous GSTM2 and provide a novel therapeutic strategy for the prevention and treatment of vascular aging.