Liraglutide prevents lupus-associated diffuse alveolar hemorrhage via inhibiting lymphocyte infiltration and promoting macrophage M2 polarization.

Epimedium polysaccharide alleviates adenine-induced oligoasthenozoospermia in mice by inhibiting ferroptosis and inflammation through TLR4/NF-κB and SLC7A11/GPX4 axis.

To investigate the mechanism by which BRD4 accelerates acute lung injury (ALI) by promoting ferroptosis mediated by ANP32B/H3K27ac/MYC/HOXB8/BACH1 in alveolar epithelial cells. Gene co-expression was analyzed with bioinformatics. Eighteen SPF-grade C57BL/6J male mice (6-8 weeks old, 20 ± 4 g) were divided into three groups: Sham, Model, and Model-JQ1. Lung function was analyzed post-anesthesia; after euthanasia, lung tissues were collected for TEM to observe structural changes. HE staining assessed cellular morphology and pathology, while Prussian blue staining evaluated iron deposition. Western Blot detected BRD4, ANP32B, MYC, HOXB8, and BACH1 protein expression. ELISA measured GSH, MDA, lipid ROS, Fe2+, and LPO levels. MLE-12 cells were cultured and divided into four groups: NC, LPS, LPS-JQ1, and LPS-JQ1-BACH1-OE. Western Blot and immunofluorescence analyzed protein expression, and TEM observed cell ultrastructure. ELISA assessed oxidative stress markers. HOXB8 regulates BACH1, impacting oxidative stress and iron metabolism genes. LPS induced significant cell damage, alleviated by BRD4 inhibition. JQ1 improved lung injury and function, reduced iron accumulation, and oxidative damage. Western Blot and ELISA showed LPS upregulated key proteins, reversed by BRD4 inhibition. JQ1 inhibited LPS-induced stress in cells; BACH1 overexpression partially reversed this. Immunofluorescence showed BRD4 inhibition suppressed BACH1 and promoted SLC7A11 expression. ALI is accelerated by BRD4 through the promotion of ANP32B/H3K27ac/MYC/HOXB8/BACH1-mediated ferroptosis in alveolar epithelial cells. A deeper understanding of the role of BRD4 in alveolar epithelial cell injury is provided by these findings, and potential therapeutic targets for the treatment of ALI may be identified.

Chlorothalonil is linked to gastrointestinal toxicity, but its underlying mechanisms remain unclear. This study investigated the chlorothalonil-induced intestinal injury in mice. Histological analysis showed epithelial damage and goblet cell depletion. Chlorothalonil disrupted the gut microbiota, including the depletion of Lactobacillus and the enrichment of Enterococcus. Untargeted metabolomics revealed a reduction in sphinganine, which is associated with oxidative stress and ferroptosis. Molecular analysis indicated ferroptosis activation via the Keap1/Nrf2 pathway, evidenced by decreased glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) expression. Prussian blue staining and transmission electron microscopy confirmed iron deposition and mitochondrial injury, hallmark features of ferroptosis. Fecal microbiota transplantation confirmed dysbiosis as a driver of injury, while ferrostatin-1 treatment partially rescued the pathological phenotype. These findings demonstrate that chlorothalonil causes microbiota dysbiosis and decreases sphinganine, which inactivates Nrf2 signaling and results in ferroptosis. Our study provides new mechanistic insights into the toxicological mechanisms of chlorothalonil.

