Prussian Blue Staining Research Articles

Electroacupuncture Inhibits Ferroptosis by Modulating Iron Metabolism and Oxidative Stress to Alleviate Cerebral Ischemia–Reperfusion Injury

Ischemic stroke (IS) is one of the leading causes of mortality and long-term disability worldwide. Electroacupuncture (EA) is commonly used in the treatment of IS, meaning that may reduce cerebral ischemia–reperfusion injury (CIRI). The middle cerebral artery occlusion/reperfusion (MCAO/R) rat models were created by the modified Zea Longa suture method. EA treatment was performed for 7 consecutive days at the acupoints Neiguan (PC6), Shuigou (GV26), and Sanyinjiao (SP6). The neurological function was assessed using the Zausinger six-point neurological deficiency score. The cerebral infarct volume was detected by 2,3,5-triphenyl tetrazolium chloride (TTC) staining. Hematoxylin and eosin (HE) staining was employed to observe the pathological changes in brain tissues. Prussian blue staining was employed to investigate iron deposition within the brain tissues. Transmission electron microscopy (TEM) was utilized to examine the morphological characteristics of mitochondria. Simultaneously, flow cytometry was utilized to detect the fluorescence intensity of reactive oxygen species (ROS). Assay kits were employed to measure the levels of Fe2+ and glutathione (GSH). Additionally, western blot (WB) and real-time quantitative polymerase chain reaction (RT-qPCR) assays were performed to evaluate the expression levels of proteins associated with ferroptosis. Compared with the MCAO/R group, both the MCAO/R + EA and MCAO/R + DFO groups exhibited significant improvements in neurological function following cerebral ischemia–reperfusion (CIR), attenuated the pathological brain tissue injury, and reduced the cerebral infarct volume and iron deposition in brain tissue. Furthermore, both the MCAO/R + EA and MCAO/R + DFO groups displayed a marked reduction in mitochondrial injury. There was a substantial decrease in Fe2+ and ROS levels, accompanied by a notable increase in GSH level and glutathione peroxidase 4 (GPX4) activity. Compared with the MCAO/R group, the levels of ferroportin1 (FPN1) protein and mRNA expression were significantly increased in the MCAO/R + EA and MCAO/R + DFO groups, and the expression levels of transferrin (TF), transferrin receptor 1 (TFR1), divalent metal transporter 1 (DMT1) protein and mRNA, as well as ferritin (FER) protein, were significantly decreased. EA inhibits ferroptosis by modulating iron metabolism and oxidative stress to alleviate CIRI, exerting neuroprotective effects.Graphical

Caffeic Acid-Biogenic Amine Complexes Outperform Standard Drugs in Reducing Toxicity: Insights from In Vivo Iron Chelation Studies.

Iron homeostasis imbalance, caused by conditions such as thalassemia, sickle cell anemia, and myocardial infarction, often results in elevated free iron levels, leading to ferroptosis and severe organ damage. While current iron chelators like deferoxamine (DFO) and deferiprone are effective, they are associated with significant side effects, including nephrotoxicity, gastrointestinal bleeding, and liver fibrosis. This creates an urgent need for safer, natural-product-based alternatives for effective iron chelation therapy (ICT). This study investigates caffeic acid (CA)-based complexes with biogenic amines, specifically spermine (CA-Sp) and histidine (CA-His), as potential ICT candidates. Initial in vitro assays on HEK-293 cells under iron dextran (ID)-induced toxicity have demonstrated their protective effects, with CA-Sp exhibiting superior efficacy. The in vivo studies in mice have further validated their potential, showing remarkable iron chelation and toxicity mitigation compared to DFO. Inductively coupled plasma mass spectrometry (ICP-MS) reveals significant iron excretion in fecal matter in the treatment group along with reductions in serum ferritin levels. The markers of nephrotoxicity (creatinine) and liver function (ALT, AST) have also been shown to be normalized in treated groups, while immunological analyses have revealed restored levels of neutrophils, T cells, and B cells. Additionally, the inflammatory cytokines, TNF-α and IL-6, have exhibited significant reductions, with the CA-based formulations surpassing the effects of DFO. Histological analyses using Prussian blue staining have further confirmed reduced iron deposition in vital organs such as the liver, kidney, and spleen. These findings highlight CA-Sp as a particularly promising candidate for ICT, offering a safer and more effective strategy for managing iron overload and its associated complications.

