Fibrosis and tumour innervation are two features of the tumour microenvironment (TME) that contribute directly to the lethality of pancreatic ductal adenocarcinoma (PDAC), but their potential interactions have not been explored. Moreover, although it is known that activated Schwann cells (SCs) stimulate cancer cell invasion, it remains unclear how SCs are activated. We determined how SCs are activated in the pancreatic fibrotic microenvironment. The correlation between physical features of the microenvironment and SC activation was assessed in human patient samples and in mice by SC c-Jun phosphorylation monitoring, atomic force microscopy and multiphoton live imaging. Several in vitro models in which forces were applied to SCs expressing a reporter for c-Jun phosphorylation and RNA-Seq analysis were used to decipher the cellular and molecular mechanisms of SC activation. Nerves surrounded by stiff stroma present higher SC activation. Intravital imaging shows a matrix-dependent SC activation. Mechanical forces on SCs induce c-Jun phosphorylation in SCs in a non-canonical manner that involves a nuclear sensing machinery with the pro-inflammatory enzyme phospholipase A2. Fibrosis enhances the protumorigenic impact of innervation by activating SCs via a mechanism in which nuclear compression triggers non-canonical activation of the AP-1 transcription factor complex. Pancreatic fibrosis alone, without cancer cells, is sufficient to activate SCs, suggesting this mechanism may be common across non-malignant pancreatic diseases. Notably, SCs are more sensitive to mechanical activation than PDAC cells. These findings reveal TME interactions that may guide future microenvironment-targeted PDAC therapies.
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Background
During nuclear disasters, shelter-in-place (SIP) is often a safer alternative to evacuation for patients in medical facilities who may be particularly vulnerable. In Japan, medical institutions located near nuclear facilities have been equipped with positive-pressure systems (PPS), designed specifically for radiation protection. However, operational protocols to execute and manage SIP and PPS remain poorly defined. This study aimed to investigate the challenges in implementing SIP at the time of nuclear catastrophe while maintaining essential clinical functions.
Methods
A case study was conducted at the Matsue Red Cross Hospital, located within an Urgent Protective Action Planning Zone. Data were gathered through participation in the hospital's Nuclear Disaster Countermeasures Committee and through analysis of meeting minutes, facility documents, and technical specifications.
Results
The findings revealed a fundamental conflict between the technical requirements of positive-pressure systems (PPS) and the continuity of clinical services. Four critical sub-challenges were identified: (1) the unavoidable presence of certain staff and patients in non-protected zones within the hospital; (2) the potential inability to maintain sufficient positive pressure in protected zones during active clinical service; (3) the difficulty in regulating indoor temperature and humidity as a consequence of PPS operation; and (4) the increased risk of external radiation exposure in the vicinity of PPS units when outdoor ambient dose rates are elevated.
Conclusion
For facilities that already have PPS installed, further investigations into PPS operations and the development of countermeasures are necessary. Furthermore, when installing PPS in the future, it is desirable for the government, hospitals, and installation companies to thoroughly discuss operations and design systems to minimise the impact on hospital functions.
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Aim: Nanobiotechnology has grown rapidly in recent years and constitutes an integral part of modern disease diagnosis and treatment. The goal of this study was to evaluate the protective effect of synthesized selenium nanoparticles (SeNPs) against ionizing radiation (IR)-induced hematopoietic system injury. Methods: An aqueous extract of Rhodiola (RHO) was used for the synthesis of SeNPs (RHO-SeNPs). The resulting nanoparticles were characterized by ultraviolet-visible spectrophotometry and transmission electron microscopy, followed by biological assessment (antioxidant and cytotoxicity). Experiments with BALB/c mice and mouse hematopoietic stem cells (HSCs) exposed to 60Co γ-rays were used to evaluate the therapeutic efficiency of RHO-SeNPs. Immunohistochemistry, ELISA, JC-1 staining, CCK-8, TUNEL, flow cytometry, and Western blot were employed to investigate the underlying mechanisms. Results: RHO-SeNPs showed excellent antioxidant performance and had no toxicity in the major organs of BALB/c mice when administered at ≤20 mg/kg. RHO-SeNPs dose-dependently improved hematopoietic function and survival rate of IR-exposed BALB/c mice, in addition to suppressing mitochondrial oxidative stress, inflammatory responses, and bone marrow cell apoptosis. In vitro studies revealed that RHO-SeNPs limited Kelch-like ECH-associated protein 1 (KEAP1) expression and induced nuclear translocation of nuclear factor erythroid 2-related factor 2 (NRF2), thereby attenuating oxidative stress and HSC apoptosis. Both in vivo and in vitro experiments demonstrated that the disruption of NRF2 nuclear translocation partly abolished the radioprotection of RHO-SeNPs. Conclusion: These findings identify the mechanisms underlying the radioprotection of RHO-SeNPs and their potential role as a medical countermeasure against IR exposure.
