Acta Biochimica et Biophysica Sinica

Subarachnoid hemorrhage (SAH) resulted from intracranial aneurysm (IA) rupture is an especially severe form of stroke. Endothelial dysfunction represents the initiating event of IA pathogenesis. Understanding the role of endothelial cells (ECs) underlying formation of IAs is helpful to seek for pharmaceutical treatment strategy. Based on single-cell RNA sequencing, proteomics, and metabolic analysis, we discovered a change in cell population in IA samples, majorly in ECs and macrophages (MPs). Abnormal ECs exhibit senescence and death in IA samples, which is absent in the control arterial samples. Cross-analysis of multi-omics revealed that CALM1, a calcium detector involved in mechanotransduction, is downregulated in the abnormal ECs. CALM1 knockdown leads to senescence and inhibits the proliferation and maturation of ECs under turbulent flow. Through high-throughput virtual screening, this work identified compound ZC04329651 as a potent CALM1 activator in maintaining the stability of endothelial cell junctions and attenuating cellular senescence. Thus, our findings showed compound ZC04329651 up-regulate the expression of CALM1 to restore ECs, which maybe a promising pharmacological treatment strategy for IAs.

Highly reactive aldehydes are generated during metabolic processes in the body, and their detoxification is essential for maintaining cellular homeostasis. Hexadecenal, a long-chain fatty aldehyde, is formed during the sphingolipid degradation pathway from the lipid mediator sphingosine-1-phosphate (S1P). However, the cytotoxicity resulting from dysregulation of hexadecenal metabolism is still unclear. To elucidate the effects of impaired hexadecenal metabolism, we analyzed the function of ALDH3A2, an aldehyde dehydrogenase in humans. Our results revealed that ALDH3A2 enzymatic activity is crucial for the suppression of DNA damage, particularly interstrand DNA crosslinks, upon S1P exposure. Furthermore, we demonstrated that hexadecenal accumulation promotes cell death accompanied by the activation of cellular stress responses and morphological abnormalities in the endoplasmic reticulum. These findings suggest that ALDH3A2 functions as a metabolic safeguard to suppress DNA damage and cell death in response to the enhanced metabolic flux of hexadecenal.

Acute lung injury (ALI) is a severe inflammatory syndrome frequently complicated by brain injury, which worsens outcomes. Here, we explored whether regulation of the lung-brain axis represents a therapeutic strategy for the management of ALI. We show that intranasal administration of Fritillaria taipaiensis-derived exosome-like nanovesicles (Ft-ELNs) ameliorates LPS-induced ALI and ALI-associated hypothalamic inflammation in mice. Ft-ELNs preferentially accumulate in the lungs, inhibiting TRPV1 in both the lungs and the hypothalamus to establish the lung-brain axis. In parallel, Ft-ELNs primarily regulate systemic humoral immunity by inhibiting the VEGFR1/PI3K/AKT/NF?B signaling pathway. In vitro experiments further confirmed that Ft-ELNs are efficiently internalized by alveolar epithelial MLE-12 cells, exerting protective effects by suppressing VEGFR1 expression. Mechanistically, we identified Ft-miR1 of Ft-ELNs as the key functional effector that directly targets the Flt1 3'UTR to inhibit VEGFR1 expression. These data suggest that Ft-ELNs attenuate lung-brain axis inflammation by targeting VEGFR1 and may hold potential as novel therapeutic agents for the treatment of severe ALI.

Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1), a key regulator of mitophagy, has been linked to the pathogenesis of Parkinson's disease (PD). PINK1 recruits Parkin, an E3 ubiquitin ligase, triggering mitophagy in response to mitochondrial damage. During mitophagy, the quantity, stability, and activity of PINK1 must be strictly regulated; however, the mechanisms governing these parameters under cellular stress are still unclear. Herein, we determined the structural basis for PINK1 maturation mediated by heat shock protein 90/cell division cycle 37/FK506-binding protein 51 (HSP90/CDC37/FKBP51) chaperone complex. We identified PINK1-associated proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and determined the structures of the complexes using Cryo-Electron Microscopy (Cryo-EM). Results showed that FKBP51 potentially interacts with a conserved leucine-proline-phenylalanine (LPF) motif on the activation loop of PINK1 and negatively regulates PINK1 functions in mitophagy. A PINK1 mutation located at the FKBP51 recognition site is linked to mitophagy deficiency, which can be partially rescued by specific inhibition of FKBP51. These findings reveal a general mechanism for PINK1 recognition by the HSP90/CDC37/FKBP51 chaperone complex and suggest a potential approach for upregulating PINK1 activity, which is impaired in PD.