Myocardial ischemia-reperfusion (MI/R) injury significantly limits the clinical benefits of coronary reperfusion therapy. Ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation, has been implicated in myocardial ischemia-reperfusion (I/R) injury. Liproxstatin-1 (Lip-1) is a potent ferroptosis inhibitor, but its dynamic, dose-dependent effects on key molecular pathways and pathological hallmarks in the heart remain incompletely characterized. To systematically investigate the dose- and time-dependent cardioprotective effects of Lip-1 against myocardial I/R injury, with a focus on the NRF2/GPX4 pathway, iron deposition, and lysosomal integrity. Ninety Wistar rats were randomly allocated to 15 experimental groups (n = 6 per group): Normal (no surgery), Sham (thoracotomy without ischemia), I/R model, and I/R + Lip-1 treatment groups. Lip-1 was administered intravenously at doses of 1, 3, or 5mg/kg at 0, 24, 48, and 72h post-reperfusion initiation, with myocardial tissue and blood samples harvested 6h after each injection. Cardiac function was assessed by echocardiography. Myocardial infarct size was determined by Evans Blue/TTC double staining. Serum levels of CK-MB and LDH were measured as markers of myocardial injury. Analyses included Western blot for NRF2 and GPX4 expression, Prussian blue staining for iron deposition quantification, and immunofluorescence for LAMP1 localization and intensity. Statistical analysis was performed using two-way ANOVA with Tukey's post hoc test for Lip-1 treatment groups, and t-tests or one-way ANOVA for model validation comparisons. Compared to Sham, I/R injury significantly decreased LVEF, increased infarct size, and elevated CK-MB and LDH levels (all P < 0.0001), confirming successful model establishment. It also downregulated GPX4 expression, induced severe iron deposition, and reduced LAMP1 levels, while triggering an adaptive upregulation of NRF2. Lip-1 treatment produced dose- and time-dependent protection across all measured endpoints. It improved cardiac function, reduced infarct size, and attenuated CK-MB and LDH release, with significant dose×time interactions for infarct size (F(6,60) = 8.338, P < 0.0001), CK-MB (F(6,60) = 6.467, P < 0.0001), and LDH (F(6,60) = 9.021, P < 0.0001). It dynamically modulated the NRF2/GPX4 axis, with peak GPX4 expression observed following the 48-hour administration (sampled at 54h post-reperfusion). Lip-1 progressively reduced iron deposition, with maximal effect observed after the 72-hour administration (sampled at 78h post-reperfusion), and rescued LAMP1 downregulation in later sampling points. Statistical analysis revealed significant dose×time interactions for NRF2 (F(6,60) = 200.8, p < 0.0001), GPX4 (F(6,60) = 34.84, p < 0.0001), and iron deposition. High-dose Lip-1 (5mg/kg) demonstrated superior and sustained efficacy across all parameters. Lip-1 confers multi-faceted cardioprotection against I/R injury through sequential mechanisms involving early potentiation of the NRF2/GPX4 antioxidant defense, progressive attenuation of pathological iron accumulation, and restoration of lysosomal membrane integrity. The strict dose and temporal dependency of these effects provide critical insights for optimizing ferroptosis-targeted therapeutic strategies in ischemic heart disease.

Pyroptosis and ferroptosis may be implicated in status epilepticus (SE)-induced neuronal damage. This study aimed to elucidate the effects of Gasdermin D (GSDMD) on pyroptosis and ferroptosis after SE, and to determine whether GSDMD inhibition ameliorates SE-induced neuronal damage and cognitive dysfunction. In the lithium-pilocarpine-induced SE rat model, we measured GSDMD and glutathione peroxidase 4 (GPX4) expression in the hippocampus via western blotting to evaluate the involvement of pyroptosis and ferroptosis after SE. After GSDMD knockdown by RNA interference, we performed Nissl staining to detect neuronal loss; quantitative reverse transcription polymerase chain reaction (qRT-PCR) to detect the hippocampal expressions of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α); Prussian blue staining to detect iron deposition; and western blotting and enzyme-linked immunosorbent assay to detect the expressions of the ferroptosis-related molecules GPX4, malonaldehyde (MDA), and glutathione (GSH). Finally, cognitive and behavioral functions were evaluated using the spontaneous-activity test and novel object-recognition test. Western blotting showed a marked increase in GSDMD expression (P < 0.050) and a notable decrease in GPX4 levels in (P < 0.050) the hippocampus of rats, providing evidence that both pyroptosis and ferroptosis are activated following SE. In addition, GSDMD knockdown significantly improved hippocampal neuronal survival and improved cognitive and behavioral functions of SE rats. Meanwhile, knockdown of GSDMD led to a significant reduction in the mRNA expression of IL-1β, IL-6, and TNF-α (pyroptosis products) (P = 0.021, 0.010, and 0.015, respectively), a decrease in iron deposition (P = 0.000) and MDA levels (ferroptosis product) (P = 0.000), and increased GSH and GPX4 expression (negative ferroptosis regulators) (P = 0.000 and 0.004, respectively). Pyroptosis and ferroptosis may jointly contribute to SE-induced neuronal damage. GSDMD may regulate both pyroptosis and ferroptosis, and inhibiting GSDMD could improve cognitive and behavioral function in SE rats.