Parkinson's disease (PD) is a common neurodegenerative disease. Our previous single-cell sequencing results suggested that Transcription Factor E3 (TFE3) was only differentially expressed in microglia in the MPTP mouse model. However, the functional role of microglial TFE3 in PD pathogenesis remains unclear. First, motor function was assessed in MPTP mice following TFE3 overexpression in substantia nigra microglia. Second, RNA-seq was used to identify the function of TFE3 in microglia, and the molecular mechanism was verified both in vivo and in vitro. Finally, we investigated whether TFE3 nuclear translocation affects its transcriptional activity and subsequently influences microglia in vitro. Overexpression of TFE3 in substantia nigra microglia could alleviate PD-related phenotypes. RNA-seq revealed that TFE3 regulates microglial phagocytosis and inflammation. Mechanistically, TFE3 affects these functions by regulating the expression of Mer receptor tyrosine kinase (Mertk) and lysosomal-associated membrane protein 1 (Lamp1). Finally, Rapamycin could activate the nuclear translocation of TFE3 and enhance phagocytosis and alleviate inflammation of microglia. Our findings demonstrate that Rapamycin activates TFE3, which in turn upregulates the expression of Mertk and Lamp1 in the substantia nigra. This TFE3-mediated pathway plays a critical role in regulating microglial phagocytosis and inflammation in the PD model.
CCR4-NOT regulates multiple steps in gene regulation and has been well studied in yeast. While the human complex is known to play an essential role in cytoplasmic mRNA degradation, its nuclear functions are poorly characterized. Here, we used auxin-induced degradation to deplete the scaffold subunit CNOT1 and the E3 ligase CNOT4 to characterize the complex's regulation of transcription. Transient transcriptome profiling (TT-Seq) revealed that depleting either subunit led to widespread activation of RNA synthesis in genic and intergenic regions. Interestingly, fewer genes were down-regulated, including KRAB-Zinc-Finger-protein (KZNF) genes. KZNFs repress genes and retrotransposable elements (rTEs), and consistent with decreased KZNF expression, rTEs, mainly Long Interspersed Nuclear Elements (LINEs), were activated. Full-length active LINEs and rTEs located outside of genes were activated too, suggesting that the increased transcription is not the direct result of transcription of the genes in which the rTEs are embedded. Most of the activated transcription events occurred near KZNF binding sites, suggesting that KZNF regulation plays a role in suppressing both genic and rTE transcription. Finally, we demonstrate that CCR4-NOT regulates the stability of rTE RNAs, indicating that the complex tightly controls transposon expression by repressing transcription and targeting their RNAs for degradation.
Uranium mononitride (UN) is a promising advanced nuclear fuel featuring high inherent uranium density and excellent thermal conductivity. Although density functional theory (DFT) has been extensively used to explore the fundamental properties of UN, systematic and comprehensive studies on the thermomechanical performances of irradiation-defective UN remain limited. Formation energies of typical point defects in UN are strongly dependent on local chemical environments, with Schottky defects exhibiting distinct energetic characteristics under U-rich, N-rich, and stoichiometric conditions. The introduction of these defects systematically reduces the elastic moduli of UN, while both Poisson's ratio (σ) and Pugh's ratio (κ) increase accordingly. Vickers hardness (HV) gradually declines with defect incorporation, as verified by empirical formula-based fracture toughness (KIC) assessments. The elastic anisotropy of defective UN is quantitatively evaluated via anisotropy factors and intuitively visualized using three-dimensional (3D) contour plots. This work further systematically calculates the melting point (Tm) and minimum thermal conductivity (κmin) of defective UN. Thermodynamically, defective UN is less stable than pristine UN at low temperatures, yet becomes stabilized at high temperatures owing to its reduced Gibbs free energy (g). The linear thermal expansion coefficient (CTE, αL) increases sharply with rising temperature and eventually plateaus at a stable value. This atomic-scale insight into radiation-induced defect mechanisms and their modulation effects on thermomechanical and thermal performances is essential for addressing key technical bottlenecks and advancing the development of next-generation nuclear reactor fuels.