Japanese Encephalitis Virus (JEV) belongs to the Flavivirus family, and the RNA dependent RNA polymerase (RdRp) domain located at the C terminus of non structural protein 5 (NS5) regulates de novo viral genome synthesis. The conserved priming loop inserted in the thumb domain of RDRP initiates de novo genome synthesis. Interestingly, the reported replication process initiates with an interaction between NS5 (methyltransferase/RDRP) and NS3 (protease/helicase), and inhibiting this interaction directly correlates with the inhibition of viral replication. The function of the priming loop in the context of the NS5 NS3 interaction is not yet known. In this study, we studied the priming loop function in the NS5 NS3 interaction and viral replication. Using a structure based drug design approach, we screened the Maybridge compound library against the RdRp priming loop and identified 15 candidate compounds based on binding energy. Among them, DSHS00151 showed significant dose dependent inhibition of the NS5 NS3 interaction with an IC50 of 3.5 micromolar in the mammalian two hybrid assay, inhibited viral infectivity with an IC50 of 2.54 micromolar, and reduced viral RNA load with an IC50 of 2.9 micromolar, compared to CD10712. Molecular Dynamics (MD) simulations revealed that DSHS00151 exhibits stronger and more stable binding with the priming loop residues (790 to 812) compared to CD10712. The molecular mechanism suggested by the docking of the NS5 and NS3 proteins showed that the priming loop tends to rotate toward the NS3 protein to stabilize the complex, and compound binding restricts this loop rotation, thus compromising the stability of the NS5 and NS3 complex and affecting viral replication. Overall, our study validated the function of the NS5 priming loop as an allosteric site, and compounds binding to this allosteric site belong to the Non Nucleoside Reverse Transcriptase Inhibitors (NNRTI) class of inhibitors, which disrupt the NS5 and NS3 interaction and could be developed as novel anti JEV therapeutics.

Melittin exhibits antitumor activity in cervical cancer models, yet the long non-coding RNA (lncRNA) response and associated regulatory networks remain poorly understood. Here, strand-specific RNA-seq data from melittin-treated and untreated U14 murine cervical cancer cells were analyzed to characterize melittin-responsive lncRNAs and explore their potential functional associations. A total of 28,162 lncRNAs were identified, including 27,307 known and 855 novel transcripts. Differential expression analysis revealed 404 differentially expressed lncRNAs (DElncRNAs), comprising 191 upregulated and 213 downregulated lncRNAs, w most of which were predicted to localize to the cytoplasm or nucleus. Cis-target analysis identified 52 neighboring mRNAs as putative targets of 46 DElncRNAs. Functional enrichment highlighted mitochondrial electron transfer and redox-related processes, including the mitochondrial electron transfer flavoprotein complex, electron-transferring-flavoprotein dehydrogenase activity, ubiquinone binding, and quinone binding. In parallel, melittin induced mitochondrial membrane depolarization and increased intracellular reactive oxygen species accumulation in U14 cells. Co-expression analysis further identified 138 lncRNAs co-expressed with 161 mRNAs, which were enriched in chromatin remodeling, DNA replication, and DNA repair. EdU incorporation decreased with increasing melittin concentrations, indicating suppression of DNA synthesis and proliferative activity. RT-qPCR analysis confirmed the expression trends of selected DElncRNAs. Collectively, these findings demonstrate extensive remodeling of the lncRNA landscape in melittin-treated U14 cells and suggest that melittin-responsive lncRNA-mRNA networks are associated with mitochondrial redox disruption and impaired DNA synthesis. This study provides a transcriptomic framework for identifying candidate lncRNA-mRNA regulatory axes underlying the antitumor response to melittin.