The spleen serves as a critical reservoir of immune cells that play a key role in chronic inflammation and malignancy. While migration of immune cells from the spleen to the tumor immune microenvironment has been described, the dynamics of this process are poorly understood. Tracking the in vivo dynamics could provide valuable insights for understanding tumor immune biology, as well as potentially improving immunotherapy-based treatment regimens. In this study, splenocytes were labeled with superparamagnetic iron oxide (SPIO) nanoparticles conjugated with protamine-indocyanine green (SPIOpICG) for noninvasive, biocompatible, and real-time tracking. Transmission electron microscopy confirmed spherical SPIO and SPIOpICG with average sizes of 9.3 ± 3.1 and 12.7 ± 2.7 nm, respectively. SPIO exhibited a hydrodynamic volume of 110.37 ± 10.56 nm with a polydispersity index (PDI) of 0.356 ± 0.075, whereas SPIOpICG showed a hydrodynamic volume of 1078.48 ± 24.23 nm with a PDI of 0.399 ± 0.540. Ex vivo labeling of splenocytes demonstrated high cell viability (96.2 ± 2.8%) and uptake efficiency (94.3 ± 2.1%) after 24 h, confirming the nanoconjugate biocompatibility. Tumor-bearing Buffalo rat models received intravenous injections of free SPIOpICG or SPIOpICG-splenocytes. MRI performed before and 24 h postinjection revealed a significant reduction in T2* signal intensity in the liver and spleen, suggesting splenocyte migration to these organs. Iron accumulation was confirmed by Prussian blue staining in histology and atomic absorption spectrophotometry. While free SPIOpICG predominantly accumulated in the liver and spleen, SPIOpICG-splenocytes exhibited localization in the tumor, as evidenced by reduced T2* MRI signal (100 ± 7.6 to 70 ± 5.6%, p < 0.001) and elevated iron content (29.8 ± 29.7 to 598 ± 26.8 ppm/g, p < 0.001). Flow cytometry analysis of tumor tissues revealed a significantly higher accumulation of SPIOpICG-positive cells in the SPIOpICG-splenocyte group (50.45 ± 6.15%) compared to free SPIOpICG (16.10 ± 0.42%). Phenotypic analysis of positive cells revealed that macrophages, particularly the M2 subtype, constitute the predominant SPIOpICG-positive population within the tumor. These findings demonstrate that splenocyte-mediated delivery is largely driven by macrophage trafficking to tumor sites. This capability provides valuable insights into splenic immune cell trafficking and highlights splenocytes, particularly macrophages, as effective natural carriers for targeted nanoparticle delivery, offering a biocompatible platform for the development and optimization of immune cell-based immunotherapies.