AMP-activated protein kinase (AMPK) is a crucial energy sensor that regulates a wide range of important processes in skeletal muscle. AMPK is present in several subcellular compartments (including the cytosol, nucleus, and mitochondria). However, the influence of physiologically relevant interventions on AMPK's localization in skeletal muscle is not well understood, especially during older age. Accordingly, this study examined AMPK signaling in skeletal muscle from aged male rats (24-25-months-old) subjected to either eight-weeks of calorie restriction (CR; consuming 65% of ad libitum intake) or a single swim-exercise session. Phosphorylation of AMPK and its substrate acetyl-CoA carboxylase (ACC), as well as abundance of AMPK subunits (α1, α2, β1, β2, γ1, γ3), were assessed by immunoblotting in whole muscle lysates and cytosolic, nuclear, and mitochondrial-enriched fractions obtained by differential centrifugation. CR increased phosphorylation of AMPK and ACC in whole muscle lysates, but not in the three subcellular fractions that were tested, suggesting AMPK-activation occurs in other, currently unidentified compartments. In contrast, exercise significantly increased AMPK phosphorylation in the cytosolic fraction and ACC phosphorylation in whole lysates and all three subcellular fractions. AMPK-γ1 abundance was greater in the mitochondrial-enriched fraction of CR versus ad libitum muscles. These findings revealed strikingly different patterns of AMPK activation within key subcellular compartments in response to two important physiological interventions. This study substantially advances current knowledge and provides a foundation for future research on AMPK's compartment-specific roles in skeletal muscle physiology and aging.
Anti-PD1 immune checkpoint inhibitors (ICI) are effective in clear-cell renal-cell carcinoma (ccRCC). Preliminary data suggest inhibition of Receptor Activator of Nuclear Factor-κB Ligand (RANKL) signaling may potentiate ICI efficacy. We evaluated the activity and safety of the RANKL-inhibitor denosumab with pembrolizumab in people with pretreated advanced ccRCC. This single-arm, multi-center phase 2 trial enrolled participants with metastatic or unresectable ccRCC progressing on or after VEGFR-targeted tyrosine kinase inhibitor (TKI) therapy. Participants received pembrolizumab 200 mg IV every 3 weeks plus denosumab 120 mg SC on days 1, 8 and 22, then 3-weekly until disease progression, unacceptable toxicity, or a maximum of 24 months. Primary endpoint was objective tumor response (ORR). Secondary endpoints: median progression free survival (PFS), progression-free survival rate at 6 months (PFS6m), duration of response (DOR), and adverse events (AE). 59 participants were recruited: male (81%), International Metastatic Database Consortium favorable risk (48%), at least one prior VEGFR-TKI in all participants, 2 or more in 16%. At a median follow-up of 40 months, the ORR was 31% (all partial; 95%CI 20%-45%). Median DOR was 17 months. Median PFS was 7.5 months and PFS6m rate was 53% (95%CI, 39%-65%). Immune-related grade ≥ 3 AEs were reported in 21% of participants; study treatment was discontinued for toxicity in 22%. One participant suffered G3 osteonecrosis of the jaw, and 1 died of myositis attributed to study treatment. The combination of denosumab and pembrolizumab is safe in pretreated clear cell renal carcinoma, with activity meriting further investigation.