The interest in compounds that protect against heat stress-induced damage has been heightened due to world global warming. We found the protective effects of a Japanese natural drug named BG, containing oriental bezoar and ginseng, against heat stress in Drosophila. BG suppressed the heat-induced shortened lifespan and reduced fertility in Drosophila. Interestingly, the protective effects of BG against heat stress were abolished in heat shock protein 70 (HSP70) mutant flies. To see the protective effects in humans, we applied BG on the cytotoxicity in heat-stressed human hepatic cell line, HepG2. BG suppressed heat stress-induced cytotoxicity at 43{degrees}C, and increased HSP70 and heat shock factor 1 (HSF1) mRNA expression in HepG2 cells. These findings indicate that BG protects against heat stress-induced damage via the HSF1/HSP70 pathway and has potential as a therapeutic agent for heat stress-induced disorders, including heatstroke even in human.

Alkaline ceramidase 2 (ACER2) catalyzes the hydrolysis of ceramides into sphingosine and free fatty acids, playing crucial roles in regulating various biological processes, including but not limited to cell proliferation, survival, and the DNA damage response. However, a pharmacological tool that regulates its catalytic and signaling functions remains to be developed. In this study, N-[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]dodecane-1-sulfonamide (KPB82) was identified as a potent and selective ACER2 inhibitor. In vitro enzymatic assays showed that KPB82 inhibited ACER2 with an IC50 of 190 nM, while displaying minimal activity against acid and neutral ceramidases (IC50> 100 {micro}M) and no significant inhibition of alkaline ceramidase 1 or alkaline ceramidase 3. Enzymatic kinetic studies revealed that KPB82 is a competitive inhibitor of ACER2. The three-dimensional structure of ACER2 was modeled in the AlphaFold3 server, revealing key Zn2+-coordinating histidine residues and a substrate-binding pocket. Molecular docking studies demonstrated that KPB82 binds ACER2 within this pocket, forming hydrogen bonds with the catalytically important amino residues, Asp93 and His215, and interacting with several hydrophobic residues. Lactate dehydrogenase release assays revealed that KPB82 significantly reduced cytotoxicity in HeLa cells overexpressing ACER2, as well as in HCT116 cells, RT4 cells, and human umbilical vein endothelial cells treated with the DNA-damaging agent doxorubicin. Lipidomic analysis showed that KPB82 inhibited sphingosine production in response to ACER2 overexpression or doxorubicin treatment. Collectively, these findings demonstrate that KPB82 is a potent and selective ACER2 inhibitor capable of suppressing ACER2-dependent biological processes and highlight its potential as a tool for studying ceramide metabolism and ACER2-mediated signaling pathways.

Hepatocellular carcinoma (HCC) is particularly aggressive and difficult to treat. Due to the lack of early clinical diagnosis and the unsatisfactory clinical treatment effect, it is particularly important to identify novel markers that can predict tumor behavior in HCC. biogenesis of ribosomes BRX1 (BRIX1) is abundant in various tissues of the human body. However, the regulatory mechanisms and its role in various tissues are not fully understood. Here, we analyzed the expression pattern of BRIX1 in HCC from public gene expression databases and tissue samples from clinical HCC. We confirmed that BRIX1 was upregulated in both HCC cell lines and HCC paraffin section samples. BRIX1 depletion significantly dicreased the capacity of cells to grow and migrate in vitro, and knockdown BRIX1 suppressed tumor growth in xenograft tumor model. Mechanistically, BRIX1 depletion suppressed the MAPK/ERK pathway, as reflected by reduced phosphorylated ERK (p-ERK) levels. In summary, we provide a rational clue for the further investigation of BRIX1 as an invaluable biological marker for diagnosing and predicting prognosis of patients with HCC.