Ability of SPP1 to Alleviate Post-Intracerebral Hemorrhage Ferroptosis via Nrf2/HO1 Pathway.

This study aimed to investigate the role of secretory phosphoprotein 1 (SPP1/OPN) in modulating iron-induced cell death (ferroptosis) following intracerebral hemorrhage (ICH). By integrating transcriptomic analysis and experimental validation, we sought to identify key molecular pathways and therapeutic targets associated with ferroptosis in ICH. The Gene Expression Omnibus Series GSE24265 dataset was analyzed using the limma package (R platform) to identify differentially expressed genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) enrichment analyses were performed to elucidate biological functions. Genes associated with iron-induced mortality were identified by cross-referencing transcriptomic profiles with the FerrDb database. A protein-protein interaction network was constructed using Cytoscape, and hub genes were identified. An experimental ICH model was developed in mice using stereotactic instrumentation, and the effects of OPN administration were evaluated through neurological assessments, biochemical assays (superoxide dismutase, glutathione, malondialdehyde), Western immunoblotting (GPX4, ACSL4), Prussian blue histochemistry, and electron microscopy. Transcriptomic analysis identified 27 hub genes, with CD44 and ITGB3 characterized as receptors for OPN. In the ICH model, OPN administration improved neurological outcomes, elevated antioxidant markers, and reduced lipid peroxidation. OPN upregulated GPX4 while suppressing ACSL4, indicating anti-ferroptotic effects. These protective effects were mediated through the Nrf2 pathway, as confirmed by inhibitor ML385. Prussian blue staining and electron microscopy demonstrated reduced cerebral iron deposition and mitochondrial damage following OPN treatment. This study provides novel evidence for SPP1/OPN as a key modulator of ferroptosis in ICH, highlighting its potential as a therapeutic target. By enhancing iron homeostasis and mitigating oxidative stress, OPN offers a promising strategy for improving outcomes in ICH patients.

Neuroprotective effects of bone marrow mesenchymal stem cells combined with mannitol on radiation-induced brain injury by regulating autophagy via the PI3K/AKT/mTOR signaling pathway.

Neuroprotective effects of bone marrow mesenchymal stem cells combined with mannitol on radiation-induced brain injury by regulating autophagy via the PI3K/AKT/mTOR signaling pathway.

The ferroptosis of sertoli cells inducing blood-testis barrier damage is produced by oxidative stress in cryptorchidism.

The ferroptosis of sertoli cells inducing blood-testis barrier damage is produced by oxidative stress in cryptorchidism.

Vicenin-2 in Suhuang antitussive capsule attenuates mitophagy-dependent ferroptosis via LRP1 for treating post-infectious cough.

Vicenin-2 in Suhuang antitussive capsule attenuates mitophagy-dependent ferroptosis via LRP1 for treating post-infectious cough.

Hemosiderin quantification in hemophilic arthropathy using quantitative magnetic resonance imaging

The goal of this study is to quantify hemosiderin deposition in the knee joint tissues of hemophilic arthropathy (HA) patients using quantitative susceptibility mapping on MRI. Knee synovial tissues from HA patients and controls without hemophilia were included. The tissues underwent ultrashort echo time quantitative susceptibility mapping (UTE-QSM) and clinical MRI. HA tissues were processed histologically with Perl’s Prussian Blue (PPB) staining to identify iron contents. Seven regions of interest were drawn in each tissue, and the susceptibility values were tested. Moreover, the association between the estimated magnetic susceptibility and the iron contents quantified by histology was investigated. Nine synovial tissues were procured from total knee arthroplasty of hemophilia patients (males, 40.8 ± 9.0 years), and three synovial tissues were harvested from cadaveric knee joints of donors without hemophilia as controls (males, 72.0 ± 12.8 years). The estimated susceptibility values (ESVs) showed significant differences between HA and control samples. Accordingly, HA tissues presented a mean ESV of 0.48 ± 1.08 ppm and control tissues of -0.13 ± 0.12 ppm (p < 0.05). A significant linear correlation was found between the iron level quantified by histology (PPB stain) and the ESV estimated by UTE-QSM (R = 0.908, p < 0.01). There was a significant difference in the susceptibility in high load (HL) tissues compared to low load (LL) tissues (ESV = 5.57 ± 1.23 ppm for HL vs. 0.57 ± 0.85 ppm for LL, p < 0.001). Reliable hemosiderin quantification in joint tissues of HA patients can be achieved using MRI based on quantitative susceptibility mapping.