Radioresistance in colorectal cancer (CRC) remains a critical clinical challenge, with underlying mechanisms involving deoxyribonucleic acid (DNA) damage repair, metabolic reprogramming, and immune evasion. Here, we employed multi-omics approaches, including ribonucleic acid (RNA) sequencing, proteomic profiling, and lactyl-proteomics of radioresistant CRC cell models, combined with co-immunoprecipitation, molecular docking, spatial transcriptomics, and single-cell RNA sequencing. Functional validation included production of an EF-hand domain family member D2 (EFHD2) lysine 9 (K9) lactylation-specific antibody, T cell and macrophage co-culture assays, and multiplex immunofluorescence analyses, performed in vitro and in vivo using immunocompetent and immunodeficient mice, alongside patient-derived rectal cancer samples collected from our institution. We found that hsa-let-7b-5p was significantly downregulated in radioresistant CRC cells and tissues, correlating with poor prognosis. Restoration of hsa-let-7b-5p inhibited EFHD2, impaired homologous recombination through reduced activation of RAD51 recombinase (RAD51) and replication protein A2 (RPA2), prolonged phosphorylated histone H2AX at serine 139 (γH2AX) foci persistence, and increased oxidative stress and apoptosis after radiation. Mechanistically, EFHD2 interacted with high mobility group box 1 (HMGB1), enhancing lactate metabolism via hypoxia-inducible factor-1 alpha (HIF-1α) stabilization, with K9 lactylation of EFHD2 being essential for its DNA repair function. Elevated EFHD2-driven lactate reshaped the tumor immune microenvironment by promoting M2 macrophage polarization, suppressing antigen presentation, activating nuclear factor kappa-B (NF-κB) signaling, and upregulating programmed death-ligand 1 (PD-L1). Flow cytometry and immunohistochemistry revealed reduced CD4⁺/CD8⁺ T cell infiltration and increased forkhead box P3 positive (Foxp3⁺) regulatory T cells (Tregs) in EFHD2-high tumors, confirmed by spatial and single-cell transcriptomics showing immunosuppressive signatures. Importantly, combining EFHD2 knockdown with immune checkpoint blockade synergistically enhanced radiosensitivity and restored antitumor T cell responses. Collectively, these findings demonstrate that EFHD2 drives lactate-mediated immunosuppression and DNA repair to promote radioresistance in CRC, suggesting that targeting the EFHD2 axis may restore antitumor immunity and improve therapeutic outcomes.
Proper orientation of the mitotic spindle is critical for a wide range of biological processes. However, the molecular mechanisms that regulate the formation of the spindle orientation machinery remain incompletely understood. In this study, we identify an essential role for ubiquitin-specific peptidase 21 (USP21)-mediated deubiquitylation in the control of spindle orientation. Histological analyses of Usp21 knockout mice revealed multiple epithelial abnormalities, including delayed corneal epithelial stratification, dilated renal tubules, and impaired skin wound healing. These defects were attributable to spindle misorientation in corresponding epithelial tissues. In vitro studies further confirmed that USP21 deficiency disrupts spindle orientation in cultured cells. Mechanistically, USP21 interacts with nuclear mitotic apparatus protein (NuMA) and removes the lysine 63-linked ubiquitylation of NuMA at lysine 1330 and lysine 1335. The deubiquitylation of NuMA triggers its interaction with Leu-Gly-Asn repeat-enriched protein (LGN), thereby recruiting NuMA to the cell cortex to form the spindle orientation machinery. Collectively, these findings uncover a previously unrecognized role for USP21 in spindle orientation and underscore its importance in epithelial development and homeostasis through the deubiquitylation of NuMA.
Crop productivity is significantly restricted by drought and salt stress. As a conserved family of calmodulin-binding proteins with transcriptional activation activity, CAMTAs are essential for modulating plant responses to a range of abiotic stressors. Nevertheless, there is still much to learn about the functional characterisation of CAMTAs in foxtail millet, which is a model plant for the study of cereal crops. In this study, we amplified and explored the molecular function of foxtail millet SiCAMTA6 gene. The result of subcellular localization showed that SiCAMTA6 was localized in both the nuclear and plasma membrane. Heterologous overexpression of SiCAMTA6 in Arabidopsis thaliana (A. thaliana) and rice improves drought tolerance but increases salt sensitivity. These results provide a basis for future molecular breeding efforts to increase foxtail millet resistance to stress.