Therapy of ischemic stroke is currently limited to pharmacological and/or mechanical recanalization. There are no neuroprotective therapies approved for use during the rehabilitative phase of ischemic stroke, which is characterized by neurodegenerative changes. Thus, the search for neuroprotective compounds capable of preventing neuronal death caused by pathogenetic cascades triggered during hypoxia is an urgent task. In this study, we demonstrate increased culture viability following pre- and post-incubation with salicyl-carnosine (SC) in a model of oxygen glucose deprivation on a primary culture of rat cortical neurons. Its neuroprotective properties were greater than that of acetylsalicylic acid and carnosine, and it was effective in lower concentrations. In addition, SC protected the culture from NMDA-induced excitotoxicity. We also showed the passage of SC into neurons, and the presence of its direct antioxidant activity in a model of paraquat-induced oxidative stress. The neuroprotective effects of SC are associated with a decrease in the level of pro-apoptotic protein Bak and a decrease in the activation of kinase p38, as well as an increase in the activation of kinase ERK1/2. The acquired data suggests that SC is a promising neuroprotective compound, and warrants further investigation in vivo.

Neurotrophins regulate neuronal survival, differentiation, and synaptic plasticity through activation of tropomyosin receptor kinase (Trk) receptors, and their dysregulation is strongly implicated in neurodegenerative disorders, including Alzheimer's disease. However, the therapeutic application of recombinant neurotrophins remains limited by poor bioavailability, rapid degradation, and inadequate blood brain barrier permeability. Here, we report the discovery of Bavachin as a dual Trk-A/Trk-B neurotrophin mimetic via an integrated computational screening and experimental validation approach. Structure-based virtual screening of a bioactive natural product library derived from Traditional Chinese Medicine, combined with a novel consensus-ranking strategy, enabled the identification of flavonoid scaffolds targeting neurotrophin-binding sites of Trk receptors. Among these, Bavachin demonstrated robust, dose-dependent activation of both Trk-A and Trk-B receptors, but not Trk-C, indicating receptor selectivity. Induced-fit docking and molecular dynamics simulations revealed stable binding of Bavachin within conserved and receptor-specific ligand-binding pockets, consistent with its dual agonist activity. Collectively, this study identifies Bavachin as a putative small-molecule neurotrophin mimetic and establishes an integrated computational and experimental framework for the discovery of dual Trk-A/Trk-B agonists for neurodegenerative disease therapeutics.

Mitochondrial dysfunction and oxidative stress represent two interconnected, primary causes for Diabetic Neuropathy (DN); however, the majority of currently available anti-diabetic therapies have focused on glucose control as opposed to neurodegenerative downstream effects. Corilagin, is an ellagitannin having high anti-oxidant properties; however, it has not been evaluated against hyperglycemia induced neuronal injury. The present study demonstrates the ability of Corilagin to protect against mitochondrial dysfunction via models of diabetic nephropathy and cerebral ischemia. High glucose (50 mM, 24 hr) was utilized to induce diabetes like conditions in the SH-SY5Y human neuroblastoma Cell Line. High glucose induced significant decreases in cell viability, increases in intracellular and mitochondrial reactive oxygen species, depletion of reduced glutathione reserves, induces apoptosis, and causes mitochondrial depolarization and fragmentation. Corilagin pre-treatment attenuated each of these high-glucose induced effects by protecting against mitochondrial membrane potential loss and maintaining mitochondrial network morphology while reducing apoptotic cell fraction relative to glucose alone. Additionally, these protective effects were accompanied by restoration of AMPK phosphorylation and up-regulation of SIRT1, PGC1 and TFAM, components that are part of the principal signaling pathway that regulates mitochondrial biogenesis; therefore, therefore, this pathway may contribute mechanistically to the cyto-protective effect of Corilagin. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/740444v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1be4a92org.highwire.dtl.DTLVardef@11d6e9org.highwire.dtl.DTLVardef@1346757org.highwire.dtl.DTLVardef@16c9f1e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Proposed mechanism underlying the neuroprotective effects of Corilagin against high glucose-induced mitochondrial dysfunction.High glucose suppresses AMPK phosphorylation, leading to downregulation of the SIRT1-PGC-1-TFAM signaling axis, increased intracellular and mitochondrial reactive oxygen species (ROS), glutathione depletion, apoptosis, mitochondrial depolarization, and mitochondrial fragmentation. Corilagin pretreatment restores AMPK activation and the downstream SIRT1-PGC-1-TFAM pathway, thereby reducing oxidative stress, preserving intracellular glutathione, preventing apoptosis, maintaining mitochondrial membrane potential, and protecting mitochondrial network integrity. C_FIG