Tiaojing Cuyun Recipe (TJCYR), a known Chinese herbal compound, has been shown to improve endometrial receptivity and embryo implantation in patients with embryo implantation dysfunction (EID). This study investigated the mechanism by which TJCYR improves endometrial receptivity. High-performance liquid chromatography (HPLC) was used to detect the compounds of TJCYR. A mouse EID model was established by subcutaneous injection of mifepristone into the neck. The rats were randomized into control, EID, Prog, TJCYR-L, and TJCYR-H groups. Hematoxylin and eosin (H&E) staining and transmission electron microscopy (TEM) were performed to assess pathological changes in the endometrium. The aggregation of Fe3+ was assessed using Prussian blue staining. Western blotting, immunofluorescence, and assay kits were used to determine the endometrial receptivity, ferroptosis indicators, and SLC7A11/GSH/GPX4 axis-related biomarkers. TJCYR-H group significantly increased the implanted sites (P<0.05), improved endometrial pathological changes and enhanced the expression of progestogen receptor (PR), estrogen receptor α (ERα), leukocyte inhibitory factor (LIF), osteopontin (OPN), integrin αV, SLC7A11, GPX4 (glutathione peroxidase 4), and ferritin, as well as the serum estradiol (E2), progesterone (Pg), reduced glutathione (GSH), glutathione peroxidase (GSH-Px), and T-GSH levels in EID. TJCYR inhibited the reduction in mitochondrial volume and membrane shrinkage, increased density, and decreased cristae in endometrial glandular epithelial cells of EID. Furthermore, TJCYR reduced the aggregation of Fe3+, downregulated serum MDA and GSSG levels, and reduced the expression of cyclooxygenase-2 (COX2), 4-hydroxy-2-nonenal (4-HNE), acyl-CoA synthetase long-chain family member 4 (ACSL4), and transferrin receptor (TFR) in EID mice. TJCYR can improve endometrial receptivity of EID mice by inhibiting ferroptosis, which may be related to the SLC7A11/GSH/GPX4 axis.

Accurate assessment of lymph node status is essential for staging and treatment planning in breast cancer. Conventional morphological imaging criteria have limited sensitivity and specificity. Superparamagnetic Iron Oxide (SPIO)-enhanced MRI offers a functional imaging alternative exploiting macrophage-mediated iron uptake. This study evaluated the diagnostic performance of SPIO-enhanced MRI in differentiating metastatic from reactive hyperplastic lymph nodes and explored the association between MRI signal characteristics and Vascular Endothelial Growth Factor C (VEGF-C) expression. Sixty female breast cancer patients (January-May 2024) underwent preoperative plain and SPIO-enhanced MRI (Resovist, 0.2 mL/kg, 12-h delay). Among 112 harvested lymph nodes, Hematoxylin and Eosin staining, Prussian Blue staining, and VEGF-C immunohistochemistry were performed. The Percentage of Signal Intensity Loss (PSIL) was calculated on T2*-weighted images. The primary diagnostic analysis was patient-level using mean PSIL with receiver operating characteristic analysis. Of 112 lymph nodes from 60 patients, 47 were metastatic and 65 reactive hyperplastic. Reactive nodes showed marked signal attenuation (mean PSIL 64.7 ± 12.0%) compared with metastatic nodes (11.6 ± 7.8%; p < 0.001). Patient-level ROC analysis yielded an area under the curve of 0.84 (95% CI: 0.73-0.95). At a data-derived optimal cutoff of 42% (Youden index), sensitivity was 83%, specificity 85%, positive predictive value 80.0%, negative predictive value 87.2%, and overall diagnostic accuracy 84.0%. Histopathological Prussian Blue grading supported differential iron uptake patterns between groups. High VEGF-C expression (≥++) was more prevalent in metastatic than hyperplastic nodes (85.1% vs. 12.3%; p < 0.001). PSIL showed a moderate inverse correlation with VEGF-C grade (Spearman r = -0.67; p < 0.001). SPIO-enhanced MRI demonstrated favorable diagnostic performance for differentiating metastatic from reactive hyperplastic lymph nodes at the patient level. The imaging characteristics were associated with supportive histopathological findings and VEGF-C expression patterns. These findings warrant validation in larger, multicenter studies using currently available iron oxide agents.