Radioactive iodine (RAI) is a crucial treatment for refractory pediatric and adolescent Graves' disease (GD) cases characterized by poor response to antithyroid drug (ATD), recurrence, adverse effects, or excessive thyroid enlargement. Compared to adults, pediatric and adolescent patients currently lack effective efficacy prediction models tailored to this specific population. Furthermore, the applicability of existing adult dose calculation standards to children remains controversial. This study aims to develop a predictive model and explore the cutoff values of key variables influencing treatment efficacy, thereby providing a new adjunctive tool for the clinical management of pediatric and adolescent GD patients. A total of 95 children and adolescents with GD who received RAI therapy at the Department of Nuclear Medicine, the Second Affiliated Hospital of Nanchang University, between January 2020 and May 2025 were initially screened. Following the exclusion of 13 patients who had undergone prior thyroid surgery, received non-initial RAI treatment, or lacked regular follow-up, 83 patients (aged 9-19 years; 58 females) were finally enrolled. Based on thyroid function status 6 months post-treatment, patients were categorized into the cured group (Clinical Cure and Hypothyroidism) and the uncured group (Ineffective and Partial Improvement). Univariate analysis and LASSO regression were employed for preliminary variable screening to identify predictive indicators. A multivariate logistic regression model was subsequently constructed based on these selected variables. Model performance was evaluated using the area under the curve (AUC), calibration curves, and decision curve analysis (DCA), with internal validation performed via Bootstrap (2000 resamplings). The optimal cutoff values were determined using the Youden index. The proposed dose per gram of thyroid tissue was a protective factor (OR = 1.048, 95% CI (1.01-1.09), P = 0.018); Thyroid weight (OR = 0.956, 95% CI (0.924-0.983), P = 0.004) and TRAb (OR = 0.914, 95% CI (0.861-0.963), P = 0.001) were risk factors. The model achieved an AUC of 0.833 on the original dataset, and an adjusted AUC of 0.810, After model establishment, further analysis of key variables revealed significantly reduced cure rates in patients with thyroid weight > 55.52 g, TRAb > 13.8 IU/L, and the proposed dose per gram of thyroid tissue < 97.5 µCi/g (3.61 MBq/g). A model incorporating the proposed dose per gram of thyroid tissue, TRAb, and thyroid weight effectively predicts treatment efficacy. For high-risk patients (thyroid weight > 55.52 g, TRAb > 13.8 IU/L), increasing the proposed dose (≥ 97.5 µCi/g or 3.61 MBq/g) is recommended to enhance treatment outcomes.
Previous efforts to link Palaeolithic cultural records to specific populations through DNA analysis have focused on materials from archaeological floor deposits such as bones, sediments, and artefacts. In this study, we explore whether rock art, a spatially distinct expression of human activity, can also preserve DNA traces from its creators. We analyse DNA preservation in pigment samples collected in and around 24 rock art panels from 11 caves across Spain and Portugal, including simple marks (from nine sites), hand stencils (Maltravieso Cave, Extremadura, Spain), and figurative paintings (Cave of Altamira, Cantabria, Spain). We recover traces of ancient human mitochondrial and nuclear DNA, unaccompanied by faunal DNA, from a pigmented calcite crust at Escoural Cave (Portugal), as well as from an unpigmented cave wall sample from the same site. The absence of faunal DNA in both samples suggests direct DNA deposition through human contact. In contrast, three additional unpigmented samples, from Escoural and Covarón Cave (Asturias, Spain), yielded mixtures of human and faunal DNA, suggesting indirect deposition. Although our results do not conclusively link ancient human DNA preservation to the generation of cave art, we show that traces of human DNA can persist on cave walls for thousands of years.
Educational tours in radiopharmaceutical production facilities are often considered difficult to implement because of strict safety regulations, confidentiality requirements, and operational constraints. However, when appropriately designed, such tours can function as structured educational activities rather than simple facility visits. Here, we describe the design and implementation of educational tours conducted in a hospital-based radiopharmaceutical production facility, with particular emphasis on training radiologic technology students and early-career technologists. A notable feature of these tours was that explanations and guidance were provided primarily by radiologic technologists who had been trained through routine 18F-FDG production activities, allowing participants to observe professional role development in a clinical setting. Rather than providing unrestricted access, the tours were intentionally structured to highlight decision-making processes, safety management, and interprofessional collaboration. Qualitative observations indicated that participants' questions shifted from generalized concerns about radiation risk toward a more operational understanding of radiopharmaceutical production and professional responsibility. These findings suggest that well-designed facility tours can serve as an effective educational framework for nuclear medicine technology training while maintaining safety and confidentiality.