Mutations in p53 and its 12 isoforms can alter its functions. As N-terminally truncated isoforms of p53 (delta40p53, delta133p53, and delta160p53) participate in tetramer formation, they are important regulators of cancer fate. Although delta40p53- and delta133p53-mediated regulation of cancer is well reported, the mechanism underlying delta160p53 production and its functional role remains unclear. We investigated the internal ribosomal entry site (IRES)-mediated translation of {Delta}160p53 and its role in cancer regulation. As differential synthesis of delta160p53 was observed under different stress conditions, IRES-mediated translation of this isoform was demonstrated using bicistronic luciferase constructs. No cryptic promoters or splicing sites were detected in the IRES sequence. Cell death and late apoptosis were significantly decreased, while proliferation, the number of cells in the S phase, and drug resistance were induced by delta160p53. Furthermore, delta160p53 did not induce p53-responsive promoters. RNA sequencing analysis of delta160p53 overexpression showed similar results, along with the inhibition of other tumor suppressor genes. Overall, our results provide insights into IRES-mediated translation of delta160p53, which can be considered a novel target for cancer treatment.

Triple-negative breast cancer (TNBC) is a highly aggressive malignancy and options for targeted therapy are limited. Although PARP inhibitors (PARPi) like olaparib (Ola) leverage synthetic lethality, their clinical utility is severely impeded by drug resistance and low frequency of BRCA mutations. In this study, 1,2,3,4,6-penta-O-galloyl-{beta}-D-glucose (PGG), a natural polyphenolic compound, can induce a state of homologous recombination deficiency (HRD) in TNBC cells by inhibiting the PALB2-BRCA2 interaction. PGG potentiated the inhibitory effects of Ola on the in vitro proliferation, clonogenic potential, migration, and invasion of TNBC cells. Mechanistically, the dual therapy triggered severe oxidative stress and mitochondrial collapse, which accelerated the classical apoptosis cascade and promoted the emission of immunogenic cell death (ICD)-associated DAMPs. The accumulated cytosolic double-stranded DNA activated the innate immune cGAS-STING pathway, thereby promoting robust intra-tumoral infiltration of CD8+ T cells. Furthermore, since cGAS-STING hyperactivation drives compensatory PD-L1 upregulation on TNBC cells, incorporating anti-PD-L1 antibody into the combination regimen further reversed the immunosuppressive barrier, achieving near-complete tumor eradication and maximum infiltration of cytotoxic T cells in vivo. Bioinformatics analyses further suggested a potential network of PGG targets associated with extracellular matrix organization, offering a bioinformatics-derived hypothesis for its theoretical involvement in modulating stromal tension and vascular homeostasis. Collectively, this study establishes PGG as a multi-functional therapeutic agent integrating intracellular synthetic lethality and innate immune sensing, while providing a preliminary bioinformatic rationale for prospective microenvironmental investigation. This study also shows PGG as a multi-functional therapeutic agent that integrates intracellular synthetic lethality, innate immune sensing, and microenvironmental remodeling to convert immunologically "cold" TNBC into "hot" tumors.

Exploring the ability of metabolic enzymes to interact with diverse small molecules can provide insights into their ligand recognition and activity regulation. Glucose-6-phosphate isomerase (GPI) is an enzyme that catalyzes the second step of glycolysis, a critical node that links glucose metabolism to other cytosolic pathways, such as pentose phosphate and hexosamine biosynthetic pathways. Despite its importance in cellular metabolism, little is known about the broad ligandability of GPI. Here, we performed a structure-based virtual screening using molecular docking to identify new modulators predicted to engage with the human GPI active site. Enzyme kinetic and binding studies confirmed eleven compounds as active-site binders of GPI, exhibiting inhibitory activities and binding affinities in upper micromolar to sub-millimolar range. The binding of several ligands to GPI active site was further supported by X-ray crystallographic data. In addition, in vitro assays were performed to evaluate the modulatory effects of the GPI hits on cellular glycolysis and viability. The identified inhibitors included the mono-phosphorylated metabolite of ribavirin, which inhibited glycolytic flux and cell viability; two compounds with phosphate bioisosteres; and endogenous phosphate-containing metabolites from the pentose phosphate, hexosamine, nucleotide, and mevalonate pathways. Collectively, these findings expanded the repertoire of human GPI ligands and revealed previously unrecognized GPI-metabolite interactions that may contribute to modulation of glycolytic flux.