To determine the prevalence of erythroid dysplasia and ring sideroblasts (RS) after peripheral blood stem cell transplantation (PBSCT), stratified by transplant type, and to assess the association with SF3B1 mutations and event-free survival (EFS). In a retrospective single-center study, 35 adult patients undergoing PBSCT (10 haploidentical, 25 HLA-identical/compatible) were compared to 10 controls without myeloid malignancy. Bone marrow smears collected≥25days post-transplant (median 82days) were analyzed for erythroid dysplasia (≥10 %) and RS (≥5 %) using Modified Wright and Prussian blue stains. SF3B1 mutation status was assessed via NGS. EFS was calculated using Kaplan-Meier analysis; multivariate Cox regression evaluated the predictors ofEFS. Erythroid dysplasia was significantly more common in PBSCT recipients than controls (p=0.011), markedly so in haploidentical recipients (8/10, 80 %) vs. HLA-identical/compatible (8/25, 32 %) and controls (2/10, 20 %). RS≥5 % occurred in 50 % of haploidentical PBSCT, 16 % of HLA-identical/compatible PBSCT, and 0 % of controls (p=0.015). However, no pathogenic SF3B1 mutations were detected. In multivariate analysis, only haploidentical transplant type (HR 10.2, p=0.016) and age (HR 1.11 per year, p=0.033), but not ring sideroblastosis or erythroid dysplasia, were independent predictors ofEFS. Erythroid dysplasia and RS are significantly more frequent after haploidentical PBSCT, yet in the absence of SF3B1 mutations and without independent prognostic significance, they likely represent reactive marrow changes rather than clonal disease. Transplant modality and patient age remain key determinants of post-transplant outcome.

To investigate the molecular mechanism by which miR-24-3p promotes spinal cord injury (SCI) repair in rats. The changes in spinal cord miR-24-3p expression was detected in a SD rat model of SCI with qRT-PCR. Using a stereotaxic apparatus, miR-24-3p agomir or a negative control reagent was microinjected at 3 mm rostral and caudal to the SCI epicenter of the rats. Motor function recovery of the SCI rats was evaluated with BBB scores, histopathological changes and Fe²⁺ content in the SCI area were examined with HE staining and a commercial assay kit, and the changes in iron deposition in the SCI area was observed using Prussian blue staining with DAB. Western blotting and immunofluorescence staining were used to detect expressions of glycogen synthase kinase-3β (GSK-3β) and ferroptosis-related proteins xCT and GPX4 in the injured tissue. The targeted regulatory relationship between miR-24-3p and GSK-3β was verified using dual-luciferase reporter assay. In PC12 cells with erastin-induced ferroptosis, malondialdehyde (MDA) content was detected and the expression levels of GSK-3β, xCT and GPX4 proteins were determined by Western blotting and immunofluorescence staining. The expression of miR-24-3p and protein levels of xCT and GPX4 were significantly decreased in the SCI area of the rat models. Treatment with miR-24-3p agomir significantly improved hindlimb motor function of the SCI rats, alleviated spinal cord pathologies, reduced Fe²⁺ content, iron deposition and GSK-3β protein expression, and upregulated xCT and GPX4 protein expressions in the SCI area. Dual-luciferase reporter assay confirmed targeted inhibition of GSK-3β by miR-24-3p. In erastin-induced PC12 cells, transfection with miR-24-3p mimics significantly decreased intracellular MDA content and GSK-3β protein expression and increased xCT and GPX4 protein levels, and these effects were enhanced by co-transfection with GSK-3β inhibitor. miR-24-3p promotes repair of SCI in rats by inhibiting ferroptosis via targeted suppression of GSK-3β.