Sepsis is associated with a pronounced but poorly understood endoplasmic reticulum stress (ERS) response. In this study, we found that death-associated protein kinase 2 (DAPK2), a calcium/calmodulin-regulated serine/threonine kinase, exhibits elevated expression in macrophages from patients with sepsis and from septic mice. Macrophage DAPK2 expression is transcriptionally upregulated through the activation of the Toll-like receptor 4 (TLR4)-myeloid differentiation primary response 88 (MyD88)-nuclear factor-κB (NF-κB) pathway. Macrophage-specific deletion of DAPK2 attenuated sepsis severity and mitigated the ERS response. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified heat shock protein family A member 5 (HSPA5) as a binding partner for DAPK2. Because DAPK2 function had been previously associated with the kinase activity, we speculated that it might control ERS of macrophages through HSPA5 phosphorylation. Further investigation indeed revealed that DAPK2 phosphorylates HSPA5 at serine-588, which promotes the proteasomal degradation of HSPA5 and subsequently leads to the activation of inositol-requiring enzyme 1α (IRE1α). Inhibition of HSPA5 exacerbated sepsis in mice with macrophage-specific DAPK2 deficiency; however, this effect was abrogated by the deactivation of IRE1α. In conclusion, our findings demonstrate that DAPK2 propagates macrophage ERS through the HSPA5-IRE1α axis during systemic infection, suggesting this pathway as a potential therapeutic target in sepsis.
Luteolin is a natural flavone with various beneficial properties. We hypothesise that luteolin could alleviate haemodynamic, vascular, and renal changes caused by renal artery stenosis in rats. Male Sprague-Dawley rats were randomly divided into five groups (n = 8/group): Sham, two-kidney, one-clip (2K1C), 2K1C + luteolin (25 or 50 mg/kg/day), 2K1C + lisinopril (5 mg/kg/day). Hemodynamic parameters, vascular-kidney function and morphology were evaluated. Luteolin treatment for four weeks markedly alleviated hemodynamic changes, mitigated sympathetic nerve overactivity and ameliorated endothelial dysfunction in 2K1C rats. Aortic hypertrophy and remodelling, evident by increases in cross-sectional areas, aortic wall-to-lumen ratio, and fibrotic areas in 2K1C rats, were relieved by luteolin treatment (p < 0.05). Luteolin alleviated renal injuries, restored glomerular filtration rate, decreased urinary α-1-microglobulin levels, and attenuated renal fibrosis in 2K1C rats (p < 0.05). It reduced superoxide production, protein carbonyl, malondialdehyde, tumour necrosis factor-α, and interleukin-6 concentrations, but raised superoxide dismutase and catalase activities in 2K1C rats (p < 0.05). Luteolin mitigated the overexpression of α-smooth muscle actin, vascular cell adhesion molecule-1, transforming growth factor-β1/Smad2/3, and nuclear factor-κB proteins in aortic tissue. In addition, the overexpression of the JAK2/STAT3 pathway in a non-clipped kidney was suppressed by luteolin treatment (p < 0.05). Lisinopril has shown advantageous effects on vascular and renal abnormalities, akin to those of luteolin. In summary, luteolin improved haemodynamic alterations and vascular and renal impairment in 2K1C rats by attenuating oxidative stress, inflammation, and fibrotic signalling pathways, suggesting its potential as a promising therapeutic candidate for renal artery stenosis-induced vascular and renal injury.
The pathological processes of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are closely associated with excessive activation of inflammasomes. HSPA8 is a constitutively expressed molecular chaperone involved in cellular signaling and immune-inflammatory regulation, but its role in EAE-associated neuroinflammation remains unclear. In this study, we found that HSPA8 was significantly upregulated in the spinal cord tissues of EAE mice and positively correlated with disease severity. Immunofluorescence co-staining showed that HSPA8 upregulation was more prominently associated with Iba1-positive microglia/macrophage-enriched regions than with GFAP- or NeuN-positive regions. Intrathecal knockdown of HSPA8 attenuated EAE progression, reduced inflammatory responses, and alleviated demyelination and axonal injury. In vitro, HSPA8 knockdown reduced NLRP3 inflammasome-mediated IL-1β/IL-18 release, caspase-1 activation, GSDMD-N formation, and pyroptosis in THP-1 cells, bone marrow-derived macrophages, and BV2 microglia, and these effects were partially restored by siRNA-resistant HSPA8 rescue. Mechanistically, HSPA8 knockdown was associated with impaired NF-κB-dependent priming, as reflected by reduced p65 phosphorylation, nuclear translocation, NF-κB transcriptional activity, and pro-IL-1β expression. HSPA8 is also associated with ASC, and HSPA8 knockdown impaired NLRP3-ASC interaction, ASC oligomerization, and ASC speck formation during inflammasome assembly. These findings suggest that HSPA8 is functionally associated with NLRP3 inflammasome activation through both NF-κB‑dependent priming and ASC‑associated assembly, thereby contributing to neuroinflammation in EAE. HSPA8 may represent a potential therapeutic target for MS-related and other inflammasome-driven neuroinflammatory disorders.