Melittin, a highly active natural antimicrobial peptide derived from honeybee venom, holds immense pharmacological potential against solid tumors. However, its specific anti-tumor efficacy and transcriptomic dynamics in cervical carcinoma remain to be systematically characterized. This study evaluated the anti-cancer properties of melittin on murine U14 cervical cancer cells following by transcriptomic investigation of the underlying mechanism. Phenotypic evaluations revealed that melittin potently inhibited U14 cell viability, while wound healing assays demonstrated a profound, dose-dependent suppression of cellular migration, culminating in near-complete migratory arrest at high concentrations. Furthermore, flow cytometry quantified a dramatic, dose-dependent surge predominantly in late apoptotic cell populations. These apoptotic events were structurally corroborated by scanning electron microscopy (SEM), which revealed severe plasma membrane perforation and morphological exhaustion. Enrichment analyses indicated that the physical membrane disruption inflicted by melittin translated into a severe metabolic crisis, marked by a global suppression of ribosomal biogenesis and mitochondrial oxidative phosphorylation. Furthermore, melittin profoundly repressed the Tie2-mediated angiogenic pathway (Etv2 down-regulation) while triggering the lethal hyperactivation of the AP-1 transcriptional stress complex (Jun, Fos, Fosb). Collectively, these findings elucidate the pharmacological networks underlying melittins cytotoxicity, providing solid molecular evidence for its development as a natural therapeutic agent against cervical cancer.

Minor introns are rare but evolutionarily conserved introns in the human genome. Defects in minor spliceosome components have been reported to cause several human diseases. Mutations in ZRSR2 are frequently observed in myelodysplastic syndrome (MDS) patients. However, the precise molecular mechanism by which these mutations cause a loss of ZRSR2 function in minor intron splicing remain elusive. In this study, we characterized the impact of MDS-causing missense mutations in ZRSR2. Most of the mutant ZRSR2 proteins we tested showed different subnuclear localization pattern from that of wild-type ZRSR2 protein. Co-immunoprecipitation (co-IP) analysis revealed that UHM missense mutations disrupted the interaction between ZRSR2 and SF3B complex. We also demonstrated that ZRSR2 preferentially immunoprecipitated minor spliceosome components over major spliceosome components and it directly interacts with SF3B1. Furthermore, co-IP analysis utilizing a variety of ZRSR2 mutants revealed that mutations in the UHM and Zn2 domains significantly attenuated the interaction with SF3B complex. Finally, UHM and Zn2 mutations markedly disrupted the supporting activity of ZRSR2 for minor intron splicing. Our results indicate that disruption of the physical interaction with SF3B complex is one of the hallmarks of MDS-causing mutations of ZRSR2, which results in aberrant splicing of minor introns.