Electroacupuncture Ameliorates Chronic Inflammatory Pain and Depression Comorbidity by Inhibiting Nrf2-Mediated Ferroptosis in Hippocampal Neurons.

Chronic inflammatory pain and depression are highly comorbid, with nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated ferroptosis in hippocampal neurons strongly associated with the onset and progression of the comorbidity. Electroacupuncture (EA), widely used to treat pain and mood disorders, may ameliorate chronic inflammatory pain and depression comorbidity (CIPDC) by inhibiting Nrf2-mediated ferroptosis in hippocampal neurons, though its mechanism of action remains partially understood. In this study, we established the CIPDC model by administering a subcutaneous injection of complete Freund's adjuvant (CFA) into the left hind paw. Evaluations of EA's effects on pain thresholds and depressive behaviors in CIPDC rats included paw withdrawal mechanical threshold, paw withdrawal thermal latency, sucrose preference test, open field test, and forced swim test assessments. HE staining was performed to assess the pathological and morphological alterations in hippocampal neurons. FJB staining was utilized to evaluate neuronal degeneration, while transmission electron microscopy (TEM) was employed to examine ultrastructural changes in hippocampal neuronal mitochondria. Prussian blue staining was conducted to visualize ferrous ion deposition in the hippocampus, andthe contents of ferrous ion (Fe2+), malondialdehyde (MDA), and glutathione (GSH)were measured using colorimetric assay kits. Western blotting (WB) was performed to determine the relative protein expression of Nrf2, FTH1, FTL, xCT, GPX4, ACSL4, LPCAT3, and LOX in the hippocampus. Additionally, the relative mRNA expression of FTH1, FTL, xCT, GPX4, ACSL4, LPCAT3, and LOX was analyzed by PCR. Flow cytometry was used to quantify ROS levels in the hippocampus, and immunofluorescence staining was applied to detect nuclear expression of Nrf2 as well as co-localization of GPX4 with the neuronal marker NeuN. Our results demonstrate that EA upregulates nuclear Nrf2 expression in hippocampal tissue, thereby alleviating iron metabolism dysregulation, enhancing antioxidant system activity, and reducing lipid peroxidation. This process inhibits ferroptosis in hippocampal neurons, promoting their repair and remodeling, and effectively treating CIPDC.

Abstract 2526: Development of ultrahigh sensitivity magnetic particle imaging for stem cell tracking

Abstract In vivo tracking of stem cells is critical to stem cell transplantation and regenerative medicine research. Achieving long-term, non-invasive imaging of transplanted cells demands highly sensitive imaging technology. Magnetic particle imaging (MPI) is an emerging modality capable of visualizing the distribution of superparamagnetic iron oxide nanoparticles (SPIOs). Previous studies have reported that MPI can detect clusters of approximately 2500 SPIO-labeled stem cells. However, more than this sensitivity level is needed to monitor the survival, migration, differentiation, and regeneration of stem cells. To address this limitation, we developed an ultrahigh-sensitivity magnetic particle imaging (US-MPI) device capable of detecting stem cells at scales of hundreds of cells. Through full-chain design and optimization, US-MPI maximizes the capture of SPIO signals from stem cells. Using a commercial SPIO (VivoTrax), mouse mesenchymal stem cells were labeled, and samples with varying cell numbers (ranging from 50 to 1×107) were tested using US-MPI and 9.4 T MRI (uMR 9.4T, United Imaging Life Science Instrument, Wuhan, China) for comparison. US-MPI demonstrated the ability to detect as few as 50 labeled stem cells in vitro and 150 cells in vivo, whereas 9.4 T MRI required at least 1×105 cells for detection in vitro. Thus, US-MPI exhibited a sensitivity 2, 000 times higher than that of 9.4 T MRI. The efficacy of US-MPI for stem cell tracking was further validated in a lipopolysaccharide (LPS)-induced mouse hind limb inflammation model. Eight hours after SPIO-labeled stem cells were injected intravenously, US-MPI detected strong signals on the inflamed limb, which remained significantly higher than those of the healthy limb during a 48-hour monitoring period. Post-mortem Prussian blue staining of inflamed tissues confirmed the presence of blue-stained SPIO particles, verifying that the US-MPI signals originated from labeled stem cells. These results highlight the ultrahigh sensitivity of US-MPI for stem cell tracking, providing a valuable tool for advancing stem cell research. Citation Format: Yimeng Li, Yu An, Yang Du, Lin Shen, Jie Tian. Development of ultrahigh sensitivity magnetic particle imaging for stem cell tracking [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2526.