Bone marrow iron staining is traditionally used to assess iron stores and identify ring sideroblasts (RS), a key feature of sideroblastic anaemia and historically important in the classification of myelodysplastic and myelodysplastic/myeloproliferative overlap neoplasms. With the adoption of the WHO 2022 classification and increasing availability of next-generation sequencing (NGS), the diagnostic relevance of routine iron staining warrants re-evaluation. We retrospectively analysed 101 consecutive adult diagnostic bone marrow examinations (51 myeloid, 50 lymphoid) performed at a regional Australian hospital between January 2023 and June 2024. Perls' Prussian blue staining was routinely performed. Ferritin was available in 76% of cases, and molecular testing in 35% of cases. Two independent reviewers graded bone marrow iron using the Gale score and enumerated RS. Diagnostic impact was categorised as either no impact, supportive but non-essential, or potential diagnostic miss. Interobserver agreement, correlation with serum ferritin, and diagnostic performance for iron deficiency were evaluated. Iron staining had no diagnostic impact in 94% of cases and was supportive but non-essential in 6%; omission would not have altered the final diagnosis in any case. RS ≥ 15% were identified in seven myeloid cases, including MDS, AML, and therapy-related myeloid neoplasms. SF3B1 mutations were present in three RS-positive cases; RS presence did not independently determine diagnosis. Interobserver agreement for iron grading was good (weighted κ = 0.77, 95% CI 0.64-0.89). Correlation between marrow iron grade and serum ferritin was weak (r ≈ 0.38). Low marrow iron grade (≤ 1) showed variable sensitivity (33% to 67%) but high specificity (> 90%) for iron deficiency. Routine bone marrow iron staining rarely influences diagnostic classification in contemporary practice. RS morphology is supportive but not determinative, and marrow iron grading correlates poorly with systemic iron status. These data support a targeted rather than routine approach to marrow iron staining, particularly where NGS is available and routinely performed in de novo diagnoses.

Push pin induced artifact: A mimic of iron pill injury in gastrointestinal pathology.

MiR-135b-5p relieves sevoflurane-induced postoperative cognitive dysfunction by inhibiting JAK2-STAT3-mediated hepcidin upregulation.

ABSTRACTKufor‐Rakeb Syndrome (KRS) is a rare neurodegenerative disease caused by homozygous mutations in the ATP13A2 gene. The ATP13A2 protein, found in lysosomal and late‐endosomal membranes, performs cellular functions such as iron‐chelating agent transport and intracellular iron homeostasis. Mutations in ATP13A2 can lead to intracellular iron accumulation; however, whether KRS caused by an ATP13A2 mutation falls under Neurodegeneration with Brain Iron Accumulation disorders has long been debated. The most fundamental reason is that magnetic resonance imaging (MRI) cannot identify iron deposits in the basal ganglia in all KRS cases. We hypothesize that analyzing iron deposition at the cellular level could be more sensitive in detecting varying levels of iron accumulation associated with different ATP13A2 mutations, and it may be more useful when conventional MRI fails to detect iron, yields inconclusive results, or cannot be performed. We identified two new ATP13A2 mutations (p.Leu518_Thr519del, and p.Leu939Pro) in this study and comparatively investigated the impacts of three distinct ATP13A2 mutations (p.Pro474fs, p.Leu518_Thr519del, and p.Leu939Pro) using KRS patients' primary fibroblasts and MCF7 cells overexpressing these mutated ATP13A2 proteins to analyze if these different mutations of ATP13A2 can cause differing levels of iron accumulation. Following the detection of iron deposits via Prussian blue staining and inductively coupled plasma mass spectrometry, the cell viability was assessed via MTT assay to ascertain the impact of iron accumulation. Each type of ATP13A2 mutation led to iron accumulation; however, frameshift and deletion mutations resulted in more iron accumulation than the missense mutation. In addition, the transient overexpression of the wild‐type ATP13A2 attenuated the cell death caused by iron accumulation. This study demonstrated that different types of ATP13A2 mutations are related to varying levels of iron accumulation and provided an explanation for the inconsistent perspectives on the association of KRS with iron accumulation.