This study aims to investigate the cardioprotective effects of 4-Hydroxybenzaldehyde (4-HBA) against isoproterenol (ISO)-induced cardiac fibrosis and to elucidate the underlying mechanisms. In vivo, cardiac fibrosis was induced in C57BL/6 mice by subcutaneous injection of ISO, and the mice were treated with 4-HBA or a TGF-{beta} inhibitor. Assessments using echocardiography, histopathology, and Western blotting demonstrated that 4-HBA significantly alleviated ISO-induced cardiac dysfunction, reduced collagen deposition, and attenuated apoptosis in mice. Mechanistically, 4-HBA inhibited TGF-{beta} expression and Smad2/3 phosphorylation. In vitro, ISO was applied to cardiomyocytes (HL-1) and cardiac fibroblasts (MCFs), with or without 4-HBA or TGF-{beta} inhibitor intervention. The results showed that 4-HBA suppressed HL-1 apoptosis and fibroblast proliferation, and significantly reduced the expression of extracellular matrix genes, TGF-{beta} levels, and Smad2/3 phosphorylation in MCFs. These findings indicate that 4-HBA reduces myocardial injury while targeting the TGF-{beta}/Smad2/3 pathway to attenuate cardiac fibrosis, highlighting its potential as a therapeutic agent for fibrotic cardiomyopathy.This study aims to investigate the cardioprotective effects of 4-Hydroxybenzaldehyde (4-HBA) against isoproterenol (ISO)-induced cardiac fibrosis and to elucidate the underlying mechanisms. In vivo, cardiac fibrosis was induced in C57BL/6 mice by subcutaneous injection of ISO, and the mice were treated with 4-HBA or a TGF-{beta} inhibitor. Assessments using echocardiography, histopathology, and Western blotting demonstrated that 4-HBA significantly alleviated ISO-induced cardiac dysfunction, reduced collagen deposition, and attenuated apoptosis in mice. Mechanistically, 4-HBA inhibited TGF-{beta} expression and Smad2/3 phosphorylation. In vitro, ISO was applied to cardiomyocytes (HL-1) and cardiac fibroblasts (MCFs), with or without 4-HBA or TGF-{beta} inhibitor intervention. The results showed that 4-HBA suppressed HL-1 apoptosis and fibroblast proliferation, and significantly reduced the expression of extracellular matrix genes, TGF-{beta} levels, and Smad2/3 phosphorylation in MCFs. These findings indicate that 4-HBA reduces myocardial injury while targeting the TGF-{beta}/Smad2/3 pathway to attenuate cardiac fibrosis, highlighting its potential as a therapeutic agent for fibrotic cardiomyopathy.

Three copper(II) complexes containing 1,10-phenanthroline derivatives, [CuCl2(phen)].0.5H2O (1), [CuCl2(neo)].0.75H2O (2), and [CuCl2(tmp)].H2O (3), were evaluated for their antitumor activity in MDA-MB-231 breast cancer cells. All complexes markedly reduced cell viability, exhibiting significantly lower IC values and higher selectivity indices than the corresponding free ligands, CuCl2, and cisplatin, highlighting the therapeutic advantage of metal complexation. Among them, only 1 induced DNA damage at sub-IC concentrations. Cytotoxicity in all complexes was associated with intracellular ROS generation and apoptosis, although 2 and 3 produced a stronger oxidative response. Complex 3 additionally promoted necrosis. Cell proliferation was inhibited in a concentration-dependent manner, accompanied by increased intracellular copper accumulation. Moreover, 2 and 3 significantly inhibited Na/H exchanger 1 (NHE1) activity, reducing cell migration and MMP-9 activity. Western blot analysis further demonstrated that all three complexes modulated the expression of NHE1, G protein-coupled estrogen receptor (GPER), and apoptosis-related proteins. Overall, our findings show that ligand methylation enhances the antitumor activity of copper(II) complexes and shifts their mechanism of action from DNA damage-driven cytotoxicity toward ROS-mediated apoptosis, while also improving inhibition of NHE1-dependent migratory pathways. These results identify NHE1 as a key molecular target underlying the enhanced antitumor activity of methylated phenanthroline copper(II) complexes.

With the rapid development of modern biotechnology, DNA vectors have become fundamental tools for inserting, transferring, and expressing specific gene sequences in various fields such as gene cloning, gene expression, gene editing, and gene therapy. However, when dealing with complex structured DNA sequences, traditional vector construction methods face challenges with low connection efficiency. This study proposes a new method for constructing recombinant vectors by employing a strategy of high-temperature treatment followed immediately by placement on ice, effectively reducing the complexity of DNA structures and enhancing the efficiency of PCR product-vector connection, thereby improving the construction efficiency of recombinant vectors. This paper describes the technical details of the method, experimental validation, and applications in gene cloning, gene recombination editing, and the preparation of gene therapy drugs, providing a new efficient tool for molecular biology experiments.