Excessive inflammation drives organ dysfunction and high mortality in life-threatening conditions such as sepsis. Baicalein, a bioactive flavonoid, possesses well-recognized anti-inflammatory properties, yet its molecular targets in macrophages and potential systemic immunomodulatory effects on peripheral blood immune cells under hyperinflammatory conditions remain poorly characterized. Our previous studies demonstrated that baicalein alleviates hepatic inflammation in mice with non-alcoholic fatty liver disease (NAFLD) and inhibits NF-κB nuclear translocation in RAW264.7 macrophages. Here, by integrating network pharmacology, molecular docking, bulk RNA sequencing of macrophages, and single-cell RNA sequencing of peripheral blood from sepsis patients, we identified JAK2, SRC, TP53, MAPK3, AKT1, HSP90AA1, and ESR1 as potential core targets of baicalein in macrophages, and validated that the JAK2-STAT3 and NF-κB pathways might be the key downstream regulatory axes of its anti-inflammatory effects. Furthermore, we revealed that baicalein may modulate, based on single-cell expression signatures, the inflammatory phenotype of multiple peripheral blood immune cell populations, including monocytes, T cells, B cells, and granulocyte-monocyte progenitors, suggesting a potential systemic anti-inflammatory effect that requires experimental validation in human cells. Collectively, our findings elucidate the potential molecular targets of baicalein in macrophages and its multi-cellular immunoregulatory mechanisms under hyperinflammation, providing novel mechanistic insights for the clinical application of baicalein in inflammatory diseases.
Colorectal cancer (CRC) is one of the most common and deadly types of cancer globally. Ferroptosis, a type of regulated cell death that relies on iron, has become a promising target for treating CRC. Alkannin, a natural compound from Lithospermum erythrorhizon, exhibits anti-tumor activity, yet its mechanism in CRC is unclear. This study investigated alkannin's role in regulating ferroptosis via the Keap1/Nrf2/GPX4 axis. Using network pharmacology and experimental validation in HCT116 and SW480 cells and a xenograft mouse model, we found that alkannin markedly inhibited the viability, proliferation, and migratory capacity of CRC cells, demonstrating significant anti-tumor activity. Network pharmacology revealed a primary association between alkannin's therapeutic effects and the induction of ferroptosis, along with the regulation of oxidative stress pathways, with a notable focus on the Keap1/Nrf2 axis. Experimental evidence confirmed that alkannin induced ferroptosis, as reflected by increased intracellular Fe2+ levels and lipid peroxidation, along with reduced glutathione (GSH) content. These effects were reversed by ferroptosis inhibitors, which also attenuated alkannin-induced cytotoxicity. Mechanistically, alkannin enhanced Keap1 protein stability by suppressing its ubiquitination. It promoted the interaction between Keap1 and Nrf2, leading to decreased Nrf2 expression and inhibition of its nuclear translocation, thereby downregulating the expression of glutathione peroxidase 4 (GPX4), a key suppressor of ferroptosis. Genetic silencing of Keap1 significantly diminished alkannin-induced ferroptotic cell death. In vivo, alkannin effectively inhibited tumor growth in xenografted nude mice. Furthermore, it induced ferroptosis in tumor tissues, as evidenced by similar biochemical changes, which were counteracted by co-administration of a ferroptosis inhibitor. Consistently, alkannin upregulated Keap1 expression while reducing the protein levels of Nrf2 and GPX4 in tumor tissues. In conclusion, alkannin induces ferroptosis in CRC by stabilizing Keap1 to inhibit the Nrf2/GPX4 pathway, supporting its potential as a CRC therapeutic agent.