Bulk RNA-seq conjoined with ScRNA-seq analysis reveals the molecular characteristics of nucleus pulposus cell ferroptosis in rat aging intervertebral discs

ObjectiveRecently, several studies have reported that nucleus pulposus (NP) cell ferroptosis plays a key role in IDD. However, the characteristics and molecular mechanisms of cell subsets involved remain unclear. We aimed to define the key factors driving ferroptosis, and the characteristics of ferroptotic NP cells subsets during IDD.MethodsThe accumulation of iron ions in NP tissues of rats caudal intervertebral discs (IVDs) was determined by Prussian blue staining. Fluorescent probe Undecanoyl Boron Dipyrromethene (C11-BODIPY) and lipid peroxidation product 4-Hydroxynonenal (4-HNE) staining were performed to assess lipid peroxidation level of NP cells. The differentially expressed genes in NP tissues with aging were overlapped with FerrDB database to screen ferroptosis driving genes associated with aging-related IDD. In addition, single cell sequencing (ScRNA-seq) was used to map the NP cells, and further identify ferroptotic NP cell subsets, as well as their crucial drivers. Finally, cluster analysis was performed to identify the marker genes of ferroptotic NP cells.ResultsHistological staining showed that, compared with 10 months old (10M-old) group, the accumulation of iron ions increased in NP tissues of 20 months old (20M-old) rats, and the level of lipid peroxidation was also enhanced. 15 ferroptosis driving factors related to IDD were selected by cross-enrichment. ScRNA-seq identified 14 subsets in NP tissue cells, among which the number and ratio of 5 subsets was reduced, and the intracellular ferroptosis related signaling pathways were significantly enriched, accompanied by enhanced cell lipid peroxidation. Notably, ranking the up-regulation fold of ferroptosis related genes, we found Atf3 was always present within TOP2 of these five cell subsets, suggests it is the key driving factor in NP cell ferroptosis. Finally, cluster cross-enrichment and fluorescence colocalization analysis revealed that Rps6 +/Cxcl1- was a common molecular feature among the 5 ferroptotic NP cell subsets.ConclusionsThis study reveals that ATF3 is a key driver of NP cell ferroptosis during IDD, and Rps6 +/Cxcl1- is a common molecular feature of ferroptotic NP cell subsets. These findings provide evidence and theoretical support for subsequent targeted intervention of NP cell ferroptosis, as well as provide directions for preventing and delaying IDD.

Nigrostriatal iron accumulation in the progression of Parkinson’s disease

Iron deposition in the nigrostriatal system plays a pivotal role in Parkinson’s disease (PD) onset and progression. This study explored the time course of nigrostriatal iron accumulation in 54 PD patients at early to moderately advanced stages and 20 age-matched healthy controls. Using multi-echo T2*-MRI and R2* relaxometry, iron content was assessed in the substantia nigra pars compacta (SNpc) and striatum. In vivo findings were contrasted with histological analyses in a progressive 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced parkinsonism model involving six non-human primates (NHPs) and two controls using Perls’ Prussian blue staining. Complementarily, dopaminergic degeneration was quantified by 6-[18F]-fluoro-L-dopa PET in humans and TH immunohistochemistry in NHPs. Results showed progressive iron accumulation in the SNpc correlating with striatal dopaminergic denervation and neuronal loss. Striatal iron followed a V-shaped progression, decreasing initially and increasing later. Iron in the SNpc may serve as a marker of neurodegeneration in PD, while decreased striatal iron may indicate pathological susceptibility to dopaminergic loss.