Objective: To evaluate the utility of synovial iron quantification using Magnetic resonance imaging (MRI) in assessing structural joint damage in the knee of patients with rheumatoid arthritis (RA). Methods: This cross-sectional study employed a two-stage design. In the initial comparative stage, 6 patients with RA and 5 patients with osteoarthritis (OA) were recruited to compare synovial R2* values, a metric derived from iterative decomposition of water and fat with echo asymmetry and least-squares estimation quantitation (IDEAL-IQ) MRI sequences representing synovial iron content. Following this, the RA cohort was expanded to a total of 51 patients to investigate the association between R2* values and clinical parameters, including disease activity and bone erosion. Synovial fluid iron levels were measured with an Iron Assay Kit and synovial iron deposits were semi-quantified via Prussian blue staining. Associations between R2* and clinical and laboratory parameters, including inflammatory factors and joint damage indices, were analyzed using Spearman's rank correlation. Univariate and multivariate ordered logistic regression models were employed to identify factors associated with bone erosion severity. An R2*-based nomogram was developed and validated using receiver operating characteristic (ROC) analysis and calibration curves. Results: Synovial R2* values were significantly higher in RA patients than those with osteoarthritis (53.66 S-1 vs. 31.38 S-1, p < 0.05), consistent with Prussian blue staining results. While synovial R2* values showed no significant correlation with systemic iron metabolic markers, inflammatory indicators, or the Disease Activity Score 28, they were positively correlated with bone erosion severity (ρ = 0.500, p < 0.001) and negatively associated with the joint space width (ρ = -0.307, p < 0.05). Multivariate analysis identified R2* as an independent indicator linked to bone erosion extent (OR = 2358.336, p < 0.001). The R2*-based nomogram demonstrated good discriminative performance. (AUC = 0.83). Conclusions: The R2* value derived from IDEAL-IQ MRI is a reliable tool for quantifying synovial iron and may represent a promising non-invasive imaging biomarker reflecting bone erosion in RA patients.

To investigate the mechanism of Yiyuan moxibustion in improving lipid peroxidation and urine retention in spinal cord injury (SCI) rats by regulating ferroptosis via the nuclear factor erythroid 2-related factor 2 (NRF2)/glutathione peroxidase 4 (GPX4) pathway. Forty-eight female Wistar rats were randomly divided into sham operation, model, moxibustion and moxibustion + inhibitor (combination) groups, with 12 rats in each group. SCI induced urinary retention model was established by using Allen's method. Moxibustion was applied to "Shenque" (CV8), "Guanyuan"(CV4) and "Zhongji" (CV3) for 15 min, once daily for 14 d. Rats in the combination group received moxibustion combined with intraperitoneal injection of the NRF2 inhibitor ML385 (30 mg/kg) once daily for 14 consecutive days. Urodynamic analysis was used to record bladder leak point pressure, maximum bladder volume, and bladder compliance to evaluate bladder function. H.E. staining was used to observe morphological changes in spinal cord tissue. Prussian blue staining was used to observe iron deposition in spinal cord tissue. Transmission electron microscopy (TEM) was used to observe mitochondrial morphological changes in spinal cord tissue. Colorimetric assays were used to measure serum Fe2+, malondialdehyde (MDA), and reduced glutathione (GSH) levels. Western blot was used to detect the protein expressions of NRF2, GPX4, and solute carrier family 7 member 11 (SLC7A11) in spinal cord tissue. Rats in the model group had disrupted spinal architecture, vacuolated neurons, swollen mitochondria with lost cristae, and brownish iron deposits, which were relatively milder in the moxibustion group. Compared with the sham operation group, the bladder leak point pressure, serum GSH content, and expression levels of NRF2, SLC7A11, and GPX4 proteins in spinal cord tissue were all significantly reduced (P<0.01), while the maximum bladder volume and bladder compliance, as well as the serum contents of Fe2+ and MDA, were significantly increased (P<0.01) in the model group. When compared to the model group and the combination group, the moxibustion group exhibited a decrease in maximum bladder volume, bladder compliance, serum Fe2+ and MDA content (P<0.01), but an increase in bladder leak point pressure, GSH content, and the expression of NRF2, SLC7A11, and GPX4 proteins (P<0.01, P<0.05). Yiyuan moxibustion may alleviate lipid peroxidation caused by ferroptosis in spinal neurons by up-regulating the NRF2/GPX4 pathway, thereby promoting the recovery of neural regulation of bladder function and improving urine retention in SCI rats.