Treatment with Manganese Porphyrin, MnTnBuOE-2-PyP5+, Suppressed the Activation of Macrophages in a Mouse Intracerebral Hemorrhage.

Background: Manganese porphyrin, MnTnBuOE-2-PyP5+ (BMX-001), improves neurologic deficits in experimental ischemic stroke and has the potential to serve as an adjunct with thrombolysis or thrombectomy in stroke patients. In 10-30% of stroke patients following thrombolysis, the hemorrhagic transformation, associated with iron release, occurs. This study aimed to examine the neurologic outcome following the BMX-001 treatment in a mouse intracerebral hemorrhage (ICH) model with relevance to prospective ischemic stroke clinical trials. Methods: Twenty C57Bl6 mice were randomly assigned to groups after surgery and received vehicle or BMX-001 treatment immediately following stereotaxic left striatum collagenase injection. Post-ICH body weight, the Corner test, neurological deficit score, and Rotarod test were examined. Six sham surgery mice serve as a control group. At 72 h, the brain histological evaluation was performed, including hemorrhage size, Prussian blue staining, and the activation of macrophages. Data were collected by a researcher who was blind to groups. Results: No significant difference in body weight, neurological deficits, and hemorrhage size was found between groups. However, BMX-001 reduced the number of macrophages in the hemorrhagic area (48 ± 10 in vehicle, 33 ± 8 in BMX-001, p = 0.008) and the number of cells stained with Prussian blue-an indicator of iron released during hemorrhage (65 ± 22 in vehicle and 41 ± 15 in BMX-001, p = 0.027). Conclusions: The results support the safe use of BMX-001 in stroke patients in combination with thrombolysis or thrombectomy and, moreover, indicate the beneficial anti-inflammatory effect of BMX-001, alike to that previously reported in stroke studies of analogous, similarly redox-active, Mn porphyrins.

Panaxadiol Attenuates Brain Damage by Inhibiting Ferroptosis in a Rat Model of Cerebral Hemorrhage.

Intracerebral hemorrhage (ICH) is the most common subtype of hemorrhage stroke, with a high disability, morbidity and mortality rate globally. Panaxadiol (PD), a triterpenoid sapogenin monomer, is isolated from the roots of ginseng, which has shown a variety of biological properties, such as anti-inflammation, anti-cancer, and neuroprotection. However, its effect and mechanism on ICH were still unknown. Thirty-six rats were randomly divided into six group (n = 6), namely, sham, ICH, ICH + 5 mg/kg PD, ICH + 10 mg/kg PD, ICH + 20 mg/kg PD, and ICH + 10 mg/kg PD + 50 mg/kg vismodegib. Rats were treated with type IV collagenase to induce an in vivo model of ICH, and then intraperitoneally injected with PD (5, 10 and 20 mg/kg) and 50 mg/kg vismodegib (an inhibitor of hedgehog signal). The effect and potential mechanism of PD on ICH were explored by behavioral test, brain water content measurement, Evans blue detection, hematoxylin-eosin (HE) staining, iron level examination, Prussian blue staining, western blot and immunohistochemistry, respectively. An increase in the mNSS (13.17 ± 1.17), and a decrease in the rotarod latency (40.67 ± 9.31), modified Garcia score (9.83 ± 1.47), forelimb use times (3.33 ± 0.82), left forepaw placements (29.90 ± 4.38) and left turns (17.34 ± 3.55) in ICH rats were reversed with the PD treatment (6.83 ± 0.75, 113.5 ± 11.95, 17.50 ± 1.87, 8.17 ± 0.98, 63.56 ± 9.84, and 42.13 ± 4.52 respectively). PD treatment reduced the brain water content (73.13 ± 3.16 vs. 86.82 ± 4.74), the level of Evans blue (2.14 ± 0.25 vs. 4.03 ± 0.20) and cerebral hemorrhage in ICH rats. Also, PD injection decreased the iron level (0.06 ± 0.005 vs. 0.17 ± 0.02) and the expression of ACSL4 (0.56 ± 0.07 vs. 1.23 ± 0.16), with the increased expression of GPX4 (1.14 ± 0.08 vs. 0.21 ± 0.03) in ICH rats. Mechanically, PD treatment restored the decreased expression of SHH (0.96 ± 0.13 vs. 0.20 ± 0.03), GLI1 (0.89 ± 0.13 vs. 0.06 ± 0.007) and PTCH (0.75 ± 0.05 vs. 0.10 ± 0.01) in ICH rats. Inhibition of SHH signaling by vismodegib reversed the ameliorative effect of PD on ICH rats. PD improved brain damage by suppressing ferroptosis via the activation of the SHH/GLI signaling pathway, which could lay a theoretical foundation for the treatment of ICH.