Tripterygium glycosides tablets (TGT) are effective against autoimmune diseases but cause significant drug-induced liver injury (DILI) that limits clinical use. While TGT disrupts hepatic iron-lipid homeostasis and co-administration with Total Glucosides of Peony (TGP) mitigates its toxicity, the multi-cellular mechanisms remain unclear. In the current study, using integrative single-cell RNA sequencing and pathological validation in controlled mouse models (TGT vs.Con and TGT + TGP vs. TGT.) we elucidated a pathogenic iron-lipid axis driving hepatotoxicity via cellular cascades. TGT initiated Kupffer cell M1 polarization, releasing pro-inflammatory cytokines (TNF-α and IL-1β) that recruited neutrophils and induced NETosis-mediated oxidative stress. Concurrently, hepatic endothelial cells developed iron overload with increased Hamp and decreased Slc40a1, alongside inflammatory damage, while hepatocytes exhibited fatty acid metabolic dysfunction and lipid peroxidation, collectively propagating adipocyte hyperplasia and perilipin-2-driven lipid accumulation. Critically, TGP rescued toxicity by reversing Kupffer cell M1-to-M2 polarization with decreased iNOS and increased CD206, suppressing NET formation with decreased Ly6G and CitH3, alleviating iron deposition with reduced Prussian blue staining, and normalizing lipid metabolism with decreased oil red O staining and perilipin-2. This study identifies the iron-lipid axis as the central driver of TGT-induced liver injury, which is mediated by a sequential multi-cellular cascade involving Kupffer cells, neutrophils, endothelial cells, hepatocytes, and adipocytes. These findings position TGP as a multi-target detoxification agent that mechanistically disrupts this axis and establishes a cellular hierarchy blueprint for metabolic toxicity intervention, supporting its potential as a rescue strategy for precision medicine and detoxification screening.

ObjectivesStroke in sickle cell disease (SCD) is often attributed to large vessel involvement in the disorder, whereas the contribution of cerebral microvascular disease has been less explored. In this study, we investigated the formation of cerebral microvascular lesions and the involvement of mast cells in a humanized SCD mouse model.MethodsWe studied hemorrhagic microvascular disease in a well-characterized mouse model of humanized transgenic sickle (HbSS-BERK) expressing >99% human sickle hemoglobin (HbS) and a control (HbAA-BERK) mouse model expressing normal human hemoglobin A (HbA). Mouse brains were analyzed by Prussian blue staining to detect cerebral microhemorrhage (CMH) formation. Mast cell identification was performed by toluidine blue staining.ResultsSCD brain sections exhibited approximately 86% more CMH than controls (mean ± SE of 1.17 ± 0.22 vs. 0.63 ± 0.13 number/cm2, P = .02). Mast cells were positively correlated with CMH number in SCD mice (Spearman r = 0.42, P < .05), but not in control mice.ConclusionSCD mice demonstrated significantly increased CMH load compared with control mice, and SCD microhemorrhages were associated with the number of mast cells. These findings highlight the significance of cerebral microvascular disease in SCD and imply that cerebral mast cells may be a novel therapeutic target in SCD.

Hepatic encephalopathy is a severe complication of liver failure, traditionally investigated through brain-liver interactions; however, the involvement of extrahepatic organs remains poorly understood. This study examined splenic histopathological changes in a mouse model of acute hepatic encephalopathy induced by ammonium acetate administration, focusing on iron metabolism and macrophage activation. Although conventional hematoxylin and eosin staining revealed no overt structural abnormalities, unstained spleen sections demonstrated abundant black deposits, predominantly in the red pulp. Prussian blue staining confirmed a significant increase in hemosiderin-positive cells; however, a subset of black deposits was iron-negative. Immunohistochemical analyses revealed that these iron-negative pigments were localized within F4/80-positive macrophages and colocalized with heme oxygenase-1 (HO-1), suggesting enhanced heme degradation. Ultrastructural observations further identified electron-dense granules consistent with hematin accumulation in splenic macrophages. Hematological analyses revealed significant reductions in red blood cell count and hemoglobin levels, indicating accelerated erythrocyte destruction. Collectively, these findings demonstrate that acute hepatic encephalopathy induces splenic macrophage activation, accompanied by disordered iron metabolism and hematin accumulation. This study highlights the spleen as a previously underappreciated extrahepatic organ involved in the pathophysiology of hepatic encephalopathy and suggests that splenic heme-iron handling may represent a novel therapeutic target.