APOE modulates ferroptosis to drive macrophage polarization toward the M2 type and enhance PTC migration and invasion.

APOE modulates ferroptosis to drive macrophage polarization toward the M2 type and enhance PTC migration and invasion.

NLRP3 Mediates NOXs-Induced Iron Overload and Inflammation but Not Oxidative Damage in Colons of DSS-Treated Mice.

Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) with chronic and recurrent characteristics caused by multiple reasons, including iron overload, intestinal inflammation, and barrier dysfunction. Here, we investigated the effects of chemical inhibition of NOXs and NLRP3 activity on colonic iron metabolism and inflammatory reactions in a murine model of dextran sodium sulfate (DSS)-induced ulcerative colitis. The mice were randomly divided into five groups: normal control group, DSS-induced ulcerative colitis model group (DSS), DSS + Dapansutrile group, DSS + Diphenyleneiodonium chloride group, and DSS + Dapansutrile + Diphenyleneiodonium chloride group. On day 14, the mice were euthanized. Tissues were collected and analyzed to determine the effects of chemical inhibition of NOXs and NLRP3 activity on colonic iron metabolism and inflammatory reactions of dextran sodium sulfate-induced ulcerative colitis. Measurements such as weight, disease activity index, HE staining, Prussian blue staining, immunohistochemical and immunofluorescence, ELISA, flow cytometry detection, Western blot, and Quantitative Real-Time PCR were conducted. Chemical inhibition of NOXs and NLRP3 in vivo could significantly reduce colonic iron overload and macrophage infiltration, thus alleviating colonic inflammatory response and tissue damage. Notably, the inhibition of NOXs significantly inhibited the expression of NLRP3 and oxidative damage, but the inhibition of NLRP3had no significant effect on the expression of NOXs and oxidative damage, suggesting NOXs may exert their effects other than oxidative damage through NLRP3. To our knowledge, this work is the first to reveal that NLRP3 mediates NOXs-induced colonic iron overload and inflammation rather than oxidative damage in ulcerative colitis murine model, suggesting that the NOXs might promote ulcerative colitis by inducing colonic iron overload and macrophage infiltration dependent or partially dependent on NLRP3, as well as oxidative damage independent of NLRP3, which imply that both NOXs and NLRP3 are attractive targets for anti-colitis therapy.

Magnetically Labeled iPSC-Derived Extracellular Vesicles Enable MRI/MPI-Guided Regenerative Therapy for Myocardial Infarction.

Stem cell-derived extracellular vesicles (EVs) offer a promising cell-free approach for cardiovascular regenerative medicine. In this study, we developed magnetically labeled induced pluripotent stem cell-derived EVs (magneto-iPSC-EVs) encapsulated with superparamagnetic iron oxide (SPIO) nanoparticles for image-guided regenerative treatment of myocardial infarction, in which EVs that can be detected by both magnetic resonance imaging (MRI) and magnetic particle imaging (MPI). iPSC-EVs were isolated, characterized per MISEV2023 guidelines, and loaded with SuperSPIO20 nanoparticles using optimized electroporation conditions (300 V, 2 × 10 ms pulses), achieving a high loading efficiency of 1.77 ng Fe/10 6 EVs. In vitro results show that magneto-iPSC-EVs can be sensitively detected by MPI and MRI, with a detectability of approximately 10 7 EVs. In a mouse myocardial ischemia-reperfusion model, intramyocardially injected magneto-iPSC-EVs (2 × 10 9 ) were imaged non-invasively by in vivo MPI for 7 days and ex vivo MRI, with the presence of magneto-iPSC-EVs confirmed by Prussian blue staining. Therapeutically, both native and magneto-iPSC-EVs significantly improved cardiac function, with a 37.3% increase in left ventricular ejection fraction and 61.0% reduction in scar size. This study highlights the potential of magneto-iPSC-EVs as a cell-free approach for cardiovascular regenerative medicine, offering both non-invasive imaging capabilities and therapeutic benefits for myocardial repair.

Mitigating Remote Organ-Induced Brain Injury in Renal Ischemia-Reperfusion: The Role of Oleuropein in Inhibiting Oxidative Stress, Inflammation, Ferroptosis, and Apoptosis in Male Rats.

Renal ischemia-reperfusion (RIR) induces brain damage as a distant organ. Oleuropein has antioxidant properties. This study aimed to explore oleuropein's protective effects against brain injury following RIR in rats. Thirty-six male Wistar rats were divided into six groups (n = 6) including sham, oleuropein (200mg/kg), RIR, and RIR groups treated with oleuropein (50, 100, and 200mg/kg). 48h after injury, blood urea nitrogen (BUN) and creatinine levels were surveyed. The western blotting analysis was performed to assay the interleukin-1 beta (IL-1β), IL-10, tumor necrosis factor-alpha (TNF-α), and nuclear factor kappa-light-chain-enhancer of activated B cells p65 (NF-κB p65), Bcl-2 associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), cleaved caspase-3, glutathione peroxidase-4 (GPX4), nuclear factor erythroid-related factor-2 (NRF2), solute carrier family 7, member 11 (SLC7A11), and anti-acyl-CoA synthetase long-chain family 4 (ACSL4) proteins in kidney and/or brain tissues. Also, malondialdehyde (MDA) and total antioxidant capacity (TAC) levels, the activity of GPx, catalase, and superoxide dismutase (SOD) were evaluated. Kidney and brain tissues damage scores (KTDS and BTDS) were determined by H&E staining method. Prussian blue staining was conducted to identify iron accumulation. RIR significantly increased BUN, serum creatinine levels, KTDS, BTDS, iron deposition, MDA concentration, Bax, cleaved caspase-3, IL-1β, TNF-α, NF-κB p65, ACSL4 proteins expression levels, while decreasing TAC content, SOD, GPx, and catalase activity, Bcl-2, GPX4, SLC7A11 and NRF2 proteins expression in kidney and/or brain tissue of RIR group versus the sham (P < 0.05). Moreover, oleuropein attenuated these indicators in the RIR + oleuropein (200mg/kg) group versus the RIR group (P < 0.05). Our study showed that RIR induced brain damage, and oleuropein exhibited protective effects against brain injury induced by RIR, through inhibiting oxidative stress, inflammation, ferroptosis, and apoptosis mechanisms.

IL-19 as a promising theranostic target to reprogram the glioblastoma immunosuppressive microenvironment

BackgroundGlioblastoma multiforme (GBM) is an aggressive brain tumor with chemoresistant, immunosuppressive, and invasive properties. Despite standard therapies, including surgery, radiotherapy, and temozolomide (TMZ) chemotherapy, tumors inevitably recur in the peritumoral region. Targeting GBM-mediated immunosuppressive and invasive properties is a promising strategy to improve clinical outcomes.MethodsWe utilized clinical and genomic data from the Taiwan GBM cohort and The Cancer Genome Atlas (TCGA) to analyze RNA sequencing data from patient tumor samples, determining the association of interleukin-19 (Il-19) expression with survival and immunosuppressive activity. Gene set enrichment analysis (GSEA) was performed to assess the relationship between the enrichment levels of immune subsets and Il-19 expression level, and Ingenuity Pathway Analysis (IPA) was used to predict immune responses. Cytokine array and single-cell RNA sequencing were used to examine the effects of IL-19 blockade on tumor immune microenvironment, including tumor-infiltrating leukocyte profiles, differentiation and immunosuppressive genes expression in tumor associated macrophages (TAM). CRISPR Il-19−/− cell lines and Il-19−/− mice were used to examine the role of IL-19 in tumor invasion and M2-like macrophage-mediated immunosuppression. Additionally, we developed novel cholesterol-polyethylene glycol-superparamagnetic iron oxide-IL-19 antibody nanoparticles (CHOL-PEG-SPIO-IL-19), characterized them using dynamic light scattering and transmission electron microscopy, Fourier-Transform Infrared spectroscopy, prussian blue assay, and conducted in vivo magnetic resonance imaging (MRI) in a human glioblastoma stem cell-derived GBM animal model.ResultGenomic screening and IPA analysis identified IL-19 as a predicted immunosuppressive cytokine in the peritumoral region, associated with poor survival in patients with GBM. Blocking IL-19 significantly inhibited tumor progression of both TMZ-sensitive (TMZ-S) and TMZ-resistant (TMZ-R) GBM-bearing mice, and modulated the immune response within the GBM microenvironment. Single-cell transcriptome analysis reveal that IL-19 antibody treatment led to a marked increase in dendritic cells and monocyte/macrophage subsets associated with interferon-gamma signaling pathways. IL-19 blockade promoted T cell activation and reprogrammed tumor-associated macrophages toward weakened pro-tumoral phenotypes with reduced Arginase 1 expression. Il19−/− M2-like bone marrow-derived macrophages with lower Arginase 1 level lost their ability to suppress CD8 T cell activation. These findings indicated that IL-19 suppression limits TAM-mediated immune suppression. Molecular studies revealed that IL-19 promotes TMZ-resistant GBM cell migration and invasion through a novel IL-19/WISP1 signaling pathway. For clinical translation, we developed a novel CHOL-PEG-SPIO-IL-19 nanoparticles to target IL-19 expression in glioblastoma tissue. MRI imaging demonstrated enhanced targeting efficiency in brain tumors, with in vivo studies showing prominent hypointense areas in T2*-weighted MRI scans of tumor-bearing mice injected with CHOL-PEG-SPIO-IL-19, highlighting nanoparticle presence in IL-19-expressing regions. Prussian blue staining further confirmed the localization of these nanoparticles in tumor tissues, verifying their potential as a diagnostic tool for detecting IL-19 expression in glioblastoma. This system offers a theranostic approach, integrating diagnostic imaging and targeted therapy for IL-19-expressing GBM.ConclusionIL-19 is a promising theranostic target for reversing immunosuppression and restricting the invasive activity of chemoresistant GBM cells.Graphical

Tracking the healing effect of human Wharton’s jelly stem cells labeled with superparamagnetic iron oxide nanoparticles seeded onto polyvinyl alcohol/chitosan/carbon nanotubes in burn wounds by MRI and Prussian blue staining

Regenerative medicine through the application of tissue engineering and cell transplantation has provided a new door for wound healing. In this study, the healing effect of human Wharton's jelly stem cells (WJSCs) labeled with superparamagnetic iron oxide nanoparticles (SPIONs) seeded onto polyvinyl alcohol/chitosan/carbon nanotubes (PVA/CS/CNTs) in burn wounds was investigated by performing magnetic resonance imaging (MRI) and Prussian blue staining. Human WJSCs were prepared from umbilical cord and characterized. PVA/CS/CNTs were fabricated via electrospinning. Forty-eight rats were divided into four groups. The control group underwent a third-degree burn injury and was left untreated. The second group received silver sulfadiazine after burn induction, the third group was treated with PVA/CS/CNTs after burn wounds, and the fourth group received WJSCs labeled with SPIONs seeded onto PVA/CS/CNTs following burn injury. Tensile strength was investigated, real-time polymerase chain reaction was used to evaluate apoptosis, and Prussian blue staining and MRI were performed to trace labeled cells. The mesenchymal properties of WJSCs were characterized. Histologically, healing was observed as complete granulation occurred and epithelial tissues were formed in the absence of inflammatory cells, with increased expression of Bcl-2 and a decrease in Bax genes in the fourth group. Internalization of SPIONs within WJSCs was confirmed by Prussian blue staining and MRI on day 14. WJSCs labeled with SPIONs seeded onto PVA/CS/CNTs could successfully participate in the healing of burn wounds and could be easily tracked by MRI as a noninvasive method, providing a new door in regenerative medicine for burn wounds.

Nrf2 protects against oxidative damage induced by hemoglobin in the liver of grass carp (Ctenopharyngodon idella)

Nrf2 protects against oxidative damage induced by hemoglobin in the liver of grass carp (Ctenopharyngodon idella)